CHRONIC CHLAMYDIA INFECTIONS IN MALES: IMPACTS ON TESTICULAR FUNCTION AND SPERMATOGENESIS
Emily Rose Bryan Bachelor of Biomedical Science (Honours)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
School of Biomedical Sciences
Faculty of Health
Queensland University of Technology
2018
Keywords
Chronic Chlamydia infection, male infertility, testicular infection, Sertoli cells, germ cells, Leydig cells, macrophages, spermatogenesis, sperm, DNA damage, transcriptome, offspring.
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis i
Abstract
Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease worldwide. Recent estimates indicate that 131 million people have genital C. trachomatis infections. This estimate does not account for unreported and asymptomatic infections. A large proportion of infections are asymptomatic; approximately 50% of male and 75% of female infections. Screening programs have largely been targeted at women, leaving men as a potential reservoir of undetected infections. Chronic infections, which are largely uncharacterized, may develop as a result of untreated, asymptomatic colonization.
C. trachomatis causes reproductive tract inflammation and damaging pathology in a large proportion of infections, potentially causing infertility in men and women.
Female models of infection are more frequent than male models of infection. This has led to knowledge gaps in understanding of (i) the male immune response to chlamydial infection, (ii) cell and tissue types that are susceptible to infection and subsequent pathology in the male reproductive tract (MRT), (iii) duration of MRT infection, and (iv) effective prevention and treatment strategies for males. Currently,
C. trachomatis infection has been associated with poor sperm quality, but not necessarily with pathology.
Rodent models of infection revealed that Chlamydia can be found in almost the entire length of the MRT including the urethra, bladder, seminal vesicles, prostate, epididymis, and testes. An acute model of infection showed that sperm immobility and malformation compromised fertility. This was likely the result of specialized
Sertoli cell and spermatogonial stem cell (SSC) death within testicular tissue. Despite
ii Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
the indications that chronic infection may be possible, the pathological effect on the
MRT and on male fertility after this point remained uncharacterized.
In this study, several in vitro and in vivo models were established to produce key pieces of MRT infection information, with a focus on chronic testicular infections.
The hypothesis was that testicular cells are functionally compromised by chlamydial infection. This impairs spermatogenesis, and therefore fertility. Each of the testicular cell lineages have critical roles in spermatogenesis. For example, Sertoli cells form the blood-testis-barrier, SSCs are the lineage from which all sperm are produced,
Leydig cells secrete androgens, and Tmφ are required for maintenance of testicular immune privilege.
In the study presented here, TM3 Leydig, TM4 Sertoli, and GC-1 germ cell lines were productively infected with Chlamydia muridarum. RAW264.7 macrophages, simulating Tmφ, were also susceptible and capable of transmitting infection to the testicular cells. Infection caused DNA fragmentation and hypomethylation within
TM3, TM4, and GC-1 cells. Differential gene expression analysis also revealed up- regulation of interferon-beta mediated pro-inflammatory and WBC chemokine transcripts in TM3 and GC-1 cells, and altered regulation of structural elements during a stress-like response in TM4 cells.
Following intra-penile infection of C57BL/6 mice, rapid dissemination of Chlamydia to the testes occurred via macrophages. Actively replicating Chlamydia was detected at six-months post infection in the testes, indicating a chronic infection had been established. Mice had testicular histological changes including (i) significant loss of
Sertoli cells, tight junctions, and Myoid cells and (ii) significantly increased testicular WBCs. Systemic anti-sperm antibodies were also detected. The testicular
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis iii
abnormalities likely contributed to the significant decreases in sperm motility, normal morphology, oocyte binding capacity, and DNA integrity.
When the chronically infected mice were bred with healthy, C57BL/6 females, the offspring had decreased viability and developmental abnormalities, including having reduced body size and stunted reproductive tract growth. The sperm isolated from the male pups was also abnormal, having reduced sperm count, motility and oocyte binding capacity similar to their infected sires.
The in vivo mouse studies were followed up with a human component. Chlamydia was detected in 43.75% of open testicular biopsies with Sertoli cell only, germ cell arrest, and hypospermatogenesis diagnoses. Chlamydia was also detected in three out of five fresh fine-needle testicular biopsies from current male infertility patients. The biopsies from these patients were aspermic, so IVF was unsuccessful. None of these patients had a history of STI.
This study demonstrates the destructive nature of testicular chlamydial infections in fertility, and the potential for multigenerational effects on male infertility. Although only emerging as an important MRT pathogen, this study highlights the inadequacy of current chlamydial detection methods and the need for alternative treatment options other than the currently available antibiotics. This likely includes the development of a male targeted vaccine. Further investigation into the pathogenic capacity of Chlamydia within the MRT is required, particularly whether treatment can recover fertility. However, this study has provided information that fills major knowledge gaps in the literature and the foundations for further investigation to commence.
iv Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
Table of Contents
Keywords ...... i Abstract ...... ii List of Figures ...... viii List of Tables ...... x List of Abbreviations ...... xi Statement of Original Authorship ...... xiv Acknowledgements ...... xv Chapter 1: Introduction ...... 1 1.1 Background ...... 2 1.2 Hypothesis and Specific Aims of the Study ...... 3 1.3 Significance of the Study ...... 4 Chapter 2: Literature Review ...... 7 2.1 Chlamydial impact on the community ...... 8 2.2 Chlamydial Infections ...... 11 2.2.1 Chlamydial lifecycle and transmission ...... 11 2.2.2 Chlamydial Pathogenesis ...... 13 2.2.3 Chronic and Persistent Infections ...... 17 2.3 The Male Reproductive Tract ...... 18 2.3.1 Testicular cells and structure ...... 20 2.3.2 Leydig cells ...... 20 2.3.3 Sertoli cells ...... 21 2.3.4 Spermatogenic cells and Spermatogenesis ...... 23 2.3.5 Semen and Spermatogenesis Dysregulation ...... 25 2.3.6 Testicular macrophages ...... 28 2.3.7 Chlamydia and macrophages ...... 30 2.4 Male infertility and Chlamydia ...... 33 2.4.1 C. trachomatis and male factor infertility ...... 33 2.4.2 C. muridarum and male rodent infertility ...... 38 2.5 Research challenges ...... 40 2.6 Research Applications ...... 40 Chapter 3: Materials and Methods ...... 43 3.1 Solutions and Buffers ...... 44 3.1.1 Phosphate Buffered Saline (PBS) ...... 44 3.1.2 PBS-Tween (PBST) ...... 44 3.1.3 Tris Buffered Saline (TBS) ...... 44 3.1.4 TBS-TWEEN® (TBST) ...... 45 3.1.5 Sucrose Phosphate Glutamate (SPG) Medium ...... 45 3.1.6 PBS Cocktail ...... 45 3.1.7 Ultravist Gradient ...... 45 3.1.8 Mammalian Cell Lysis Buffer ...... 46 3.1.9 Red Blood Cell (RBC) Lysis Buffer ...... 46 3.1.10 Biggers, Whitters and Whittingham (BWW) Medium ...... 46 3.1.11 Sperm Capacitation Medium ...... 47
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis v
3.1.12 Oocyte Collection and Storage ...... 47 3.1.13 Heat-inactivated Fetal Calf Serum (HI-FCS) / Horse Serum (HI-HS) ...... 47 3.1.14 Immortalised Cell Line Growth Medium ...... 48 3.1.15 Primary Cell Growth Medium ...... 48 3.1.16 Tris Borate Ethylenediaminetetraacetic acid (EDTA) Buffer (TBE) ...... 48 3.2 Cell Culture and Experimentation ...... 48 3.2.1 Immortalised Cell Line Culture ...... 48 3.2.2 Primary Cell Culture ...... 49 3.2.3 Monolayer Co-culture ...... 50 3.2.4 Transwell® Co-culture ...... 51 3.2.5 Live Cell Imaging ...... 51 3.2.6 Comet Assay ...... 51 3.2.7 Transcriptomics ...... 52 3.2.8 Methylation Detection Mass Spectrometry ...... 53 3.2.9 Detection of Testosterone ...... 53 3.3 Chlamydia Culture and Experimentation ...... 54 3.3.1 Chlamydia Bulk-up ...... 54 3.3.2 Semi-purification ...... 54 3.3.3 Ultra-purification ...... 55 3.3.4 Chlamydia Titration ...... 55 3.3.5 Chlamydia Infection ...... 56 3.3.6 Chlamydial extrusion isolation ...... 56 3.3.7 Chlamydial Polymerase Chain Reaction (PCR) ...... 56 3.3.8 Chlamydial Immunocytochemistry (ICC) Staining ...... 57 3.3.9 Chlamydial Immunohistochemistry (IHC)...... 57 3.3.10 Flow Cytometric Isolation and Infection of Testicular Macrophages ...... 60 3.3.11 Chlamydial Progeny Quantification ...... 61 3.3.12 Detection of anti-Chlamydial Antibodies ...... 61 3.4 Animal Experimentation ...... 62 3.4.1 Mice 62 3.4.2 Anaesthesia ...... 62 3.4.3 Intra-penile Infection ...... 63 3.4.4 Cardiac Bleeding and Serum Isolation ...... 63 3.4.5 Penile Tissue Collection ...... 63 3.4.6 Testicular Tissue Collection ...... 64 3.4.7 Epididymis Tissue Collection ...... 64 3.4.8 Vas Deferens Removal ...... 64 3.4.9 Sperm Collection and Analysis ...... 64 3.4.10 Female Reproductive Tract ...... 65 3.4.11 Spleen and Splenocyte Collection ...... 66 3.4.12 Mouse Weight and Organ: Body Weight Ratio ...... 66 3.4.13 Detection of Anti-Sperm Antibodies ...... 67 3.4.14 Euthanasia ...... 67 3.4.15 Breeding protocol ...... 67 3.4.16 Mouse Tissue Homogenization ...... 68 Analysis ...... 68 Ethics Statement ...... 69 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells ...... 70 4.1 Introduction ...... 71 4.2 Materials and Methods ...... 74
vi Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
4.3 Results ...... 77 4.4 Discussion ...... 91 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection99 5.1 Introduction ...... 100 5.2 Materials and Methods ...... 103 5.3 Results ...... 105 5.4 Discussion ...... 122 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 137 6.1 Introduction ...... 138 6.2 Materials and Methods ...... 141 6.3 Results ...... 144 6.4 Discussion ...... 167 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 176 7.1 Introduction ...... 177 7.2 Materials and Methods ...... 180 7.3 Results ...... 182 7.4 Discussion ...... 192 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies .. 198 8.1 Introduction ...... 199 8.2 Materials and Methods ...... 202 8.3 Results ...... 204 8.4 Discussion ...... 217 Chapter 9: General Discussion ...... 224 9.1 General Discussion ...... 225 9.2 Future Directions ...... 230 9.3 Significance and Conclusion ...... 233 Appendices 235 Appendix A ...... 235 Appendix B ...... 237 Appendix C ...... 238 Appendix D ...... 258 Appendix E ...... 271 Appendix F...... 272 Appendix G ...... 273 Bibliography ...... 275
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis vii
List of Figures
Figure 2.1 Chlamydia muridarum Lifecycle ...... 12 Figure 2.2 Male reproductive tract ...... 19 Figure 2.3 Human Testicular Structure ...... 20 Figure 2.4 Spermatogonium and Spermatogenesis ...... 23 Figure 4.1 Co-culture configurations for chlamydial transmission ...... 75 Figure 5.1 Neutral comet assay of untreated, irradiated, and infected TM3, TM4, and GC-1 cells...... 106 Figure 6.1 Detection of C. muridarum in mouse testes...... 145 Figure 6.2 Changes to Sertoli cell populations during chronic C. muridarum infection...... 148 Figure 6.3 Changes to Leydig cell populations during chronic C. muridarum infection...... 150 Figure 6.4 Changes to Myoid cell populations during chronic C. muridarum infection...... 152 Figure 6.5 Changes to testicular immune cell populations during chronic C. muridarum infection...... 155 Figure 6.6 Testicular cleaved-caspase 3 abundance during chronic C. muridarum infection...... 157 Figure 6.7 Titre of circulating testosterone during chronic C. muridarum infection...... 158 Figure 6.8 Sperm vitality, motility, and morphology during chronic C. muridarum infection...... 160 Figure 6.9 Oocyte binding capacity of sperm from mice chronically infected with C. muridarum...... 162 Figure 6.10 DNA fragmentation present in sperm from mice chronically infected with C. muridarum...... 164 Figure 6.11 Anti-sperm antibody production and sperm agglutination present in mice chronically infected with C. muridarum...... 166 Figure 7.1 Breeding study timeline...... 181 Figure 7.2 Viability, numbers, sex, and weight gain of pups bred from sires chronically infected with C. muridarum...... 183 Figure 7.3 Developmental delays displayed in pups born to chronically infected male mice...... 184 Figure 7.4 Low reproductive tract weight of female pups born to sires chronically infected with C. muridarum...... 186 Figure 7.5 Low testis weight of male pups born to sires chronically infected with C. muridarum...... 189 Figure 7.6 Low sperm quality of male pups born to sires chronically infected with C. muridarum...... 191 Figure 8.1 Primary antibody-, secondary antibody-, and DAB only-controls for detection of inclusions in testicular biopsies...... 204 viii Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
Figure 8.2 Detection of chlamydial inclusions in human testicular biopsies of patients with three diagnoses of male infertility...... 207 Figure 8.3 Immunohistochemical detection of MOMP in human testicular biopsies...... 210 Figure 8.4 PCR amplification of C. trachomatis DNA extracted from human testicular biopsies...... 212 Figure 8.5 Gel electrophoresis of C. trachomatis amplicons produced by PCR...... 214 Figure 8.6 Detection of anti-chlamydial antibodies found in serum from male infertility patients...... 216 Figure 9.1 Hypothetical model of monocyte/macrophage transmission of Chlamydia around the male reproduction tract...... 226
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis ix
List of Tables
Table 1: List of Abbreviations ...... xi Table 2: Global methylation status of C. muridarum infected versus non-infected TM3, TM4, and GC-1 cells...... 108 Table 3: Top five most significant DEGs for infected vs non-infected TM3 cells...... 111 Table 4: Top five most significant DEGs for infected vs non-infected TM4 cells...... 112 Table 5: Top five most significant DEGs for infected vs non-infected GC-1 cells...... 113 Table 6: Number of significant DEGs common between TM3, TM4, and GC-1 cells...... 113 Table 7: Top five most significantly altered pathways for TM3 cells infected with C. muridarum versus non-infected cells...... 114 Table 8: Top five most significantly altered pathways for TM4 cells infected with C. muridarum versus non-infected cells...... 115 Table 9: Top five most significantly altered pathways for GC-1 cells infected with C. muridarum versus non-infected cells...... 115 Table 10: Global DNA methylation status in sperm from mice chronically infected with C. muridarum...... 163 Table 11: MOMP positivity rate in human testicular biopsies...... 205 Table 12: Identification of C. trachomatis positive biopsies via melt curve analysis...... 213 Table 13: Correlation of C. trachomatis positivity with IVF outcome ...... 215 Table 14: Full DEG list for C. muridarum infected vs non-infected TM3 cells...... 238 Table 15: Full DEG list for C. muridarum infected vs non-infected TM4 cells...... 242 Table 16: Full DEG list for C. muridarum infected vs non-infected GC-1 cells...... 251 Table 17: Common DEGs between infected TM3, TM4, and GC-1 cells...... 255
x Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
List of Abbreviations
Table 1: List of Abbreviations 8-Hydroxy-guanosine 8-OHG
Aberrant Body AB
Anti-Müllerian Hormone AMH
AP Endonuclease 1 APE1
Apurinic/Apyrimidinic Site AP
Assisted Reproductive Technology ART
Base Excision Repair BER
Boron-dipyrromethene BODIPY
Cluster of Differentiation CD
Dendritic Cells DC
Deoxyribonucleic Acid DNA
Double stranded Deoxyribonucleic acid dsDNA
Early Upstream Open-reading-frame Gene euo
Elementary Body EB
Filamentation, Temperature Sensitive Gene ftsK
Follicle Stimulating Hormone FSH
H2A Histone Family Member X γH2.AX
Heat Shock Protein-60 Hsp-60
Histone-like Protein Gene hctB
Immunoglobulin A IgA
Immunoglobulin G IgG
In Vitro Fertilisation IVF
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis xi
Inbred albino laboratory mouse strain BALB/c
Inbred black-coated laboratory mouse strain C57BL/6JArc or C57BL/6
Indoleamine dioxygenase IDO
Interferon Beta IFNβ
Interferon Gamma IFNγ
Interleukin IL
Intracytoplasmic Sperm Injection ICSI
Lipopolysaccharide LPS
Major Histocompatibility Complex MHC
Major Outer Membrane Protein MOMP
Messenger Ribonucleic Acid mRNA
Multiplicity of Infection MOI
Natural Killer cells NK cells
Non-coding Ribonucleic Acid ncRNA
Outer Membrane Cytochrome B omcB
Outer Membrane Protein A Gene ompA
Oxidative Stress OS
Oxoguanine Glycosylase 1 OGG-1
Pathogen Associated Molecular Patterns PAMPs
Pathogen Recognition Receptor PRR
Pelvic Inflammatory Disease PID
Phosphatidylserine PS
Quantitative Polymerase Chain Reaction qPCR
Reactive Nitrogen Species RNS
Reactive Oxygen Species ROS
xii Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
Reticulate Body RB
Reverse transcription Polymerase Chain RT-PCR
Reaction
Ribonucleic Acid RNA
Ribosomal Subunit 16 Ribonucleic Acid 16S rRNA PCR
Polymerase Chain Reaction
Sexually Transmitted Infection STI
Sperm Chromatin Dispersion Assay SCDA
Spermatogonial Stem Cell SSC
Terminal Deoxynucleotidyl Transferase TUNEL dUTP Nick End Labeling
Testicular macrophage Tmφ
Toll-Like Receptor TLR
Transfer Ribonucleic Acid tRNA
Tumour Necrosis Factor Alpha TNFα
Type Three Secretion System T3SS
Tyrosine phosphorylation TP
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis xiii Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.
QUT Verified Signature Signature:
Date: 30/05/2018
xiv Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis
Acknowledgements
There is an extensive list of people who have collaborated on this project. First and foremost, I’d like to thank my principal supervisor, Professor Kenneth Beagley, for giving me the opportunity, support and guidance to undertake this PhD project. It has been an absolute privilege to work with Ken, an experience that I will always be grateful for and inspired by.
My associate supervisors, Dr Alison Carey, Professor Eileen McLaughlin, and
Professor Robert McLachlan have provided invaluable support for which I am incredibly grateful. I would also like to thank key team members Dr Charles
Armitage, Dr Avinash Kollipara, and Dr Kate Redgrove for their patience and assistance. The Infection and Immunity program members, particularly Professor
Louise Hafner, Dr Emma Sweeney, Dr Peter Mulvey, Chandan Mangar and Logan
Trim, who have provided much appreciated support. Also, Dr Anusch Yazdani and other members of the Queensland Fertility Group staff for their contribution to the project, and the Monash IVF and Monash SPH staff for their contribution.
I’d like to thank my partner, Dr Jacob Tickner, for the never-ending love and support, and always sticking it out with me. Thank you, to my mum and dad, my family and friends, for all the love, cheer, and support.
I would not have gotten far in this adventure without all of these people. Thank you everyone.
Chronic Chlamydia infections in males: Impacts on testicular function and spermatogenesis xv
Chapter 1: Introduction
Chapter 1: Introduction 1
1.1 Background
Causing approximately 131 million human reproductive tract infections globally each year, Chlamydia trachomatis is the major bacterial pathogen of the human reproductive tract [5]. C. trachomatis research has primarily focussed on women and the links between these infections and pathology, especially infertility, are relatively well established. However, the role of C. trachomatis in development of male reproductive tract disease is poorly understood. C. trachomatis infected men have been reported as having abnormal semen parameters including; low sperm count, abnormal sperm morphology, decreased sperm motility, as well as increased sperm
DNA damage [6-9]. These abnormalities may lead to decreased fertilizing ability and infertility [9]. Underlying this subfertility is that sperm do not possess the cellular mechanisms for DNA repair, thus “damaged” DNA may enter the oocyte during fertilization [10], resulting in poor embryonic development and survival.
A likely contributing factor to these abnormal sperm is the disruption of their production and maturation process (spermatogenesis), which occurs in the testis and epididymis. There are several testicular cell types that are critical for spermatogenesis, and each cell type appears to be vulnerable to infection in a different way. Leydig cells produce factors that promote spermatogenesis, including testosterone, and they reside in the interstitial space of the testes exposing them to infection via the blood and epididymis [11]. Similarly, Tmφ reside in the interstitium. They contribute to the immunosuppressive environment and tissue homeostasis of the testes and as such, are M2-like macrophages that are susceptible to infection [12]. Spermatogonial stem cells (SSC) are the cells from which all sperm are derived. While their differentiated forms reside protected behind the blood-testis- barrier, this SSC population is exposed to infection via the interstitium [13]. Sertoli
2 Chapter 1: Introduction
cells are a non-renewing cell type that is particularly susceptible to infection due to their phagocytic abilities [14]. They also form an immune privileged, nutrient rich, and protective environment, the blood-testis-barrier, necessary for spermatogenesis
[14]. All these specialized cell functions may be compromised by Chlamydia infection, and result in dysregulation of spermatogenesis.
1.2 Hypothesis and Specific Aims of the Study
To investigate the potential for Chlamydia to establish a chronic testicular infection and the downstream impacts of the infection on testicular function, the following hypothesis and aims have been developed.
Hypothesis: Chlamydia infection in males can result in chronic testicular infection, damage to testicular cells, and impaired spermatogenesis.
Aim 1: To investigate the susceptibility to, and infection kinetics of Chlamydia within testicular cells.
Aim 2: To determine the functional changes induced by Chlamydia infection within testicular cells, and the corresponding effect on sperm health and fertility.
Aim 3: To determine the frequency and effect of C. trachomatis infection in testicular biopsies from men donating sperm for assisted reproductive technologies
(ART) and correlate infection presence with reproductive outcomes.
This study will utilise both primary cells and immortalized cell lines in a range of in vitro experiments and in vivo mouse model infection experiments to determine the impact of Chlamydia infection on male fertility. Sertoli, Leydig, spermatogonial, and
Tmφ cell infection kinetics will be characterized to establish their susceptibility to infection and role in transmission of Chlamydia to, and within, the testes. These cells
Chapter 1: Introduction 3
will also be assessed for changes to viability, genomic integrity, epigenetic, and transcriptomic changes. A range of immuno-assays and molecular techniques will be used to determine cellular changes associated with cell function and specifically with a role in the regulation of spermatogenesis.
The corresponding effect on fertility resulting from dysfunctional testicular cells will be assessed in an in vivo mouse model of infection. This will be achieved by characterizing the health and number of offspring produced from breeding with a healthy female, as well as the health of the male offspring sperm cells.
Finally, human testicular tissue will also be assessed to determine the frequency of
Chlamydia trachomatis infection in the infertile male population seeking ART.
Tissue, harvested from current fine-needle, and archived open testicular biopsies taken from male factor infertility patients presenting at IVF clinics, will be examined for the presence of C. trachomatis. The needle biopsies will be assessed via real-time
PCR for the presence of C. trachomatis. These results will be correlated with patients’ clinical history and the outcome of their concurrent IVF cycle. As a larger population survey the open testicular biopsies will be examined via histological techniques for the presence of C. trachomatis, spermatogenic and associated somatic cell and tissue damage, and the localisation of immune cells.
1.3 Significance of the Study
The World Health Organization estimates 2.7% of men aged 16 – 49 years old have active Chlamydia infections [5]. This is likely an underestimate as up to 50% of male infections are undiagnosed due to their asymptomatic nature. While the prevalence of genital Chlamydia infections is similar in men and women, to date it is predominantly considered to cause the most significant pathogenesis in females.
4 Chapter 1: Introduction
Therefore, this study aims to challenge these preconceptions, by providing information about the poorly understood disease sequelae and pathogenesis in males.
The results of this study will provide insight into the role of Chlamydia in male infertility, an emerging and controversial field. Infection profiles and the functional characterization of cellular and molecular changes within infected male reproductive tract cells is critical in determining how spermatogenesis is dysregulated, which cell types are responsible, and if this can be prevented. This will inform future development of an anti-chlamydial vaccine for males or other prophylactic or therapeutic approaches targeting male Chlamydia infections.
Chapter 1: Introduction 5
Chapter 2: Literature Review
Chapter 2: Literature Review 7
2.1 Chlamydial impact on the community
Approximately 131 million people acquire a chlamydial infection each year, making
Chlamydia the most common sexually transmitted bacterial infection worldwide [5].
Recent estimates indicate C. trachomatis infection rates in 16 – 49-year-old women to be 4.3%, and 2.7% in men in the same age group [5, 15]. This represents around
4% of the global population and a majority of individuals in the reproductive age group [16]. The asymptomatic nature of many infections (up to 75% of female and
50% of male infections are asymptomatic) leads to low detection rates and continued transmission of infection and thus these figures are acknowledged to be underestimations of the chlamydial disease burden [17].
As male screening programs are uncommon, asymptomatic male infections may constitute a large proportion of the reservoir of unrecognised infections in the community. This may increase rates of transmission, subsequently increasing rates of pathology in men and women. Screening trials or programs have been established in several European counties, Australia, and the USA that include men and women [18,
19]. However, these represent some of the only male-inclusive programs in the world. Comparatively, female-targeted programs are more commonly adopted [20-
23]. Although these may not strictly be male-excluding, by virtue of the information about infections that they provide, they do target females. This disparity in attitude towards detecting infections is an issue that must be rectified if we are to reduce
Chlamydia transmission rates and identify more accurate prevalence statistics.
Additionally, as most screening programs are opportunistic, rather than active, there is some doubt as to the worth and effectiveness of screening programs in significantly reducing the community chlamydial burden [24-26]. This signifies the need for additional prevention methods such as a vaccine [27, 28].
8 Chapter 2: Literature Review
As infections are often asymptomatic, medical intervention and treatment are not commonly sought. Instead, infections are usually detected opportunistically during routine health checks, although these are performed more commonly for women via
Pap smear [18, 29] than the equivalent tests are for men [30, 31], which reduces the opportunity for detection in men. Diagnoses of symptomatic infections utilising nucleic acid amplification testing is relatively sensitive and most commonly used
[32]. However, this technique predominantly relies on collection of urethral swabs and urine specimens, which will only detect infections of the lower reproductive tract. Unfortunately, the severe pathology leading to infertility sequelae, often occurs in the upper reproductive tract [33, 34] where swab and urine based diagnostic methods can be ineffective [35-38]. Current treatment of recognised infections is limited to antibiotics, and treatment failure has been documented [39, 40]. Reported cases of chlamydial antibiotic resistance are few [39, 41] and instances of treatment failure are commonly attributed to patient non-compliance [42]. However, the lack of understanding of the upper reproductive tract susceptibility and response to chlamydial infection also impacts treatment efficacy and pathology prevention.
Specifically, several key knowledge gaps exist in our understanding of Chlamydia infections. These include identifying the differences in susceptibility in regions of the reproductive tract during chlamydial infection, between symptomatic/asymptomatic, or first exposure/recurrently infected people, and determining the efficacy of antibiotics in each of these different cases.
This complex and dynamic challenge is often answered by the call for development of an anti-chlamydial vaccine [27, 28, 43, 44]. Currently, no prophylactic or therapeutic vaccines exist that target Chlamydia. However, one vaccine candidate has recently entered phase 1 clinical trials [45]. Several experimental models of
Chapter 2: Literature Review 9
vaccination have been explored with limited success. These studies have trialled multiple vaccine strategies for clearance of infection including the use of different chlamydial antigens [46-48], vaccination schedules [49, 50], vaccination routes [51-
53], adjuvant compositions [54-56], and the targeting of different components of the host immune system [1, 57, 58]. The most successful strategy for preventing transmission was dual vaccination of both males and females [50], whereas previously, less-effective methods utilized vaccination of only a single-sex, predominantly female. However, a vaccination regime consistently resulting in sterilizing immunity to Chlamydia is yet to be achieved. This is due to the dichotomy that exists between the different and often opposing host immune responses that must be invoked to clear infection as opposed to preventing pathology development [1].
Regardless of these challenges, vaccine development and implementation remains the most highly recommended option for prevention of chlamydial disease.
The predominant and severe chlamydial disease sequelae of concern to the community are infertility related, which affect both men and women. Despite this, research has predominantly been aimed at women, as the burden of disease is principally thought to affect females [44, 59]. On average, approximately 25% of female infections result in upper reproductive tract infection [60-62]. Around 25% of those infections result in pelvic inflammatory disease (PID), and PID is heavily associated with infertility issues including fallopian tube scarring and ectopic pregnancy [5, 15]. The risk of PID also increases with recurrent infection [63].
Comparative statistical evidence is not present for male infections or male infertility.
The lack of investigation into male disease is likely a consequence of the historical view of infection being less frequent, more likely to be identified, and being linked with only female infertility.
10 Chapter 2: Literature Review
2.2 Chlamydial Infections
2.2.1 Chlamydial lifecycle and transmission
Chlamydiae are Gram-negative, non-motile bacteria with a unique biphasic developmental cycle. Infection is initiated by the binding of an infectious elementary body (EB) to host cell membranes; this is commonly thought to occur via some combination of heparin and mannose receptors, clathrin, and dynamin, although, some studies have shown that these pathways can be utilized by EBs independently as well as in combination [64-67]. Chlamydia mediates the remodelling of the cell cytoskeleton and endocytosis of the EB [68]. The EB differentiates into the reticulate body (RB, see Figure 2.1), which produces between 100 and 1000 metabolically active RBs by binary fission [69].
The RBs are contained within the endosome, which is modified using the type three secretions system (T3SS) possessed by chlamydial species, and called an inclusion
[69]. Inclusions prevent host cell detection of the Chlamydia and inhibit endogenous phagosomal degradation pathways through use of the T3SS [69]. Bacterially derived inclusion membrane proteins (Incs) with large hydrophobic domains, and other anti- host factors constitute a large portion of the inclusion surface [70]. Anti-host factors, for example effector proteins that interact with host Golgi apparatus, are also secreted into the host cell cytosol by the T3SS [70]. The T3SS is composed of cytoplasmic ancillary proteins that start within the inclusion, a basal apparatus which anchors in the inclusion membrane, the needle and connecting needle tip, and the translocator which passes secreted factors from the needle to the host cell cytoplasm
[71, 72]. Together, these components form a complete molecular needle structure.
Chapter 2: Literature Review 11
After 24-48 hours post infection for C. muridarum in mice, and 56-72 hours post infection for C. trachomatis in humans, the RB replication is complete and RBs revert to EBs where they escape the host cell by lysis, or extrusion, to initiate re- infection [69, 73].
Chlamydia mediated cell lysis has been documented in a variety of cell types, most commonly in epithelial cells [74]. Under some conditions, lysis has been observed in infections of immune cells. However more recently, extrusion of inclusions has been shown to occur from cells, at rates more frequently than previously predicted [75,
76]. Extrusion does not necessarily kill the host cell [76], though the intra-cellular changes that occurred during infection may persist. Interestingly, after extrusion an intracellular inclusion is still extant and produces viable progeny.
Transmission of Chlamydia from an infected individual to a partner usually occurs
Figure 2.1 Chlamydia muridarum Lifecycle Figure 2.1 shows the commonly accepted process of entry of elementary bodies (EB), inclusion development and replication of reticulate bodies (RB), and eventual exit of EBs via lysis from host cells [1].
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via reproductive tract mucosal secretions/fluids containing EBs. In males, semen composed predominantly of prostatic fluid and sperm, is the most likely source of transmission. C. trachomatis genital serovars (D-K) infect epithelial cells in the male reproductive tract and this can lead to a range of disease states including urethritis
(inflammation within the urethra), prostatitis (inflammation within the prostate), epididymitis (inflammation within the epididymis), and orchitis (inflammation within the testis) [17, 77, 78]. The primary infection site in the male is generally the penile urethra. As with other male urogenital infections including E. coli, chlamydial infection is then thought to ascend from the lower to the upper reproductive tract [17,
79, 80]. However, the human epididymis is 5-6 metres long [81] and an infection ascending via epithelial cells, against the flow of seminal fluid(s) seems unlikely; suggesting an alternate route of dissemination. Although models of epididymitis, prostatitis and orchitis do exist, there is no clear indication of the mechanism by which Chlamydia invades these tissues [17].
2.2.2 Chlamydial Pathogenesis
As stated, much of what is known about chlamydial pathogenesis has been elucidated in female models of infection in animals and, therefore, has inherent shortcomings when applied to human in vivo infections in males. The mechanism for pathogenesis in the male reproductive tract is likely to be different to the female for several reasons including but not limited to; (i) presence of immune privilege in the testes,
(ii) anatomical differences, and (iii) the impact of testosterone as the major hormone
(in contrast to female estrogen and progesterone) on the immune response [82]. An overview of the proposed model of pathogenesis is generally as follows.
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The presence of Chlamydia in the reproductive tract may elicit an uncontrolled inflammatory immune response in many symptomatic cases, which is primarily responsible for pathology development (immunopathology) [83]. Chlamydial antigen-dependant immune responses take place. The C. trachomatis Pathogen
Associated Molecular Patterns (PAMPs) e.g. Heat Shock Protein-60 (hsp-60) and lipopolysaccharides (LPS) are recognised and bound by Pattern Recognition
Receptors (PRRs) e.g. Toll-like Receptor (TLR)-2 and TLR-4, activating both innate and adaptive arms of the host immune response [84-87]. PAMPs are recognised by innate cells including neutrophils, macrophages, Natural Killer cells (NKs), and dendritic cells (DCs), as they attempt killing of the Chlamydia via phagolysosome and inflammasomal activity [86, 88-90].
Neutrophils, macrophages, NKs and DCs, in combination with infected host epithelial cells, secrete pro-inflammatory cytokines and chemokines. These include interleukin (IL)-6, IL-8, IL-12, IL-1β, interferon gamma (IFNγ), and tumour necrosis factor alpha (TNFα), anti-microbial reactive oxygen and nitrogen species (ROS and
RNS respectively), and indoleamine dioxygenase (IDO, in humans), which promote clearance of Chlamydia [84, 85, 91-94]. The presence of the chemokines e.g. chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL2, chemokine (C-C motif) ligand 2 (CCL2), CCL4, and CCL5, and Human Leukocyte Antigen (HLA, in humans)/major histocompatibility complex (MHC, in mice) molecule presentation on the surface of infected epithelial cells promotes recruitment and activation of adaptive immune system components predominantly in the T helper (Th) 1 phenotype [95, 96]. Chlamydiae also down-regulate HLA/MHC presentation as a protective adaptation, which limits the efficiency of this antigen presentation method
[97-100]. The inflammatory response from infected epithelial cells contributes
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heavily to pathophysiology and may be responsible for scarring associated with chlamydial infections, a classical example from females is PID resulting in fallopian tube scarring [101].
Cluster of Differentiation (CD) 4+ T and CD8+ T cells produce cytokine signals for further immune cell recruitment and promote clearance of the infection. Both T cell types have a role in protection of the host against infection [102]. CD8+ T cells have the capability to respond and specifically lyse Chlamydia infected cells, killing the infection and providing some protection [103-105]. However, cell lysis also contributes to inflammation at the infection site [106]. Antigen specific, TNF-α producing CD8+ T significantly contribute to upper FRT pathology, so these cells are not considered the major protective type of T cell [106]. Instead, CD4+ T cells, particularly those which produce IFNγ and have a more T helper (Th) 1 phenotype, have a protective ability during re-infection challenges, and depletion of these cells increases both infectious and disease burden [107-109]. IFNγ mediated depletion of intracellular tryptophan through the IDO system in humans is thought to be responsible for limiting the replication capacity of Chlamydia [110-112].
Unfortunately, this may also induce or contribute to chlamydial persistence. Rapidly responding, tissue-resident memory T cells also contribute to re-infection protection
[113]. More recently, the Th17 response has been investigated with limited successes, as an additional mechanism for chlamydial clearance with the idea that it may clear infection and prevent immunopathology [58, 114]. However, the role is still being defined as IL-17 has also been implicated in exacerbation of immunopathology [114, 115]. Modulation of both the Th1 and Th17 responses by T regulatory cells remains key in limiting host immunopathology sequelae.
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B cells produce anti-Chlamydia antibodies to further assist in the clearance of infection. Immunoglobulin (Ig) G and IgA antibody production commonly occurs targeting the major outer membrane protein (MOMP), as the immunodominant chlamydial antigen [107]. Other inclusion proteins including outer membrane complex protein B (OmcB) and inclusion protein A (IncA) and T3SS complex proteins are also immunogenic [116-118]. Antibody responses can include neutralising antibody production to aid in chlamydial clearance and protect against re-infection [119, 120]. In the male mouse reproductive tract, poly-immunoglobulin- receptor mediated transport of neutralizing IgA into prostatic fluid correlates with reduced infectious burden [120]. However, antibody production can also enhance immunopathology of the reproductive tract. Hsp-60 is highly conserved across species including chlamydial and human hsp-60 homologues, which contain approximately 48% amino acid homology [121]. Antibodies targeting chlamydial hsp-60 are cross-reactive and can target host cells, exacerbating pathogenesis [122,
123]. Furthermore, infectivity of non-neutralising IgG-coated Chlamydia EBs is enhanced, as IgG transcytosis through FcRn-expressing epithelial layers enhances infection [124].
Infections that are cleared by the host immune response, or with assistance from antibiotic treatment (azithromycin or doxycycline), are referred to as acute infections
[125-128]. For example, many models of female infection exist that indicate the duration of natural infection in female mice is acute, lasting approximately 35 days.
Fewer studies have investigated acute male infections. Although importantly, two studies conducted in mouse models showed infection was possible up to 8-10 weeks and resulted in poor testicular, epididymal, and sperm health [50, 129].
Comparatively, little is known about the duration of infections in men and women.
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Some studies have found acute infections can be cleared with antibiotics after several weeks, while some have found that infections lasted longer than one year [130].
2.2.3 Chronic and Persistent Infections
In addition to the immunopathology which commonly occurs in symptomatic cases, asymptomatic infections also often occur [17]. Weak, non-protective Th1 and Th17 responses may be responsible for these asymptomatic infections, but little is known about what influences the outcome of an infection [131-133]. It has been hypothesised that differences in both host genetics and chlamydial strain virulence are factors in the severity of the pathology [34, 134, 135]. It is also unclear whether an asymptomatic infection is in fact the development of a low level chronic infection
[130]. If chronic infection occurs, it could indicate that the infected host is continuing to shed EBs and that sexual transmission could still occur. As with many other community-acquired infections including C. difficile, malaria, Ebola, hepatitis C virus, and N. gonorrhoeae, establishment of chronic asymptomatic states can lead to a reservoir of infection in the community, which, as screening programs are few and detection methods are flawed, perpetuates the infection cycle [136-140].
The asymptomatic infections which do not resolve can be called either chronic or persistent infections. In vivo and in vitro persistence of C. muridarum (the mouse specific strain) has been characterized in female BALB/c mice and McCoyB (mouse fibroblast derived) cell lines, respectively [141]. In vitro, persistent infections were found to be morphologically distinct, and contain enlarged Aberrant Bodies (AB), which differ from both RBs and EBs [141].
ABs are produced when dedifferentiation of RBs to EBs is suppressed due to stress from the host immune response or the presence of antibiotics [142]. Significant
Chapter 2: Literature Review 17
down-regulation of euo, ftsK, hctB, omcB, and ompA genes was observed in induced persistent infections compared to active infections [141]. ABs reverted to RBs when the stressing factor was removed from the system [141, 142]. Persistence is thought to account for a portion of asymptomatic and recurrent infection cases, as these dormant inclusions avoid immune detection. However, there is currently no evidence of in vivo persistence from the male reproductive tract.
Chronic infections are non-resolving, long-term infections that are known to occur in vivo [137, 143-147]. They may differ from persistent infections and involve active chlamydial replication, but it is currently unknown whether they share characteristic of AB formation with the in vitro persistent infections. Both types of long term infections are infrequently studied. Very little evidence exists for either persistent or chronic chlamydial infections in humans. What does exist consists of women infected genitally with C. trachomatis long term [148-150], people with reactive arthritis cause by C. trachomatis [151, 152], people with ocular C. trachomatis infection [143], and people with respiratory C. pneumoniae infections and associated heart diseases [145, 153, 154]. There is some thought that gastric chlamydial infection creates a reservoir of infection in the body that is unable to be cleared by immune response or antibiotic therapy [155]. The gastric infection disseminates and causes re-infection of the genital tract, which results in recurrence [155]. However, in general, chronic chlamydial disease is not well understood [144, 145]. There is evidence of asymptomatic infections in males, but the duration of these infections is unknown [35, 96, 156].
2.3 The Male Reproductive Tract
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In part, the knowledge gaps surrounding male infections are due to the incomparability of the female and male reproductive tracts (FRT and MRT respectively) (Figure 2.2). The MRT contains a greatly longer urethra than the FRT
[2]. The MRT contains accessory glands in separate organs, i.e. the prostate and seminal vesicles, which are absent in the FRT [2]. The blood supply throughout the
MRT and FRT are structurally distinct after each diverges from the aorta [2]. The
MRT contains the extensive length of the epididymis and seminiferous tubules [81].
To some degree the epididymis is immune privileged [157], and the testes contain complete immune privilege, a phenomenon absent from the FRT [158]. Each of these differences has the potential to alter the infection kinetics. Of particular interest though, are the testes. The kinetics of infection within an immune privileged tissue,
Figure 2.2 Male reproductive tract Figure 2.2 shows the anatomy of the MRT; 1. the testes as the site of sperm production and the flow of sperm through the MRT from the testes, 2. to the epididymis, 3. to the vas deferens, 4. and out through the urethra (arrows) [2].
Chapter 2: Literature Review 19
and particularly within the specialized testicular cell types, are unknown although infection is associated with inflammation (orchitis) [17]. Therefore, this represents a major knowledge gap within the literature.
2.3.1 Testicular cells and structure
The testicular structure, as it gives rise to a unique function, is particularly distinctive
(Figure 2.3). The purpose of the testes is to produce sperm via spermatogenesis [2].
This cannot occur without the specialized testicular Sertoli and spermatogonial cells in the seminiferous tubules, and Leydig cells and testicular macrophages (Tmφ) in the interstitial space.
2.3.2 Leydig cells
Leydig cells support spermatogenesis and are located within the interstitial space of
Figure 2.3 Human Testicular Structure Figure 2.3 shows the normal structure of the adult human testis. The testicular parenchyma (TP) contains seminiferous tubules and interstitial tissue, the seminiferous tubules drain to ducts (tubuli recti, arrows), tubuli recti collate in the mediastinum testis (MT) in channels called the rete testis (RT), the RT becomes the ductuli efferentes (asterisks), which drains into the epididymis [3].
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the testes [159]. The major Leydig cell function is the production of male sex hormones, called androgens [11]. Testosterone, androstenedione, and dehydroepiandrosterone are all secreted under the stimulation of interstitial-cell- stimulating hormone and Luteinising hormone (LH), which are produced by the pituitary gland [160, 161]. Testosterone in particular, is essential for spermatogenesis as it promotes maturation of dividing germ cells [162]. Leydig cells are proliferative; they have a progenitor population resident within the testes [159]. Mesenchymal-like stem cells divide into Leydig cell progenitors, which proliferate into immature
Leydig cells that over time mature into adult Leydig cells [161]. Each stage of
Leydig cell expresses the specific marker 3-β-hydroxysteroid dehydrogenase
(3βHSD) and LH receptors [160, 163]. The adult Leydig cell population is not generally proliferative but this is the population that primarily produces testosterone.
It is unclear how susceptible each population is to chlamydial infection and how each population will respond. As Leydig cells are in close proximity to Tmφ they may have some protection from infection generally [164]. However, models of HIV-2 and mumps virus infection and of exposure to LPS and ROS, for human Leydig cells, exist showing deregulation of the cell function including a decrease in testosterone production [165-167]. Deregulation of Leydig cell function after chlamydial infection may occur in a similar way, which will impact on male fertility.
2.3.3 Sertoli cells
Conversely to Leydig cells, in the adult testis, Sertoli cells are a non-renewable population [14]. Sertoli cells line the seminiferous tubules and have several essential activities that support spermatogenesis. Firstly, each Sertoli cell forms tight junctions with neighbouring Sertoli cells to form the blood-testis-barrier, which shields immunogenic germ cells [168]. The blood-testis-barrier creates part of the protective
Chapter 2: Literature Review 21
immune-privileged environment for spermatogenesis [158]. The blood-testis-barrier prevents movement of almost all substances into the seminiferous tubules. Sertoli cells do not possess the Fc Receptor to allow antibody transcytosis and Tmφ and other interstitial cells are not able to cross the blood-testis-barrier [168]. The exception are spermatogonia, which mediate temporary release of tight junctions to allow meiotic germ cells to traverse the barrier [169].
Sertoli cells are phagocytic and normally function to clear apoptotic sperm cells [14].
This may also give them the ability to actively take up infectious particles, increasing their risk of infection. The phagocytic ability is unconventional though and not mediated by normal antigen binding mechanisms, instead it is mediated by phosphatidylserine binding to class B scavenger receptor type 1 [170, 171]. It is unclear how Sertoli cells will respond to chlamydial binding and infection.
Sertoli cells are secretory cell types that produce hormones and other soluble factors.
These include; (i) AMH, which is produced during fetal development, (ii) inhibin and activins, which are produced after puberty, (iii) transferrin, which assists in iron ion movement, (iv) androgen binding protein, which increases the concentration of intra-tubular testosterone to stimulate spermatogenesis, (v) oestradiol-aromatase, which converts testosterone to oestradiol to direct spermatogenesis, and (vi) GDNF and ERM transcription factors, which are needed for spermatogonial stem cell maintenance [172-174].
One study found that Sertoli cells became apoptotic when infected in vivo with
Chlamydia in a pro-inflammatory environment, in the presence of IFNγ. If the Sertoli cell population was damaged by chlamydial infection, reduction in available secretions could disrupt spermatogenesis in multiple ways [129]. Furthermore,
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transplantation of Sertoli cells into mouse testes with supporting-cell deficiency dramatically changed the testicular environment to one that would support spermatogenesis, showing that Sertoli cell absence inhibits spermatogenesis [175].
Each stage of spermatogenesis requires a form of the immune-privilege, phagocytic protection, and hormonal and nutrient stimulation from Sertoli cells [14]. Chlamydia induced Sertoli cell damage may eliminate or reduce this.
2.3.4 Spermatogenic cells and Spermatogenesis
Spermatogenesis is the process by which functional, mature spermatozoa are generated (Figure 2.4). This process begins during male puberty and is regulated by the expression of bone morphogenic protein 8b (BMP8B) [176]. When BMP8B reaches a specific concentration, male germ cells begin to divide, and spermatogenesis commences. The dividing germ cells constantly produce BMP8B to
Figure 2.4 Spermatogonium and Spermatogenesis Figure 2.4 shows the localisation of spermatogonium A and B on the basement membrane of the seminiferous tubule, the location of primary and secondary spermatocytes behind the blood-testis-barrier (BTB), the spermatids, and the final stage of spermatogenesis, the spermatozoa in the lumen of the seminiferous tubule [4].
Chapter 2: Literature Review 23
continuously promote spermatogenesis [176].
Spermatogenesis is supported by both Leydig and Sertoli cells as previously described and cannot take place without these support cells. Spermatogenesis also critically relies on the presence of spermatogonial stem cells (SSCs), as these are the progenitor cell type from which all sperm are made [177]. The SSC population consists of Spermatogonium A, which continuously divide to both replenish the SSC population and provide the precursor sperm cells, called Spermatogonium B [13].
These cells are commonly identified by the Plzf marker, which is a transcription factor that is essential to their population maintenance [178].
Type B spermatogonia are committed to differentiation into primary spermatocytes
[176]. As these cells divide, they modulate the temporary release of the tight junctions of the blood-testis-barrier to allow the primary spermatocytes to pass through [169]. These cells rapidly divide and are commonly identified by proliferating cell nuclear antigen (PCNA), which specifically localizes to mitotically active germ cells within the testis [179]. Primary spermatocytes are the first stage of spermatogenesis that occurs behind the blood-testis barrier and these cells meiotically divide to produce secondary spermatocytes [176]. The secondary spermatocytes again divide via the second stage of meiosis to produce the haploid round spermatids [176]. Round spermatids develop into elongated spermatids, which mature into the spermatozoa [176]. Spermatozoa are the final stage of spermatogenesis, they are positioned on the luminal surface of the seminiferous tubules [176].
Chlamydial infection of the testis has been shown in an acute mouse model to cause damage and apoptosis of the germ cell population, resulting in decreased viable
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sperm cell numbers [129]. However, human germ cells are relatively unstudied and the pathway leading to infection of germ cells is currently unknown. Spermatogonial stem cells are self-renewing, so may regenerate if infection damages only a portion of the population, although they may also form a reservoir of infection in the seminiferous tubules, leading to infection of surrounding cells including sperm.
Chlamydia infection of germ cells could potentially cause damage directly to the sperm DNA and contaminate semen.
2.3.5 Semen and Spermatogenesis Dysregulation
Sperm constitutes the major cellular fraction of semen, the liquid fraction is composed of prostatic and seminal vesicle secretions [180]. Abnormalities that occur within the two components can influence fertility and can include changes to semen liquefaction/viscosity/appearance/volume/pH, and changes to sperm count/motility/morphology/vitality/DNA integrity [180]. Several infectious agents have been detected in semen from sub-fertile men including human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes simplex virus
(HSV), papillomavirus, Escherichia coli, Neisseria gonorrhoeae, and C. trachomatis
[181, 182]. These agents are predominantly associated with changes in sperm parameters. The interactions between the infections and the sperm, and the mechanisms for parameter changes, are fundamentally uncharacterized. This represents a major knowledge gap within the literature.
One mechanism that could lead to sperm parameter changes is the dysregulation of spermatogenesis by infections occurring within the testes, as this may lead to production of sperm with the outlined abnormalities. Infectious agents that have been identified within the human testes include mumps virus, HIV-1, EBV, and HSV,
Chapter 2: Literature Review 25
usually in the context of testicular oncogenesis [181]. Some sperm abnormalities such as low sperm count or low motility can generally be overcome by ART [183,
184]. However, some abnormalities, particularly severe sperm DNA fragmentation, cannot always be overcome by ART and this is rapidly becoming one of the most important markers of male infertility [10, 185, 186].
DNA fragmentation observed within sperm would indicate gross chromosomal aberrations that may originate from complex DNA lesions and breaks. This damage would normally be repaired primarily via non-homologous end joining and homologous recombination [187]. However, these repair mechanisms do not exist within spermatozoa, as these cells are terminally differentiated haploid cells and have extremely limited DNA repair capabilities. They are only able to perform the first step in base excision repair (BER), a repair mechanism with limited functionality
[10]. During this process, the enzyme OGG-1 removes single inappropriate or damaged nucleotides/bases and re-joins the DNA strand leaving an AP site (an apurinic/apyrimidinic/abasic site) [188]. Sperm lack the following crucial stages of
BER, where the APE1 enzyme recognises and creates a 3’ hydroxyl group at the AP site, which opens the DNA strand for input of correct nucleotides and closure of the repaired strand [10, 188]. Sperm are reliant on oocyte machinery to complete the process following fertilization.
The extent to which sperm cells undergo BER in the presence of Chlamydia infection is unknown. However, in an FRT cell infection model, Chlamydia was shown to inhibit the repair of fragmented DNA induced by ionising radiation [189]. As these cells are diploid somatic cells, they have full DNA repair capability, so it could be inferred that sperm would be unable to undergo BER if they are infected and/or if their DNA was damaged.
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Cellular DNA damage can originate from many endogenous sources, e.g. somatic/meiotic recombination, stalled replication forks, and exogenous sources, e.g. cigarette smoking, Hepatitis C virus infection, and H. pylorii infection [190-192].
One of the most common and well-studied events caused by these factors is oxidative stress (OS), commonly in the form of reactive oxygen species (ROS) and resultant formation of 8-hydroxyguanosine (8-OHG) adducts [193]. Normal cellular function
(e.g. capacitation of sperm cells) requires production of OS, which is beneficial and counteracted by naturally occurring antioxidants [194]. Imbalance in this system that results in increased OS can promote cell death (apoptosis or necrosis) and autophagy
(catabolic mechanism causing cell dysfunction) [195]. It is unknown whether chlamydial infection skews this balance.
In infected Sertoli or germ cells, excessive production of ROS could damage DNA and affect normal cellular functions [195-197]. Similar effects may occur in sperm cells either directly via infection, although there is no consensus in literature regarding chlamydial adherence and infection of sperm in vivo [6, 8, 9, 198] compared to in vitro [199-203], or indirectly from a lack of developmental support from abnormal Sertoli cells. Cellular DNA damage may result in changes to the transcriptome, i.e. changes to amounts or types of RNA that is transcribed from
DNA. If abnormal transcriptional changes are reflected in the proteome (protein component of a cell) or the non-coding RNA, this is highly likely to be responsible for abnormal cell function [204, 205].
Further transcriptome changes with similar consequences to above may be induced by epigenetic changes. This involves addition or removal of acetyl, ubiquitin, or methyl groups to/from DNA or histones (control long DNA strands to condense into heterochromatin or loosen to euchromatin, and present gene sections to transcription
Chapter 2: Literature Review 27
enzymes), which can cause chromatin remodelling and masking of transcribable regions of genes or directly alter gene transcription respectively [206, 207].
Chlamydiae possess a methyltransferase gene which may have this function. Some epigenetic changes can also be inherited and this may influence the success of embryogenesis or the health of any offspring that are produced [206].
Oxidative stress, epigenetic changes and DNA damage/fragmentation may be markers of, or contribute to poor Sertoli, spermatogonium and sperm cell health.
Spermatozoa DNA fragmentation is a major cause of male infertility [186, 208-211] and Chlamydia is a suspected aetiological agent [6, 212]. There are currently no studies available which show the mechanistic pathways leading to this type of infertility.
2.3.6 Testicular macrophages
Tmφ constitute the majority of testicular immune cells. They are predominantly of an
M2, CD163+ phenotype and provide a large contribution to the immune suppressive or immune-privileged environment within the testes that is required for spermatogenesis to occur [213, 214]. Murine Tmφ have been fairly well characterized, these are generally the resident
F4/80+/CD206+/Ym1+/Fizz1+/Mrc1+/Arg1+/CD163+/MHCII+ cells [213]. These cells originate during embryogenesis and have a self-renewing capability [215]. There is also a smaller population of Tmφ that most likely originate from the circulating population that are CD163-/CD86+ [216]. Tmφ retain their phagolysosomal ability, however they have reduced expression of pro-inflammatory cytokines such as IL-1β,
IL-6, and TNF-α to prevent inflammatory cell recruitment and immunopathology within the testis [157, 217]. As Tmφ are also responsible for protecting the testis
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from infection this becomes a difficult balance between effective clearance of infection and avoiding immunopathology [217]. Regardless, M2 macrophages are not able to clear chlamydial infection [218, 219].
Tmφ contribute heavily to the immune-privileged status of the testis. They create the tolerance for immunogenic germ cell derived antigens that would otherwise elicit an autoimmune response against the germ cells [220]. These antigens are expressed on all meiotic and post-meiotic germ cells produced during spermatogenesis [157].
These include secondary spermatocytes, spermatids, and spermatozoa. This occurs through the suppressive effects and truncated inflammatory abilities of the Tmφ.
When this process is deregulated it contributes to infertility, with autoimmune responses detected in approximately 10-20% of idiopathic male infertility [221-223].
This process works in concert with the tight junctions between intra-tubular Sertoli cells, which provide the blood-testis-barrier to shield the germ cell antigens more effectively.
Tmφ are intrinsically linked with Leydig cells [164, 224]. Some evidence suggests that Leydig cells are actually responsible for maintenance and recruitment of Tmφ populations, possibly by expression of monocyte chemoattractant protein 1 (MCP-1
[225, 226]. The Tmφ grow in close proximity to Leydig cells. They project digitations into the Leydig populations indicating close communication between the two cell types [227] and macrophage/monocyte colony stimulating factor (M-CSF) deficient mice possess dysfunctional Leydig cells [228].
A small Tmφ portion also resides in close proximity with the seminiferous tubule basement membrane. This population communicates with the peritubular Myoid cells of the basement membrane and the SSCs around the outside of the tubules. Perhaps
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their most important role is the secretion of factors including colony stimulating factor 1 (CSF1), which promote SSC division and differentiation [229]. This function demonstrates again, the important role of specialized Tmφ in formation of the particular environment required for spermatogenesis.
2.3.7 Chlamydia and macrophages
Macrophages (mφ) are generally thought to provide an effective innate immune mechanism for detecting and destroying harmful microorganisms that would otherwise colonise the body and cause pathology. Macrophages phagocytose bacteria into a phagosome, which upon entry to the cytoplasm fuses with the endogenous lysosome to form the phagolysosome [230, 231]. The phagolysosome contains the components to enzymatically digest the bacteria, or alternatively kill by production of reactive oxygen or nitrogen species that degrade the bacteria [232-235]. The bacterial antigens produced in this process are presented on the membranes of macrophages through the MHC pathway in mice or the equivalent HLA in humans
[231]. Other immune cells such as B and T cells respond to the antigen presentation to mount an antigen specific and more comprehensive defence against the bacterial invasion [236].
Many bacterial species are known to survive within macrophages under specific conditions and using various mechanisms, including Streptococcus pneumoniae and
Group A streptococci [237, 238], Haemophilus influenzae [239], Treponema pallidum [240], Klebsiella pneumoniae [241], Pseudomonas aeruginosa [242],
Escherichia coli [243], Salmonella species [244, 245], Staphylococcus aureus [246],
Listeria monocytogenes [247], Shigella flexneri [248], Yersinia pestis [249], Brucella species [250], Legionella pneumophila [251], Rickettsiae [252], several
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Mycobacterium species [253], Helicobacter pylori [254], and Chlamydia species
[219]. When bacteria evade phagolysosomal degradation, they may replicate within the macrophage, which continues to circulate throughout the body, potentially exposing other anatomical sites to infection. This has been highlighted by several studies, including one study of the systemic spreading of Coxiella burnetii, including to the epididymis [255].
In the case of Chlamydia, this has been most thoroughly characterized during C. pneumoniae infection, less so during C. trachomatis and C. muridarum infections
[219, 256-258]. Both C. trachomatis and C. muridarum form inclusions within macrophages, which can survive when infected in vitro for up to 48 hours, suggesting that infection subverts the macrophage killing mechanisms [218, 259,
260].
Specific subsets of macrophages are more able to support chlamydial growth than others. Macrophages, as leukocytes, display the common leukocyte antigen CD45 so are CD45+ [261]. However, they display other CD markers depending on the phenotype. The phenotype can initially be characterized as whether macrophages are polarised towards pro-inflammatory M1 or anti-inflammatory M2 [262]. Generally,
M1 macrophages express higher levels of MHC II, CD68, CD80, CD86, IL-1β, TNF-
α, IL-12, IL-18, IL-23, and reactive nitrogen than M2 macrophages [263-265]. These may have a suppressive effect on chlamydial replication, although not on the initial uptake of EBs. However, M2 macrophages both take up EBs and allow productive inclusion formation. M2 macrophages express higher levels of IL-10, EGF, VEGF,
TGF-β and arginase 1, than M1 macrophages, which could increase their susceptibility to infection [266].
Chapter 2: Literature Review 31
Macrophages are highly plastic in nature, being able to polarise towards M1 or M2 with the appropriate stimulus, and this may be taken advantage of by Chlamydia
[219]. Chlamydiae possess the T3SS, which interacts with host cells on transcriptional and molecular levels to create a more favourable environment [72]. In macrophages, this means that where bacterial stimulation would normally cause M1 polarisation, this may not occur during chlamydial infection. Chlamydia affects the transcription of many genes and repurposes host proteins and lipids to promote its replication, to prevent host cells dying during replication, and that contribute to detection of infection [189, 267, 268]. This has not been characterized within macrophages but could influence plasticity so that survival and replication are easier.
The macrophages that Chlamydia may access initially after infection would be in the penile tissue. There are likely to be both resident and circulatory macrophages in the penile tissue. Few models exist of penile macrophage infection. However,
CCR5+/CD4+ resident penile macrophages have been implicated in establishment of
HIV-1 infection [269] and penile monocytes in development of HSV-2 infection
[270]. As previously described, many models exist for circulating macrophage infection. Within the penis, there is a rich blood and lymphatic supply including; dorsal artery, cavernous artery, circumflex artery, bulbourethral artery, internal pudendal artery, periprostatic plexus, bulbourethral vein, deep dorsal vein, circumflex vein, internal pudendal vein, cavernous vein, bulbar vein, subtunical venous plexus, and retro coronal venous plexus [2]. Both resident and circulatory macrophages would have extensive access to the circulation to migrate to other anatomical sites. For example, the internal and external pudendal arteries cross both the penis and the testes, which could provide direct access to the testes for the
Chlamydia-infected macrophages.
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Infected macrophages may migrate into the testis and contribute to the Tmφ population, or they may transfer the infection to the resident testicular cell populations [216]. It seems likely that infection could transfer to the Tmφ population as these cells uniquely polarise to M2 with microbial stimulus, with the purpose of maintaining an immunosuppressive environment within the testis [213, 217, 271].
2.4 Male infertility and Chlamydia
Chlamydial infections of the male reproductive tract are known to occur in the urethra, epididymis, accessory glands, and importantly the testis [17]. Testicular infection is contentiously associated with impaired testicular function, which, as the site of spermatogenesis, includes reduced sperm health [129]. Sperm parameters, including total count, motility, morphology, and DNA integrity have been investigated with varying results in acute mouse and human infections [6-9, 35-37,
199-201, 203, 272-282]. The cause of the reduced sperm health is unknown; however, the abnormalities are likely to contribute to male infertility.
2.4.1 C. trachomatis and male factor infertility
As Chlamydiae are non-motile bacteria and they are limited to survival within host cells, the convention suggests that a route of ascending infection is followed [69].
The penile urethra is the initial site of infection. Then the infection ascends through the urethra, accessory glands, vas deferens, epididymis, and then infection gains access to the testes. There is evidence of infection in most of these tissues that have been correlated with potential sequelae.
Infection of the urethra can cause urethritis; characterized by discharge and dysuria during symptomatic infections [42]. Urethral infection may provide direct contact
Chapter 2: Literature Review 33
between sperm and Chlamydia, however brief, upon ejaculation of semen. If the infection ascends further within the MRT, this leads to the accessory glands including the prostate.
Infection of the prostate can cause prostatitis, which is characterized by pelvic pain and inflammation. The role of Chlamydia in human prostatitis is still uncertain due to inconsistency in detection methods and debate over tissue samples being contaminated with urethral flora [147, 283]. The pathophysiology of chlamydial prostatitis is also not well understood in human or animal models. Prostate and prostatic fluid infections have been demonstrated in a rodent model of infection and this is hypothesised to have a role in sexual transmission of Chlamydia [50, 282].
While not providing direct contact with sperm, prostate infection could introduce
EBs into seminal plasma components.
Ascending further, infection of the vas deferens and epididymis may also provide direct contact between sperm and Chlamydia. Chlamydial epididymitis is evident in human and rodent models although the pathogenesis mechanism remains uncertain
[272, 273, 275]. Post-spermatogenesis in the testes, sperm is matured and stored in the epididymis and then moves to the vas deferens prior to ejaculation. These locations, particularly the epididymis, have specific structures that could be compromised by chlamydial infection. The epididymis is comprised of three segments, (i) the tail/cauda, which is proximal to the vas deferens, (ii) body/corpus, which links to the distal end, and (iii) head/caput, which is distal to the vas deferens/proximal to the testis [3]. Each segment provides a specialized role in sperm maturation and storage. The plausibility of the ascending route of infection is questionable here, as the non-motile bacteria would be required to travel through a minimum of five meters of tubule length in the human epididymis (one meter in the
34 Chapter 2: Literature Review
male mouse) [81] against the flow of sperm. This suggests infection could not spread rapidly and would need to combat the flow of sperm in the opposite direction. The flushing of the reproductive tract, in this case with semen and urine, is well established as a method for clearing extracellular infections, which may include the
EBs shed after cell lysis, and inducing biofilm and intracellular bacterial community formation in those bacteria capable to evade this [284, 285].
After gaining access to the testes via the epididymis or an alternative route,
Chlamydia would have direct access to sperm if infection was to breach the seminiferous tubules. The specialized cell types, Leydig, Sertoli and SCCs, residing in the testes that are paramount to spermatogenesis may become infected and their function altered. The resulting impact on spermatogenesis could be the abnormal sperm parameters observed in infected men. Infectious, and particularly chlamydial orchitis, is frequently not viewed differentially to epididymitis, leading to the grouped condition of epididymo-orchitis [286, 287]. Chlamydial epididymo-orchitis has not been extensively modelled in animals or humans, leading to uncertainty in the ability of Chlamydia to actively cause the disease [278, 281, 288]. This may be due to a lack of understanding in the pathogenesis pathways leading to chlamydial epididymo-orchitis or orchitis alone.
The ability of Chlamydia to directly contact and affect sperm has been investigated from several functional standpoints. The adherence of C. trachomatis to sperm is debatable; it has been evidenced through immunofluorescent co-localisation of EBs and sperm, visualisation via electron microscopy, and through changes that occurred in sperm after co-incubation with C. trachomatis [199-201, 212, 277]. Decreased sperm motility and viability are associated with co-incubation with C. trachomatis, and more convincingly, increased sperm tyrosine phosphorylation [200]. Tyrosine
Chapter 2: Literature Review 35
phosphorylation is normally induced during sperm capacitation [289], so C. trachomatis-mediated premature capacitation may decrease rates of natural conception. The reduced sperm concentration and motility decreases the likelihood of successful oocyte fertilisation. This compromised fertility may increase the need for assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) or intra-cytoplasmic sperm injection (ICSI) to achieve fertilisation [183, 186].
C. trachomatis DNA has been directly identified in semen, rather than adhered to sperm, in several studies and has been associated with abnormal sperm parameters including; low sperm count, decreased motility, abnormal morphology, low volume, and defective acrosome reaction [35-37, 198, 274, 276, 290]. This could be the result of direct contact between Chlamydia and sperm, or the result of invasion into MRT tissues that contribute to semen; testes, epididymis, prostate, and seminal vesicles.
Conversely, multiple studies have found no correlation between infection and poor sperm health [9, 198, 279]. The disparity in the literature has been attributed to multiple factors including; (i) inconsistency in chlamydial detection methods (IgG and IgA based ELISA, PCR, immunofluorescent assay, culture), (ii) differences in sample being tested (serum, seminal plasma, semen, isolated sperm, tissue biopsy),
(iii) inconsistency in sperm testing methods that are generally laboratory specific,
(iv) and variability in the populations being tested (ethnicity, age, amount and type of sexual activity, presence of symptomatic or asymptomatic infection). For example, the rate of chlamydial infection in infertile men is debatable, ranging from 0-90.3% in literature produced in different studies from around the world [291]. To date, there are no definitive answers or mechanisms linking Chlamydia infection and low sperm quality.
36 Chapter 2: Literature Review
More recently, sperm isolated from Chlamydia positive men were found to have
DNA fragmentation increased by approximately 3.2 times when compared to sperm from non-infected men [6]. Sperm DNA damage and fragmentation is considered to be disadvantageous for, and a prognostic marker of reduced male fertility and poor pregnancy outcomes [186]. In ART extensive DNA damage correlates with delayed onset of a fertilized oocytes’ first cleavage following both IVF and ICSI. This is considered a marker of unsuccessful embryonic growth (embryogenesis) and poor pregnancy outcomes (e.g. miscarriage) [186, 292]. In these cases, a high proportion of sperm have DNA damage, which has likely occurred during spermatogenesis.
Sperm do not possess the cellular machinery to repair the fragmented DNA; this is carried out by the oocyte after fertilisation [209]. This repair process is associated with an increased time to the first oocyte cleavage and with risk of adverse pregnancy outcomes [209].
Although the correlation between infection and sperm DNA damage is an important finding that furthers the link between infection and infertility, there are conflicting reports. These reports show sperm DNA fragmentation occurs to a different extent or not at all. The major concern regarding the conflict in this field is the great variation in methods for testing of sperm DNA fragmentation [210, 293]. Testing of sperm can occur via comet assay, terminal deoxyuridine nick end labelling (TUNEL) assay, sperm chromatin structure assay (SCSA), and sperm chromatin dispersion assay
(SCDA). Each method quantifies DNA fragmentation uniquely and is not necessarily comparable to the others. Each method also has many laboratory or clinic specific modifications that have been implemented, further reducing any comparative power between studies. It has been suggested that the TUNEL and SCDA methods that directly measure whether DNA fragmentation has occurred and minimise sample
Chapter 2: Literature Review 37
handling may be most effective [210]. However, there is no clear view on which method should be the gold standard for the field, so clarification on the negative effect of infection on sperm DNA fragmentation are still required.
2.4.2 C. muridarum and male rodent infertility
The development of animal models has promoted progress in the field of male infection and infertility. Varying reports exist for the kinetics of infection, location of infection after dissemination, and whether fertility is adversely affected as measured by sperm parameter abnormalities [129, 281, 282]. It is generally accepted that C. muridarum leaves the urethra rapidly (within 1 week) after intra-penile infection, but views on the classification of acute versus chronic time of infections differ. There is a large range, from three to 12 weeks post-infection, that defines the difference between acute and chronic [58, 281, 294]. This broad definition may need to be narrowed down to provide a definitive concept of acute and chronic infections.
The location of the disseminated infection also varies, particularly with respects to the epididymis and testes in the upper reproductive tract, where different animal models have generated conflicting results [129, 281]. Like human infections, there is little consensus on sperm parameter changes. This could be attributed to large variation in the techniques use to harvest and analyse sperm, and inconsistencies in the parameters being analysed.
A rat model of infection showed that C. muridarum could be detected by PCR in the urethra (67% of rats), bladder (50%), prostate (33%), and seminal vesicles (33%) 15 days post intra-penile infection, which was termed an acute infection. At 80 days, termed a chronic infection, infection was still detectable by PCR in the prostate (63% of rats) and seminal vesicles (38%) [281]. Notably, despite the apparent
38 Chapter 2: Literature Review
dissemination of the infection from the urethra, at both acute and chronic time points
0% of rats had infected epididymis or testis tissue determined by PCR. At neither time points were the motility, viability, or total count of sperm isolated from the epididymis significantly different between the infected and sham-infected control rats [281]. This was supported in part by a second study by the same group, with the urethra (50%), bladder (60%), testis (20%), and prostate (100%) infected at acute time points, and only the prostate (45%) remained chronically infected [282].
Variation in results between studies from this group and between other models in the literature may be due to the use of PCR as the sole mode of chlamydial detection, as opposed to confirmation via culture or direct detection within the tissue using immunohistochemistry (IHC) techniques. This rat model contrasts with mouse models, which show that infection does have an impact on fertility measured directly by abnormal sperm parameters, and indirectly hypothesised by declining testicular health [50, 53, 129]. These differences may be influenced by using C. muridarum, a natural mouse pathogen, in rats [295, 296]. The kinetics of infection supported dissemination from the urethra in acute timeframes of 4 and 8 weeks, this time to the vas deferens, epididymis, and testes in one study. The testes were PCR and culture positive (100%), and infected mice had a significant reduction in Sertoli cells and
SSCs [129]. In this case, the sperm were isolated from the vas deferens, there was reduced total count, and sperm was significantly more amorphic and immotile in infected mice compared to naïve mice [129].
Regardless of the extensive variability in animal model findings, the rapid dissemination of Chlamydia throughout the male reproductive tract is consistent.
This would suggest a systemic route of infection rather than the classical ascending route due to, as previously mentioned, the likely retrograde action of fluids exiting
Chapter 2: Literature Review 39
the extensive length of the tract. The possibility of systemic spreading of Chlamydia has been investigated in circulating immune cells due to the complex intracellular requirement of the infection. Monocytic and dendritic cell infection models have been established and these immune cells are able to harbour actively replicating
Chlamydia for several days [90, 256, 297, 298]. The idea of systemic infection gains credence when considering the emerging role for Chlamydia in other disease states at anatomically distinct sites to the initial infection. These include; C. trachomatis in synovium during reactive arthritis [151, 152], and C. pneumoniae moving from the respiratory tract to the brain during dementia/Alzheimer’s and atherosclerotic legions during coronary heart disease/stroke [145, 154, 299].
2.5 Research challenges
Some studies have found that sperm from men with reproductive tract chlamydial infections have poor health and increased DNA fragmentation compared to non- infected men [6, 7, 36]. This would provide a tangible explanation for chlamydial infertility. However, the question of where this sperm and DNA damage occurs remains controversial and unresolved; could it occur during spermatogenesis in the testes, or only during storage in the epididymis? This study will develop the concept that the damage is the result of chronic testicular infections, which disrupt spermatogenesis. This leads to the next research challenge; identifying the origin of spermatogenesis disruption. This is likely to result from damage to Sertoli cell,
Leydig cell, and SSC populations, as they are critical to spermatogenesis.
2.6 Research Applications
Specialized testicular cells have an irreplaceable role in spermatogenesis. For example; transplantation of Sertoli cells into mouse testes with this deficiency
40 Chapter 2: Literature Review
dramatically changed the testicular environment to one that would then support spermatogenesis, showing that Sertoli cell absence is inhibitory to spermatogenesis.
This study will determine the potential for testicular cells to be damaged or destroyed by Chlamydia and if this damage is mirrored in sperm cells health. This damage may include DNA fragmentation and epigenetic changes. These factors could be detrimental to the viability or fertility of any offspring produced by affected males.
This may provide information for future treatment schemes of chlamydial infertility in males by providing a target for prevention of disruption to this pathway. Such information would be important for development of a much-needed anti-chlamydial vaccine for males.
Chapter 2: Literature Review 41
Chapter 3: Materials and Methods
Chapter 3: Materials and Methods 43
This chapter describes the detailed methods used in achieving the results found in the results chapters of four, five, six, seven, and eight. An overview of the methods that are specific to each of the results chapters are identified in the Materials and Methods sections of each of the results chapters. This study was funded by the
National Health and Medical Research Council (NHMRC) of Australia.
3.1 Solutions and Buffers
3.1.1 Phosphate Buffered Saline (PBS)
PBS was made following the manufacturer’s instructions for product code
BR0014G (Thermo Fisher Scientific, Victoria, Australia) in Milli-Q® ddH2O
(QTUM0TEX1, Merk, Victoria, Australia). PBS was sterilised by autoclaving at
121°C for 15 minutes, stored at room temperature, and used for experimentation as required.
3.1.2 PBS-Tween (PBST)
PBST was made by the addition of TWEEN® 20 detergent (P9416, Sigma-Aldrich,
New South Wales, Australia) to PBS (section 3.1.1) to a final concentration of 0.1% v/v, stored at room temperature, and used for experimentation as required.
3.1.3 Tris Buffered Saline (TBS)
TBS was prepared to a final concentration of 50 mM Tris base (10708976001, Roche
Biochemical Reagents, New South Wales, Australia) and 150 mM NaCl (AJA465-
5KG, Univar, Victoria, Australia) in Milli-Q® ddH2O (Merk) and adjusting the pH to 7.6 with 1 M HCl (256-2.5L PL, Univar). TBS was made as required as it is stable for up to three months when refrigerated.
44 Chapter 3: Materials and Methods
3.1.4 TBS-TWEEN® (TBST)
TBST was made by the addition of TWEEN® 20 (Sigma-Aldrich) to TBS (section
3.1.3) to a final concentration of 0.1% v/v, stored at room temperature, and used for experimentation as required.
3.1.5 Sucrose Phosphate Glutamate (SPG) Medium
One litre of SPG was made at a time by dissolving 74.6 g sucrose (530-500G,
Univar, New South Wales, Australia), 0.512 g KH2PO4 (AJA391-5KG, Univar), and
1.237 g K2HPO4 (2221-500G, Univar) in Milli-Q® ddH2O (Merk) and adjusting the pH to 7.2 with HCl (256-2.5L PL, Univar). SPG was sterilised by autoclaving at
121°C for 15 minutes, cooled and then frozen in 45 mL aliquots. Directly before use, an aliquot was thawed and 5 mL of Gibco® GlutaMAXTM (35050061, Thermo
Fisher Scientific) was added.
3.1.6 PBS Cocktail
Heparin sulphate (100 µg/mL, H4784, Sigma-Aldrich), DNaseI (100 µg/mL,
4536282001, Roche Biochemical Reagents), and MgCl2 (5 mM, M4880, Sigma-
Aldrich) were made in a solution of PBS (section 3.1.1) to a total volume of 100 mL when required.
3.1.7 Ultravist Gradient
For ultra-purification of Chlamydia EBs a gradient of Ultravist® (370, Bayer, New
South Wales, Australia) diluted in PBS (section 3.1.1) was layered into 14 mL ultracentrifuge tubes (344060, Beckman Coulter, New South Wales, Australia) from
36% at the bottom of the tube, to 29%, to 25%, and 18% at the top of the tube.
Chapter 3: Materials and Methods 45
3.1.8 Mammalian Cell Lysis Buffer
Cell lysis buffer requires 20 mM Tris base (Roche) at pH 7.5 – 8.0, 120 mM NaCl
(Univar), 1% v/v IGEPAL® CA-630 (18896, Sigma-Aldrich), 1 mM EDTA
(AJA180, Univar) and mammalian protease inhibitor cocktail (PIC, P8340, Sigma-
Aldrich) at 1x concentration. These were made to the volume required per experiment in Milli-Q® ddH2O (Merk) directly before use, and chilled to 4°C before use.
3.1.9 Red Blood Cell (RBC) Lysis Buffer
RBC lysis buffer was made up to 1 L by dissolving 8.3 g of NH4Cl (A9434, Sigma-
Aldrich), 1.0 g NaHCO3 (S5761, Sigma-Aldrich), and 37.0 mg EDTA (Univar) in
Milli-Q® ddH2O (Merk), and adjusting the pH to 7.4 using HCl (Univar). The buffer was stored at 4°C, and filter sterilised using a 0.2 µm filter before use.
3.1.10 Biggers, Whitters and Whittingham (BWW) Medium
The BWW base medium was used as the sperm isolation, non-capacitating medium.
The stock was prepared to a 1 L volume by making a solution in Milli-Q® ddH2O
(Merk) of NaCl (2.77 g, Univar), KCl (175 mg, P9541-500G, Sigma-Aldrich),
CaCl2.2H2O (125 mg, 31307-500G, Sigma-Aldrich), KH2PO4 (8.1 mg, Univar), and
MgSO4.7H2O (147 mg, 63138-250G, Sigma-Aldrich). Then 50 mL of the solution was used as required with additional D-Glucose (25 mg, 1083370250, Merk),
Sodium Pyruvate (25 mg, P5280-25G, Sigma-Aldrich), Sodium Lactate (92.5 µl of
60% w/v stock, L1375-100ML, Sigma-Aldrich), Streptomycin (250 µl, S9137-100G,
Sigma-Aldrich), HEPES Buffer (500 µl, H4034-25G, Sigma-Aldrich), Polyvinyl
Alcohol (25 mg, P8136-250G, Sigma-Aldrich). The osmolarity was adjusted to be
46 Chapter 3: Materials and Methods
290-310 Osm. The BWW was warmed to 37°C directly before use and maintained at
37°C as often as possible during use.
3.1.11 Sperm Capacitation Medium
The capacitation media uses the BWW base media (section 3.1.10) as the major component with the addition of NaHCO3 (52.5 mg, S6297-250G, Sigma-Aldrich),
1mM pentoxifyllinene (P1784-10G, Sigma-Aldrich), and 1mM dbcAMP (D0627-
250MG, Sigma-Aldrich) per 50 mL of BWW. The osmolarity was adjusted to 290-
310 Osm, and the media was warmed to 37°C directly before and maintained during use. Due to degredation of components, the capacitation media was unable to be stored and was used only on the same day as preparation.
3.1.12 Oocyte Collection and Storage
The ovary hyperstimulation, oocyte collection, and storage in the salt storage buffer were carried out as per previously published methods [300], this was carried out at
Newcastle University by Dr Kate Redgrove. Briefly, female, 6-week old, C57BL/6 mice were injected via the intraperitoneal route with equine chorionic gonadotropin to promote oocyte maturation. After 48 hours, mice were euthanized, ovaries were collected, and ovarian follicles were then punctured to release oocytes, which were collected in M2 medium supplemented with milrinone. Cumulus cells surrounding oocytes were mechanically removed via pipetting before use in experimentation.
3.1.13 Heat-inactivated Fetal Calf Serum (HI-FCS) / Horse Serum (HI-
HS)
Inactivation of the calf and horse serums (10099141, Thermo-Fisher Scientific) used in cell culture media was carried out by heating in a water bath at 56°C for 30 minutes. FCS was then stored at -20°C in 25 mL aliquots until required.
Chapter 3: Materials and Methods 47
3.1.14 Immortalised Cell Line Growth Medium
To create the complete media, 500 mL of Roswell Park Medical Institute (RPMI)
1640 (22400105, Thermo-Fisher Scientific) was supplemented with 10% HI-FCS v/v
(section 3.1.13), 2 μg/mL gentamycin sulfate (15750078, Thermo-Fisher Scientific), and 100 μg/mL streptomycin sulfate (S9137-100G, Sigma-Aldrich). Complete media was made as required, refrigerated at 4°C for storage, and heated to 37°C before use in experimentation.
3.1.15 Primary Cell Growth Medium
Phenol red-free Dulbeco’s Modified Eagle’s Medium/F-12 (DMEM/F-12, 11039021,
Thermo Fisher Scientific) supplemented with 5% HI-HS v/v (section 3.1.13), 2
μg/mL gentamycin sulfate (Thermo Fisher Scientific), and 100 μg/mL streptomycin sulfate (Sigma Aldrich). Complete media was made as required, refrigerated at 4°C for storage, and heated to 37°C before use in experimentation.
3.1.16 Tris Borate Ethylenediaminetetraacetic acid (EDTA) Buffer (TBE)
Concentrated 10 x TBE was made by dissolving Tris Base (108 g, Roche), boric acid
(55 g, A0387165215, Merk), and EDTA (9.3 g, Univar) in 1 L of Milli-Q® ddH2O
(Merk). For use in experimentation, 1 x TBE was made by diluting one part of concentrate with nine parts of Milli-Q® ddH2O (Merk).
3.2 Cell Culture and Experimentation
3.2.1 Immortalised Cell Line Culture
During this project the TM3 Leydig (ATCC® CRL-1714TM), TM4 Sertoli (ATCC®
CRL-1715TM), GC-1 Germ (ATCC® CRL-2053TM), and RAW 264.7 macrophage
(ATCC® TIB-71TM) cell lines were cultured in complete RPMI 1640 (section
48 Chapter 3: Materials and Methods
3.1.14), at 37°C, 5% CO2, and passaged to a maximum of P30. TM3, TM4, and GC-
1 cells were split using 0.5% trypsin-EDTA (15400054, Gibco®) as per the
American Type Culture Collection (ATCC) guidelines available for each line. RAW
264.7 cells were split using mechanical lifting with a scraper (83.1830, Sarstedt,
South Australia, Australia).
3.2.2 Primary Cell Culture
During this project primary C57BL/6 mouse Leydig, Sertoli, spermatogenic cells, and Tmφ cells were cultured in complete DMEM/F-12 (section 3.1.15), at 37°C, 5%
CO2, and were not passaged. Cells were isolated as required using the following method. Mouse testes were isolated (section 3.4.7), decapsulated of the tunica albuginea, and tubules were gently loosened but not fragmented using forceps.
Tubules were incubated in collagenase (0.5 mg/mL, C0130-100MG, Sigma-Aldrich) for 15 minutes, 34°C, and at 80 rpm. Tubules were then washed three times in Hanks
Balanced Salt Solution (HBSS, 24020-117, Thermo Fisher Scientific) by centrifugation at 1000 rpm, for 3 minutes each. The supernatant from the washes contains the interstitial cells; this was layered over Percoll® (P4937, Sigma-Aldrich), incubated for 20 minutes, then the top fraction containing Leydig cells and Tmφ was pelleted by centrifugation (1000 rpm, 3 minutes), sorted by flow cytometry (3.3.10), and resuspended in complete medium for use in experimentation. The pellet containing intra-tubular cells was incubated in 0.5 mg/mL trypsin-EDTA (Sigma-
Aldrich) for five minutes at 37°C. Tubules were then washed twice in HBSS
(Thermo Fisher Scientific) and once in trypsin inhibitor (0.3 mg/mL, 10109886001,
Roche) by centrifugation at 1000 rpm for 3 minutes each. Tubules were then enzymatically digested in collagenase (Sigma-Aldrich), hyluranidase (H1136,
Sigma-Aldrich), DNaseI (Roche), and trypsin inhibitor (Roche) for 40 minutes, at
Chapter 3: Materials and Methods 49
34°C, 80 rpm. The digested product was washed twice in HBSS by centrifugation at
1000 rpm for three minutes each. The pellet was resuspended in HBSS, passed through a 70 µm cell strainer, then subjected to a hypotonic shock to enrich for
Sertoli cells, before being washed in HBSS for a final time. Cells were resuspended in complete medium and seeded at 1 x 105 cells per well onto sterile glass coverslips
(round, 8 mm diameter, G401-08, ProSciTech, Queensland, Australia) in a 48-well microtitre plate. Cells were grown for 24 hours before use in experiments and the media was changed immediately before use. Spermatogenic cells were isolated by Dr
Kate Redgrove at the University of Newcastle [301]. Cultures were stained using
ICC (3.3.8) to confirm enrichment of each cell lineage.
3.2.3 Monolayer Co-culture
Monolayers of RAW 264.7 cells were infected at Multiplicity of Infection (MOI) of
1, the second cell type remained non-infected, and both were incubated for 48 hours
(section 3.2.1) in separate T75 cell culture flasks (CLS430641, Corning®, New
South Wales, Australia). After 24-hours the infected cells were dislodged from the flask by scraping, and washed three times via centrifugation (1000 x g) in a heparin sulphate (Sigma-Aldrich) in PBS (section 3.1.1) solution to remove any extracellular
Chlamydia. The second cell type (either TM3, TM4, or GC-1) was dislodged by trypsanisation. The two cell types were then mixed in a 1:1 ratio in RPMI 1640
(section 3.1.14) and seeded in triplicate in a 48-well cell culture plate (Corning®).
Every 24-hours for two weeks, cells were fixed in 100% v/v methanol and stained using immunocytochemistry techniques for Chlamydia (3.3.8), F4/80 to show the
RAW264.7 macrophages, and Alexa Fluor™ 555 Phalloidin (Thermo Fisher
Scientific) to shows the second testicular cell type.
50 Chapter 3: Materials and Methods
3.2.4 Transwell® Co-culture
The Transwells® had uncoated membranes with 0.4 μm pore-sizes and were for a
24-well format (BD Diagnostics, New South Wales, Australia). They were used in corresponding 24-well plate holders (BD Diagnostics). Transwell®-containing plates were inverted, testicular cell line cells TM3 Leydig, TM4 Sertoli, or GC-1 germ were seeded onto the basolateral side of the membrane, covered with the plate, and incubated at 37 °C, 5% CO2 for 2 hours, then washed to remove unbound cells. Then
600 µL of RPMI 1640 (section 3.1.14) was added into the basolateral well.
Macrophage cell line RAW 264.7 cells were infected 48 hours previously (as per section 3.2.3), then seeded onto the apical membrane in 200 µL of RPMI 1640.
Wells were fixed at appropriate time points using 100% v/v methanol (AJA318-
2.5LGL, Univar).
3.2.5 Live Cell Imaging
The cells visualised using live cell imaging were the TM3 and TM4 cells. These were seeded into 48-well microtitre plate (Corning®) in complete RPMI 1640
(section 3.1.14) containing DAPI (1:40000 v/v, D9542-1MG, Sigma-Aldrich), which was used to indicate dead cells, 36-hours before the imaging took place. Then 12- hours prior to imaging, the cells were infected at MOI 0.1 with C. muridarum Nigg
(GFP producing strain made by Dr Charles Armitage). Imaging was conducted at
37°C, 5% CO2, for 96 hours in total, with an image captured every 30 minutes
(AF6000, Leica Microsystems, New South Wales, Australia).
3.2.6 Comet Assay
Cells were seeded into three 48-well microtitre plates (Corning®), one plate for irradiation (positive control, 6 Gy), one plate for non-treated cells (negative control),
Chapter 3: Materials and Methods 51
and one plate for infection (test condition). Cells were trypsinised to dislodge them from wells at the appropriate time points. Cells (50 μL of cells at 2 x 105 cells/mL) from each well were mixed in individual microfuge tubes with 0.6% w/v low-melting point agarose (1613111, Bio Rad, New South Wales, Australia, molten, made in 1 x
TBE, 500 μL, at 37°C). Then, 50 μL of cell/agarose mixture was applied to two-well
CometSlidesTM (Trevigen, New South Wales, Australia) and spread to cover the sample areas. Slides were placed at 4°C in the dark for 10 minutes, and then immersed in pre-chilled lysis buffer at 4°C for 30 minutes. Lysis buffer is made by dissolving 2.5 M NaCl (Univar), 100 mM EDTA (Sigma-Aldrich), 10 mM Tris
(Roche, pH 10), and 1% Triton™ X-100 (T8787-250ML, Sigma-Aldrich) in Milli-
Q® ddH2O (Merk). Slides were immersed in 1 x TBE for 15 minutes at 4°C. An electrophoresis tank was filled with 1 x TBE to cover slides on the electrophoresis gel tray. The power supply was set to 70 volts (~90 mA) and slides were electrophoresed for 30 minutes. Slides were immersed Milli-Q® ddH2O (Merk) for 5 minutes at 4°C, then 80% v/v ethanol for 5 minutes, dried at room temperature for 30 minutes. Slides were then stained with 1 x SYBR® Safe (S33102, Thermo Fisher
Scientific) for 10 minutes, dried, and viewed using epiflourescent microscopy (Nikon
Instruments, New York, United States of America).
3.2.7 Transcriptomics
TM3 Leydig, TM4 Sertoli, and GC-1 germ cells were used to study transcriptional changes induced by C. muridarum during infection. Cells were mock infected with
SPG or infected at MOI 1 with C. muridarum Weiss for 30 hours. RNA was extracted using an RNeasy kit (QIAGEN, Victoria, Australia) as per the manufacturer’s instructions. Extracted RNA was quantified using the NanoDrop™
(Thermo Fisher Scientific). RNA was then sent to the Beijing Genomics Institute
52 Chapter 3: Materials and Methods
(BGI) where validation (Agilent 2100 Bioanalyzer) reverse transcription, library preparation, and RNAseq (BGISEQ-500 Platform, Hongkong, China) were carried out. Sequenced reads were then analysed with the assistance of BGI bioinformatics pipeline. The programs StringTie (transcript assembly), HISTAT (genome mapping),
Cuffcompare (a tool of Cufflinks, annotation), Bowtie2 and RSEM (gene mapping and expression), DEseq2 (differential gene expression analysis), and Kyoto
Encyclopaedia of Genes and Genomes (KEGG) pathway classification, were used in the bioinformatics pipeline to provide a range of RNAseq analyses.
3.2.8 Methylation Detection Mass Spectrometry
DNA was extracted from cells using DNeasy Blood and Tissue DNA extraction kit
(QIAGEN) as per the manufacturer’s instructions for cultured cells. Extracted DNA was sent to QUT Central Analytical Research Facility (CARF) where it was tested by
NanoDrop™ (Thermo Fisher Scientific) and determined to be >500 ng/25 µL reaction. DNA was then digested using DNA Degradase Plus™ (E2021, Zymo
Research, distributed by Integrated Sciences, New South Wales, Australia) and methylated cytosine residues were detected by mass spectrometry (LCMS-8050,
Shimadzu, Tokyo, Japan) by weight differential from demethylated cytosine. The number of methylated cytosine residues was quantified and compared between the infected and non-infected samples.
3.2.9 Detection of Testosterone
An enzyme linked immunosorbant assay (ELISA) was used to detect testosterone produced by TM3 Leydig cells in cell culture and in mouse serum. The TM3 cell culture media supernatant was centrifuged at 1000 x g to pellet contaminating cells, then applied to the ELISA plate wells. Serum was harvested (section 3.4.4) from 6-
Chapter 3: Materials and Methods 53
month old, male, C57BL/6 mice that had been infected with C. muridarum Weiss or mock infected with SPG (section 3.1.5). The ELISA was performed in-house at the
School of Agriculture and Food Sciences (University of Queensland) by Dr Tamara
Keeley.
3.3 Chlamydia Culture and Experimentation
3.3.1 Chlamydia Bulk-up
Mouse specific strains used in this project include C. muridarum Weiss (wild type) and C. muridarum Nigg (plasmid cured, transformed to produce GFP by Dr Charles
Armitage) originally sourced from the ATCC (ATCC® VR-123™). Both strains were grown in McCoyB cell monolayers for 28 hours. Low titre chlamydial seeds were mixed with complete RPMI 1640 (section 3.1.14) and placed on monolayers growing in T175 flasks (Corning®). After approximately four hours of incubation
(37°C, 5% CO2) media was replaced with complete RPMI 1640 supplemented with cycloheximide (1 µg/mL, C7698, Sigma-Aldrich) and incubated for a further 24 hours.
3.3.2 Semi-purification
After infected monolayers had be incubated for the required time, they were trypsanised and collected into 50 mL tubes, centrifuged at 1000 x g, 10 minutes, 4°C, sonicated (Microson Ultrasonic Cell Disruptor, MISONIX, New York, United States of America) on ice at mid-high range, 5 x 15 second bursts, centrifuged at 1000 x g,
30 minutes, 4°C, then the supernatants were centrifuged at 14000 x g, 15 minutes,
4°C. The pellets obtained from the last centrifugation are the semi-pure Chlamydia
EBs and RBs with minimal cell debris, and are resuspended in SPG (section 3.1.5) and stored at -80°C until required for experimentation.
54 Chapter 3: Materials and Methods
3.3.3 Ultra-purification
Semi-pure Chlamydia was passed through an Ultravist® gradient (section 3.1.7) using ultra-centrifugation. The semi-pure EB solution was layered over the 18% solution and then tubes were centrifuged at 100 000 x g, for 2 hours, at 4°C. The pure
EBs formed a layer between the 36% and the 29% solutions, this was pipetted into microfuge tubes, washed in SPG (section 3.1.5) by centrigugation at 18 000 x g, for
15 minutes, at 4°C, and then stored at -80°C until required. The RBs and any remaining cell debris from the semi-pure is caught by other dilutions of the
Ultravist®.
3.3.4 Chlamydia Titration
Semi-pure and ultra-pure EBs were titrated by thawing an aliquot from -80°C storage, placing a 10-fold dilution series on a McCoyB cell monolayer, and growing the Chlamydia for the required time (section 3.3.1). The infected monolayers were then fixed in 100% v/v methanol, stained (section 3.3.8), and inclusions were viewed using epiflourescent microscopy (Zeiss Axio Vert.A1, Göttingen, Germany) across
10 non-overlapping fields of view (FOV) per well for the dilution with a reasonable number of inclusions present (less than 100). The number of inclusions per FOV were counted using MetaMorph® software (Molecular Devices, California, United
States of America). The average number of inclusions (measured as inclusion forming units – IFU) per FOV, was converted to average IFU per millilitre by multiplying the IFU/FOV by the number of FOV per well and the dilution factor for the seed being titrated.
Chapter 3: Materials and Methods 55
3.3.5 Chlamydia Infection
Monolayers of cells were grown to a maximum of 80% confluency before being infected. The spent culture medium was aspirated from the monolayer immediately before infection. Titrated Chlamydia (section 3.3.4) was added to fresh complete cell culture medium at the correct MOI, placed onto cell monolayers, and then incubated
(37°C, 5% CO2, 4 hours). The infectious medium was then replaced with fresh medium for all testicular and macrophage cells, or with fresh media containing cycloheximide (1 µg/mL) for McCoyB cells. Infections were incubated at 37°C, 5%
CO2 until appropriate timepoints, when they were fixed in 100% v/v methanol.
3.3.6 Chlamydial extrusion isolation
Chlamydial extrusions were isolated from macrophage and testicular cell line culture by evaporation of cell culture media containing the extrusions onto StarFrost poly-L- lysine coated glass microscopy slides (G312P-G, ProSciTech) at 37°C in a Class II
Biosafety Cabinet. Slides were then fixed by applying 100% v/v methanol and allowing the methanol to evaporate at room temperature. The extrusions were then detected by ICC staining methods and viewed using epifluorescent microscopy techniques (see section 3.3.8 for staining procedure).
3.3.7 Chlamydial Polymerase Chain Reaction (PCR)
C. trachomatis was detected in human testicular tissues collected from IVF clinics during this project. DNA was extracted from the biopsies using the DNeasy Blood and Tissue kit (QIAGEN) as per manufacturer’s instructions. DNA extracted
(DNeasy, QIAGEN) from C. trachomatis serovar D (ATCC® VR-855™) EBs was used as a positive control. Primers targeting C. trachomatis 16S rRNA DNA were used as previously described [302]. The protocol was modified to include detection
56 Chapter 3: Materials and Methods
of low-abundance targets by extending the cycle number to 50, and a melt curve analysis was added to the assay. Melt curves were considered to be of the same species origin when peaks fell within 4°C on each side, of the control. No-template controls were used in all assays. All C. trachomatis DNA detection was completed on the Rotor-Gene Q platform (QIAGEN). Amplicons were then used in 2% agarose gel electrophoresis (120 V, 38 minutes, 1 x TBE buffer (section 3.1.16), 11 µL amplicon volume). The amplicon size was approximately 76bp, so the molecular weight marker HyperLadder™ 50bp (BIO-33054, Bioline, Teletech Park, Singapore) was used to estimate amplicon sizes within the gel.
3.3.8 Chlamydial Immunocytochemistry (ICC) Staining
Infected cell monolayers were fixed using 100% v/v methanol (Univar) and then stained and viewed using epiflourescent microscopy (Zeiss Axio Vert.A1). The staining procedure included blocking with 5% FCS for 1 hour at room temperature, application of primary antibody (anti-MOMP IgG raised in sheep, made in-house) for 1 hour at room temperature, washing 4 x 5 minutes in PBS (section 3.1.1), application of the secondary antibody (anti-sheep IgG raised in donkey conjugated to
Alexaflour 488, Thermo Fisher Scientific) mixed with DAPI (1:40000 dilution of 1 mg/mL stock solution, Thermo Fisher Scientific) for 1 hour at room temperature, and washing 4 x 5 minutes in PBS (section 3.1.1).
3.3.9 Chlamydial Immunohistochemistry (IHC)
3.3.9.1 Mouse Tissue
Infected mouse tissues were harvested as per methods in Section 3.4, 7 µm sections were cut at -22°C using a cryostat (Leica CM1850 Cryomicrotome, New South
Wales, Australia) onto Superfrost™ Plus glass microscope slides (4951PLUS4,
Chapter 3: Materials and Methods 57
Thermo Fisher Scientific), and placed into -80°C storage. Slides were processed for
IHC using the following protocol as required.
Sections were removed from -80°C storage, immersed in 4°C 100% v/v acetone for
20 minutes, air dried at room temperature for 20 minutes, immersed in 4°C 100% v/v acetone for 10 minutes, air dried for 10 minutes, washed 2 x 5 minutes in PBS
(section 3.1.1), immersed in 10% neutral buffered paraformaldehyde for 5 minutes, washed 2 x 5 minutes in PBS (section 3.1.1), immersed in 0.3% H2O2 for 10 minutes, washed 2 x 5 minutes in PBS (section 3.1.1), and immersed in 5% FCS in PBS
(section 3.1.1) for 1 hour at room temperature. Dako Pen (S2002, Aligent, Victoria,
Australia) wells were then drawn tightly around tissue sections. The primary antibody was added to each well and incubated for 1 hour at room temperature then washed 2 x 5 minutes in PBS (section 3.1.1). The secondary antibody was then added to each well and incubated for 1 hour at room temperature. Chromogenic DAB substrate was applied for 7 minutes at room temperature then washed off by 2 x 5 minutes rinses in PBS (section 3.1.1). A methyl green counter stain was added to each well for 2 minutes, then washed in Milli-Q® ddH2O (Merk) for 2 x 2 minutes.
Finally, slides were dipped in 95% v/v ethanol 10 times, dipped in 100% v/v ethanol
10 times, dipped in 100% v/v xylene 2 x 1.5 minutes, mounted in DPX Mountant for histology (44581-100ML, Sigma-Aldrich), and left to dry in a fume hood overnight.
Stained slides were then imaged using slide scanner (Aperio AT Turbo – Digital
Pathology Scanner, Leica Biosystems, New South Wales, Australia) and relevant parameters were quantified.
Antibody combinations used included (i) primary mouse anti-α-Tubulin (T9028,
Sigma Aldrich) with secondary goat anti-mouse IgG-HRP (1030-05, Southern
Biotech, Victoria, Australia) to detect Sertoli cells, (ii) primary goat anti-3βHSD (sc-
58 Chapter 3: Materials and Methods
30820, Santa Cruz Biotechnology, Texas, United States of America) with secondary anti-goat IgG-HRP (Southern Biotech) to detect Leydig cells, (iii) primary mouse anti-PCNA (NA03, Merck Millipore) with secondary goat anti-mouse IgG-HRP
(Southern Biotech) to detect germ cells [129], (iv) primary rabbit anti-PLZF
(ab39354, Abcam, Cambridge, United Kingdom) with secondary donkey anti-rabbit
IgG-HRP (Southern Biotech) to detect germ cells, (v) mouse anti-ZO-1 (33-9100,
Thermo Fisher Scientific) with goat anti-mouse IgG-HRP (Southern Biotech) to detect tight junctions, (vi) mouse anti-SMA (CD001C, Biocare Medical, Queensland,
Australia) with goat anti-mouse IgG-HRP (Southern Biotech), (vii) rabbit anti- cleaved caspase 3 (CP229C, Biocare Medical) with donkey anti-rabbit IgG-HRP
(Southern Biotech), and (viii) primary rabbit anti-F4/80 (Thermo Fisher Scientific) with secondary mouse anti-rabbit IgG-AF594 (Thermo Fisher Scientific) to detect macrophages.
3.3.9.2 Human Tissue
Ethical approval to work with these tissues was given by Monash University Human
Research Ethics Committee (RES-16-0000-559L). Human testicular open biopsy tissues were collected during 2017 and were stored at Monash Medical Centre
(Monash Health Anatomical Pathology department) archives of past patients. A selection of 200 biopsies were sectioned at Monash and sent to QUT for analysis.
The sections were processed by the Histology core facility at Queensland Institute of
Medical Research (QIMR) Berghofer Medical Research Institute, and the following protocol was provided.
Sections were dewaxed and rehydrated through descending graded alcohols to water using standard protocol (program 3 Sakura DRS stainer). Endogenous peroxidase
Chapter 3: Materials and Methods 59
activity was blocked by incubating the sections in 2.0% H2O2 in TBS for 10 minutes.
Sections were washed in three changes of water. Sections were transferred to Biocare
Medical Diva antigen retrieval solution and subject to 10 minutes of heat antigen retrieval using the decloaking chamber (Biocare Medical) at 115°C. After completion of the cool down cycle, the container of slides was removed and allowed to cool for a further 20 minutes before transferring back to TBS. Sections were in three changes of TBS. Nonspecific antibody binding was inhibited by incubating the sections in
10% FCS+10% donkey serum in Biocare Medical Background sniper for 30 minutes.
In a humidified chamber, excess FCS was decanted from the sections and the sheep anti-MOMP primary antibody (diluted 1:100) in blocking buffer was applied for 60 minutes at room temperature. Sections were washed in three changes of TBS.
Jackson Immunoresearch Donkey anti-sheep HRP (diluted 1:300) in TBS was applied for 30 minutes at room temperature. Sections were washed in three changes of TBS. Signals were developed in betazoid DAB for 2 minutes. Sections were washed in water three times to remove excess chromogen. Sections were lightly counterstained in Hematoxylin (program 7 DRS), washed in water, dehydrated through ascending graded alcohols, cleared in xylene, and mounted using DePeX or similar.
3.3.10 Flow Cytometric Isolation and Infection of Testicular Macrophages
Mouse testicular interstitial cells were isolated as per the protocol in section 3.2.2.
The interstitial cells were stained for cell-identifying markers using fluorophore conjugated anti-CD45.2, anti-F4/80, and anti-CD11b. The FACS Aria III (BD
Diagnostics) was used to sort out the Tmφ using firstly a live/dead gate (Zombie
Aqua™ Fixable Viability Kit, 423101, BioLegend, California, United States of
America), then single cell and lymphocyte gates were applied, then a CD45+ gate,
60 Chapter 3: Materials and Methods
finally cells that were double positive for F4/80 and CD11b were isolated. The isolated Tmφ were seeded into a 48-well microtitre plate and incubated overnight
(37°C, 5% CO2). After the incubation, cells were infected with C. muridarum Weiss as per section 3.3.5. The infection was allowed to develop for 24, 48, and 72 hours.
At each time point, cells were fixed, stained, and viewed under epifluorescent microscopy as per section 3.3.8.
3.3.11 Chlamydial Progeny Quantification
The TM3 Leydig, TM4 Sertoli, GC-1 germ, and RAW 264.7 macrophage cell lines were cultured as per section 3.2.1. The cells were infected at MOI 1 with C. muridarum Weiss as per section 3.3.5. At 30-hours post infection, the cultures were sonicated with a probe sonicator (MISONIX) on a moderate setting for 4 x 15 second bursts alternated with 4 x 15 second rest periods. The now-infectious supernatant containing the progeny of each cell lineage was then subcultured onto an 80% confluent, McCoyB cell monolayer. The quantification was treated as per the titration of Chlamydia described in section 3.3.4.
3.3.12 Detection of anti-Chlamydial Antibodies
Anti-chlamydial antibodies were detected in human serum provided by Monash IVF
(ethics provided by Monash IVF HREC under 666-15) and Queensland Fertility
Group (ethics provided by QFG HREC under QFG12.15), and QUT HREC
(1500000394). Whole blood was collected into SST vacutainers (BD Diagnostics), clotted, and spun at the clinics then shipped to QUT for testing. At QUT the serum was placed onto an in-house made ELISA plate. The plate was coated with C. muridarum MOMP. The detection antibody was anti-human IgG conjugated to HRP.
Chapter 3: Materials and Methods 61
3.4 Animal Experimentation
3.4.1 Mice
Ethics approval was acquired from the Queensland University of Technology Animal
Ethics Committee (QUT AEC, approval number 1400000250) and the QIMR
Berghofer Medical Research Institute AEC (approval number A1603-609M) for the use of mice in this project. Mice were housed and fed ad libitum in accordance with
Australian Standards, and procedures were performed in physical containment level
2 (PC2) conditions at the QUT Medical Engineering Research Facility (MERF). For the chronic infection and breeding studies, male C57BL/6 that were 6-weeks old were sourced from the Animal Research Centre (ARC, South Australia, Australia), infected via the intra-penile route (section 3.4.3) and housed until mice reached six months of age. Mice were then either euthanized (section 3.4.14) and had tissues collected (section 3.4.4-11) or were bred with females first (section 3.4.15) and then euthanized (section 3.4.14) and tissues harvested (section 3.4.4-11). Female mice were C57BL/6 aged between 6 – 12 weeks as they were proven breeders and had dropped one litter previously (ARC). Mice used during vasectomy experiments were sourced from the ARC with either intact or vasectomised reproductive tracts, were 6
– 12 weeks old, and were infected via the intra-penile route (section 3.4.3) before tissues were harvested (section 3.4.4-11) at appropriate time points.
3.4.2 Anaesthesia
Anaesthetisation of mice was required for intra-penile infection (section 3.4.3). This was done by intraperitoneal (IP) injection of ketamine (100 mg/kg) (Parnell
Laboratory, New South Wales, Australia) mixed with xylazine (10 mg/kg) (Bayer) in
62 Chapter 3: Materials and Methods
PBS (section 3.1.1). Ketamine was used by drug license holder in accordance with the Queensland Health (Drugs and Poisons) Regulation Act (1996).
3.4.3 Intra-penile Infection
Anesthetized male C57BL/6 mice (section 3.4.2) were placed on their backs, gentle pressure was applied to penis area exposing the barb. A 5 μL volume of SPG (section
6 3.1.5) containing 1 x 10 IFU of C. muridarum (Weiss) was gently applied to the barb with a micropipette.
3.4.4 Cardiac Bleeding and Serum Isolation
Mice were euthanized (section 3.4.14), the abdominal and chest skin was pulled back, and ribs bisected to reveal the heart. The right ventricle was punctured with a
26-gauge needle and blood drawn into a 1 mL syringe. Blood was aspirated into microfuge tubes and clotted at 4°C overnight. The microfuge tubes were then centrifuged at 1000 x g for 5 minutes to separate the serum from the clotted cells.
The serum was removed to a separate microfuge tube using a micropipette and both tubes were stored frozen at -80°C until required for experimentation.
3.4.5 Penile Tissue Collection
Mice were euthanized (section 3.4.14), the abdominal tissue was bisected downward to expose the lower reproductive tract. The encasing tissue was removed using curved surgical scissors and the penile tissue was dissected from the reproductive tract. The penile tissue was either frozen at -80°C in Tissue-Tek® O.C.T Compound
(OCT, ProSciTech) or fixed in Bouin’s solution (HT10132-1L, Sigma-Aldrich) immediately after removal.
Chapter 3: Materials and Methods 63
3.4.6 Testicular Tissue Collection
Mice were euthanized (section 3.4.14), the abdominal tissue was bisected downward to expose the lower reproductive tract, and upward to expose the abdominal fatty tissue. The abdominal fatty tissue was carefully pulled upward using forceps until the connected testes were exposed. Once the testes were exposed, the combined testes and epididymis were dissected out. Using a dissecting microscope, the epididymides were separated from the testes. The testes either underwent tissue processing for primary cell isolation (section 3.2.2), were frozen at -80°C in OCT (ProSciTech), or fixed in Bouin’s solution (Sigma-Aldrich) immediately after removal.
3.4.7 Epididymis Tissue Collection
As stated in section 3.4.6, the epididymides were removed in conjunction with the testes and separated using a dissecting microscope. Immediately after removal, epididymides were either fixed in Bouin’s solution (Sigma-Aldrich) or frozen at
-80°C in OCT (ProSciTech).
3.4.8 Vas Deferens Removal
After the MRT had been exposed (section 3.4.6), the vas was dissected out of the abdominal cavity by cutting the tissue directly adjoining the epididymis on the distal end and the seminal vesicles on the proximal end. The vas was immediately placed into mineral oil (M8410, Sigma-Aldrich) that had been pre-warmed to 37°C.
3.4.9 Sperm Collection and Analysis
Sperm was collected from mice six-months post infection (section 3.4.3) by dissecting out the Vas Deferens (section 3.4.9) and then gently pushing the compacted sperm out of the tubules into BWW media (section 3.1.10) with the
64 Chapter 3: Materials and Methods
magnification assistance of a dissecting microscope (Leica MZ6, Leica
Microsystems). Sperm were incubated for 10 minutes in BWW medium (section
3.1.10) at 37°C, 5% CO2 to allow any cell debris or aggregated sperm to sink and for viable sperm to swim up. Immediately following swim-up, sperm count and motility were assessed using a haemocytometer viewed using inverted light microscopy
(Zeiss Primo Vert, Göttingen, Germany). Sperm in BWW were mixed with eosin Y
(1:1 v/v) to assess percent vitality and the percent forward progressively motile.
Approximately 1 x 106 sperm were then moved to capacitating BWW media (section
3.1.11) and incubated for 10 minutes, 37°C, 5% CO2. Following capacitation, sperm from each mouse was incubated with 8 x C57BL/6 female mouse oocytes for 20 minutes, 37°C, 5% CO2, and then viewed using inverted light microscopy (Zeiss
Primo Vert) to count the number of sperm bound to each oocyte (ZP binding) [303].
Sperm were heat and methanol (100% v/v) fixed onto glass microscope slides and stained with eosin Y and methylene blue (to assess morphology (IHBI histology microscope). Sperm were set into low-melting point agarose on chromatin dispersion assay (CDA) slides at 4 ºC for five minutes, then slides were placed in lysis buffer for five minutes, washed in RO water for five minutes, fixed in 80% v/v ethanol for two minutes and 100% v/v ethanol for two minutes, then stained with SYBR Green
II (Invitrogen, Australia) mixed with VECTASHIELD antifade mounting medium
(Vector Laboratories, CA, USA) (1:1 v/v). Sperm cells were viewed and counted using an epifluorescent microscope (Zeiss Axio Vert.A1).
3.4.10 Female Reproductive Tract
Female pups were euthanized (section 3.4.14) during the breeding study for histological examination. The abdominal tissue was bisected downward to expose the lower reproductive tract, and upwards to expose the upper reproductive tract. The
Chapter 3: Materials and Methods 65
fatty and connective tissue was removed where possible using forceps and curved surgical scissors and then the entire reproductive tract was dissected from the abdominal cavity. Immediately after removal, the reproductive tract was either fixed in Bouin’s solution (Sigma-Aldrich) or frozen in OCT (ProSciTech).
3.4.11 Spleen and Splenocyte Collection
Mice were euthanized (section 3.4.14), the abdominal tissue was bisected upwards to expose the spleen. The spleen was mechanically disrupted by being pushed through a
70 µm cell strainer to achieve a single cell suspension of splenocytes. The splenocytes were treated with RBC lysis buffer (section 3.1.9) to remove contaminating erythrocytes, then cultured for the required time in complete RPMI
1640 (section 3.1.14). Where splenocytes were not immediately required, they were frozen at -80ºC in a solution of HI-FCS (section 3.1.13) with 10% v/v HYBRI-
MAX® DMSO (D2650, Sigma-Aldrich).
3.4.12 Mouse Weight and Organ: Body Weight Ratio
Individual male and female, adult and juvenile mice were weighed in the project.
Live mice were contained in an opaque, high sided container on scales to measure the total body weight. After euthanasia (section 3.4.14) the organs of interest were dissected out of the body, cleaned of any additional material using a dissecting microscope and then weighed on a fine balance. The organ: body weight ratio can then be calculated by dividing the weight of the mouse by the weight of the organ
(ratio), dividing 1 by the obtained value and multiplying by 100 to obtain the percentage of total body weight for graphing purposes.
66 Chapter 3: Materials and Methods
3.4.13 Detection of Anti-Sperm Antibodies
An enzyme linked immunosorbant assay (ELISA) was used as per the manufacturer’s instructions (MyBioSource.com, California, United States of
America) for the detection of anti-sperm antibodies in mouse serum. Serum was harvested (section 3.4.4) from 6-month old, male, C57BL/6 mice that had been infected with C. muridarum Weiss or mock infected with SPG. The ELISA plate that was provided by MyBioSource was coated with mouse sperm antigen, this was used to capture the anti-sperm antibodies from the serum samples that were applied to the plate.
3.4.14 Euthanasia
A lethal dose of sodium pentobarbitone (200 mg/kg, 47815, Virbac, New South
Wales, Australia) in PBS (section 3.1.1) solution was given to mice via the intra- peritoneal route, using a 26-gauge needle attached to a 1 mL syringe.
3.4.15 Breeding protocol
The breeding study for this project was carried out at QUTs MERF. Six-week old, male, C57BL/6 mice were infected or mock infected with SPG (section 3.1.5) via the intra-penile route (section 3.4.3) to create the infected (n = 6) and non-infected (n =
6) breeding groups respectively. Female C57BL/6 mice that had previously successfully dropped one litter of pups, were sourced from the Animal Resource
Centre (n = 12, Adelaide, South Australia). For both breeding groups, each male was housed with two females once weekly, until all female mice were impregnated.
Females were housed separately when they showed vaginal plugging to indicate successful mating, this was checked one day after co-habitation. Females had the minimum possible handling/disturbance to minimize stress during pregnancy and one
Chapter 3: Materials and Methods 67
week following birth, to reduce risk of resorption and killing of pups. One week after litters were dropped, pups were weighed, sexed, and tagged with identifiers. Pups were then weighed every two-three days until the appropriate time point for their euthanasia (CO2 pumped into closed container used at seven, 11, and 21 days post birth, sodium pentobarbitone as per section 3.4.14 used at 42 days post birth). At the time of euthanasia, the reproductive tracts were harvested (section 3.4.10 for FRT, section 3.4.6-9 for MRT and sperm) and the organ:body weight ratio was determined for each individual (section 3.4.12). At 21-days post birth, pups were divided by sex and weened into their own cages. Some pups from the infected breeding group were unhealthy (> 20% lighter than non-infected counterparts, and remaining dependant on dams for food) at weaning, so were euthanized after ethical consideration. After litter euthanasia, dams were weighed and euthanized (section 3.4.14). The sires were also euthanized (section 3.4.14) after the breeding study concluded.
3.4.16 Mouse Tissue Homogenization
Immediately after being harvested as previously described, mouse tissues were placed in 500 µL of SPG (section 3.1.5) and mechanically disrupted using a tissue homogenizer with a blade attachment (TH220, DMNI International, Atlanta, United
States of America).
Analysis
The analyses that relate to specific experiments are contained within the Materials and Methods sections of the relevant chapters. In general, data was compiled, graphed, and analysed using GraphPad Prism (version 7). Data was graphed as the sample mean and error bars in all figures represent the standard deviation. Single time-points where non-infected and infected conditions were compared were
68 Chapter 3: Materials and Methods
analysed using an unpaired, non-parametric, Students T test. For single time points where multiple comparisons between more than two cell lines or conditions were required, analysis was achieved using Sidak’s multiple comparisons two-way
ANOVA test. For time course experiments which required analysis of multiple conditions over multiple time points, a Tukey’s multiple comparisons two-way
ANOVA was used. For high-volume data gathered through quantification of histology parameters including ZO-1, α-Tubulin, 3βHSD, SMA, caspase 3, and
WBCs, data was analysed using IBM SPSS statistical analysis program. A bootstrapped, independent samples T test was applied in this situation. P values for all tests where P < 0.05 were considered to be significant (*P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001).
Ethics Statement
The experiments performed for the completion of this project were approved to be conducted by the relevant institutional safety and ethics committees. The project was approved by QIMRB Safety Committee (P2184 for molecular, in vitro, and animal work), QIMRB Animal Research Ethics Committee (A1603-609M), QIMRB Human
Research Ethics Committee (waived approval, safety under P2320 for fresh biopsies and P2322 for fixed biopsies), QUT Animal Ethics Committee (1400000250), QUT
Human Research Ethics Committee (1500000394 for fresh biopsies and 1700000362 for fixed biopsies), QFG Human Research Ethics Committee (QFG12.15), Monash
University Human Research Ethics Committee (666-15 for fresh biopsies and RES-
16-0000-559L for fixed biopsies), and Newcastle University Animal Research
Committee (A-2014-412). All experiments were conducted within the guidelines set forth by each of the approving institutions.
Chapter 3: Materials and Methods 69
Chapter 4: Infection Kinetics of
Chlamydia in Testicular Cells
70 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
4.1 Introduction
The immunosuppressive environment in the testes provides an ideal niche for chlamydial infection and replication, but the susceptibility to, and infection kinetics of Chlamydia within testicular cells is unknown. Mapping the kinetics of infection in testicular cells; if they are susceptible to infection, how long they may sustain inclusion growth, whether viable progeny is produced and can be transmitted, and how testicular cells might become infected to begin with, is the initial step in delineating the impacts of Chlamydia in the testes. The testicular cells being investigated here include the interstitial cells; macrophages and Leydig cells, and the seminiferous tubule cells; Sertoli cells and spermatogonia.
Macrophages and monocytes have previously been investigated as hosts of C. pneumoniae infection in the context of atherosclerosis development and heart disease, as these cells are prevalent in atherosclerotic plaques. C. pneumoniae has also been isolated from atherosclerotic plaques, although the cell type it resides in in vivo remains unconfirmed. In vitro, C. pneumoniae infection of monocytes and macrophages produces viable progeny and induces host cell changes consistent with promoting arthrosclerosis development [256, 257, 298, 304].
Similarly, this chapter aims to explore the capability of macrophages as carriers of C. muridarum after intra-penile infection, which is the initial site of natural infection in an MRT infection. It is hypothesised that infected macrophages systemically spread from the urethra to the testes. This provides an alternate route of infection than the conventional ascending route through the urethra, vas deferens, and epididymis (> 5 metres in a human [81]) against the flow of urine and semen. Survival and replication of many micro-organisms inside macrophages, including several viral and bacterial
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 71
species, is documented. Of particular interest are the HIV, HSV, C. burnetii, and E. coli infection of macrophages, which are either known pathogens of the MRT or resulted in MRT infections [79, 243, 255, 269, 270].
Chlamydia infected macrophages may travel systemically from the urethra, to colonise the interstitial compartment, transmitting infection to the Tmφ after lysis or via extrusion, as macrophages are prone to extrusion uptake [76]. A portion of the
Tmφ population is known to be recruited from the circulatory system via MCP-1 expression from interstitial cells and other mechanisms [226]. The resident Tmφ maintain an anti-inflammatory environment to preserve the critical blood-testis- barrier [305]. This means that Tmφ have a truncated anti-bacterial response and that they may have increased susceptibility to infection [213]. Additionally, as predominantly M2-like macrophages, Tmφ may be able to sustain a chlamydial infection and are likely to produce viable chlamydial progeny. M2 macrophages, although not specifically Tmφ, are susceptible to C. trachomatis and C. muridarum infection [219].
In the same way, Leydig cells residing in the interstitial compartment may become infected. Productive HIV-2 and mumps virus infections are possible within Leydig cells in vitro but the in vivo susceptibility and route of transmission of infections to
Leydig cells remains unknown [165, 306]. Functional changes to Leydig cells have been linked with viral infection, including changes in testosterone secretion, which would have an adverse effect on spermatogenesis and fertility [165, 306]. Mumps virus infection has also been linked with testicular atrophy, spermatogenesis arrest, and azoospermia, which may be the downstream result of decreased testosterone secretion by Leydig cells [307]. This highlights the importance of understanding the interactions between Leydig cells and Chlamydia.
72 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
Less information still, is available for the remaining testicular cells that develop, and reside within, the seminiferous tubules. Seminiferous compartment cell infection is also an uncertain process. Sertoli cell infection by Zika virus, Ureaplasma urealyticum, and C. muridarum, and germ cell infection by retrovirus and HIV-1, have been achieved in vitro and in some cases in vivo, but the in vivo mechanism for this infection has not been characterized [308-311]. An acute mouse model of testicular C. muridarum infection, including Sertoli cells, resulted in spermatogenesis dysregulation and decreased sperm quality [129]. This underscores the importance of understanding the infection kinetics within the seminiferous compartment as this appears to have a direct effect on male sub-fertility.
In this chapter, it is hypothesised that testicular cell types are susceptible to infection, that transmission between testicular cell types is possible, and that this contributes to a chronic testicular infection. Each testicular cell type; Tmφ, Leydig cells, Sertoli cells, and spermatogonia, have a unique and irreplaceable role in spermatogenesis and therefore in male fertility. The infection kinetics of the individual testicular cells, their susceptibility to infection, and their ability to support inclusion development and progeny production, will be investigated. In addition, the ability of macrophages
(simulating circulating macrophages) to transmit C. muridarum in both contact- dependent and contact-independent manners to other macrophages (simulating Tφ) and to testicular cells will be investigated. This chapter works towards the completion of Aim 1 of this study: To investigate the infection kinetics of Chlamydia within testicular cells.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 73
4.2 Materials and Methods
The detailed materials and methods used in this chapter can be found in Chapter 3.
The important materials and methods relevant to this chapter include: media preparation for immortalized cell lines (Section 3.1.14), immortalized cell line culture (Section 3.2.1), media preparation for primary cells (Section 3.1.15), primary cell isolation and culture (Section 3.2.2), live cell imaging of immortalized cells
(Section 3.2.5), monolayer co-culture (Section 3.2.3), Transwell® co-culture
(Section 3.2.4), Chlamydia infection (Section 3.3.5), extrusion isolation (Section
3.2.9), progeny quantification (Section 3.3.11), and detection of Chlamydia using immunocytochemistry (Section 3.3.8).
Briefly, primary mouse Leydig cells, Sertoli cells, spermatogonia, and Tmφ and their immortalized counterparts (TM3 Leydig cells, TM4 Sertoli cells, GC-1 germ cells, and RAW 264.7 macrophages) were cultured individually in monolayers to determine whether each cell lineage is susceptible to infection with C. muridarum
Weiss. The cells were fixed in methanol at 24-hours post infection, stained using fluorescent immunocytochemistry techniques to show cell specific markers and
Chlamydia, then viewed under epifluorescent microscopy to detect chlamydial inclusions within cells.
The immortalized cell lines were then used in further experimentation to explore the infection kinetics within the different cell lineages. The infection kinetics of an interstitial lineage, TM3 Leydig cells, and an intra-tubular lineage, TM4 Sertoli cells, were investigated using live-cell imaging techniques. TM3 and TM4 cells were infected at MOI 0.1 with a GFP-producing strain on C. muridarum, and then viewed for 96 hours. This established the developmental timeline of inclusions within these
74 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
cells. Next, TM3, TM4, GC-1, and RAW 264.7 cells were infected for 30 hours with
C. muridarum, then the cells were lysed using sonication, the progeny were titrated on a McCoyB monolayer, and infectious progeny were quantified using fluorescent immunocytochemistry techniques.
Using monolayer (Figure 4.1 A) and Transwell® co-culture systems (Figure 4.1 B and C), the ability of infected RAW 264.7 macrophages to transmit infection to the different cell lineages was investigated. RAW 264.7 macrophages were infected at an MOI 1 with C. muridarum for 48 hours before being introduced into co-culture with either TM3, TM4, or GC-1 cells. The co-cultured cells were fixed in methanol, stained for both cell specific markers and Chlamydia using immunocytochemistry techniques and then viewed using epifluorescent microscopy, every 24 hours for two weeks. This time-course was used to determine if/when transmission would occur from RAW 264.7 macrophages and the testicular cell lines.
Figure 4.1 Co-culture configurations for chlamydial transmission Figure 4.1 shows the three different co-culture configurations trialled in Chapter 4 during the exploration of in vitro macrophage to testicular cell transmission of C. muridarum. Each panel shows the infected RAW 264.7 macrophages in blue and the testicular cells, either of TM3 Leydig/TM4 Sertoli/GC-1 germ, in purple. The cells are contained within 48-well microtiter plates in RMPI 1640 supplemented with 10% FCS. Panel A shows the contact-dependent transmission, monolayer co-culture configuration. Panel B shows the contact independent transmission, Transwell® co- culture configuration, where a membrane divides cells completely. Panel C shows the contact semi-dependent transmission, Transwell® co-culture configuration, where a membrane still divides cells incompletely.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 75
Subsequently to the set of cell-cell transmission experiments, extrusions produced by
RAW 264.7, TM3, TM4, and GC-1 were also investigated. Supernatant was taken from each transmission configuration, including the chambers of the Transwells®.
The supernatant was evaporated into glass microscope slides, fixed with methanol, stained for the presence of Chlamydia using immunocytochemistry techniques, and viewed using epifluorescent microscopy.
Statistical analysis was carried out on quantitative data gathered in Chapter 4.
Infection kinetic parameters were quantified, graphed, and analysed using GraphPad
Prism (version 7) software. To compare the differences in kinetics parameters between cell types at a specific time point, unpaired, non-parametric, Student T-tests were applied. To compare the differences in kinetics parameters between cell types over a time course, a Tukey’s multiple comparisons two-way ANOVA test was applied. The level of statistical significance was set at P ≤ 0.05 (*P < 0.05, **P <
0.01, ***P < 0.001, ****P < 0.0001).
76 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
4.3 Results
The mouse testicular cell lines TM3 Leydig, TM4 Sertoli, GC-1 germ, and
RAW264.7 macrophages were infected with C. muridarum Weiss at MOI 1 and all cell lines were shown to be susceptible to infection, as seen in Figure 4.2 Cell lines panel. Primary Leydig, Sertoli, spermatogonium, and macrophages were isolated from 6-week old C57BL6 male mouse testes. These cells were infected with C. muridarum Weiss at MOI 1 and were all shown to be susceptible to infection as seen in figure 4.2 Primary Cells panel. An example of a chlamydial inclusion from each cell type is identified by a white arrow in all the infected conditions (24h PI), no chlamydial inclusions were identified in any of the non-infected conditions (No infection). The chlamydial inclusions are shown in green throughout the infected conditions. Cell specific markers, 3βHSD for Leydig cells, γ-Tubulin for Sertoli cells, PCNA for spermatogonia, and F4/80 for macrophages, are shown in red where applicable. The cells all show a nuclear counter stain, DAPI, which is represented as blue.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 77
Figure 4.2 Direct testicular cellular infections with C. muridarum. This figure shows two panels of individual testicular cell types. The first panel shows TM3 Leydig, TM4 Sertoli, GC-1 germ, and RAW 264.7 macrophage cell lines infected with Chlamydia muridarum Weiss. The second panel shows primary Leydig, Sertoli, germ, and Tmφ cells that were isolated from six-week-old, C57BL/6, male mice, and then infected with C. muridarum Weiss. Chlamydial inclusions were stained using MOMP (green, examples highlighted by white arrows), cell specific markers were used (red), and a DAPI nuclear counterstain was also used (blue). These are representative images of triplicates.
78 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
The initial susceptibility to infection (at 24-hours post infection) of TM3 Leydig and
TM4 Sertoli cell lines was calculated by quantifying the number of inclusions per field of view (FOV) in an 80% confluent monolayer by immunofluorescent microscopy detection, as can be seen in Figure 4.3 A, TM3 cells had a lower susceptibility to infection (4.333 inclusions/FOV) compared to the TM4 cells (10.67 inclusions/FOV). Although more than twice the number of inclusions were formed in the TM4 cells, this was not a statistically significant difference in infection rate (P =
0.1).
Figure 4.3 B and C show the full C. muridarum lifecycle within the TM3 and TM4 cells. This was observed using live cell imaging techniques with a strain of GFP- producing C. muridarum Nigg. This revealed several key similarities and differences in the replication of Chlamydia within the different cells. TM3 and TM4 cells support inclusion development, have a replication cycle of between 48-56 hours, after which time they rupture and cause re-infection of surrounding cells. The rupture of cells was observed by the cells gaining DAPI staining as the dye permeates the dead or dying cells. Spreading of infection to neighbouring cells was observed by the approximate 12-hour post-rupture formation of new inclusions. This can be seen on the graphs by the secondary inclusion formation at 64 hours post-infection.
Interestingly, TM4 cells displayed a highly significant increase (P < 0.0001) in new inclusion formation compared to the TM3 cells, supporting the finding of their increased susceptibility to infection. This can be seen in panel C, at 80 hours post- infection. TM4 cells also developed significantly larger inclusions than the TM3 cells (P < 0.01) at 80 hours post infection, as can be seen in Figure 4.3 C.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 79
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80 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
Figure 4.3 Chlamydial growth characteristics in TM3 and TM4 cell lines. Figure 4.3 shows the resulting infections kinetics mapped using live cell imaging techniques. TM3 Leydig and TM4 Sertoli cells were infected with C. muridarum Nigg (GFP producing strain) and the growth of the chlamydial inclusions was visualised and quantified as A. the number of inclusions that were formed per field of view after 24 hours, B. the size of the inclusions that were formed, C. an example of the imaging time course at approximately 80 hours post-infection, and D. quantification of the susceptibility to infection at 24 hours post-infection. The red arrows represent examples of the original inclusions that are formed from time-zero. These can be seen on the graphs between zero and 64 hours post-infection. The white arrows represent examples of secondary infections that have resulted from inclusion lysis and re-infection of surrounding cells. Examples of lysed cells marked by DAPI are indicated by blue arrows. This can be visualised on the graphs during the 64-96 hours post-infection period. The white scale bar represents 100 µm in each image. Images were captured at 600X magnification. Graphs were generated using GraphPad Prism (version 7), 2-way ANOVA was used to determine differences between the different cell lines and timepoints assayed. P values generated were considered to be significant when P<0.05 (*P<0.05, **P<0.01, ***P<0.001). These represent an average of technical triplicates. Images represent examples of the 80- hour time-point of triplicate experiments.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 81
Figure 4.3 D shows an example of the live cell imaging time course that was used to determine the aforementioned infection kinetics. There were no inclusions identified in the non-infected conditions (uninfected panel). Chlamydial inclusions are shown in green owing to the GFP production of this chlamydial strain in the infected conditions (infected panel). Examples of inclusions lasting from the primary infection are indicated by red arrows, and examples of secondary inclusions are indicated by white arrows, at 80 hours post-infection. Examples of the DAPI marked cells are indicated by the blue arrows.
This finding led to the testing of the number of viable progeny produced by each cell type, as a potential reason for the differences in inclusion size. This was tested by lysis of mature inclusions at 30-hours post infection and the titration of EBs produced by each cell type on a McCoyB cell monolayer. This experiment also included GC-1 germ, and RAW264.7 macrophage cell lines. The titration, shown in
Figure 4.4, revealed that TM4 cells produced the largest number of progeny (4.5x106
IFU/mL), followed by GC-1 (3x106 IFU/mL), TM3 (1.85x106 IFU/mL), and lastly
RAW264.7 cells (1.8x106 IFU/mL). Although there is a trend evident in the data shown in Figure 4.4, there was no statistical significance between any of the cell lines.
82 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
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Figure 4.4 Chlamydial progeny production from testicular cell lines. This figure shows the number of chlamydial progeny produced after a 24-hour infection period of each of TM3 Leydig, TM4 Sertoli, GC-1, and RAW 264.7 macrophage cell lines. Each cell line was infected with C. muridarum Weiss, then lysed using sonication and the supernatant used to subculture the progeny onto McCoyB fibroblast cells. The stained McCoyB cells were imaged using epifluorescent microscopy. The inclusions were quantified using MetaMorph software and quantifications were adjusted to represent the number of inclusion forming units (IFU) per mL of infectious culture media supernatant produced after sonication. The graph represents the mean and SD of duplicates, each with technical duplicates. The graph was generated and data analysed (Students T test, significance set at P < 0.05) using GraphPad Prism (version 7) software.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 83
Having established the infection kinetics within the singular cell types, the potential for macrophage-mediated transmission to testicular cells was investigated. This firstly occurred in a cell-cell contact-dependent monolayer co-culture experiment.
RAW264.7 macrophages were infected with C. muridarum Weiss at an MOI 1, and then introduced into a non-infected monolayer of either TM3, TM4, GC-1 or
RAW264.7 cells. After immunofluorescent microscopy analysis of the monolayers at
24-hour intervals for two-weeks post co-culture, it was established that C. muridarum was transmitted from infected RAW264.7 cells to each of the different non-infected cell types. This had occurred by two-weeks post infection, evidenced by a small number of inclusions that have formed and are visible within non-infected cell types (Figure 4.5).
The images were captured two-weeks post co-culture of infected RAW264.7 macrophages and non-infected TM3, TM4, or GC-1 testicular cells in a monolayer.
Macrophages were stained to show F4/80 in red, yellow marks all cell cytoskeletons with Phalloidin-AF594, and MOMP was stained in green. When co-localised, the red-yellow-green make light yellow indicating infected RAWs, and yellow-green make light green indicating infected testicular cells. Individual cells were also identifiable by their nuclei, which were stained blue with DAPI.
84 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
Figure 4.5 Transmission of C. muridarum from macrophages to testicular cell in vitro. The images represent the result of 14 days post co-culture of infected RAW264.7 macrophages and non-infected TM3, TM4, or GC-1 testicular cells in a monolayer. Red staining marks macrophages with F4/80, yellow marks all cell cytoskeletons with Phalloidin-AF594, and green marks chlamydial inclusions with MOMP. When co-localised the red-yellow-green make light yellow indicating infected RAWs, and yellow-green make light green indicating infected testicular cells. Images are representative of triplicates.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 85
At approximately 80-hours post-co-culture, the cell culture monolayers were also assayed for the presence of extruded inclusions, or extrusions, using brightfield
(Monolayer panel) and immunocytochemistry techniques (Monolayer Supernatant panel) as seen in Figure 4.6. Both cell-bound inclusions (indicated by blue arrows) and extrusions (indicated by red arrows), were identified in each of the TM3, TM4, and GC-1 cells. Multiple extrusions per FOV were identified for each cell line and the extrusions ranged in size between 20-50 µm, as can be seen in green in the
Monolayer Supernatant panel of Figure 4.6.
86 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
Figure 4.6 Chlamydial extrusion production from testicular cell lines in monolayer co-culture. This figure shows the extrusions that were isolated after co-culture of C. muridarum Weiss infected RAW 264.7 cells with either TM3 Leydig, TM4 Sertoli, or GC-1 germ testicular cell lines. At approximately 80-hours post infection, the cells were imaged using bright field microscopy as seen in the monolayer panel. Cell culture supernatant was evaporated onto poly-L-lysine coated glass microscope slides and stained for MOMP (green), as seen in the monolayer supernatant panel. The blue arrows indicate inclusions that were growing within cells, and the red arrows indicate inclusions that were not contained within cells. The white scale bar represents 100 µm in each image. Images were captured using epifluorescent microscopy at 400X magnification. Images are representative of triplicates.
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 87
To determine whether the production of extrusions from testicular cell lines was a contact-dependent phenomenon, contact semi-dependent or contact-independent
Transwell® co-culture systems were also established (depicted in Figure 4.1).
Infected RAW264.7 cells were placed into the apical chamber of the Transwell®, while the non-infected testicular cells were placed either on the basal surface of the
Transwell® membrane (contact semi-dependent Transwell® supernatant panel), or within the basal chamber (contact independent Transwell® supernatant panel). This replicates the possibility that Tmφ are relatively stationary within the testis and instead release EBs to infect surrounding cells. Samples of cell culture supernatant were taken from both the contact-dependent and contact-independent co-culture systems and analysed for the presence of chlamydial extrusions.
In figure 4.7, C. muridarum inclusions were visualised by brightfield microscopy
(Transwell® panel) approximately 96-hours post-co-culture, when both cell-bound inclusions (shown with blue arrows) and extrusions (red arrows) can be seen.
Extrusions were visualised by immunofluorescent microscopy (green extrusions indicated by red arrows) in the basal compartment supernatant of both Transwell® systems at the assayed time of 96 hours post-co-culture. This showed that transmission has occurred from RAW264.7 macrophages to TM3, TM4, GC-1, and to other RAW264.7 macrophages in a manner unconnected to cell-cell contact. As extrusions were visualised in the supernatant of both co-culture systems, this showed that extrusions are produced by TM3, TM4, GC-1, and RAW264.7 cells after macrophage-mediated transmission of Chlamydia. Extrusions ranged in size between
5-20 µm, and multiple extrusions were identified per FOV in each condition, whereas the Transwell® membrane pore size was 4 µm.
88 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
Figure 4.7 Chlamydial extrusion production from testicular cell lines in Transwell® Co-culture. This figure shows the extrusions that were isolated after co-culture of C. muridarum Weiss infected RAW 264.7 cells with either TM3 Leydig, TM4 Sertoli, or GC-1 germ testicular cell lines. RAWs were seeded onto the apical membrane of the Transwell®, testicular cells were seeded either onto the basal membrane of the Transwell® (contact semi-dependant) or the culture surface of the microtitre culture plate (contact independent). At approximately 80-hours post infection, the cells were imaged using bright field microscopy as seen in the Transwell® panel. Cell culture supernatant was evaporated onto microscope slides and stained for MOMP (green),
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 89
as seen in the Contact independent Transwell® supernatant panel, and the Contact semi-dependant Transwell® supernatant panel. The blue arrows indicate inclusions that were growing within cells, and the red arrows indicate inclusions that were not contained within cells. The white scale bar represents 100 µm in each image. Images were captured using epifluorescent microscopy at 400X magnification and are a representative of triplicates.
90 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
4.4 Discussion
This chapter was designed to investigate the infectivity and growth kinetics of
Chlamydia within specific testicular cell types. This is the precursor to studying the health and viability of the testicular cells. Specialized Leydig, Sertoli, germ and Tmφ cells have a critical role in spermatogenesis. Discovering their ability to harbour infection and the effects of infection on their function would be paramount to establishing the causal link between chlamydial infection and male infertility. This chapter specifically investigates the first part of this hypothesis, with the intent to establish whether infection of these cell types may contribute to testicular dysfunction and eventually to male infertility.
Interesting but non-significant differences were found in the susceptibility to infection between testicular cell types and there may be several cell-specific reasons for the differences. Sertoli cells are phagocytic and therefore may actively uptake infectious organisms including Chlamydia [14]. TM4 cells retain their phagocytic ability, so are an accurate model in this respect [312]. The usual function of Sertoli cell phagocytosis is to clean-up cell debris and apoptotic sperm produced during spermatogenesis [170]. A small amount of sperm cell death normally occurs during spermatogenesis and this would be detrimental to the proper sperm maturation if not eliminated by Sertoli cells [171]. As with immune cell phagocytosis, Sertoli cell phagocytosis is triggered by a target, in this case by phosphatidylserine (PS) rich debris [313]. Interestingly, a recent study showed that chlamydial extrusions produced by HeLa and McCoyB cells are coated in host cell membrane, approximately 15% of which contain high levels of PS [76]. The extrusions found in this study were produced by all of the TM3, TM4, GC-1, and RAW264.7 cell lines
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 91
after infected RAW264.7 cells transmitted the infection. In the future, the PS component of extrusions produced by testicular cells could be investigated.
If testicular cells including macrophages, produce extrusions that also contain PS in vivo, this likely enhances the Sertoli cell infectivity of the extrusions. While not likely to be a selective capability of Chlamydia growing within the testes, as PS coated extrusions that have previously been described from HeLa and McCoyB cells, which are not testicular cells, this finding does give credence to the idea of the testes, particularly Sertoli cells, being an ideal niche for chlamydial infection.
A crucial aspect of the PS coated extrusions may include promoting an anti- inflammatory response within the cells that takes them up, as this is a function of PS influx [314]. PS coating is a proposed mechanism of several viral and protozoan infections in achieving in vivo persistence [314]. This is important as Sertoli, Leydig, and spermatogonia cells have a functional NALP1/3 inflammasome and some cytokine secretion abilities [315, 316], which could inhibit inclusion development.
PS influx accompanying chlamydial invasion into cells could destabilize these endogenous degradation and detection pathways.
Based on monolayer infection, germ cells and Sertoli cells were comparatively susceptible to infection. Both the GC-1s and primary germ cells represent a mixed population of spermatogonia and primary spermatocytes, rather than a pure population of SSCs. These type of germ cells may be generally more susceptible to, and less able to defend against, infection as they would not naturally encounter micro-organisms while protected by the tight junctions of the blood-testis-barrier.
This is evidenced by the reduction in inflammatory cytokine secretion by spermatogonia/spermatocytes that reside behind the blood-testis-barrier [317]. As
92 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
SSCs reside within the interstitium, they may have reduced infectivity in vivo and this remains to be established.
Also, yet to be established, is the mechanism for infection of germ cells residing behind the blood-testis-barrier. One possibility is that Chlamydia transmits through
Sertoli cells to the germ cells growing between them. However, there may be a second potential route for intra-tubular infection through SSCs. Spermatogonia mediate the temporary release of the tight junctions that form the blood-testis-barrier during spermatogenesis as they differentiate towards spermatozoa [169]. If SSCs are infected during the early stages of development outside the blood-testis-barrier, they could carry the infection through the blood-testis-barrier to within the seminiferous tubule during normal differentiation. This could lead to infection of neighbouring
Sertoli cells and other spermatogonia.
TM3 and RAW264.7 cells and their primary cell counterparts had a similar but slightly lower susceptibility to infection compared to TM4/Sertoli and GC-
1/spermatogonia cells, as seen by the direct cellular infection images. This may reflect an innate resistance to infection of cells present in the interstitial space, as a defence mechanism against testicular infection. Many factors could influence the infectivity, for example, low availability of the chlamydial binding ligand the heparin sulfate proteoglycans (HSPGs) [318], which in Leydig cells are utilised for FGF-2 and LH binding to promote steroidogenesis [319-321]. Alternatively, some forms of
Leydig cells e.g. the newly formed adult Leydig cells, have very little cytoplasm which may impede inclusion formation [159, 322].
Similarly, and as previously described, Tmφ retain their expression of innate infection suppressive mechanisms including a cytotoxic effect even though they have
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 93
a diminished pro-inflammatory effect [217, 271]. The primary isolated macrophages shown in Figure 4.2 shows the ex vivo infection and inclusion formation within a pure flow cytometry isolated Tmφ population, Dr Charles Armitage primarily conducted this experiment. This indicates that Tmφ are susceptible to infection and
RAW264.7 are an appropriate representative model, despite the differences in the inclusion morphology within the different macrophage types.
The EB progeny produced by each of the TM3, TM4, GC-1, and RAW264.7 cell lines reflected the pattern displayed by the susceptibility experiment. TM4 and GC-1 cells produced similar amounts of EBs during the chlamydial replication cycles, and more than both the TM3 and RAW264.7 cells. This most likely reflects the respectively non-suppressive and suppressive cellular environments. It may be possible that within TM4 and GC-1 cells there is more uncontrolled chlamydial proliferation to result in a larger number of progeny. It is unclear at this stage what factors promote the excessive proliferation. Factors could include (i) increased abundance of growth factors, (ii) elongated replication cycles, (iii) rapid reversion of
RBs to EBs, or perhaps (iv) the large size of the cells with increased cytoplasmic space [322], (v) lack of suppressive molecules such as interferons [317, 323], and
(vi) enhanced uptake of EBs by TM4 cell phagocytosis leading to multiple inclusions/inclusion fusions [324]. Excessive proliferation may also mean that production of extrusions is increased, subsequently increasing infectivity within the testis.
One of these potential factors for increased proliferation was investigated; the length of replication time. However, the results from this experiment did not show a noticeable difference in replication time between the TM3 and TM4 cell types. There may be small differences that were not picked-up by the four-hour incremental time-
94 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
points. The experiment was also limited by having to follow one FOV for the duration of the experiment to capture the full length of the replication. Cells outside the FOV, or motile cells that moved out of the original FOV were not viewed so only a portion of the population was captured and this could skew the results. Regardless, based on the triplicate average there does not seem to be a significant difference in the length of replication of Chlamydia within TM3 or TM4 cells. This indicates that another factor influences the increased EB production and this requires further investigation.
The live cell imaging model also showed that TM3 and TM4 cells died resultant from infection, most likely due to chlamydial-mediated lysis of cells. This was shown by the uptake of DAPI into the dead cells as the cell membranes become permeable to the dye. It is unknown whether this accurately reflects primary cell infection, particularly considering the potential for chlamydial extrusion, which generally leaves host cells intact but with a residual inclusion.
Transmission of infection within the testes could result from both extrusions and cell lysis. The in vitro transmission model was unable to clarify which mode of transmission is most important. In monolayer and Transwell™ systems, either mode would result in transmission to the non-infected cell type. Transmission was slow in all co-culture configurations, taking a minimum of three days of co-culture. Contact independent in vitro transmission of Chlamydia from DCs takes an extended time also [298]. This may be a feature of immune cell infections. However, as the
Transwell™ pore size was 4 µm, and extrusions from this study and in literature were generally larger, it may be individual EBs that primarily transmit infection from macrophages. Further investigation is required to understand this. Further investigation into whether Chlamydia within extrusions is live and replication-
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 95
capable would also be required (e.g. ICC targeting an active replication marker such as TC0500, as opposed to MOMP which is expressed in all stages of chlamydial development).
Another component of the co-culture models that requires further investigation and optimisation, is the difficulty in detecting transmission of infection to the testicular cell types. Detachment of the testicular cells, most frequently the TM4 cells, from the culture surface upon infection was commonly encountered. Attempts to capture detached cells from culture supernatant were unsuccessful. This led to the detection of extrusions as evidence of transmission, particularly in the Transwell™ systems where a membrane separates the two cell types. A possible reason for cell detachment, at least for TM4 cells, is suggested in Chapter 5 where RNAseq revealed that structural components of TM4s are down-regulated during infection. In the future, addition of extracellular matrix may improve adherence and assist in optimising the in vitro transmission models.
The validation of preliminary findings from the in vitro model by replication in an in vivo model of transmission was next investigated. Mice were infected via the intra- penile route and the whole blood and the testes was tested for the presence of
Chlamydia-infected macrophages. These were detectable in both blood and testes, at three-days post infection. This data is shown in Appendix A. The systemic route of infection was confirmed by vasectomising mice and titrating the EBs present in the testes. No significant difference was found in the amount of Chlamydia that reached the testes two-, four-, and eight- weeks after vasectomy, which indicates that infection travels through the systemic route. This experimental data can be seen in
Appendix B and the experiment was primarily performed by Dr Avinash Kollipara and Logan Trim.
96 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
While more difficult to show in vivo cell-cell transmission, the initial avenue of investigation was to establish whether the number of macrophages increased in the testes over time. This was in an effort to understand whether a progressive accumulation of infected macrophages contributes to establishment of chronic infection, as well as whether the circulating infected macrophage become resident in the testes which would also increase the quantity in the interstitium. The number of macrophages in the interstitium was increased at six-months post infection compared to two weeks post-infection. However, whether the Tmφ were infected at this time has not been established and this requires further investigation. This data is shown in
Chapter 6.
The increase in macrophages over time within the testes also leads to another avenue of investigation; whether Tmφ provide a reservoir of ongoing infection, which allows the infection to avoid activating a severe inflammatory immune response within the testes. One study suggested that expression of IFNγ and IL-10 are crucial in controlling chlamydial replication within macrophages [325]. A proportion of Tmφ constitutively express both IFNγ and IL-10, and IFNγ can be induced with LPS and viral infection to increase the expression [317, 326, 327]. However, IL-10 is not constitutively expressed in the CD163-negative Tmφ population [327], and is downregulated in Tmφ in response to LPS stimulation [328], so perhaps these cells act as a reservoir of infection.
Regardless of the transmission timeline from Tmφ, these cells have an intrinsic role in maintaining the testicular environment as previously described. Damage to these cells either through death by lysis or alteration by chlamydial T3SS would be disadvantageous to spermatogenesis [229]. The same holds true for the remaining
Leydig, Sertoli, and spermatogonial testicular cell types. The accumulation of
Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells 97
varying subtypes of macrophages could similarly have a negative effect on spermatogenesis by increasing the risk of breakdown of immune privilege within the testes.
The results of this chapter indicate that each of the specific Leydig, Sertoli, spermatogonia, and Tmφ cells, is susceptible to chlamydial infection. These cells support development of chlamydial inclusions and infectious progeny production, to different extents specific to each cell type. The infection is transmitted from macrophages via both Chlamydia-mediated cell lysis and extrusion formation, to other testicular cell types. Macrophages have been shown to be a novel mechanism involved in transmission of Chlamydia to different sites within the MRT, and within the testicular environment. This chapter has laid the foundation for further study into the cellular and molecular alterations within testicular cell types that are caused by chlamydial infection and the impacts of these on spermatogenesis are investigated in
Chapter 5 and Chapter 6.
98 Chapter 4: Infection Kinetics of Chlamydia in Testicular Cells
Chapter 5: In vitro Changes to
Testicular Cells caused by
Chlamydia Infection
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 99
5.1 Introduction
Having established the in vitro infectivity of Chlamydia within testicular cells, the characterization of changes that are induced after infection is possible. The major changes that are of interest are those that induce functional changes to the testicular cells. Each of the testicular cell types have highly specialized functions, corresponding to their individual roles in spermatogenesis. For example, Leydig cells produce testosterone which is a major driver of spermatogenesis [159], Sertoli cells form the tight-junctions of the blood-testis-barrier which protects sperm cells from autoimmune destruction [14], and SSCs are the cell from which all sperm are derived
[176]. There are numerous mechanisms that could influence cell function including the presence of molecular inhibitors, post-transcriptional or post-translational modifications, and the focus of this chapter is transcriptional modification and dysregulation.
The transcriptome arises via the process of generating RNA from the genome through transcription [197]. The messenger RNA (mRNA) within a cell is a small portion of the transcriptome [197]. The mRNA is translated into the protein content of a cell (the proteome) [197], with the assistance of transfer RNA (tRNA).
Individual tRNA molecules bind and carry amino acids to the ribosome where polypeptides are being translated from mRNA [329]. Alterations in the transcriptome can therefore affect the proteome. The full transcriptome does not equate to the full proteome, however changes to mRNA and non-coding RNA (ncRNA) can alter the proteome [196, 204]. The ncRNA engages is transcriptional and post-transcriptional regulation but is not translatable [330]. However, the transcriptome can give an indication of the health and function of a cell as a representation of gene transcription. For example, genome instability, DNA fragmentation, or epigenetic
100 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
modification can alter the transcriptome, which in turn alters the proteome and the competency of the cell function [331, 332].
Genome integrity and DNA fragmentation are of interest in this study as C. trachomatis causes double stranded DNA (dsDNA) fragmentation in female reproductive tract cells [189]. Although the mechanism that allows Chlamydia to cause DNA damage is unclear, infection of MRT cells will likely cause similar dsDNA fragmentation. Destabilization and DNA breakages within the coding regions of the genome disrupt gene expression if the breakages are not repaired. This alters the transcriptome and potentially the cell function.
Examples of this process of functional alteration can be observed during several infection models. C. trachomatis infection of epithelial cells causes histone modification, inhibits DNA repair, and can cause oncogenic-like cell proliferation
[189]. C. pneumoniae infection of monocytes causes differentiation to macrophages
[256], and macrophages to foam cells [257]. Helicobacter pylori infection of gastric cells results in oncogenic genomic and proteomic changes [190].
Another important method of altering the transcriptome is via epigenetics. An epigenetic factor changes gene expression, without altering the genome sequence or stability [207]. There are many mechanisms that can achieve this, including the addition or removal of methyl and acetyl groups to DNA and/or histones. For example, in a cancer biology setting, excessive removal of methyl groups from DNA
(DNA hypomethylation) is frequently associated with uncontrolled gene expression leading to oncogenesis [207]. Conversely, DNA hypermethylation is generally associated with reduced gene expression [207]. Methylation changes can also occur on histones, changing their ability to perform chromatin remodelling, which impedes gene expression as transcription enzymes cannot access the DNA [333].
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 101
Interestingly, epigenetic changes can occasionally be heritable, so multiple generations may be affected by a parent’s disease state [206]. Furthermore, chlamydial species possess a methyltransferase enzyme, which as the name suggests is responsible for transferring methyl groups to and from DNA [334]. After secretion of the methyltransferase through the T3SS, Chlamydia would have access to the host nucleus and may perform host epigenetic modification influencing gene expression and the transcriptome.
The relationship between epigenetics and transcriptional regulation has been most thoroughly characterized in a cancer biology setting, and as such several infectious agents have been identified as causing oncogenesis by epigenetic dysregulation. H. pylori [335, 336] and Epstein-Barr virus [337] infections can result in gastric cancer, hepatitis B and C virus infections can result in liver carcinoma [338, 339], and HPV infection can result in head/neck carcinoma and cervical cancer formation [340-342].
The mechanism allowing infectious agents to modify their host epigenomes remains uncharacterized in most cases. Some hypotheses include viruses creating modifications during the insertion of their genetic material into the host genome, and bacteria like H. pylori, which possesses a type four secretion system (T4SS), utilizing secretion systems to introduce enzymes like the methyltransferase to modify host genomes. Regardless of the mechanism, the result is always that the infectious agent creates a more favourable environment for their survival and replication, usually at the expense of host cell functionality.
Chapter 5 aims to establish an in vitro model of C. muridarum dysregulation of testicular cell function, by investigating the effects of infection on DNA integrity, the methylome, and the transcriptome. This chapter is working towards the completion of the second experimental aim identified for this project.
102 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
5.2 Materials and Methods
The full and detailed materials and methods used in this chapter can be found in
Chapter 3. The materials and methods relevant to this chapter include: immortalized cell line media preparation (Section 3.1.14) immortalized cell line culture (Section
3.2.1), SPG preparation (Section 3.1.5), Chlamydia infection (Section 3.3.5), immunocytochemistry (Section 3.3.8), Comet assay (Section 3.2.6), global methylation mass spectrometry (Section 3.2.8), RNAseq (Section 3.2.7), and testosterone detection (Section 3.2.9).
Briefly, immortalized cell lines TM3 Leydig, TM4 Sertoli, and GC-1 germ were cultured individually in monolayers. The cells were either irradiated, mock infected with SPG, or infected at an MOI of 1 and then assayed. Genome integrity in the form of DNA fragmentation was investigated first using the comet assay. Cells were assayed at three time-points, two-hours, eight-hours, and 24-hours post treatment.
Cells were harvested at each time-point and set into low-melting point agarose.
Electrophoresis was performed on these gels, which separates the intact portions of the genomes (forms a comet head) from fragmented DNA pieces (forms the comet tail). After electrophoresis was performed, the cells were stained with green fluorescent DNA binding dye, so they were able to be viewed by epifluorescent microscopy. By measuring the comet tail relative to the head, the amount of DNA fragmentation was quantified and compared between cell lineages.
The global methylation status of infected versus non-infected TM3, TM4, and GC-1 cells was investigated as an indicator of epigenetic changes. DNA was extracted from the cell lines after 24-hours of infection with C. muridarum. Mass spectrometry of cytosine residues present in the whole genome determined the methylation status.
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 103
RNA was also extracted from infected and non-infected TM3, TM4, and GC-1 cell lines. The RNA was reverse transcribed into cDNA for a transcriptomic and differential gene expression (DGE) analysis approach to defining cellular changes induced by infection. The RNAseq (BGISEQ-500 Platform) and DGE analysis were performed by BGI (Hong Kong).
The amount of testosterone produced by infected versus non-infected TM3 cells was measured as a functional marker of Leydig cell health. The TM3 cell culture supernatant was collected, centrifuged to remove contaminating cells and debris, and used for ELISA. This was performed in-house, by Dr Tamara Keeley, at the
University of Queensland.
The comet assay was quantified using the ImageJ OpenComet plugin, then data was graphed and analysed using GraphPad Prism (version 7) software. To compare the differences in infected versus non-infected versus irradiated cells at each time point a
Tukey’s multiple comparisons two-way ANOVA test was applied. Mass spectrometry data was analysed using Sidak’s multiple comparisons two-way
ANOVA. ELISA data was analysed using an unpaired, non-parametric, Student T- test. The level of statistical significance for all tests was set at P ≤ 0.05 (*P < 0.05,
**P < 0.01, ***P < 0.001, ****P < 0.0001). The transcriptome and DGE analysis was generated by BGI bioinformatics pipeline and pathway analysis was conducted using Kyoto Encyclopaedia of Genes and Genomes (KEGG).
104 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
5.3 Results
DNA fragmentation was investigated within testicular cells using the neutral comet assay as seen in Figure 5.1. The comet assay is a single cell, gel electrophoresis- based assay that allows visualisation of the intact and fragmented components of individual cell genomes. The intact component forms the comet ‘head’ and the fragmented component is drawn away from the head by electrophoresis and forms the comet ‘tail’ (examples shown in Figure 5.1 D). The length of the tail can be measured, relative to the head size, to produce the relative tail moment (RTM), which is graphed for analysis of the amount of DNA fragmentation a cell has sustained.
The neutral comet assay was performed on TM3 (Figure 5.1 A), TM4 (Figure 5.1 B), and GC-1 (Figure 5.1 C) cells at two-, eight-, and 24-hours post treatment. The non- treated control (white bars) in each cell line and time point, showed that over the time-course the amount of DNA fragmentation stayed at a relatively stable background level of approximately 50 U of RTM in each of the TM3, TM4, and CG-
1 cell lines.
The fragmentation-inducing control used was ionising radiation (IR, grey bars). IR induced DNA fragmentation in each of the cell lines, significantly peaking above the background at two-hours post treatment for TM3 (P < 0.0001), TM4 (P < 0.0003), and GC-1 (P < 0.0001) cells. This generally decreased back towards background levels at eight-hours for TM3 (P < 0.0001), TM4 (P = 0.1624), and GC-1 (P < 0.001) cells. Then at 24-hours there was a further decrease in the difference between the irradiated and non-treated conditions for TM3 (0.0007), TM4 (P = 0.2604), and GC-
1 (P = 0.4513) cells.
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 105
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Figure 5.1 Neutral comet assay of untreated, irradiated, and infected TM3, TM4, and GC-1 cells. Figure 5.1 shows the DNA fragmentation, measured by neutral comet assay, that resulted from ionising radiation (IR, grey), C. muridarum Weiss infection (black), and for normal cell culture (white) of TM3 Leydig cells (A), TM4 Sertoli cells (B), and GC-1 germ cells (C). Comet relative tail moments were quantified using the OpenComet software plug-in (version 1.3, OpenComet) for ImageJ, and then graphed using GraphPad Prism (version 6). Analysis was conducted using 2-way ANOVA to compare between the untreated, irradiated, and infected cells. Differences were considered to be statistically significant when P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). These graphs represent the mean and standard deviation of triplicate experiments. Examples of the comets that were imaged at 24-hours post treatment are shown in D.
106 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
Similarly to the IR, at two-hours post infection with C. muridarum fragmentation levels increased above the non-treated control in all TM3 (P < 0.0001), TM4 (P <
0.1977), and GC-1 (P < 0.0001) cells. However, dissimilar to the IR treatment, infection resulted in sustained fragmentation at eight-hours post infection in all TM3
(P < 0.0.0001), TM4 (P = 0.0168), and GC-1 (P < 0.0001) cells. The peak of fragmentation in the measured time-points was generally at 24-hours post infection, this was highly significant in TM3 (P < 0.0001), TM4 (P < 0.0001), and GC-1 (P <
0.0001) cells.
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 107
The global DNA methylation status (percent of methylated cytosine residues) of each testicular cell lineage was investigated next by mass spectrometry at QUTs Central
Analytical Research Facility (CARF). The methylation statuses of cells in their normal state were relatively equal; TM3 cells had 5.4%, TM4 cells had 5.36%, and
GC-1 cells had 5.37% as seen in Table 2. After 30-hours of infection, each cell lineage displayed decreased methylation (hypomethylation). TM3 cells had the greatest loss of methylation, of 0.7% (P < 0.0001). TM4 and GC-1 cells had similar loss of 0.16% (P = 0.6791) and 0.1% (P = 0.9848) respectively. Therefore, the infected TM3 cells had 4.7%, TM4 cells had 5.2%, and GC-1 cells had 5.27% methylation remaining after 30 hours of infection with C. muridarum. This data is representative of triplicate mass spectrometry experiments.
Table 2: Global methylation status of C. muridarum infected versus non-infected TM3, TM4, and GC-1 cells. Sample type %Total average % Average Difference TM3 Non-Infected 5.399728131 Infected 4.700849233 -0.698878897* TM4 Non-Infected 5.36429936 Infected 5.201609448 -0.162689912 GC-1 Non-Infected 5.369494729 Infected 5.269238957 -0.100255772 *: P < 0.0001, Sidak’s multiple comparisons two-way ANOVA test
To establish a role for C. muridarum infection in testicular cell transcriptional dysregulation, whether a result of DNA fragmentation and epigenetic change or otherwise, RNAseq was conducted. A differential analysis of gene expression approach was taken to understand the transcriptional differences between infected and non-infected TM3, TM4, and GC-1 cells.
108 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
Figure 5.2 shows the scatter plots summarizing the individual genes detected during
RNAseq. The grey points represent genes without altered expression during infection. Blue points represent genes that were significantly (P < 0.05) under- represented in the transcriptome during infection. The red points represent genes that had significantly (P < 0.05) increased representation in the transcriptome during infection.
Figure 5.2 A shows that TM3 Leydig cells produced a total of 18396 genes that were identified. Of those, 18155 genes were unchanged during infection (grey), 16 were significantly down-regulated (blue), and 225 were significantly up-regulated (red). A total of 241 genes were changed during infection.
Figure 5.2 B shows that TM4 Sertoli cells produced a total of 18154 genes that were identified. Of those, 17708 were unchanged during infection (grey), 338 were down- regulated during infection (blue), and 108 were up-regulated (red). This equates to
446 genes that were altered during infection.
Figure 5.2 C shows that GC-1 cells produced a total of 18068 identifiable genes. Of those, 17810 genes were unchanged (grey), 30 were down-regulated (blue), and 228 were up-regulated (red). This equates to 258 genes with altered expression during infection.
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 109
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Figure 5.2 Differential expression of genes for TM3, TM4, and GC-1 cells infected with C. muridarum versus non-infected cells. Figure 5.2 shows the scatter plots of genes detected during RNAseq of TM3 Leydig (A), TM4 Sertoli (B), and GC-1 germ cells (C). The red points represent genes that had significantly (P < 0.05) increased representation in the transcriptome during infection. The blue points represent genes that were significantly (P < 0.05) under- represented in the transcriptome during infection. The grey points represent genes without altered expression during infection. The x-axis shows the amount of gene expression from non-infected cells and the y-axis shows the amount of gene expression from the infected cells.
110 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
The five genes with the most significantly altered expression during infection of
TM3 cells are seen in Table 3. These were Ubl carboxyl-terminal hydrolase 18
(usp18, P = 1.10x10-290), Tripartite motif protein (trim30a, P = 1.18x10-275),
Interferon inducible GTPase 2 (irgm2, P = 4.30x10-272), C-C motif chemokine 2 precursor (ccl2, P = 2.26x10-249), and Ubiquitin-like protein ISG15 precursor (isg15,
P = 8.88x10-243). These genes were all up-regulated during infection. The full list of significantly altered genes can be found in Appendix C.
Table 3: Top five most significant DEGs for infected vs non-infected TM3 cells. Symbol P value Expression Nr Description usp18 1.10x10- Up NP_036039.2|//ubl carboxyl-terminal hydrolase 290 18 [Mus musculus] trim30a 1.18x10- Up AAG53470.1|//tripartite motif protein [Mus 275 musculus] irgm2 4.30x10- Up NP_062313.3|//interferon inducible GTPase 2 272 [Mus musculus] ccl2 2.26x10- Up NP_035463.1|//C-C motif chemokine 2 249 precursor [Mus musculus] isg15 8.88x10- Up NP_056598.2|//ubiquitin-like protein ISG15 243 precursor [Mus musculus] Usp18, Ubl carboxyl-terminal hydrolase 18; Trim30a, Tripartite motif protein; Irgm2, Interferon inducible GTPase 2; Ccl2, C-C motif chemokine 2 precursor
The five genes with the most significantly altered expression during infection of
TM4 cells are seen in Table 4. These were Glutamic pyruvate transaminase (alanine aminotransferase) 2 (gtp2, P = 3.98x10-298), Actin, cytoplasmic 2 (actg1, P =
1.69x10-288), Corneodesmosin precursor (cdsn, P = 3.12x10-278), Protein NDRG1
(ndrg1, P = 1.26x10-263), Tribbles homolog 3 (trib3, P = 9.96x10-257). Gtp2, cdsn,
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 111
ndrg1, and trib3 genes were down-regulated during infection. Actg1 was up- regulated during infection. The full list of significantly altered genes can be found in
Appendix C.
Table 4: Top five most significant DEGs for infected vs non-infected TM4 cells. Symbol P value Expression Nr Description gtp2 3.98x10- Down EDL11016.1|//glutamic pyruvate transaminase 298 (alanine aminotransferase) 2, isoform CRA_c, partial [Mus musculus] actg1 1.69x10- Up XP_008692076.1|//PREDICTED: LOW 288 QUALITY PROTEIN: actin, cytoplasmic 2 [Ursus maritimus] cdsn 3.12x10- Down NP_001008424.2|//corneodesmosin precursor 278 [Mus musculus] ndrg1 1.26x10- Down NP_032707.2|//protein NDRG1 [Mus 263 musculus] trib3 9.96x10- Down NP_780302.2|//tribbles homolog 3 [Mus 267 musculus] Gtp2, Glutamic pyruvate transaminase (alanine aminotransferase) 2; actg1, Actin, cytoplasmic 2; cdsn, Corneodesmosin precursor; ndrg1, Protein NDRG1; trib3, Tribbles homolog 3
The five genes with the most significantly altered expression during infection of GC-
1 cells are seen in Table 5. These were Receptor-transporting protein 4 (rtp4, P =
7.17x10-302), Interferon gamma induced GTPase (igtp, P = 2.49x10-285), Guanylate- binding protein 4-like isoform X1 (gbp3, P = 1.62x10-273), Guanylate-binding protein
1 (gbp2b, P = 6.00x10-263), and Poly [ADP-ribose] polymerase 14 isoform X1
(parp14, P = 1.29x10-264). These genes were all up-regulated during infection. The full list of significantly altered genes can be found in Appendix C.
112 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
Table 5: Top five most significant DEGs for infected vs non-infected GC-1 cells. Symbol P value Expression Nr Description rtp4 7.17x10- Up NP_075875.3|//receptor-transporting protein 4 302 [Mus musculus] igtp 2.49x10- Up NP_061208.3|//interferon gamma induced 285 GTPase [Mus musculus] gbp3 1.62x10- Up XP_007652527.1|//PREDICTED: guanylate- 273 binding protein 4-like isoform X1 [Cricetulus griseus] gbp2b 6.00x10- Up NP_034389.2|//guanylate-binding protein 1 267 [Mus musculus] parp14 1.29x10- Up XP_006522455.1|//PREDICTED: poly [ADP- 264 ribose] polymerase 14 isoform X1 [Mus musculus] Rtp4, Receptor-transporting protein 4; igtp, Interferon gamma induced GTPase; gbp3, Guanylate-binding protein 4-like isoform X1; gbp2b, Guanylate-binding protein 1; parp14, Poly [ADP-ribose] polymerase 14 isoform X1
Table 6 shows the number of DEGs that were common between the three cell lines.
The ‘total’ line represents the tallied number of common DEGs, the ‘Up’ line shows the number of those DEGs that were consistently up-regulated, and the ‘Down’ line shows the number of DEGs that were consistently down-regulated. Where the up + down do not equal the total, the DEGs were inconsistently regulated when compared between the cell lines. The list of these DEGs and related information can be found in Appendix C.
Table 6: Number of significant DEGs common between TM3, TM4, and GC-1 cells. TM3 vs TM4 TM3 vs TM4 TM3 vs GC-1 TM4 vs GC-1 vs GC-1 Total 17 10 86 1 Up 1 3 86 0 Down 0 0 0 1
Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection 113
The genes that had significantly altered expression during infection were mapped to known biological, disease, or infection pathways using KEGG. Many pathways contained multiple altered genes that were mapped to the same pathway. The five pathways that were most significantly (P < 0.05) impacted by infection are shown for each cell line. The most significantly altered pathway is also displayed, with up- regulated genes displayed within red boxes and down-regulated genes displayed in green boxes. The four other highly significantly altered pathways can be seen in
Appendix D.
For TM3 cells, the five pathways included the herpes simplex infection (P = 5.00x10-
38), NOD-like receptor signalling pathway (P = 2.30x10-31), influenza A infection (P
= 1.88x10-22), measles infection (P = 3.12x10-18), and TNF signalling pathway (P =
1.82x10-17). This can be seen in Table 7.
Table 7: Top five most significantly altered pathways for TM3 cells infected with C. muridarum versus non-infected cells. KEGG Pathway ID Pathway DEGs with P value pathway annotation ko05168 Herpes Simplex 53 (23.87%) 5.00x10-38 infection NOD-like receptor 44 (19.82%) 2.30x10-31 ko04621 signalling ko05164 Influenza A 33 (14.68%) 1.88x10-22 infection ko05162 Measles infection 24 (10.81%) 3.12x10-18 ko04668 TNF signalling 22 (9.91%) 1.82x10-17
For TM4 cells, the five pathways included the nitrogen metabolism pathway (P =
1.17x10-4), Phototransduction – fly (P = 3.35x10-4), Legionellosis (P = 3.76x10-4),
Rap1 signalling pathway (P = 5.49x10-4), and renin secretion (P = 2.68x10-3). This can be seen in Table 8.
114 Chapter 5: In vitro Changes to Testicular Cells caused by Chlamydia Infection
Table 8: Top five most significantly altered pathways for TM4 cells infected with C. muridarum versus non-infected cells. KEGG Pathway ID Pathway DEGs with P value pathway annotation Nitrogen 5 (1.15%) 1.17x10-4 ko00910 metabolism Phototransduction 8 (1.84%) 3.35x10-4 ko04745 – fly ko05134 Legionellosis 25 (10.55%) 3.76x10-4 ko04015 Rap1 signalling 30 (12.66%) 5.49x10-4 ko04924 Renin secretion 22 (9.28%) 2.68x10-3
Interestingly, for GC-1 cells, the five pathways included the NOD-like receptor signalling pathway (P = 3.31x10-31), Herpes Simplex infection (P = 8.03x10-28), TNF signalling pathway (P = 2.17x10-20), influenza A infection (P = 1.78x10-18), and measles infection (P = 2.16x10-15), which were the same pathways targeted during
TM3 infection. This can be seen in Table 9.
Table 9: Top five most significantly altered pathways for GC-1 cells infected with C. muridarum versus non-infected cells. KEGG Pathway ID Pathway DEGs with P value pathway annotation ko04621 NOD-like receptor 45 (18.99%) 3.31x10-31 signalling ko05168 Herpes Simplex 45 (18.99%) 8.03x10-28 infection ko04668 TNF signalling 25 (10.55%) 2.17x10-20 ko05164 Influenza A 30 (12.66%) 1.78x10-18 infection ko05162 Measles infection 22 (9.28%) 2.16x10-15
Figure 5.4 shows the most highly significantly altered KEGG pathway maps for each cell line. Figure 5.4 A shows the Herpes Simplex infection pathway for TM3 cells, containing the up-regulated gene products in red boxes. These included IFNα/β,
TLR2, TLR3, RIG-1, MDA5, PKR, 2’-5’OAS, STAT1/2, STAT1, IRF9, IRF3/7,
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TAP, MHC-I, MCP-1, RANTES, IL-6, IL12, ISG56, Sp100, hDaxx, and NXF1.
There was also one down-regulated gene product, AP1, shown in the green box, which was affected during infection.
Figure 4.5 B shows the nitrogen metabolism pathway for TM4 cells. This pathway map contains the down-regulated gene products 4.2.1.1 and 6.3.4.16 displayed in green boxes, which were targeted during infection.
Figure 5.4 C shows the NOD-like receptor signalling pathway for GC-1 cells. This pathway map contains the up-regulated gene products displayed in red boxes that have been affected during infection. The gene products include OAS, GPBs,
CARD6, c1AP, A20, IFI16, IκB, IRF3/7, CASP4, CASP11, pro-CASP11, TNFα,
CXCL, MCP-1, RANTES, IRF9, and STAT1/2.
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A
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B
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C
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Figure 5.4 Pathway analysis of DEG from TM3, TM4, and GC-1 cells infected with C. muridarum versus non-infected cells. Figure 5.3 shows the most significantly affected, known biological/disease/infection pathways, which were targeted during infection of TM3, TM4, and GC-1 cells versus non-infected controls. A shows the Herpes Simplex infection pathway for TM3 cells (P = 5.00x10-38), displaying the altered genes (up-regulated in red boxes, down- regulated in green boxes). B shows the nitrogen metabolism pathways for TM4 cells (P = 1.17x10-4), displaying altered genes (down-regulated, green boxes). C shows the NOD-like receptor signalling pathway from GC-1 cells (P = 3.31x10-31), displaying the up-regulated (red boxes) genes. This data was derived from the mean of duplicate RNAseq experiments.
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To begin to follow up on the downstream effects of differences in gene expression, the testosterone production from TM3 cells was investigated as an important part of the Leydig cell proteome and the essential role it plays in controlling spermatogenesis. TM3 cells produced more testosterone while infected with C. muridarum, an average of 0.0400 ng/mL, than the non-infected counterparts which produced 0.0295 ng/mL. This was a difference of 26.25%. However, with n = 3, this was not a statistically significant difference (P = 0.5714).
Figure 5.5 Production of testosterone from TM3 cells. This figure shows the concentration of testosterone (ng/mL) produced by naïve or C. muridarum Weiss infected TM3 Leydig cells. The data was graphed using GraphPad Prism (version 6). The data represents the mean and standard deviation of triplicate experiments.
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5.4 Discussion
The results of this chapter, which investigated the effects of chlamydial infection on genome integrity, the transcriptome, and the methylome of testicular cells, provides insight into the mechanism for chlamydial disruption of cellular function.
Understanding this mechanism provides a foundation for further study into prevention or reversal of the adverse effects of infection within testicular cells and the testis.
The comet assay demonstrated that in the presence of Chlamydia, genome integrity was compromised, leading to a significant increase in DNA fragmentation in each of the TM3, TM4, and GC-1 cell lines compared to the non-infected controls. The DNA fragmentation accumulated over the 24-hour time period as shown by the increase in the RTM between the two-hour and later time-points. The mechanism for how chlamydial species cause DNA damage is unknown. However, this phenomenon has previously been observed within HeLa cells. Chumduri et al. (2013) showed that a strong γH2.AX signal is produced in infected HeLa cell nuclei up to six-hours post- infection which indicates DNA damage has occurred, but also that DNA repair mechanisms had not been initiated [189]. This may indicate that the γH2.AX signal and the repair mechanisms that it initiates are interfered with by infection.
The γH2.AX signal is created by a phosphorylation event on histone H2 (variant
AX). Through the T3SS, Chlamydiae are able to achieve host cell modification.
Olive et al. (2013) showed alteration of AP-1 phosphorylation during infection of
HeLa cells with C. trachomatis, demonstrating the ability of chlamydial species to perform phosphorylation modifications [267].
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The comet assay also showed that the damage caused by infection was equal to or more than the IR positive control when comparing the peaks of damage across the time course. This indicates that a small amount of DNA fragmentation is produced during the initial hours of chlamydial infection, increasing to the level caused by irradiation as chlamydial replication continues. The location of the fragmentation sites will be important for the health and function of the cell. Fragmentation occurring within coding regions, for example, will be detrimental to cell function.
Additionally, Chlamydia harvests several factors from host cells that they are unable to synthesise. Depleting the host cell transcriptome and proteome by causing prolific
DNA damage would also be disadvantageous to inclusion development. The reason for Chlamydia inducing DNA damage is unknown, but it may be incidental to inclusion growth. Alternatively, it may weaken the cell in preparation for Chlamydia- mediated cell lysis. Unrepaired DNA fragmentation is pro-apoptotic but chlamydial species combat this by down-regulating apoptotic pathways. This may be a mechanism for Chlamydia to regulate the timing of cell death until after the peak of replication within the host cell.
A compounding factor to transcriptional deregulation is alteration to the epigenome.
The in vitro hypomethylation observed in TM3, TM4, and GC-1 cell line DNA during infection indicates that this does occur. This is likely to be Chlamydia- mediated in part; chlamydial species possess at least one methyltransferase enzyme, these enzymes are able to function through the T3SS to interact with host chromatin
[334]. C. pneumoniae SET domain protein is a functional histone methyltransferase capable of methylating murine histones H3 and Hc1 and chlamydial Hc1 and Hc2
[343]. Similarly, C. trachomatis SET domain protein methylates human cell histones
H2B, H3, and H4 [343]. Although the C. muridarum SET domain is uncharacterized,
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it likely does contain one, as it is highly conserved in all bacteria [344]. It is yet unknown whether these enzymes interact with host DNA or are limited to histones, or whether Chlamydiae possess additional uncharacterized methyltransferase enzymes that specifically target DNA rather than histones. Histone methylation is important for transcription as it assists in chromatin remodelling to allow transcription enzymes access to DNA [207]. Chlamydial interference may block transcription of some genes to promote its survival. For example, differential histone and DNA methylation can alter the expression of genes encoding MHCI/II and other antigen presentation pathways [345], and IFNγ production [346].
Although the global hypomethylation observed in the testicular cells was small, less than one percent, this can have biological and pathological impact [347-349]. In the future, gene regions affected by methylation changes would need to be investigated to determine the true functional impact. Methylation loss from gene promotor regions, gene bodies, or from repeating sequences for example, have differential effects on gene transcription [350]. Additionally, the net effect of infection was methylation loss as the global status was measured by mass spectrometry. However, some genes may have been hypermethylated, which lead to the small net change observed. A more gene specific approach, such as bisulfite or pyrosequencing of the genome would need to be employed to understand this in more detail [351, 352].
The changes that occurred within the infected cell transcriptome were analysed by the differential gene analysis obtained after RNAseq. RNAseq shows the RNA species and their expression levels within cell at the time of harvest [197]. In this case, cells had either been infected with C. muridarum for 30 hours, close to a full round of chlamydial replication, or remained non-infected as a control. By
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comparing the mRNA expression in the infected cells, relative to the non-infected cells, the effect of infection on the transcriptome can be delineated [353].
The changes to gene expression examined in this chapter represent only a small portion, the five most significantly altered genes. For TM3 cells these included Ubl carboxyl-terminal hydrolase 18 (usp18), Tripartite motif protein (trim30a), Interferon inducible GTPase 2 (irgm2), C-C motif chemokine 2 precursor (ccl2), and Ubiquitin- like protein Isg15 precursor (isg15).
Interestingly, isg15 and irgm2 are involved in cellular responses to type I interferons and IFNγ, respectively [354, 355]. This would suggest that the TM3 cells are both secreting and responding to interferons produced during infection. IFNγ particularly, has an established role in chlamydial clearance [95, 109]. However, in this case, when interrogating the full DEG list (Appendix C), IFNβ is the significantly
(3.17x10-5) over-expressed interferon. There may be co-stimulation of transcription for these genes, or perhaps IFNγ is non-significantly induced. This could be investigated in the future, both by exploring the gene expression list and by detection of the proteins in the culture supernatant. Leydig cells are known to produce IFNα and IFNγ [317], but IFNβ has not consistently been detectable [356]. This result supports Leydig cells being capable of IFNβ production.
The effect of interferon production on spermatogenesis is also variable, with Sendai virus infection concomitantly inducing IFNα and IFNγ and stimulating testosterone production, while Mumps virus stimulates IFNγ but supresses testosterone secretion
[165]. The Mumps model is more in line with dogma suggesting that exogenous interferon supresses testosterone production from Leydig cells [357-360]. However, the increased testosterone in cell culture seen here, and in serum (Chapter 6) mirrors
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the Sendai model. Therefore, there may be pathogen specific influence on testosterone. Increases and decreases in testosterone abundance can be harmful to spermatogenesis [361, 362].
TM3 cell production and response to interferons may be an attempt to limit chlamydial replication. An IFNβ response is inducible in epithelial cells during chlamydial infections, but its purpose or any role in clearance of infections is undefined [100, 363]. Isg15 has a role in activating intracellular responses to bacterial infection [354, 364]. Isg15 and other closely functionally related genes are activated by interferon production and have previously been identified as up- regulated during persistent C. pneumoniae and C. trachomatis infections of epithelial and monocyte cell lines [365-367]. Isg15 transcription is likely activated in this case by IFNβ secretion. Similarly, irgm2 is induced by infections that utilize a phagosome
[355] and it is involved in anti- chlamydial defense through autophagy mechanisms
[368].
Usp15 is also a gene involved in immune and inflammatory pathways, as it encodes the USP15 protein which regulates IκB ubiquitination and consequently NF-κB activity [369]. During many infections the NF-κB pathway is used by cells in host defence and to regulate interferon production [370-372]. This may be an upstream factor in the possible IFNβ production from TM3 cells [363, 373]. However, over- expression of USP15 can dampen IFNβ production and anti-viral responses [374,
375]. So, the up-regulation of usp15 in infected TM3 cells could conflict with the previous results. If this is the case, it may be indicative of a balanced cellular response to inflammation. The testes are an immunosuppressed site, so perhaps
Leydig cells are programmed to limit their pro-inflammatory capacity. Alternatively, chlamydial species (except C. pneumoniae) possess two proteins, ChlaDUB1 and
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ChlaDUB2, which are structurally similar to mammalian ubiquitin-like proteases
[376]. These have previously shown binding affinity with mammalian ubiquitin-like proteins, again indicating the ability of Chlamydia to interact with its host [376].
Perhaps the ChlaDUBs interfere with the NF-κB pathway, for example by competitively binding ubiquitin sites on IκB, which prevents its degradation and activation of NF-κB [377]. This could disrupt the regulation of usp15 and possibly result in its overexpression as a compensatory process.
Similarly to usp15, trim30a is involved in the ubiquitination of STING, a cytosolic
DNA sensing system that is involved in initiating interferon production and inflammasome activity [378]. C. trachomatis and STING are linked; STING disruption abrogates chlamydial-mediated cell death, Chlamydia induced STING promotes IFNβ production, and Chlamydia deficient in STING recognition molecules have decreased survival in immune cells [379, 380]. In a viral infection context, over-expression of trim30a was inhibitory to the STING pathway as it was targeted for proteasome degradation by ubiquitination [381]. The up-regulation seen during TM3 infection, again, could be the result of (i) the host cell balancing its inflammatory response to infection to maintain an anti-inflammatory environment in the testes, or (ii) chlamydial interference via its ChlaDUBs or other mechanisms.
Dysregulating the host cell inflammasome would be beneficial in both of these contexts, in maintaining an anti-inflammatory environment by avoiding production of pro-inflammatory cytokines such as IL-1β [382], thereby limiting host response to infection.
The final gene that will be discussed for TM3s is ccl2, which encodes the chemokine
CCL2, also known as MCP-1 [383]. MCP-1 is normally expressed by interstitial cells in the testis as a mechanism of circulatory monocyte/macrophage recruitment to
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replenish the Tmφ population [226]. Over-expression of MCP-1 by infected Leydig cells could be an attempt at combating infection by recruiting Tmφ or extra circulatory monocytes/macrophages to themselves. It is debatable what the effect of this would be in vivo. Normally, recruited monocytes/macrophages would be differentiated/polarized to the M2 phenotype by interstitial components including corticosterone [384]. M2 macrophages ineffectually kill Chlamydia [219], so instead of promoting their clearance of infection, Leydig cells may simply be providing additional host cells. However, if the recruited cells respond to any antigen presentation or interferon production and polarize to M1, this may be more responsive to infection but would be detrimental to the testis. This scenario is characteristic of experimental autoimmune orchitis [225]. Recruited M1 macrophages would have full capability to stimulate other immune cells including T cells, which Tmφ generally cannot [328, 385]. This would promote a pro- inflammatory environment as those cells respond to infection, which is detrimental to blood-testis-barrier integrity and subsequently to sperm viability. Additionally, the recruited macrophages are not tolerized to germ cell antigens and may present these to T cells, which can also result in orchitis [385, 386].
The dichotomous analysis of DEGs is reflected in HSV infection pathway that was most highly significantly altered during TM3 infection. There were 21 genes/gene clusters up-regulated and one gene cluster down-regulated. The upregulated genes in this pathway coded for predominantly pro-inflammatory molecules including MCP-
1, RANTES, IL-6, and IL-12. This appears to be stimulated by PAMP binding to upregulated TLR2 and TLR3, with antigen presentation occurs through MHC-I.
Inflammation and antigen presentation is supportive of MCP-1/RANTES production to promote chlamydial clearance through immune cell recruitment.
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Conversely, the AP-1 gene cluster is down-regulated. This would be inhibitory to inflammatory processes, so protein content of the cells would need to be examined to accurately predict the outcome of these opposing transcriptome changes.
Moving to GC-1 cells next, as they too predominantly displayed up-regulation of pro-inflammatory genes in response to infection. The five most significantly over- expressed genes were Receptor-transporting protein 4 (rtp4), Interferon gamma induced GTPase (igtp), Guanylate-binding protein 4-like isoform X1 (gbp3),
Guanylate-binding protein 1 (gbp2b), and Poly [ADP-ribose] polymerase 14 isoform
X1 (parp14).
Rtp4 is a known anti-viral effector protein, working upstream in type I interferon production pathways [387]. It is categorized as an interferon-stimulated gene, similarly to the genes stimulated by interferons in the TM3 cells [387]. Many other interferon responsive genes were also seen to be altered when interrogating the full
DEG list (Appendix C), including igrm as seen in TM3s. Conversely to TM3s, there were no interferons present in the DEGs list, suggesting it is responding to a different stimulus, or fulfilling an alternative function in this case.
Similarly, tgtp is also an interferon stimulated gene. Tgtp appears to function principally as irgm2 does, as seen in TM3 cells [355]. It would presumably fulfil a similar role in host cell defence, after induction by a pro-inflammatory stimulus
[387].
Gbp3 and gbp2b fall in the family of guanylate-binding proteins (GBPs), which in the context of infection and immunity, are induced by interferons to act as cellular effectors that help restrict growth of intracellular pathogens [388]. Several examples including different infections exist. Gbp1 assists in control of Toxoplasma gondii
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infection of mouse macrophages, but can be interfered with by pathogen virulence factors (kinases in this case) [389]. Secondly, C. muridarum and C. trachomatis infection of macrophages in vitro shows a role for GPBs in activation of NLRP3 and
AIM2 inflammasomes [388]. The results of that study showed activation of caspases and pro-inflammatory cytokines after GPB induction of host defences [388].
Next, parp14 produces PARP-14, which has several interesting functions. Firstly
PARP14 acts as a transcriptional regulator for several genes including stat6 [390].
Stat6, in turn is a transcription factor for IL-4, which activates Th2 responses in T cells, and stimulates proliferation in activated T and B cells [390]. This response in the testes would be sub-optimal for fertility as previously stated, due to the consequent breakdown of the blood-testis-barrier that would follow. Stat6 and IL-4 could be investigated in GC-1 cells to determine the extent to which parp14 influences this pathway during infection. PARP14 also functions to promote DNA homologous recombination and in combination with PCNA, creates tolerance for
DNA damage [391]. The up-regulation of parp14 in germ cells may be induced in response to the DNA fragmentation that occurs during infection. The sperm progenitor cells may be programmed to tolerate DNA damage as they undergo large amounts of homologous recombination, rather than to apoptosis as many somatic cells are programmed, to preserve reproductive potential [392].
The most significantly altered pathway for GC-1 cells was the NOD-like receptor pathway. All genes mapped to this pathway were up-regulated, and included several caspases, GPBs, STAT, interferon inducible genes, and pro-inflammatory cytokines gene clusters. This is in line with the most highly significantly over-expressed genes identified and would indicate that a generally pro-inflammatory response is generated and being responded to during C. muridarum infection of GC-1 cells.
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The TM3 and GC-1 cells both produce and respond to pro-inflammatory stimuli during infection. In vivo, this may indicate that Leydig cells and SSCs residing in the interstitium respond by actively attempting to suppress infection through interferon related pathways. There was common interferon inducible and GTPase gene activities between the two cell lineages, but also several unique aspects to consider.
The last cell lineage investigated was the TM4 Sertoli line. TM4s responded differently to infection than TM3 or GC-1 cells. Genes were predominantly down- regulated in response to infection. The five most altered genes that included
Glutamic pyruvate transaminase (alanine aminotransferase) 2 (gtp2), Actin, cytoplasmic 2 (actg1), Corneodesmosin precursor (cdsn), protein NDRG1 (ndrg1),
Tribbles homolog 3 (trib3).
Firstly, gtp2 encodes an alanine transaminase enzyme that functions to generate pyruvate and glutamate, key metabolites in the gluconeogenesis energy production pathway [393]. Lactate is also produced, which is important for developing germ cells, so this represents down-regulation of gtp2 may result in low abundance of metabolites necessary for germ cell development [394]. A similar response of low lactate production was recorded from hormone treated Sertoli cells in culture [395].
Interestingly, changes in trib3 expression may also affect Sertoli cell metabolism.
Trib3 products can bind enzymes involved in the insulin metabolic pathway, preventing their activity [396]. Down-regulation of trib3 may be a cell-mediated compensatory measure to promote glucose uptake and metabolism. Insulin/glucose starvation has been shown to decrease lactate output [394]. A secondary role of trib3 involves interaction with NF-κB and apoptosis regulation [397]. Over-expression of trib3 in β-cells resulted in increased apoptosis [397], so perhaps an anti-apoptotic
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effect is seen here. Whether this could be cell- or chlamydial-mediated is debatable.
Infection of gastric cells with H. pylori (a T4SS containing organisms), which stimulates hosts cells through TLR2 and MyD88 similarly to chlamydial infection, also resulted in decreased expression of trib3 [398]. However, knock-down of trib3 resulted in increased host defence, leading the authors of the study to hypothesise that modulation of trib3 by H. pylori is conducted as part of the pathogenesis [398].
Down-regulation of trib3 may be a chlamydial pathogenesis mechanism also.
Cdsn is an unusual gene to be upregulated in Sertoli cells. It encodes corneodesmosis, a protein found in corneodesmosomes, which is involved in the maturation of cornified squamous epithelium [399]. Dysregulation of this gene, which resides in the MHC chromosome locus, may result in autoimmune skin conditions potentially due to loss of immune capability and interaction from keratinocytes [400]. Cdsn deletion caused destabilisation of the coreodesmosome and detachment of cells from their skin layers [401]. When viewed in combination with the up-regulation of the Gamma actin 1 gene, actg1, also found in this experiment, it would suggest that Sertoli cell cytoskeletons are rearranging, and likely losing their structural integrity. It seems likely that this could be a stress response. Cellular morphology is highly susceptible to change when environmental conditions are altered [402-404].
The down-regulation of ndrg1 may also support the idea that Sertoli cells are undergoing a stress response to infection. NDRG1 has a role as both cancer metastatic and autophagy/apoptosis regulatory molecule [405, 406]. Down-regulation of NDRG1 could promote loss of cell-cell adhesion [406], which is in-keeping with the loss of cell structure previously suggested. It could also promote apoptosis [407], which could be a defence mechanism of infected Sertoli cells without the over-
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expression of pro-inflammatory molecules that would adversely impact tubule immune privilege.
The nitrogen metabolism pathway was identified as the most significantly altered during infection of TM4 sertoli cells. Two gene-clusters comprising five down- regulated genes were classified to this pathway. More genes were classified to all the other pathways identified as highly significantly changed. However, the nitrogen metabolism pathway genes were most statistically significantly altered. Regardless, dysregulation/regulation of the nitrogen metabolism pathway is associated with both stress and stress responses from bacterial and eukaryotic cells as nitrogen is an essential nutrient for most organisms [408-412]. This is consistent with the stress response-like reaction that is suggested to occur within infected cells.
Overall, the TM4 cells respond differently to infection than the TM3 and GC-1 cells.
TM4 cells had a stress-like response, which altered their cellular structural components and adhesion. Whereas TM3 and GC-1 cells underwent predominantly interferon induced pro-inflammatory responses. This may represent the difference between cells that regularly interact with the interstitium that may be programmed to recruit immune assistance in combatting infection, and tubular cells that are programmed to detach themselves and self-destruct in response to stress caused by infection. Although two different types of responses to infection were observed, both have the capacity to affect spermatogenesis.
One method would occur by disrupting the blood-testis-barrier. Cytokine-mediated regulation of blood-testis-barrier integrity has been demonstrated on several occasions [413]. Interferons and TNFα have relatively well characterized roles in promoting leakiness of tight junctions, adherence junctions, and other cell-cell
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adhesions like those found in the blood-testis-barrier [413-415]. Although the responsiveness of the blood-testis-barrier to molecules like IFNβ is currently unknown, the pro-inflammatory effects from Leydig and germ cells may have a similar weakening effect. Reorganization of Sertoli cell structure and apoptosis would also result in loss of blood-testis-barrier junctions. Combined, these would enhance the probability of germ cell antigen detection, and subsequently autoimmune orchitis [220]. It would be interesting to develop a co-culture model to understand how the responses of individual cell lineages affects each other lineage, to reflect a more realistic environment.
There are disadvantages to investigating cell functionality using only a transcriptomics approach. Chlamydiae can alter the host proteome independently of the transcriptome [267]. This indicates that they carry out post-transcriptional and/or post-translational modifications in addition to any transcriptional modification. For example, C. pneumoniae induces post-translational modification of host cytoskeleton proteins keratin K8/K18, vimentin, and β-tubulin, resulting in truncation of these proteins, and this is beneficial to inclusion development [416]. These modifications will not be represented in a transcriptomics approach. More investigation with a proteomic (as well as metabolomic and lipidomic etc.) approach would be required to establish a full profile of modifications that Chlamydiae cause within host cells
[268]. As would differentiating between the chlamydial interaction with different
RNA species, e.g. between mRNA and ncRNA. The RNAseq results should also be confirmed using secondary qPCR, immunoblotting, or immunocytochemistry techniques.
Nevertheless, an example of the downstream effects of infection on cell function is shown in the quantification of testosterone production from TM3 Leydig cells.
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Increased secretion of testosterone occurred during infection as measured from the cell culture supernatant by ELISA. Testosterone availability is linked with spermatogenesis in several ways, as previously mentioned. Serum testosterone also exists in a feedback loop with follicle stimulating hormone (FSH) via the pituitary gland [162]. Excessive testosterone production exerts negative feedback within the pituitary gland and decreases FSH production [361, 417]. This would normally signal for Leydig cells to reduce testosterone production. However, this does not seem to occur during infection as can be seen in Chapter 6. Decreased FSH is linked with decreased testicular volume, which is the amount of sperm produced in a testis [417].
There may be other factors as well, such as lack of appropriate hormone receptors due to cell death, which will be explored in Chapter 6.
The results of Chapter 5 show that there may be both cell- and chlamydial- mediated changes that occur during infection of testicular cell lines. These changes occur in the genome, epigenome, transcriptome, and potentially the secretome. This was evidenced by changes to DNA integrity, DNA methylation, transcribed inflammatory and stress markers, and increased testosterone production, all of which can negatively impact spermatogenesis in different ways. The next stage of this investigation is to move from an in vitro model of infection to an in vivo model.
Chapter 6 investigates the downstream impacts on spermatogenesis that may occur both through the disrupted functionality of infected cells investigated in this chapter, as well as other aspects.
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Chapter 6: The Effects of Chronic
C. muridarum Infection on Mouse
Testes
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 137
6.1 Introduction
Testicular tissue and sperm health are the major factors in clinical determination of male fertility and diagnosis of infertility [418, 419]. Histological examination of testicular tissue can assist in determining a cause of infertility, for example genetic disorders such as Klinefelter syndrome or Sertoli cell only syndrome have characteristic features displayed within testis tissues [418]. These features are used as diagnostic tools to determine reasons for spermatogenic failure, for instance, testes with Sertoli cell only syndrome display an absence of spermatogonia within the seminiferous tubules, which leads to azoospermia or the complete absence of spermatozoa in semen [420].
Other infertility or subfertility aetiologies can result in lowered spermatogenesis
(hypospermatogenesis), rather than azoospermia [421]. These can result in sperm abnormalities that include, but are not limited to, oligospermia (fewer than five million sperm per millilitre in ejaculate – low sperm count), teratozoospermia (sperm with abnormal morphology), and asthenozoospermia (sperm with low motility)
[180]. Having one altered semen parameter may not be enough to prevent natural conception given enough time. However, cumulative parameter changes, for example low sperm count combined with abnormal morphology (oligoteratozoospermia), are commonly associated with male factor infertility [422, 423].
This is particularly true for DNA integrity within sperm, as this is frequently associated with asthenozoospermia and directly linked with embryogenesis failure and adverse pregnancy outcomes [292, 422]. Sperm DNA damage, although somewhat difficult to measure as previously discussed in section 2.4, is becoming an
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increasingly important parameter and a powerful tool to consider in the diagnosis of male factor infertility [209].
Poor sperm quality occurs in approximately 2% of all men worldwide, and often leads to the diagnosis of male factor infertility (7% of all men), which accounts for
40 – 50% of all infertility diagnoses [424]. Poor sperm quality can prevent natural and assisted conception. Abnormal sperm are less likely to be able to traverse the
FRT to encounter an oocyte or successfully bind or properly fertilize an oocyte, the result of which is preventative to conception [425]. The aetiologies of poor sperm quality are many, ranging on a spectrum from genetic, nutritional, and injury-based factors, which are relatively well understood, to those resulting from infections which are not well characterized [426-429]. These are all commonly grouped as idiopathic.
Several bacterial (Ureaplasma urealyticum, Mycoplasma genitalium, E. coli, N. gonorrhoea, and C. trachomatis) and viral (HIV, HSV, CMV, HPV, and Zika) infections have been found in semen and tissues of the MRT and associated with male infertility [181, 430, 431]. The mechanisms for how these infections lead to male infertility are not well characterized, generally only being associated with sperm parameter changes [6, 276, 432]. Hypotheses for mechanisms include direct interactions between the infectious agent and spermatozoa, disruption of embryogenesis, and dysregulation of MRT tissue health and functionality.
In the case of Chlamydia, the hypothesis being explored in this chapter is that changes to testicular health are caused by infection. The sperm parameter changes that are linked with infection occur during spermatogenesis in the testes and involves disruption of specific testicular cells. This is based on a mouse model of infection
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that was established for C. muridarum up to 12 weeks post intra-penile infection
[129]. This model determined that the viability of Sertoli cells and SSCs is compromised during infection and the downstream effect was decreased sperm count and motility (oligoasthenozoospermia) and increased morphologically abnormal sperm (teratozoospermia) [129]. This correlates with several studies of human semen with findings of oligospermia [276, 277, 280], teratozoospermia [7, 276], and asthenozoospermia [7, 276], although there is a large amount of variability in the literature surrounding the effects of C. trachomatis infection on human sperm health
[8].
This chapter aims to determine whether a chronic chlamydial infection can be established in a mouse model, particularly within the testis, and whether infection affects testicular cells and the process of spermatogenesis. Perhaps a Chlamydia specific etiology for male infertility could be established.
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6.2 Materials and Methods
The materials and methods used in this chapter can be found in Chapter 3. The materials and methods relevant to this chapter include: mice (Section 3.4.1), mouse anaesthesia (Section 3.4.2), intra-penile infection (Section 3.2.1), SPG preparation
(Section 3.1.5), mouse euthanasia (Section 3.4.14), cardiac bleed (Section 3.4.4), vas deferens dissection (Section 3.3.8), testis dissection (Section 3.4.6), mouse sperm analysis (Section 3.4.9), mouse tissue IHC (Section 3.3.9.1), mouse tissue homogenization (Section 3.4.16), global methylation mass spectrometry (Section
3.2.8), testosterone ELISA (Section 3.2.9), and anti-sperm antibody ELISA (Section
3.4.12).
Briefly, 6-week old, male, C57BL/6 mice were infected with 1 x 106 IFU of C. muridarum Weiss or sham-infected with SPG via the intra-penile route. Mice were housed for six months and then tested for the presence of a chronic testicular infection. The mouse testes were harvested and then either homogenised for culture of live EBs or fixed for detection of inclusions using immunohistochemistry techniques. Changes to the different cell populations and their viability within the testes, between the infected and non-infected mice, were also analysed using immunohistochemistry techniques. Sertoli cells were examined using α-Tubulin and
ZO-1, Leydig cells were examined using 3βHSD, Myoid cells were examined using
SMA, germ cells were examined using PCNA, and WBCs were examined using
H&E and F4/80. Cleaved caspase 3 was used as an apoptosis marker.
Next, the sperm was isolated from the vas deferens of infected and non-infected mice. The sperm were extracted into BWW basic medium. The sperm vitality, morphology, forward progressive motility, and DNA damage were assessed as
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 141
clinically relevant parameters. The vitality and forward progressive motility were assessed using a haemocytometer and light microscopy. The morphology was assessed by fixation of sperm smears to glass microscope slides with methanol, staining with haematoxylin and methylene blue, and then light microscopy. The
DNA damage was investigated using a sperm chromatin dispersion assay (SCDA).
This involves sperm being set into agarose, lysis of the nuclear membrane which releases the DNA, then staining with green fluorescent DNA binding dye for viewing with epifluorescent microscopy. A portion (1 x 106) of the sperm were also chemically capacitated in BWW+NaHCO3+pentoxifyllinene+dbcAMP. These sperm were used to investigate the oocyte binding capacity of the infected and non-infected sperm. The capacitated sperm were incubated in droplettes of BWW with C57BL/6 female mouse oocytes for 10 minutes. The sperm-oocyte aggregates were washed to dislodge unbound sperm, then the remainder was viewed using light microscopy to quantify the number of bound sperm.
Changes to the methylation status of sperm DNA was also investigated. An aliquot of both the non-capacitated and capacitated sperm underwent DNA extraction. The cytosine residues were tested via mass spectrometry to establish the whole genome methylation status.
Finally, ELISA was used to determine the titres of both testosterone and anti-sperm antibodies from the serum of the infected and non-infected mice. Whole blood was collected via cardiac bleed, it was clotted, then centrifuged to separate the serum from the clot. The serum was added to the two ELISA plates. Dr Tamara Keeley at the University of Queensland performed the in-house testosterone ELISA. The anti- sperm antibody ELISA was sourced commercially from MyBioSource.com
(MBS2603817).
142 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Chlamydial culture, IHC, and sperm parameters were quantified using microscopy techniques, then graphed and analysed using GraphPad Prism (version 7) software.
To compare the differences in infected versus non-infected mice, unpaired, non- parametric, Student T-tests were applied to sperm parameters. The T test was also applied to the mass spectrometry and ELISA data. A bootstrapped, independent samples T test was applied to histological parameters using IBM SPSS statistical analysis program. The level of statistical significance for all tests was set at P ≤ 0.05
(*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 143
6.3 Results
Figure 6.1 shows that C. muridarum can be found within mouse testes after intra- penile infection. This detection was possible by both culture of live EBs from homogenised testis tissue and by histological detection of chlamydial inclusions. The two-, four-, and eight- weeks post infection time-points for EB culture were performed in conjunction with Dr Avinash Kollipara. The six-month post infection time-point was performed in conjunction with Alison Mooney during the completion of her honours project.
The culture shows that an average of 1475 IFU can be found per testis pair from one mouse, at the chronic infection time-point of six-months post infection as seen in
Figure 6.1 A. The data also shows that an average of 2250 IFU/mouse can be cultured from testes 12-months post infection.
Using immunohistochemistry techniques, C. muridarum MOMP staining was achieved. Figure 6.1 B shows multiple inclusions exist throughout each testis section.
In Appendix E, further immunohistochemistry can be seen targeting TCO500, which is an active replication marker expressed in inclusions. These markers confirm the presence of chlamydial inclusions in the testis sections.
No EBs or inclusions were identified in any of the histological sections or homogenised testicular tissues from the non-infected mice. Non-infected mice were tested for the presence of testicular infection at the same two-, four-, and eight- weeks, and six- and 12-months post infection time-points as infected mice.
144 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
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Figure 6.1 Detection of C. muridarum in mouse testes. Testes were harvested from male, C57BL/6 mice, 6-months post intra-penile infection or mock infection with SPG. This figure shows that C. muridarum can be detected in both live EB culture (A) and as formed inclusions in testis tissue by immunohistochemistry (B). Inclusions were stained for MOMP (orange) and DAPI nuclear counterstain was used (blue). Scale bars are representative of 100 µm. Images are representative of n = 5 mice. Data was graphed and analysed (Students T- test) using GraphPad Prism (version 7).
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 145
Histological examination of testicular cell populations and testicular structure revealed several important findings. Quantification of tubule diameter, Sertoli cells,
Leydig cells, Myoid cells, WBCs, and PCNA+ germ cells were investigated in chronically infected mouse testicular tissue by immunohistochemistry techniques.
The tubule diameter and PCNA were investigated with Alison Mooney during the completion of her honours project and these results can be found in Appendix F. The parameters were measured as the number per mm2 of testis tissue, and each was reduced in the infected mouse testes compared to non-infected testes.
In Figure 6.2, the number of Sertoli cells/mm2 of testis tissue was investigated by immunohistochemical detection and quantification of α-Tubulin and of tight junctions (marked by ZO-1). Quantification shows that both α-Tubulin (Figure 6.2 A,
P < 0.001) and ZO-1 (Figure 6.2 B, P < 0.004) were significantly decreased in infected mouse testes compared to non-infected tissue by 44.14% and 37.33% respectively. Representative images of the immunohistochemistry can be visualised in Figure 6.2 C. Sertoli cells were detected in both infected and non-infected testis sections, only in different abundance between the two conditions.
146 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
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Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 147
Figure 6.2 Changes to Sertoli cell populations during chronic C. muridarum infection. This figure shows the changes to the Sertoli cell population during chronic infection, using the markers α-tubulin (n = 2) and ZO-1 (tight junction marker, n = 4). The α- tubulin (A) and ZO-1 (B) markers were quantified per mm2 of testis tissue and an example of the histological staining is provided (C, positive staining indicated by black arrows). Graphs were created with GraphPad Prism (version 7) and analysed using IBM SPSS statistical analysis program (bootstrapped, independent samples T test) with differences between infected and non-infected conditions considered to be significant when P < 0.05 (**P < 0.01, ***P < 0.001).
148 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Figure 6.3 shows the investigation of the Leydig cell population, which was determined by the cell specific marker 3βHSD. Leydig cells were identified in both infected and non-infected testis sections. However, conversely to the Sertoli cells, there was a small increase of 22.77% in Leydig cell abundance within infected testes, when compared to the non-infected testes/mm2 of tissue. This increase was not statistically significant (P = 0.284). This data is graphically represented in Figure 6.3
A, with examples of histological staining provided in Figure 6.3 B.
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Figure 6.3 Changes to Leydig cell populations during chronic C. muridarum infection. This figure shows the change in Leydig cell population during chronic infection, using the marker 3βHSD (n = 2). The 3βHSD marker was quantified per mm2 of testis tissue (A) and an example of the histological staining is provided (B, positive staining indicated by black arrows). Graphs were created with GraphPad Prism (version 7) and analysed using IBM SPSS statistical analysis program (bootstrapped, independent samples T test) with differences between infected and non-infected conditions considered to be significant when P < 0.05.
150 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
The peritubular Myoid cells were also quantified (Figure 6.4 A) and visualised
(Figure 6.4 B) in the chronically infected mouse tissue and the non-infected counterpart. Myoid cells were identified using the marker smooth muscle actin
(SMA), which in the testis is localised to the Myoid cells. SMA abundance was significantly reduced (P < 0.003) in the infected testis tissue when compared to the non-infected tissue. Myoid cells were reduced by 48.27% within the infected testes
(n = 2). SMA was successfully detected in both the infected and non-infected conditions.
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Figure 6.4 Changes to Myoid cell populations during chronic C. muridarum infection. This figure shows the change in Myoid cell population during chronic infection, using the marker SMA (n = 2). The SMA marker was quantified per mm2 of testis tissue (A) and an example of the histological staining is provided (B, positive staining indicated by black arrows). Graphs were created with GraphPad Prism (version 7) and analysed using IBM SPSS statistical analysis program (bootstrapped, independent samples T test) with differences between infected and non-infected conditions considered to be significant when P < 0.05 (***P < 0.001).
152 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Figure 6.5, shows the quantification of WBCs (A) and macrophages (B) within testicular tissue. Representative examples of the staining of WBCs, and F4/80+ macrophage (completed with Dr Avinash Kollipara) populations in testis tissue is also seen in Figure 6.5 C. WBCs and macrophages were detected in both non- infected and infected testis tissue sections in different abundances. Both WBCs (n =
3) and macrophage populations (n = 1) of cells were increased (significantly, P =
0.008 for WBCs) in infected testes when compared to non-infected testes.
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 153
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154 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Figure 6.5 Changes to testicular immune cell populations during chronic C. muridarum infection. This figure shows the quantified changes to the testicular immune cell population using haematoxylin and eosin staining to show WBCs (n = 3, A) and fluorescent immunohistochemistry to show F4/80+ macrophages (n = 1, B). An example of the histological staining is also provided (C, positive WBC staining indicated by black arrows, positive F4/80 staining indicated in red). Graphs were created with GraphPad Prism (version 7) and PMNs were analysed using IBM SPSS statistical analysis program (bootstrapped, independent samples T test) with differences between infected and non-infected conditions considered to be significant when P < 0.05 (**P < 0.01).
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 155
There were a greater number of damaged cells in infected testis tissue sections compared to the non-infected controls as seen in Figure 6.6 A. This was measured by histological detection of the cellular apoptosis marker, cleaved caspase 3, per mm2 of testis tissue. Representative images (n = 3) can be seen in Figure 6.6 B. Although there was an increase in the number of apoptotic cells/mm2 of testis tissue in the infected condition, this was not statistically significant (P = 0.1932). There were also apoptotic cells present in both infected and non-infected tissues.
156 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
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Figure 6.6 Testicular cleaved-caspase 3 abundance during chronic C. muridarum infection. This figure shows the change in apoptotic cell numbers during chronic infection, using the marker cleaved caspase 3 (n = 3). The caspase marker was quantified per mm2 of testis tissue (A) and an example of the histological staining is provided (B, positive staining indicated by black arrows). Graphs were created with GraphPad Prism (version 7) and analysed using IBM SPSS statistical analysis program (bootstrapped, independent samples T test) with differences between infected and non-infected conditions considered to be significant when P < 0.05.
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 157
To accompany changes within the Leydig cell population, the titre of testosterone in mouse serum was measured by ELISA as seen in Figure 6.7. Testosterone was detected in both infected and non-infected mouse serum samples. The mean titre was measured to be 0.2384 ng/mL in non-infected mouse serum, and the titre was increased to 0.2653 ng/mL in infected mouse serum. This represents an increase of
10.14% in the infected samples, compared to the non-infected samples, which was non-significant (P = 0.5714).
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Figure 6.7 Titre of circulating testosterone during chronic C. muridarum infection. This figure shows the titration of testosterone present in infected and non-infected mice at six-months. The graph, created with GraphPad Prism (version 7), represents the mean of the biological replicates (n = 5) and standard deviation. A Students T- test was applied to the quantification with differences between infected and non- infected conditions considered to be significant with P < 0.05.
158 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
To determine the downstream effects of testicular cell and tissue changes during chronic infection, the spermatozoa produced by these testes was assessed next in
Figure 6.8. Figure 6.8 A shows the sperm vitality, which was not statistically significantly reduced (P = 0.2991). Figure 6.8 (B) shows the sperm forward progressive motility, which was significantly reduced (P < 0.05) in infected mice.
Figure 6.8 C shows the sperm morphology, which was significantly more abnormal
(P < 0.001) in infected mice. The abnormal morphology types can be seen in Figure
6.8 D. These included (i) head defects, (ii) tail defects, (iii) principle piece defects,
(iv) head-tail connection defects, and (v) combinations of each type of defect. Sperm from infected mice also had a higher rate of capacitation present within both non- capacitating and capacitating BWW media conditions compared to the non-infected controls. The amount of tyrosine protein phosphorylation measured the sperm capacitation. However, this experiment was completed in collaboration with Alison
Mooney during the completion of her honours project and the results can be found in
Appendix G.
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 159
Figure 6.8 Sperm vitality, motility, and morphology during chronic C. muridarum infection. Figure 6.8 shows the amount vitality (A), forward progressive motility (B), and amorphic sperm present in sperm samples (C). Examples of morphological abnormalities are also shown (D). Sperm were isolated from mice 6 months post- infection with C. muridarum Weiss and compared to the non-infected control. Data was graphed and analysed using GraphPad Prism (version 6). A Students T-test was used to analyse each of the different sperm parameters comparing the infected to the non-infected control samples (n = 10), P < 0.05 was considered to be significant (*P < 0.05, ***P < 0.001).
160 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
The ability of the sperm to bind an oocyte was measured using a zona-pellucida binding assay (Figure 6.9). Sperm were significantly less able to bind an oocyte when isolated from the vas deferens of chronically infected mice compared to non- infected mice (P < 0.05). There were, on average, approximately 5 sperm bound per oocyte in the non-infected condition, compared to approximately 1 sperm bound per oocyte in the infected condition. Graphical representation of this data is seen in
Figure 6.9 A, with representative images in Figure 6.9 B.
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 161
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Figure 6.9 Oocyte binding capacity of sperm from mice chronically infected with C. muridarum. Sperm were isolated from mice six-months post infection with C. muridarum Weiss and compared to the non-infected control (n = 10). This figure shows the number of sperm that can bind oocytes in vitro (A). Representative images of the sperm-Zona Pellucida binding assay are also shown (B). The oocytes with bound sperm (indicated by black arrows) were imaged at X20 magnification. Data was graphed and analysed using Students T-test (GraphPad Prism, version 6), P < 0.05 was considered to be significant (*P < 0.05).
162 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
The genetic aspects of sperm health were also investigated in the form of DNA fragmentation and global DNA methylation status. Figure 6.10 shows that in sperm samples isolated from infected mice, there was a significant increase in the amount of DNA fragmentation (P < 0.05), compared to the sperm isolated from non-infected controls. There were sperm with DNA fragmentation present in the non-infected samples. However, these remained below ~12% and within the normal levels of detection in healthy sperm [6]. This was measured with a sperm chromatin dispersion assay.
Additionally, the infected group sperm samples had a higher proportion of sperm with hypermethylation when under in non-capacitating BWW, and hypomethylation when in capacitating BWW. There was an average increase in cytosine methylation of 0.3% in non-capacitated sperm, and an average decrease of 0.2% in capacitated sperm, within the infected group sperm samples. This is shown below, in Table 10.
Table 10: Global DNA methylation status in sperm from mice chronically infected with C. muridarum. % Methylation Change from Non-
infected to Infected
Non-capacitated 0.3 %
Capacitated -0.2 %
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 163
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Figure 6.10 DNA fragmentation present in sperm from mice chronically infected with C. muridarum. This figure shows quantification of the DNA fragmentation present in the sperm isolated from mice six-months post-infection with C. muridarum Weiss, compared to the non-infected control (A). Representative images the sperm chromatin dispersion assay used to test this are shown (B), where the dotted line indicates the outline of the DNA which has been stained with SYBR Green II (Sigma Aldrich, AUS). The sperm were imaged at X40 magnification. GraphPad Prism (version 6) was used to generate the graph and apply a Students T-test to analyse levels of DNA fragmentation present in the sperm samples (n = 10), P < 0.05 was considered to be significant (**P < 0.01).
164 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Lastly, the impact of changed testicular tissue conditions in relation to the maintenance of immune privilege within the testis was investigated in Figure 6.11.
Sperm isolated from the vas deferens of infected mice were seen by light microscopy to be aggregated, generally by the head pieces. This can be visualised in Figure 6.11
B. The sperm aggregation was associated with the presence of anti-sperm antibodies present within infected mouse serum (titrated to 100 ng/mL, Figure 6.11 A). Both aggregations of sperm and anti-sperm antibodies were greatly reduced or absent in non-infected control mice (antibodies titrated to 25 ng/mL). This represented a statistically significant production of anti-sperm antibodies within chronically infected mouse serum (P < 0.05).
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 165
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Figure 6.11 Anti-sperm antibody production and sperm agglutination present in mice chronically infected with C. muridarum. This figure shows the high titre of anti-sperm antibodies present in mouse serum six- months post infection with C. muridarum Weiss, compared to the non-infected control (A). ASABs were measured by ELISA (MyBioSourse.com, CN, MBS2603817). Representative images are of aggregated sperm isolated from the vas deferens of mice viewed by brightfield microscopy (X20 Mag, B). GraphPad Prism (version 6) was used to generate the graph and a Students T-test was used to analyse anti-sperm antibody titres, P < 0.05 was considered to be significant (*P < 0.05).
166 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
6.4 Discussion
Chapter 6 shows that Chlamydia can be found in the testes of mice six-months after intra-penile infection. The health of the testicular environment appears compromised by infection. Significant loss of key testicular cells, including Sertoli cells in the blood-testis-barrier and Myoid cells in the peritubular membrane, occurred during infection. This may be due to apoptosis as cleaved caspase 3 also had increased abundance in the infected testes. Conversely, Leydig cells and Tmφ populations were unaffected and increased respectively. Of particular interest are the WBCs as macrophages have a role in chlamydial transmission to the testes and could form a reservoir of infection. They may also be of inflammatory phenotypes, which could disrupt the immunosuppressed testicular environment. The major downstream effect of altered testicular histology was significant decrease in sperm quality. Sperm morphology, motility, DNA integrity, and oocyte binding capability were all significantly adversely impacted by infection. Such systemic aberration in sperm parameters implicates spermatogenesis dysregulation and failure.
In more detail, C. muridarum can be found within mouse testes six-months post- infection. The chlamydial inclusions identified by histology, contain actively replicating RBs, as shown by the MOMP/TC0500 histology staining. Additionally, live EBs were cultured from the chronically infected testes. This indicates that a low- grade but productive chronic infection has been established. The distinction between chronic and persistent infections is important in this scenario. Chronic infections are likely to remain sexually transmittable, whereas persistent infections most likely cannot be sexually transmitted as ABs are not infectious [141]. It is unclear at this time which testicular cell types are perpetuating the chronic infection and this requires further investigation.
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 167
It may be inferred that the Myoid, Sertoli and germ cells have become infected, and died as a result, during the six-month timeframe, due to the decreased numbers of the cell types during chronic infection. Particularly given the increased susceptibility to infection displayed by Sertoli cells shown in Chapter 4. Secondary methods could be used to confirm these decreases, such as flow cytometric quantification of total cells per testis and use of alternative IHC markers including vimentin for Sertoli cells. The decreased numbers of these cells may indicate that the populations have undergone lysis at the end of the chlamydial lifecycle. However, these cells are also apoptotic in the presence of pro-inflammatory conditions [129]. The apoptosis staining positivity within the infected testes may indicate that the populations are responding to inflammatory factors being released by surrounding cells.
Cells including the WBCs that have infiltrated the testis tissue may induce inflammatory conditions to promote apoptosis in Myoid, Sertoli, and germ cells.
While Tmφ are normally able to maintain an anti-inflammatory environment, other recruited macrophages or PMNs may counteract this effect [225, 385, 386].
Phenotyping the infiltrating immune cells may shed light on this hypothesis.
Phenotyping the macrophages further than F4/80 positivity may reveal an influx of pro-inflammatory M1 macrophages. Phenotyping the infiltrate identified by H&E staining may reveal the presence of pro-inflammatory/anti-bacterial cells including neutrophils and T cells. CD4+ T cells are known responders to chlamydial infection in both the male and female reproductive tracts, and CD8+ T cells are known to enhance pathology in the FRT [106, 107, 109].
Alternatively, the infiltrating immune cells may be responsible for maintaining the infection within the testes. Chlamydial species are known to survive within DCs [90,
302], neutrophils [433], monocytes [304], and macrophages [219, 298]; including in
168 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Chapter 4, which showed C. muridarum survival within macrophages in vitro and in vivo. Tmφ may be proliferative, or be actively recruiting circulating macrophages/monocytes, to compensate for losses in the population due to infection.
It’s likely that the results are a reflection of a combination of both of these scenarios.
The small increase in Leydig cell populations within testis tissue sections was an interesting development as the other specialized testicular cell populations were decreased. As these cells are proliferative in all stages, conversely to the Sertoli and germ cells, Leydig cells may be able to compensate for cell lysis after infection [11].
Additionally, the Leydig cells may actively have increased proliferation in response to failing spermatogenesis in some tubules. Alternatively, shrinkage of tubular space in some areas may have biased the amount of interstitial space viewed during quantification of the Leydig cells, causing the appearance of increased cell numbers.
Adjusting for tubular shrinkage and quantification of total Leydig cells per testis could be performed in the future.
Increased Leydig cell numbers may explain the increased testosterone concentration present within the serum of infected mice. As discussed in Chapter 5, testosterone concentration is linked with FSH production, which in turn is linked with testicular volume [160, 162, 417]. The levels of circulating FSH could be investigated to further this line of enquiry. However, this is outside the scope of the project at this time. This finding also correlates with the increased testosterone produced by infected TM3 cells found in Chapter 5, so it may be the result of a combination of both Leydig cell proliferation and epigenetic/transcriptomic dysregulation during infection. Regardless, any changes in hormone levels must be regarded seriously as hormones exist in an extremely fine balance within the body. Dysregulation of steroidogenesis can also have off-target or systemic effects [434].
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 169
Each of the histological results indicates that infected testicular tissue has undergone changes during chronic infection. Therefore, the functional consequences of these changes should be investigated. Firstly, loss of immune privilege is indicated by the low abundance of the ZO-1+ tight junctions in infected testes. Breakdown of the blood-testis barrier, in this case it seems via Sertoli cell death, may lead to production of anti-sperm antibodies that were observed [435]. These anti-sperm antibodies are likely to be responsible for the agglutination of sperm heads that frequently occurred within infected sperm samples [436]. Several alternatives may be possible for the cause of the anti-sperm antibody production including breakdown of the tubular structure of the epididymis. The epididymal caput has some immune privilege and has been shown to break down during chlamydial infection; this may expose sperm being stored here to the immune system in a similar method to within the testis [158, 437]. The corpus and cauda lack immune privilege, and as such have a resident immune cell population including macrophage and DC subtype populations [157]. These normally function to remove apoptotic sperm from the tubules. However, this function may be disrupted either by infection, breakdown of tissue structure, or simply being overwhelmed by an increased number of apoptotic sperm needing to be cleared [171].
Another possibility is that the sperm agglutination is unrelated to anti-sperm antibody presence. Escherichia coli interaction with sperm causes an interesting phenomenon, which results in agglutination of sperm via mannose and mannose binding structures that are expressed on both E. coli and sperm cells [438]. Chlamydial species also have a mannose-binding lectin that is involved in binding and gaining entry to host cells [65]. Therefore, direct contact between C. muridarum and spermatozoa may
170 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
also be responsible for the agglutination found. An agglutination assay could be developed in the future to investigate this.
The cause of the agglutination could be somewhat reduced by centrifugation, suggesting that the interaction is reversible/fragile. This removal of agglutination enabled sperm parameters to be measured in greater detail. However, in the setting of natural conception, sperm agglutination would most likely be inhibitory as it would impede or probably negate forward progressive motility through the FRT and then oocyte binding.
Multiple sperm parameters were investigated during Chapter 6. Firstly, sperm count and vitality were found to be non-significantly reduced from infected mice compared to the controls. This is in line with some previous findings from humans, which found that chlamydial infection was not associated with significant decreases in sperm concentration in semen or sperm vitality [198]. It is possible that performing a live/dead analysis only by cell permeability simplifies the viability of the sperm and further analysis may be required. However, many other techniques centre on DNA integrity as a marker of sperm death, which was also examined by SCDA in this study.
The SCDA showed that infected mice have significantly greater numbers of sperm with DNA fragmentation than healthy mice. DNA fragmentation is a precursor and a hallmark of cellular apoptosis, including within sperm [188]. It can be measured by several other methods besides SCDA, including TUNEL and comet assays [210].
These assays could be employed in future as a secondary confirmation of the SCDA results. However, the SCDA is a commonly used and robust approach [439]. It
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 171
requires minimal sample handling, which reduces the chance of stressing the sperm and causing false positive results [439].
Finding evidence of DNA fragmentation in both the GC-1 cells in Chapter 5 and in sperm cells in Chapter 6 is an interesting result. The GC-1 germ cell biological equivalent are the precursors of mature sperm. It may be possible that these cells are dividing/maturing with fragmented DNA. They have an increased tolerance to DNA damage as discussed in Chapter 5 RNAseq results. This is further evidence supporting faulty spermatogenesis as a result of infection, this time possibly transmitting unstable DNA to mature spermatozoa.
Sperm DNA integrity is also important for fertility as sperm with damaged DNA are more likely to produce embryogenesis failure and adverse pregnancy events, for example miscarriage [186]. As previously discussed, sperm do not have DNA repair capability [209]. The oocyte carries out any DNA repair that is required after fertilisation [209]. However, in some cases the great extent of damage to be repaired causes a delay in the time for the first oocyte cleavage [440]. This cleavage delay is associated with adverse pregnancy outcomes [209, 440].
Asthenozoospermia is also associated with DNA fragmentation. This is hypothesised to be the result of metabolic deregulation as a downstream effect of functional gene fragmentation [422]. This trend was observed in this study; infected mice were significantly more likely to produce sperm without forward progressive motility.
Sperm that lack forward motility will have reduced capability to traverse the FRT and compete for binding of an oocyte. Determining the percentage of fragmentation within the sperm genomes would be interesting in the future. Sperm with greater than
8% fragmentation have a significantly decreased ability to participate in
172 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
embryogenesis as repair of greater amounts of fragmentation is outside the capability of the oocyte in many cases [441].
Sperm morphology will also influence the motility and oocyte binding capacity
[442]. Sperm have a specialized and specific morphology that is often species specific [443]. The morphology is advantageous to both traversing the FRT and binding an oocyte [443]. Head defects may cause disorientation if not directly affecting the motility and reduce oocyte binding. Tail defects, such as the folded principle piece or incorrect connection to the head that were observed, would directly affect the motility. The sperm produced by the infected mice in this study had significantly higher amounts of amorphic sperm compared to the healthy mice. This correlates with some literature that also found that C. trachomatis infections in men resulted in higher concentration of amorphic sperm present in semen [7].
Capacitation is also an important factor in oocyte binding; sperm require the capacitation event to occur directly preceding fertilisation [444]. Sperm lacking this ability will not be able to complete an acrosome reaction and fertilisation [444]. The sperm isolated from infected mice displayed premature capacitation, marked by significantly increased levels of tyrosine phosphorylation compared to the healthy sperm. Capacitation has a finite timeframe, once the process has started it is not normally able to be halted and once it is spent it cannot be naturally replicated [445,
446]. The premature activation of the infected group sperm will likely also contribute to other abnormal parameters.
The mechanism for Chlamydia-mediated induction of premature sperm capacitation is yet unknown. One hypothesis that has emerged through this study is that infection compromises the progenitor germline, and all subsequent sperm produced are
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 173
compromised in the same way. This potentially occurs through epigenetic modification. As seen in Chapter 5, GC-1 germ cells experience hypomethylation during infection. If this also occurs in vivo within SSCs, this will likely be reflected in the spermatozoa. To investigate this, the global DNA methylation status of the non-capacitated and capacitated sperm was tested. The altered methylation present in the sperm from the infected mice supports this theory. Irregular germ line methylation does have a role in infertility [447]. In this case, genes that promote capacitation may be switched on by demethylation, as a result of infection. As there is no evidence within the literature for chlamydial infection directly within spermatozoa, it may not be possible to occur at this end stage.
However, it may also be possible that Chlamydia adheres to the outside of sperm, as suggested by some studies, and the changes resulting from the adhesion induce capacitation [199, 200]. This could be investigated in the future by measuring capacitation before and after direct incubation between Chlamydia and spermatozoa.
In vitro, tyrosine phosphorylation (TP) in human spermatozoa was induced by co- incubation with C. trachomatis [200]. TP is involved with sperm capacitation, which is why it is a useful marker in establishing sperm capacitation status [289]. The mechanism by which C. trachomatis causes TP is unclear. Modification through the
T3SS does not seem likely as there is no evidence of inclusion formation on the surface of, or within, spermatozoa. Some studies show that chlamydial species alter the redox balance with host cells as a result of ROS production [167, 448]. Redox- mediated regulation of sperm capacitation is also well characterized [444]. If chlamydial interaction with sperm also causes ROS production, this could induce capacitation.
174 Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes
Regardless of the mechanism for chlamydial infection altering sperm parameters, the cumulative effect of the changes seems to have resulted in a reduced oocyte binding capability for that sperm. The ZP-binding assay showed that after inducing capacitation in both infected and non-infected group sperm samples, only the healthy sperm are able to bind oocytes in significant numbers. In comparison, the infected group sperm could bind minimally to oocytes. In the context of fertility, this certainly indicates sub-fertility. Even during an IVF procedure, the inability of sperm to bind an oocyte is detrimental and prohibitive. When viewed in combination with the DNA fragmentation present in these sperm, ICSI seems less likely to overcome these infertility challenges also [210].
To summarise Chapter 6, focal destruction of seminiferous tubules and their resident cellular populations resulted in anti-sperm antibody production and decreased sperm quality in infected mice. Most likely through combinations of abnormal morphology,
DNA fragmentation, low motility, and premature capacitation, these sperm had significantly reduced capacity to bind oocytes. Each factor can contribute to sub- fertility and as multiple parameters are altered, this likely indicates that a lack of conception and therefore infertility is a possible outcome of chronic chlamydial infection. The chances of natural conception after chronic infection are greatly reduced and would rely on the reduced numbers of remaining healthy sperm, or potentially sperm with only a single parameter change present.
Further investigation into the downstream effects of the disadvantageous changes to sperm that chronic Chlamydia infection induces is required. This will be explored in the form of a breeding study, in Chapter 7.
Chapter 6: The Effects of Chronic C. muridarum Infection on Mouse Testes 175
Chapter 7: The Effects of Chronic
C. muridarum Testicular Infection
on Offspring
176 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
7.1 Introduction
The impacts of paternal infection on offspring health are underestimated and understudied, as is common within emerging fields of research. Conception and childbirth have historically been considered to be a woman’s problem. This is despite approximately 40-50% of infertility factors being attributed to men, and ART treatments being sought for male factor infertility [424]. As such, maternal models of infection that result in offspring mortality or morbidity have predominated.
Models of maternal infection include; viral (HIV [449, 450], enteroviruses [451,
452], hepatitis B/E [453, 454], Japanese encephalitis [455], influenza [456, 457],
HSV [458], CMV [459], parvovirus B19 [460], rubella [461], Varicella zoster [462], coxsackie B virus [463], mumps [464], and Zika [465]), parasitic (Toxoplasma gondii [466], Trypanosoma cruzi [467, 468], and Plasmodium species [469, 470]), and bacterial (Group B streptococci [471], Listeria monocytogenes [472, 473],
Escherichia coli [474, 475], Leptotrichia amnionii [476, 477], Treponema pallidum
[478], Ureaplasma urealyticum [479], Neisseria gonorrhoea [480, 481], and C. trachomatis [482]). Many of these studies included longitudinal and mechanistic aspects to uncover the effects of maternal infection on offspring health and development.
Conversely, paternal models of infection include H. pylori transmission between parents and children [483], and potential and unconfirmed links between paternal
HIV [484] and hepatitis B virus [485] infection and offspring health. These studies are separate to those showing links between infection and male sub- fertility/infertility. However, they are also correlative rather than causative as no
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 177
mechanisms have been identified that indicate how disturbances within MRT cause offspring abnormalities.
A consideration in the development of this field is that many human based studies have been unable to conclusively show that infections present in semen are not contaminants originating from elsewhere in the reproductive tract, e.g. the urethra, concomitantly to ejaculation and the subsequent isolation of sperm. Furthermore, studies commonly focus on infections causing poor sperm health, examples of C. trachomatis (and C. muridarum in a mouse model), M. hominis, and Ureaplasmas [6,
9, 129, 198] as previously mentioned in Section 6.1, or conception failure [281, 486].
Longitudinal studies on the downstream effects on offspring are infrequent. To date, the only evidence linking paternal infection mechanistically with offspring health, was completed by this project and collaborators at The University of Newcastle.
One study using a rat model of infection investigated the link between paternal C. muridarum infection and conception, where fecundity and embryonic loss were studied [281]. The fertility of the rats was tested at 15 days and 80 days post- infection [281]. There were no differences between the infected and sham-infected rat fecundity and embryonic loss before or after implantation [281]. There were also other key differences in this model that are in opposition to other models in the literature, as previously discussed in Section 2.4.2. However, importantly, this study did not conduct a longitudinal study on the health and viability of offspring post- partum.
Evidence does exist connecting the paternal impacts of several other disease states including metabolic diseases [487, 488], diabetes [489, 490], alcoholism [491, 492], and cigarette smoking [493] with offspring abnormalities or reduced
178 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
health/developmental outcomes. The advantage that these studies hold over current infection studies is the longitudinal aspect whereby following up on the health of the children born to affected men was achieved. These studies made meaningful contributions to the field of paternal impacts on offspring health. This chapter similarly aims to open the investigation into the currently completely uncharacterized effects of paternal Chlamydia infection on offspring health, which represents a major knowledge gap in the literature.
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 179
7.2 Materials and Methods
The full and detailed materials and methods used in this chapter can be found in
Chapter 3. The important materials and methods relevant to this chapter include: mice (Section 3.4.1), mouse anaesthesia (Section 3.4.2), intra-penile infection
(Section 3.2.1), mouse euthanasia (Section 3.4.14), breeding protocol including pup sex determination and weaning (Section 3.4.15), mouse weighing and calculating the organ:body weight ratio (Section 3.4.12), vas deferens dissection (Section 3.3.8), testis dissection (Section 3.4.6), mouse tissue IHC (Section 3.3.9.1), sperm analysis
(Section 3.4.9), and FRT dissection (Section 3.4.10).
Briefly, 6-week old, male, C57BL/6 mice were infected with 1 x 106 IFU of C. muridarum Weiss or sham-infected with SPG via the intra-penile route. Mice were housed for four-five months and then bred with non-infected C57BL/6 females. The females, sourced from the ARC, were proven breeders. The presence of vaginal plug formation verified successful mating.
Approximately 20-22-days after mating, litters dropped and 7-days post birth the pups were tagged for later identification, weighed, and sexed. Pups were then weighed every 2-3-days until the appropriate time points. The male and female reproductive tracts of the pups were investigated for functional aspects at various time points including 11, 21, and 42-days post birth. The FRTs were fixed, sectioned, stained with haematoxylin and eosin, then visualised with light microscopy. An overview of the timeline of evens can be seen in Figure 7.1.
The testes were taken from the male pups. Testes were fixed, sectioned, stained with haematoxylin and eosin, then visualised with light microscopy. The sperm from male pups was also isolated from the vas deferens. This was analysed when they reached
180 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
sexual maturity at 42-days post birth. Sperm was extracted into BWW medium and several sperm parameters were investigated. The sperm count, vitality, and motility were assessed using a haemocytometer viewed with light microscopy. The time- points investigated can be seen in the timeline below.
Figure 7.1 Breeding study timeline. Figure 7.1 shows the timeline of events that were investigated in the breeding study. This includes the mating and day seven, 11, 14, 21, and 42 post-birth events.
The reproductive tracts were examined using a dissecting microscope to remove surrounding fatty tissue. After the fatty tissue was removed, the tracts were weighed on a fine balance. Their proportionality to the total body weight of the animal they originated from was then calculated, resulting in the organ: body weight ratio.
IHC and sperm parameters were quantified using microscopy techniques, then graphed and analysed using GraphPad Prism (version 7) software. To compare the differences in infected versus non-infected mice, unpaired, non-parametric, Student
T-tests were applied. The T test was also applied to the mass spectrometry data. The level of statistical significance for all tests was set at P ≤ 0.05 (*P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001).
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 181
7.3 Results
Dams were successfully mated with either infected or non-infected sires. There was no noticeable difference between the time to conception or the rates of vaginal plug formation between the two breeding groups. Pregnancies progressed on a normal timeline and there was no difference between the group times to delivery.
On the day of birth, pup viability was checked non-intrusively so as not to cause undue stress. There were two deceased litters found within the infected breeding group. Pup viability was checked again seven days post birth at the first available time that pups could ethically be handled (Figure 7.2 A). The two deceased litters were recorded as zero viable at day seven. In all viable litters, there was no significant difference between the number of pups per litter (P > 0.05).
The pups were sexed at day seven with the assistance of Donna West (MERF animal technician), there was no difference between the number of males and females born between the groups (Figure 7.2 B). The non-viable litters were not able to be included in this data set.
Also at seven days post-birth, pups were weighed for the first time. Pups were then weighed every 2-3 day until day 42, revealing that there was a consistently lower total pup weight in the infected breeding group on average until day 21 (Figure 7.2
C). The weight difference between the two groups was non-significant throughout the time-course. At day 21, weaning occurred in both groups. At weaning, it was determined that ethically, two litters from the infected breeding group were required to be euthanized. Several criteria determined this; each pup in the two litters was lighter by approximately 20% than the average non-infected breeding group, each
182 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
pup showed signs of being unable to provide food and water for themselves, and each pup had shown increasingly static development compared to the controls.
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Figure 7.2 Viability, numbers, sex, and weight gain of pups bred from sires chronically infected with C. muridarum. This figure shows the outcome in terms of viability and number (A), sex (B), and weight gain of pups (C) of breeding male C57CL/6 mice (n = 6) that had been chronically infected with C. muridarum with healthy C57BL/6 female mice (n = 12). Non-viable pups were recorded as zero and were not included in the sex determination graph. The weight gain curves start seven-days after birth and continue until weaning at 21-days after birth, graphed points represent the mean and SD of all pups. Graphs and analysis were generated with GraphPad Prism (version 6), differences were considered to be significant when P < 0.05.
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 183
At 14 days post-birth a developmental time point for the pups was observed. As seen in the photographic examples in Figure 7.3, at day 14 non-infected group pups opened their eyes whereas the infected group pup did not. Similarly, at day 14, the pup lengths were measured and within the infected group the pups were shorter.
These abnormal development trends were observed within all litters born to the infected group, not limited to the particularly unhealthy litters.
Figure 7.3 Developmental delays displayed in pups born to chronically infected male mice. Figure 7.3 shows the outwardly observable developmental delays in pups born from breeding of male C57CL/6 mice (n = 6) that had been infected (intra-penile) for 4-5 months with C. muridarum with healthy C57BL/6 female mice (n = 12). The right column shows these pups. Age matched, non-infected C57BL/6 male mice bred with healthy females were used as the control. The left column shows these pups. The top row of pictures shows examples of different pup body lengths. The bottom row of pictures shows examples of whether pups eyes were open at 14-days after birth. These pictures are representative of the total 24 litters born during this breeding trial.
184 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
At specific time-points, pups were euthanized and their internal organs were evaluated with focus on the reproductive tracts. At day 42 post birth, the FRTs were weighed; there was a significant difference (P < 0.01) between the infected and non- infected breeding group pups at this time-point (Figure 7.4 A). The trend was toward a low reproductive tract weight in the infected breeding group. When normalised to the body weight to achieve an FRT:body weight ratio, this trend was less pronounced where the trend approached significance (P = 0.0682) but did not reach statistical significance (Figure 7.4 B). The representative images in Figure 7.4 C show histological sections of the FRTs at day 42, after being stained with H&E. These images show the vaginal glands, which are a healthy functioning indicator, in each breeding group. There were no structural differences distinguishable between the groups.
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 185
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Figure 7.4 Low reproductive tract weight of female pups born to sires chronically infected with C. muridarum. Reproductive tracts were harvested from female pups born to C57BL/6 sires that were chronically infected with C. muridarum and then bred with healthy C57BL/6 female mice. This figure shows graphs of the FRT weight (A) and the FRT:body weight ratio (B), and histological sections of the FRT stained with H&E of female pups born to each breading group at 42 days after birth (C). Graphs and Students T- tests were generated with GraphPad Prism (version 6), differences were considered to be significant when P < 0.05 (**P < 0.01).
186 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
The testes of the male pups were harvested at days 11, 21, and 42. There was a significant difference in the weight (Figure 7.5 A) and the testis:body weight ratio
(B) between the breeding groups at day 11, where the infected group were significantly smaller testes (P < 0.001) and a significantly reduced testis:body weight ratio (P < 0.01). There was non-significant difference between the groups at the later time-points at day 21 (Figure 7.5 C and D, P > 0.05) and day 42 (Figure 7.5 E and F,
P > 0.05). However, these time-points were unable to include the previously euthanized pups, so they represent only a selected portion of the population.
Histological sections from the testes harvested at day 21 were stained with H&E.
These showed that there was a significant decrease in the number of seminiferous tubules/mm2 in the infected group at this time-point. Defined seminiferous tubules were present from both groups though.
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 187
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188 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
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Figure 7.5 Low testis weight of male pups born to sires chronically infected with C. muridarum. Testes were harvested from male pups born to C57BL/6 sires that were chronically infected with C. muridarum and then bred with healthy C57BL/6 female mice. This figure shows graphs of the testis weight and the testis: body weight ratio at 11 (A and B respectively), at day 21 (C and D respectively), and 42 days (E and F respectively) after birth. Histological sections were stained with H&E, imaged, and then the number of tubules per mm2 of tissue was quantified, as seen in the graph in G (i). Representative histological images of the testes day 21 after birth are included in G (ii). Graphs were generated with GraphPad Prism (version 6) and a Students T-test was applied, differences were considered significant when P < 0.05 (**P < 0.01, ***P < 0.001).
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 189
The sperm from the male pups was also analysed at day 42, as the earliest time-point when sperm are functionally mature (Figure 7.6). The infected breeding group pups had reduced sperm counts compared to the healthy breeding group (Figure 7.6 A).
While this was not a statistically significant difference, the trend approached significance (P = 0.0556). The infected breeding group also had small, non- significant reductions in sperm vitality (Figure 7.6B, P = 0.5746) and forward progressive motility (Figure 7.6 C, P = 0.1255). When sperm from infected breeding group male pups was incubated with mouse oocytes, there was a non-significant increase in oocyte binding in the capacitated group compared to the non-capacitated control (Figure 7.6 D, P = 0.1247).
190 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
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Figure 7.6 Low sperm quality of male pups born to sires chronically infected with C. muridarum. Figure 7.4 shows count (A), vitality (B), motility (C) and oocyte binding (D) of sperm isolated from the vas deferens of six-week old, C57BL/6, male mice that were born to either healthy or chronically C. muridarum infected sires. The sperm were isolated in 1 mL of BWW medium, then analysed using a haemocytometer viewed under light microscopy. Eosin Y was used as a viability stain. The pups were from ten separate litters. Data was graphed and analysed (Students T-test) using GraphPad Prism (version 6). Differences between the breeding groups were considered to be significant when P < 0.05).
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 191
7.4 Discussion
Chapter 7 shows that the offspring born to chronically infected sires are negatively affected in several ways. Offspring displayed developmental delays; on average they were of consistently lower weight and size than healthy breeding group counterparts.
There were also delays or restrictions to reproductive tract development. Female offspring had smaller, though histologically normal appearing, reproductive tracts.
Male offspring also had smaller testes and produced decreased quality of sperm.
Reductions in sperm count, motility, and capacitation ability were the major abnormalities observed. This may be due to testicular abnormalities, as the number of seminiferous tubules present in the testes of these mice was significantly reduced.
There was no difference between the conception rates of the infected and non- infected breeding groups. This may be attributed to less than 100% of sperm being affected by the abnormal parameters as seen in Chapter 6. Therefore, some completely healthy sperm are still present, and the healthy sperm will still be able to produce normal conception and offspring. This hypothesis accounts for the proportion of pups from the infected breeding group with normal weight and reproductive tract development. If this was not the case, the low-quality sperm would have needed to outcompete the healthy sperm for oocyte fertilization, which seems unlikely.
Also present in the semen may be sperm with single parameter changes, or perhaps not severely deleterious changes. For example, some sires may have sperm with small amounts of DNA fragmentation that were able to be repaired by the oocyte they fertilize [441]. Some sires may have sperm with increased levels of DNA fragmentation or a higher proportion of sperm with single or multiple parameter
192 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
changes. These factors could explain the presence of the non-viable litters within the infected group.
It is unfortunate that it could not be determined whether the pups in the non-viable litters were born dead or if they died after birth. Dams are recognised as having altered maternal behaviour, including cannibalizing their neonates, if the pups are stillborn, particularly unhealthy, or the environment is overly stressful [494, 495]. As multiple other healthy litters were born to both groups on the same day as the non- viable litters, the options seem limited to the pups being stillborn or particularly unhealthy.
There were also two of 12 litters with very low birthweight within the infected breeding group that were not cannibalized by the dams but were euthanized before weaning. These unhealthy pups give credence to the possibility of the non-viable litters being stillborn or grossly unhealthy. Currently, the four out of 12 litters in the infected group being unhealthy forms an interesting trend. With a greater total number of litters this trend may gain significance and there is merit in expanding this pilot study. Correlating the extent of sperm parameter abnormalities of each individual sire with offspring health could also provide insights for future experiments.
The averaged litter weights of each group showed that there was a trend toward low weight in the infected breeding group until day 21. This was not continued past day
21 and becomes inconclusive until day 42 at the end of the experiment. Pre-weaning the two low-weight/unhealthy litters were euthanized as it was considered that they would not survive separately from the dam and had drastically delayed development.
However, this also biases the post-weaning weight gain curves as it will only include
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 193
the remaining eight healthy-looking litters. Additionally, when weaning occurs at day 21, the pups are separated from the dams and are placed in sex- specific cages.
This introduces many confounding aspects to the weight gain curves [496, 497].
These factors may include development of behavioural and hierarchical aspects within the new cages, the individual health of the animals, and how many pups were in a litter all previously in one cage now being housed at a maximum of five per cage
[496, 497].
The abnormalities present in at least two of the infected breeding group litters could have been the product of several factors. Firstly, improper repair and/or replication of fragmented DNA present within infected sire sperm may contribute [498, 499]. DNA damage within sperm that reach the fertilisation stage, as previously discussed, can contribute to delayed oocyte cleavage and adverse pregnancy outcome [440].
However, although these events are strongly associated, this is not always the case.
In some cases, embryogenesis still occurs, and foetal malformation is the result
[440]. This has been studied extensively with both sperm apoptosis and ROS as the aetiological agents of sperm DNA fragmentation [500]. This is usually in the context of paternal transmission of genetic mutations/diseases including Y chromosome deletion and achondroplasia, and the predisposition of childhood cancer development
[500].
Genetic diseases such as achondroplasia are particularly relevant to the two abnormal litters produced from breeding infected sires as these litters showed delayed development initially and then by weaning, development was altogether stunted. This resulted in the litters containing small pups around half the size of the average healthy pup. These abnormalities are somewhat reflective of the human achondroplasia (dwarfism) condition. Of course, species specific characterization of
194 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
genetic disorders would be required to find a more conclusive match between the abnormalities observed in these mouse litters and a human condition.
An additional factor that may contribute to the abnormal litters is epigenetic changes within the germ line and the sperm itself. This is a recent development in the field of epigenetics and it was previously thought that all inheritance originated in DNA. It is now accepted that epigenetic changes (epimutation) including to ncRNA, within the germline have the potential to be carried within gametes leading to epigenetic inheritance [206, 501, 502]. Depending on their position/function, epigenetic tags are usually retained, eliminated or reprogrammed during cell division, including those associated with fertilisation and this is normal [206]. However, in some cases tags that are changed in the germ line by exogenous forces are heritable [206].
Several human studies have been conducted on epimutation based diseases resulting from famine/malnutrition [503], cigarette smoking [504], and stress [505] in a range of maternal and paternal based models. Specific diseases including Prader-Willi syndrome and Beckwith-Wiedemann syndrome have also been linked to epigenetics
[506, 507]. In each case, the health of the offspring was compromised in a specific way and correlated with differential DNA methylation patterns compared to healthy children. However, it is not yet known whether these are a result of epigenetic inheritance or other genetic mechanisms.
From a reproduction-specific viewpoint, there is some evidence in the literature that men who were conceived via IVF due to male factor infertility, went on to have infertility problems of their own later in life [508, 509]. This too may be the result of epimutation specifically within the germ line. Although this is currently hypothetical
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 195
as there is no mechanism for this phenomenon that has been identified, the characteristics of the state fit with that of an epigenetic based problem.
A more conclusive example of epigenetic inheritance can be found in an agouti mouse model of coat colour [510]. The wild type agouti mouse has a brown coat established early in life by the methylation pattern on the agouti gene [510]. Changes to this from nutritional alterations result in obesity and shift to yellow coats. When these changes were induced maternally, the offspring DNA reflected the maternal methylation changes, were yellow-coated, and prone to obesity [510].
In Chapter 5 hypomethylation of the GC-1 germ cells indicates that epigenetic changes have occurred, and additionally, hypomethylation was found within sperm
DNA in Chapter 6. It could be hypothesised that these changes follow the pathway of epigenetic inheritance. Although no visible abnormalities were observed in the sires, as in the agouti mouse model, they may still be the origin of epigenetic abnormality.
This could be investigated through pyrosequencing or bisulfite sequencing of the germ and sperm cell genomes. This would give an indication as to the specific genes effected by infection.
It would also be interesting and advantageous to determine the extent of the transgenerational capability of any epimutations. The breeding of the F1 and F2 etc. generations of offspring with healthy mice may be able to determine this. One study showed that a maternal smoking mouse model produces a transgenerational effect for three generations [511]. After the F3 generation, offspring development was more normal. The effects from this smoking study also seemed to target intergenerational female fertility.
196 Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring
There does not seem to be the same obvious sex-discrimination resulting from chlamydial infection in this study. The significant reduction in the number of seminiferous tubules per mm2, combined with the low-quality sperm (reduced forward progressive motility, count, and viability), produced by the male sires shows that the F1 generation of males was affected. The female F1 generation had small reproductive tracts although functionally, the tracts looked similar to the controls at
42-days after birth. If the female F1 tracts were completely functional then there would be a male-specific effect, therefore more investigation is required. Regardless, the results are comparable to the human IVF study previously mentioned where male factor infertility had an intergenerational effect [508].
The combination of DNA fragmentation and epimutation in the male germ line could be key determinants in the role of Chlamydia in offspring mortality and morbidity.
This study does indicate that there are adverse effects of chronic paternal chlamydial infection on offspring health as hypothesised. Further investigation is required to expand the limits of this study and identify a mechanism for this role. However, preliminarily, there also appears to be a multigeneration effect possible. This is particularly strongly indicated by the low sperm quality observed in the F1 males.
Given the frequency of the diagnosed Chlamydia infections in the human population, these results indicate that there is cause for concern past those immediate issues normally associated with infection. When the asymptomatic infections are then taken into consideration, there is an alarming potential for chlamydial infections to reduce the infertility of a large portion of the male population. The frequency of C. trachomatis infection in the infertile male population within Australia will be investigated in the next chapter of this thesis.
Chapter 7: The Effects of Chronic C. muridarum Testicular Infection on Offspring 197
Chapter 8: The Prevalence of C.
trachomatis in Human Testicular
Biopsies
198 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
8.1 Introduction
The current rate of C. trachomatis infection in the global population is drastically underestimated owing to the asymptomatic nature of the majority of both male and female infections. Approximately 50% of male and 75% of female infections are asymptomatic [17]. These asymptomatic infections can perpetuate infection in the community as they form an unrecognised reservoir of Chlamydia [35]. This phenomenon has been observed in the epidemiology of several other infectious diseases including C. difficile [136], malaria [137], Ebola [138], hepatitis C virus
[140], and N. gonorrhoeae [139]. Asymptomatic chlamydial infections have also been suggested as contributing factor to idiopathic male factor infertility [512].
Male factor infertility means that the cause of infertility within a couple attempting to conceive has been narrowed down to solely within the man [424]. There are many factors that make up the known causes of male factor infertility. As discussed in
Section 6.1, there are many disorders of sperm function that are commonly identified during semen analysis including, but not limited to, oligospermia, asthenozoospermia, teratozoospermia, and azoospermia [422]. These are frequently caused by testicular abnormalities that result in spermatogenesis failure, including
Sertoli cell only syndrome, spermatogonial cell deficiency, and germ cell arrest.
These histological abnormalities and their broad ranges of etiologies are comparatively well characterized when considering the alternative, which is idiopathic infertility [421]. Idiopathic infertility describes infertility without a known cause.
Factors thought to contribute to idiopathic infertility, which effects up to 15% of couples, include environmental and chemical mutagens like those found in cigarettes,
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 199
and injuries inflicted to the testes [421, 424]. However, infectious diseases could play a part as well, for example mumps virus [307] and HIV-1 [311] have been found in human testes in association with abnormal sperm parameters. Detection of bacteria including E. coli [513], and mycoplasmas and C. trachomatis [6] in human semen have also been associated with abnormal sperm parameters. Mouse models of testicular Chlamydia infection also resulted in abnormal sperm parameters both in this study (Chapter 6) and previously [129].
There are no clear or consistent markers for male chlamydial infertility in humans.
There is evidence from animal models correlating infection with development of infertility, but no mechanism has been elucidated and there is constant debate over conflicting results from humans. Infection is loosely associated with sperm DNA fragmentation and oligo-, terato-, and astheno- zoospermia in some literature, and previously findings of this thesis support that evidence [6-8, 35, 36, 198, 276, 279,
290, 432]. However, the literature and this thesis also show how variable sperm parameter changes can be during infection.
It has been highlighted recently that multiple factors must be present to diagnose/predict male chlamydial infertility with more accuracy [514]. A combination of two or more of the following should be present for increased accuracy of diagnosis; (i) anti-chlamydial antibodies in serum, (ii) culture or immunofluorescent detection of Chlamydia within urine, semen or swab, (iii) PCR amplification of chlamydial DNA from urine, semen or swab, and (iv) sperm DNA fragmentation or sperm parameter changes. However, this also highlights a major flaw in the current dogma surrounding infections.
200 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
The convention suggests that first-void-urine testing by PCR can non-invasively and accurately detect chlamydial infection [156, 515]. This may not be accurate for chronic or asymptomatic infections; there has been no characterization of chlamydial infection duration within the lower MRT tissues in humans. Testing of semen may be more accurate, positive in more cases where urine testing has produced negative results [35, 512]. This suggests that while the lower MRT may clear infection to below detectable levels, other tissues in the upper MRT remain infected, producing sexually transmissible Chlamydia [35, 38]. This has led to the idea that semen testing should be included in chlamydial detection in men. This is highlighted by the detection of C. trachomatis in semen donated for artificial insemination [37, 274].
Although, even this may not provide an absolute answer. If shedding is low or if persistence can develop, then the levels of infection in semen may fall below the limit of detection.
Investigating the source of the sperm, the testes, may provide clarity in the development of a profile of male chlamydial infertility. This chapter aims to follow this line of inquiry by detecting Chlamydia within testicular biopsies. This is a novel approach and will provide new insights into both the frequency of asymptomatic infection and the standards and applicability of chlamydial detection. A sub- population – infertile men presenting at IVF clinics for male factor infertility – will be investigated in this chapter. Although limited to those presenting at IVF clinics, this is a good representation of the infertile male population most at risk of harbouring asymptomatic infection. The difference in infection rates between men presenting with a known (low expected detection rate) versus an idiopathic (high expected detection rate) male factor infertility will likely give an indication as to the rate of asymptomatic chlamydial infection. Additionally, a portion of the testicular
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 201
biopsies will have accompanying information relating to the success of the IVF cycle their donors participated in.
8.2 Materials and Methods
The full and detailed materials and methods used in this chapter can be found in
Chapter 3. The important materials and methods relevant to this chapter include: human testicular biopsy IHC (Section 3.3.9.2), DNA extraction from biopsies, chlamydial PCR, and agarose gel electrophoresis (Section 3.3.7), anti-chlamydia antibody detection ELISA (Section 3.3.12).
Briefly, an archive of Bouin’s fixed, paraffin embedded, testicular biopsies taken from men attending Monash Medical Centre for male-factor infertility was accessed.
A range of 120 biopsies from patients diagnosed with Sertoli cell only appearance, germ cell arrest, and idiopathic hypospermatogenesis were retrieved, sectioned onto glass microscope slides, and tested for the presence of Chlamydia using immunohistochemistry techniques. The IHC was performed by the QIMR Berghofer
Medical Research Institute core histology facility. The slides were imaged and studied for the presence of chlamydial inclusions in each of the different diagnoses.
Examples of inclusions in different and distinct testicular locations were then also found. These fixed biopsy infections were quantified using microscopy techniques, then tabulated, and analysed using GraphPad Prism (version 7) software. To compare the differences in infection rate between each of the different groups a chi-squared test was used. The level of statistical significance was set at P < 0.05.
Fresh micro-TESE and fine-needle testicular biopsy samples were also collected from non-obstructive, azoospermic men during testicular sperm recovery attempts at
202 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
Monash IVF and Queensland Fertility Group for male-factor infertility treatment.
These biopsies underwent DNA extraction and real-time PCR to detect C. trachomatis 16S rRNA DNA, using C. trachomatis serovar D EBs as a positive control. The amplicons resulting from both the biopsy DNA and the EB DNA were further assayed using melt curve analysis and agarose gel electrophoresis to confirm their identity. The amplicons were considered to have originated from C. trachomatis when the melt curve peaks were within 4°C of the EBs on either side. The size of the amplicon was predicted to be 76 base pairs, a 2% gel electrophoresis was used to make visual comparisons between band sizes.
After confirmatory testing, the infection status of the fresh biopsies was correlated with the IVF outcome provided by the clinic. Where possible, sperm parameter information (e.g. whether sperm was able to be collected from the biopsy, whether collected sperm was motile) and disease diagnosis were also correlated with infection status.
Blood samples matching the fresh biopsies were tested for the presence of anti- chlamydial antibodies via ELISA. The in-house made ELISA plate was coated with
C. muridarum MOMP, the serum samples were applied to the plate, then detected with anti-human IgG conjugated to HRP. The colorimetric detection was using TMB, with a sulfuric acid stop solution. The ELISA was then red on a spectrophotometer at
450 nm. The absorbance data was transformed and graphed using GraphPad Prism
(version 7) software.
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 203
8.3 Results
The Monash Medical Centre tissue bank provided 120 human testicular biopsies from 100 individual male-factor infertility patients. Sections of these biopsies were sent to QUT for Chlamydia detection. Sections were stained using IHC techniques for the presence of MOMP. Where both left and right biopsies were provided, the infection status was always consistent and thus recorded as a single result for that patient. Six patient samples were eliminated due to insufficient tissue size, leaving 94 samples in total. Staining was validated through the use of primary antibody only, secondary antibody only, and DAB detection agent only controls, which each showed little to no positive staining. Positive MOMP staining was determined by the presence of chlamydial inclusions, which coloured brown-black due to the use of
DAB staining. Examples of each of the controls use can be seen in Figure 8.1.
A
Figure 8.1 Primary antibody-, secondary antibody-, and DAB only-controls for detection of inclusions in testicular biopsies. Figure 8.1 shows the primary antibody-, secondary antibody-, and DAB only- controls that were developed for use with a novel assay that detects chlamydial inclusions in human testicular biopsy tissue. Positive staining would be visualised in brown from the DAB colorimetric development. Tissue have been counterstained with haematoxylin. Scale bars represent 200 µm.
204 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
Chlamydial inclusions were detected in 42 out of 94 samples, a 43.75% rate of infection overall as can be seen in Table 11. The samples were then categorically divided by the diagnosis made by the Monash Medical Centre pathologist who assessed the biopsies during the patient’s treatments. The pathology reports were provided for categorization of the samples. The rates of infection per diagnosis are shown in the table in panel B and were as follows: (i) Sertoli cell only appearance had 21 of 52 positive samples (40.38%), germ cell arrest had 6 of 11 (54.55%), hypospermatogenesis had 16 of 37 (43.24%). The hypospermatogenesis group can also been broken down into sub-categories. The rates of positivity in these were; mild hypospermatogenesis had 7 out of 17 (41.18% positivity), moderate had 2 of 5
(40%), marked had 2 of 6 (33.33%), and severe had 4 of 6 (66.67%). At 66.67% positivity, the diagnosis of severe hypospermatogenesis had the highest rate of positivity. A chi-squared statistical analysis was applied to the complete data set and no significant difference (P = 0.1) was found.
Table 11: MOMP positivity rate in human testicular biopsies. Diagnosis Number with MOMP Positivity (%)
Sertoli cell only appearance 21/52 (40.38%)
Germ cell arrest 6/11 (54.55%)
Mild hypospermatogenesis 7/17 (41.18%)
Moderate hypospermatogenesis 2/5 (40%)
Marked hypospermatogenesis 2/6 (33.33%)
Severe hypospermatogenesis 4/6 (66.67%)
Combined hypospermatogenesis 16/37 (43.24%)
Combined diagnoses 42/94 (43.75%)
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 205
Representative examples of non-infected and infected biopsies with the three major diagnoses have been identified. Figure 8.2 shows (i) non-infected Sertoli cell only appearance tissue, (ii) inclusions in Sertoli cell only appearance, and (iii) enlarged view of the inclusions, which are indicated by the arrows. Figure 8.2 also shows (iv) non-infected hypospermatogenesis tissue, (v) infected hypospermatogenesis, and (vi) an enlarged inclusion indicated by an arrow. Lastly, Figure 8.2 shows (vii) non- infected germ cell arrest tissue, (viii) infected germ cell arrest, and (ix) enlarged inclusions indicated by arrows.
206 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
Figure 8.2 Detection of chlamydial inclusions in human testicular biopsies of patients with three diagnoses of male infertility. Figure 8.2 shows that chlamydial inclusions can be detected using staining of MOMP (dark brown) in patients presenting with male infertility diagnoses of Sertoli cell only appearance, hypospermatogenesis, and germ cell arrest using immunohistochemistry techniques. Examples of non-infected tissues are shown (i, iv, and vii). Examples of infected tissues are shown (ii, v, and viii), with magnified views of inclusions (indicated by black arrows) provided also (iii, vi, and ix). Scale bars represent 200 µm.
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 207
The inclusions were present in different structures within the tissue. Representative images of the different locations of inclusions can be seen in Figure 8.3. Figure 8.3
(i) shows inclusions in the interstitum, with Figure 8.3 (ii) showing an enlarged view of inclusions indicated by arrows. Figure 8.3 (iii) shows inclusions in the basement membrane, which were thickened in many infected cases, and Figure 8.3 (iv) shows the enlarged inclusions indicated by arrows. Figure 8.3 (v) shows an inclusion in the intra-tubular compartment and Figure 8.3 (vi) shows the enlarged inclusion indicated by an arrow. The structure of the tissue in many biopsies was abnormal and disorganized, causing difficulty in determining the localization of the inclusions.
This was categorized as ‘disorganized’ tissue and an example of this type of infection can is shown in Figure 8.3 (vii), with the enlarged view of inclusions indicated by arrows in Figure 8.3 (viii).
208 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 209
Figure 8.3 Immunohistochemical detection of MOMP in human testicular biopsies. Figure 8.3 shows immunohistochemical detection of chlamydial MOMP (dark brown) in human testicular biopsy sections. The inclusions were present in different structures within the tissue; (i) shows inclusions in the interstitum, (ii) an enlarged view of inclusions indicated by arrows, (iii) inclusions in the basement membrane, (iv) enlarged inclusions indicated by arrows, (v) shows an inclusion in the intra- tubular compartment, (vi) enlarged inclusion indicated by an arrow, (vii) inclusions in disorganized tissue, and (viii) enlarged view of inclusions indicated by arrows.
210 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
In addition to the archived biopsies, five fresh testicular biopsy specimens were obtained from current and recent Monash IVF and QFG male-factor infertility patients. The total DNA was extracted from the biopsies and assayed for the presence of C. trachomatis DNA by real-time PCR as seen in Figure 8.4. The DNA was amplified using a primer set specific to C. trachomatis 16S rRNA DNA. There was
C. trachomatis DNA found within three out of the five specimens; QFG001, Monash
#3, and Monash #4. This gives an infection rate of 60%. These are indicated by the circle in Figure 8.4 A.
Each of the biopsies were assayed in triplicate by PCR. In each assay, a minimum of one triplicate was amplified between cycles 30 and 40 of the PCR. Negative replicates showed minimal amplification after 40 cycles indicative of primer dimerization. The positive control used during these assays was DNA extracted from
C. trachomatis serovar D ultra-purified EBs. In each assay, and in each replicate, the
EB DNA showed amplification no later than cycle 20. The EB DNA amplification is indicated by the arrow in Figure 8.4 A. Also included in each assay was a no- template control, as a negative control. The negative control showed no amplification within 40 cycles, and minimal amplification after 40 cycles indicative of primer dimerization. A representative image of the PCR assay can be seen in Figure 8.4 A.
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 211
A
B
Figure 8.4 PCR amplification of C. trachomatis DNA extracted from human testicular biopsies. Figure 8.4 shows the analysis of fresh testicular biopsies (n = 5) taken from men attending Monash IVF and Queensland Fertility Group for male-factor infertility. A shows amplification of C. trachomatis 16S rRNA DNA from the biopsy material by real-time PCR, where QFG001, Monash#3, and Monash#4 are positive (circle). B shows the subsequent melt curve with the accompanying analysis in (ii) which identifies the same three samples as positive. C. trachomatis serovar D EBs were used as the positive control (arrow). No-template controls were also included. The PCR based assay images are representative of triplicate assays.
212 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
The amplicons from each replicate underwent a melt curve analysis directly following the PCR. Each melt curve that resulted from a specimen was compared to the EB curve to determine the similarity between the samples. The samples were considered to be significantly similar when the melt curve peaks were within 4°C of each other. Therefore, the specimens were considered to have true C. trachomatis
DNA amplification when their melt curve peaks were within 4°C of the EB melt curve peaks. This occurred for each of the three positive samples identified, and not for the two PCR negative samples. A representative image of the melt curve can be seen in figure 8.4 B. The color codes and identification of C. trachomatis positivity produced by the Rotor-Gene PCR software have also been included in Table 12.
Table 12: Identification of C. trachomatis positive biopsies via melt curve analysis. Color Name Peak 1 QFG001 83.7 (Ctr)
QFG001 83.7 (Ctr)
Monash#1
Monash#1
Monash#2
Monash#2
Monash#3 83.5 (Ctr)
Monash#3 83.0 (Ctr)
Monash#4 83.5 (Ctr)
Monash#4 83.5 (Ctr)
EBs 83.7 (Ctr)
EBs 83.7 (Ctr)
NTC
NTC
Ctr: C. trachomatis; EBs: C. trachomatis serovar D elementary bodies; NTC: no template control.
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 213
The PCR products for each five samples were also assayed using agarose gel electrophoresis to identify the samples based on amplicon size as seen in Figure 8.5.
The QFG001 and Monash #4 samples were conclusively the correct size amplicon when compared to the positive control amplicons, they were 76bps. The Monash #3 sample was inconclusive as there was a difference in the positioning on the gel for this sample. The Monash #1 and Monash #2 samples were not of a similar size to the positive control amplicons. A representative image of the gel electrophoresis can be seen in figure C. The negative samples are denoted by ‘—’, the positive samples are denoted by ‘+’, and the ambiguous sample is denoted by ‘?’. The size of the amplicons of interest are indicated on the molecular weight ladder by the red arrow.
Figure 8.5 Gel electrophoresis of C. trachomatis amplicons produced by PCR. Figure 8.5 C. trachomatis 16S rRNA specific PCR of DNA extracted from human testicular biopsies was used to produce amplicons that were electrophoresed in agarose gel. The positive samples that are correctly sized amplicons when compared to the positive control amplicons (denoted by “+”), they were 70bps (indicated by red box and arrow). The ambiguous sample is denoted by “?”. The samples that were not of a similar size to the positive control amplicons are denoted by ‘—’.
214 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
After confirming the infection status of the patients, the status was correlated with the outcome of the IVF cycle. There was a correlation between infection and several parameters measured during the patient’s treatment as seen in Table 13 below. The first was the absence of sperm in the testicular biopsy, which links to the second parameter of failure to conceive. All three C. trachomatis positive patients were azoospermic at the time of their testicular biopsy, while the negative patients had sperm retrieved and successful IVF cycles. None of the five patients declared any previous suspected or diagnosed STIs, including Chlamydia.
Table 13: Correlation of C. trachomatis positivity with IVF outcome Sample ID Sperm Status IVF Outcome Previous STI Ctr Status
QFG001 Azoospermia No pregnancy None Positive
Monash #1 Oligospermia Pregnancy None Negative
Monash #2 Sperm from Pregnancy None Negative
CF patient
Monash #3 Azoospermia No pregnancy None Positive
Monash #4 Azoospermia No pregnancy None Positive
Lastly, serum samples from each of the 5 patients were tested via ELISA for anti-
MOMP IgG. As seen in Figure 8.6, all five of the samples showed some level of positivity. The highest titre of anti-MOMP IgG was found in the Monash #2 sample, followed by Monash #4, Monash #3, QFG001, and lastly Monash #1 had the lowest titre. None of the serum samples reach the negative control threshold (5% FCS in
PBS, indicated by the horizontal dashed line). There was no significant difference between the IgG titres though, tested by the Students T test.
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 215
0 .8
0 .6
m
n 0
5 0 .4
4
D O 0 .2
0 .0
1 1 2 3 4 0 # # # # 0 h h h h s s s s G a a a a F n n n n Q o o o o M M M M
S e r u m S a m p le (1 : 6 4 0 d ilu tio n )
Figure 8.6 Detection of anti-chlamydial antibodies found in serum from male infertility patients. Figure 8.2 shows the analysis of serum samples (n = 5) taken from men attending Monash IVF and Queensland Fertility Group for male-factor infertility. Use of an ELISA shows the detection of anti-chlamydial (MOMP) antibodies in the serum samples.
216 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
8.4 Discussion
The results of this chapter show that Chlamydia can be found in human testicular tissue by both PCR and IHC. Depending on the histological diagnosis being investigated the prevalence of infection can reach above 60% positivity as seen in the severe hypospermatogeneis/azoospermia diagnosis of fixed/fresh testicular biopsies.
If examined as a proportion of non-obstructive, azoospermic men with male-factor infertility including Sertoli cell only, germ cell arrest, and hypospermatogenesis, the rate of infection was still found to be high at 43.75% positivity. Although the number of samples analysed in the study are relatively small, this result represents a frequency of detection on the high end of the range reported in literature for other sample types [35, 432]. There are no other studies that have identified testicular chlamydial infection.
The proportions of positivity between the fresh and fixed biopsies are similar. The similarity may be different with larger sample size particularly from the fresh biopsy group. However, the current findings could provide insight into the epidemiology of chlamydial infections in infertile men. The fixed biopsies were retrieved from an archive reaching back at least ten years and the fresh biopsies were collected over the course of the last three years. This indicates that the rate of infection may be stable but longitudinal studies would be required to confirm this hypothesis.
These samples do represent a population that is potentially enriched for chlamydial positivity and the rate is most likely lower in the general population, which includes reproductively healthy men. The sample set lacks diagnoses including testicular cancer, injury, and confirmed genetic diseases like cystic fibrosis and Klinefelter’s that can also result in male-factor infertility [418, 426, 427]. Including these
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 217
diagnoses in the sample set may change the prevalence of infection in the male-factor infertility group for several reasons. For example, men with different testicular conditions may have different sexual activity patterns, which changes their risk of contracting Chlamydia and other STIs [516, 517]. Patients with diseases like testicular cancer may have had otherwise healthy testes before the malignancy necessitated use of ART. Some patients may have congenital abnormalities in hormone production (FSH/LH/testosterone), which may lead to male infertility, but may also alter the immune response to infection and the likelihood of developing testicular infection [361, 417]. These factors could impact the prevalence rate.
Regardless of the limitations of the group tested, there was no statistically significant difference in infection prevalence demonstrating that, with the current sample size, infection is not associated more with one diagnosis above any other. Therefore, no causal link can be made for the role of Chlamydia in one particular type of male infertility, such as hypospermatogenesis, at this time. What the lack of statistical significance suggests though, is that a chlamydial infection can be established indiscriminately and persist undetected in testicular tissue that contains the host cells required. These infections are actively replicating, as seen in Figure A8, where
Chlamydia was detected by the marker TC0500, an inclusion membrane protein only produced during active replication [518, 519]. As inclusions were present in the interstitium, the basement membrane, and within the seminiferous tubules of the fixed biopsy sections, any of the Leydig cells, macrophages, myoid cells, germ cells, or Sertoli cells may have carried the infection.
Defining the infected host cell lineage(s) in the future, may be useful in advancing our understanding of human testicular infections and their impact on fertility. If the host cell locations were identical to the mouse model or another animal model that
218 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
may be developed in the future, it would expand researcher’s capacity to study male chlamydial infertility. Additionally, in the human tissue, inclusions were most frequently identified in the interstitium or in disorganized regions of the tissue. If the cell lineage that maintains the infection in these tissue types could be identified, it may suggest an intervention strategy for chlamydial infertility. For example, Tmφ are common in the interstitium and could be the host, but in vitro, macrophages are also resistant to antibiotic clearance of chlamydial infection. RAW 264.7 macrophages required an azithromycin dosage approximately 10 times greater to clear infection than that needed to clear from McCoyB fibroblast cells and HeLa epithelial cells (Armitage et al., unpublished). The mouse model developed in previous chapters and the in vivo isolation of infected macrophages shown in
Appendix A, suggests that macrophages are carriers of chlamydial infection in males.
This represents a difficulty in treatment of male infections, as current antibiotic therapies may not reach the required dosage within macrophages to clear infection and prevent its dissemination throughout the testes.
A second limitation of the archived samples is that, due to ethical considerations, no patient history, sperm quality information, or IVF outcomes were available to correlate with infection status. However, this information was available to accompany the fresh biopsies, which comprise the second part of the human section of this study.
The fresh biopsies were C. trachomatis positive in three out of five cases. These three biopsies were azoospermic, and consequently the IVF cycles were unsuccessful. The sample size is too small to accurately posit a causative role for C. trachomatis in development of azoospermia. An interesting trend is emerging in this data though. The remaining two samples, which were C. trachomatis negative,
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 219
successfully had sperm retrieved from the biopsies and gave rise to pregnancies through IVF despite coming from patients with male-infertility issues.
One factor to consider when using fine-needle and micro-TESE biopsies is the miniscule cross section of the testes that is sampled [520]. If human testicular infections follow a comparable focal pattern of tissue damage seen in the mouse model (Chapter 6), fine-needle and micro-TESE biopsies may bias the detection by avoiding infected areas of the testis. This may be further complicated by the concentration of infection within the testes. Low-grade infections may be more difficult to detect and there is currently no known range for the concentration of
MRT infections.
Real-time PCR determined the C. trachomatis positivity of the biopsies. This shows that a PCR approach to detection is sensitive when modified, as in this project, for low-abundance targets. Perhaps this assay could be used as a confirmatory tool on an independent platform, for the infections identified by immunohistochemistry. Also, as PCR is utilized as a clinical tool for diagnosis of Chlamydia in other sites, a testicular Chlamydia detection assay may be a powerful instrument for infertility clinics in the future. Screening testicular biopsies from male-factor infertility patients for Chlamydia could have multiple positive impacts. It may inform patients about their health, helping them understand the cause of their infertility issues. It may also inform clinicians about the course of action required for patients to have successful
IVF outcomes. For example, multiple biopsies may need to be taken if focal destruction of tissue has occurred [521]. It may be possible that antibiotic therapy will improve sperm quality, as several human studies suggested [522, 523].
Additionally, the partners of infected men may also have contracted Chlamydia unknowingly and could also be treated.
220 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
Transmission between partners may occur unknowingly, as the testicular infection does seem to be asymptomatic. Also seen in the correlation table, none of the three positive patients have previously had a suspected or a diagnosed STI. The patients were surveyed on whether they had previously displayed symptoms of an STI, and whether they had ever been diagnosed with an STI including HSV, HIV, gonorrhoea, and Chlamydia. There is an inherent degree of uncertainty in assessing self- evaluation surveys [524, 525]. This is particularly true of socially taboo topics such as STIs, and even more so when considering that historically men are less likely to seek medical interventions than women. However, in this cohort the patients were able to consult with their andrologist if they were uncertain about answering the survey questions, which may improve the quality of the responses.
Following on from the detection of Chlamydia by PCR, the results were confirmed by two methods, the melt curve and gel electrophoresis, which both assess amplicon size. Potentially a second independent confirmatory method, such as Sanger sequencing of the amplicons produced by the PCR, could take place in the future. If these methods can validate the PCR assay initially in the laboratory, they may not be required for clinical diagnostics. Excluding extra tests would streamline a potential biopsy screening process.
Sequencing of amplicons that produced extraneous bands present on the gel electrophoresis would also be required in the future. High molecular weight bands were identified in the lanes from Monash#1, Monash#3, and Monash#2 patients. In the case of Monash #1/#2, these bands were mutually exclusive with the 76bp C. trachomatis positive bands but not in the case of Monash#3. Gel electrophoresis cannot determine the identity of these bands and their presence requires further
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 221
investigation. They may represent a flaw in the PCR assay in non-specific implication of host or chlamydial genetic material.
Finally, serum samples that matched the fresh biopsies were provided. These were tested via ELISA for the presence of anti-MOMP antibodies. Somewhat surprisingly, all the five serum samples tested positive for anti-MOMP antibodies. This may not indicate that all five patients have C. trachomatis infections. The ELISA plate was coated with recombinant C. muridarum MOMP. There is homology between C. muridarum, C. trachomatis, and C. pneumoniae MOMP and cross-reactivity of anti-
C. pneumoniae MOMP antibodies has been observed previously [526, 527]. C. pneumoniae frequently colonizes people asymptomatically in the respiratory tract, so anti-C. pneumoniae antibodies may be commonly developed [528-530]. The highest titre of anti-MOMP IgG was found in the Monash #2 sample, which was not a PCR positive biopsy. This patient may have an active C. pneumoniae infection, or perhaps the testicular biopsy did not sample an infected section of the testis. The Monash #4,
Monash #3, QFG001 serums had a range of mid titre anti-MOMP IgG. These were the PCR positive samples, so the patients likely have current C. trachomatis infections. Lastly, Monash #1, which was a PCR negative biopsy, had the lowest titre. This patient may be transiently colonized with C. pneumoniae, or perhaps had a past Chlamydia infection and antibody titres are diminished. The long serum half-life of IgG complicates its use in diagnosis of chlamydial infections [531].
In the future, the ELISA of anti-chlamydial antibodies in human serum can be repeated using C. trachomatis MOMP to compare the tires detected. If titres remain the same, this would indeed suggest cross-reactivity between different species of anti-chlamydial antibodies. If this is the case, MOMP may not be an appropriate antigen for use in this ELSIA and increased stringency would be required by
222 Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies
selection of a C. trachomatis specific antigen. The antibodies detected in this ELSIA are likely to be true anti-MOMP antibodies. The use of proteinaceous FCS in the negative control wells, which showed minimal colorimetric activity, supports the antibodies being MOMP specific. The species of the MOMP is questionable though.
In summary, after finding no evidence of these assays in the currently available literature, this chapter represents the development of two novel assays in the field.
Chlamydia has not previously been detected within human testes and in this chapter, both immunohistochemistry techniques and real-time PCR detected infection.
Chlamydia was detected in 43.75% of archived open testicular biopsies (n = 96) and
60% of fine-needle biopsies from current patients (n = 5). Despite the results being limited by small sample size and narrow range of male-factor infertility diagnoses being included, an interesting trend has emerged correlating Chlamydia with male infertility and unsuccessful IVF outcomes. The implications for both the chlamydial research and male infertility fields of identifying inclusions within the testes are broad. Further investigation into treatment strategies in particular will be necessary.
Chapter 8: The Prevalence of C. trachomatis in Human Testicular Biopsies 223
Chapter 9: General Discussion
224 Chapter 9: General Discussion
9.1 General Discussion
This study was designed to fill several knowledge gaps that currently exist including
(i) whether chronic chlamydial testicular infections can be established, (ii) what effect chronic infections have on testicular tissue health and function, which leads to
(iii) what effects chronic infections have on sperm quality, and (iv) on the health of offspring produced from those sperm. The significance of filling these knowledge gaps could be far-reaching. Beginning with redefining the current view on male chlamydial infections having a lesser impact on community health and extending into changes in clinical practice (whether male chlamydial infection should be routinely screened for in the community and in ART settings, and how they should be treated).
When viewed as a whole, the results of this study represent several important developments in the fields of Chlamydia-host interaction and male chlamydial infertility. The results support the project hypothesis; Chlamydia does have the potential to establish chronic testicular infections, and this can damage testicular cells, and impair spermatogenesis. During the exploration of this hypothesis, three distinct project aims were introduced:
Aim 1: To investigate the susceptibility to, and infection kinetics of Chlamydia within testicular cells.
Aim 2: To determine the functional changes induced by Chlamydia infection within testicular cells, and the corresponding effect on sperm health and fertility.
Aim 3: To determine the frequency and effect of C. trachomatis infection in testicular biopsies from men undergoing sperm retrieval for assisted reproductive technologies (ART) and correlate infection presence with reproductive outcomes.
Chapter 9: General Discussion 225
While fulfilling these aims, Chapter 4 established that primary and immortalized murine testicular cell lineages are susceptible to infection and can propagate infection. Resulting from Chapter 4 and collaborations with research group members, a novel role for macrophages as carriers of infection to and within the testes was established. Figure 9.1 shows a summary of the hypothesized model that resulted. It is proposed that monocytes/macrophages become infected in the penile urethra after sexual transmission of the infection. Testicular interstitial cells recruit the infected cells from circulation via of normal chemokine signalling. Dysregulation of the infected cell function may also impact how and where they travel to through the circulatory system.
Figure 9.1 Hypothetical model of monocyte/macrophage transmission of Chlamydia around the male reproduction tract. Figure 9.1 shows the hypothetical model developed during this project to show the transmission of Chlamydia to the testes via circulating monocytes/macrophages, and within the different compartments of the testes.
226 Chapter 9: General Discussion
Some cells may colonize the gut, which is also purported to harbor chronic chlamydial infection [155, 532]. Some cells may travel to the highly vascularised testes, where they find an intensely immune-suppressive environment, enabling chlamydial propagation.
This mode of systemic spreading may not be specific to chlamydial infection. Other pathogens including E. coli and N. gonorrhoeae can be sexually transmitted, have the capacity to survive in macrophages, can colonize the MRT, and can adversely impact sperm quality [243, 513, 533]. Zika and Mumps viral infections also cause testicular infections and impact sperm quality [307, 309]. This may be a common mechanism utilized by pathogens to access the testes as a replicative niche and should be explored in the future. Both in terms of preventing establishment of chronic asymptomatic infections and treatment of infections.
Testicular colonisation by Chlamydia-infected macrophages would then lead to infection of the specialized testicular cell lineages, the Leydig cells, Sertoli cells, and germ cells. The transmission from the infected macrophages to the testicular cells may occur through several means. Firstly, production of extrusions from infected
Tmφ which now act as a reservoir of continuous infection. Or, it may occur through lysis of infected macrophages and re-infection of surrounding testicular cells
(Chapter 4), the immunosuppressed environment ablating the recruitment of adaptive immune cells to combat the initial stages of infection.
As testicular cells become infected, their function can be altered as seen in results from Chapter 5. DNA fragmentation, methylomic and transcriptional dysregulation occurs during infection. The pro-inflammatory response produced by infected TM3
Leydig and GC-1 germ cells in vitro now represent a conflict with maintenance of
Chapter 9: General Discussion 227
testicular health. Over time, production of interferons and enhanced secretion of immune cell chemoattractant proteins may initiate recruitment of cells including monocytes and T cells, which would be detrimental to the anti-inflammatory testicular environment [225].
Normally, resident Tmφ are unable to engage in efficient T cell activation [213].
This is seemingly an evolutionary constraint to prevent sperm antigen detection and promote sperm antigen tolerance. If recruited monocytes recognize the infection, these may be able to engage in T cell activation [213]. This increases the likelihood of further inflammation, disruption of the blood-testis-barrier, sperm antigen recognition, and an anti-sperm immune response.
This scenario does appear possible, as seen in Chapter 6, ZO-1 marking the tight junctions of the blood-testis-barrier was significantly reduced in infected testes. This could have occurred due to down-regulation of Sertoli cell structural components or apoptosis during infection. The interference with immune privilege by infections is not limited to the blood-testis-barrier, although this also occurs during Zika virus infection [534]. This has also been observed in the blood-brain-barrier by West Nile virus [535], and tight junctions in the gut by pathogenic E. coli, Clostridium spp., and
Salmonella spp [536].
The blood-testis-barrier breakdown was accompanied by production of systemic anti- sperm antibodies in mouse serum. However, it is unclear whether this was an active response at the chronic six-months post infection time-point as a product of ongoing exposure of sperm. The histological damage may have been the result of an acute response to infection that has not repaired. Some histological damage is incurred as early as four weeks after infection. So many weeks later, exhaustion of the immune
228 Chapter 9: General Discussion
response by constant stimulation with both chlamydial and testicular antigens is a possibility [141]. The anti-sperm IgG may have been produced in the acute phase of infection and the long serum half-life of IgG enabled its detection at six-months post infection [531].
The continuing decline of the testicular environment and sperm quality may be self- perpetuating. Each testicular cell lineage has a highly specialized role in testicular homeostasis and spermatogenesis. Dysregulation of the cell populations that occurs after infection may irrevocably alter the feedback between the cells and their unique functional abilities. This could occur through hormonally- or cytokine-mediated events that occur whilst remaining healthy testicular tissue attempts to compensate for tissue lost to infection/immunity-mediated destruction and continue spermatogenesis [414, 417].
The unique testicular environment represents a major challenge for treatment of infections at this site. Traditionally, chlamydial infections are treated with antibiotics
[39]. With macrophages playing a major role in both infection and testicular health, treatment of macrophage-born infections must be considered. Levels of azithromycin required to clear chlamydial infection from macrophages in vitro cannot be achieved in patients on standard antibiotic therapy (Armitage et al., unpublished). If Tmφ infections are unable to be cleared by antibiotic therapy, this represents a major flaw in current dogma on chlamydial control.
Secondly, as adaptive immunity within the testis is not desirable as evidenced by multiple models of experimental auto-immune orchitis, a therapeutic approach to vaccination may also be ineffective [220, 225]. Previously, antigen-specific CD4+ T cells have shown their capability of decreasing infectious burden in the MRT
Chapter 9: General Discussion 229
generally [129]. However, histological damage remained in the testes [129]. Instead a prophylactic approach was more effective. Adoptively transferred before infection, antigen specific, Th2-like, CD4+ T cells prevent major histological damage [129].
This indicates the type of response that needs to be elicited by a prophylactic vaccine.
This highlights yet another difficulty with this type of infection. In the mouse model developed in Chapter 6 and in the human study in Chapter 8, testicular infections proved to be asymptomatic. Therefore, people would need to be vaccinated before their first sexual encounter, before they are at higher risk of contracting STIs [537].
An anti-chlamydial vaccine could be integrated into the national vaccination schedule at high school age, similarly to the HPV/cervical cancer vaccine. This would hopefully maximize the number of people exposed to the vaccine before contact with Chlamydia.
9.2 Future Directions
There are several chapter specific experiments that can be suggested to follow on from the foundations laid by this study. Chapter 4, which investigated the susceptibility to infection of testicular cells and the growth kinetics of Chlamydia within these cells, provides the foundation for development an in vitro model of infection transmission across the blood-testis-barrier. Development of biologically relevant in vitro models is important in research as it can reduce the use of animals.
Development of a more biologically accurate, 3D culture system, would be beneficial to the study of many testicular diseases such as auto-immune orchitis and Zika infections. Using the Transwell™ system as the basis, it is possible to more closely replicate the two separate compartments of the testis [538]. The challenges of cell
230 Chapter 9: General Discussion
detachment from the basal surface of Transwells™ may be overcome by seeding into a substance like Matrigel, a biologically active basement membrane matrix that can be layered onto a cell growth surface [539]. Primary human testicular and immune cells could be substituted into the system to compare between the mouse and human infection kinetics and cellular responses.
Development of a 3D culture model could also assist in unravelling questions raised in Chapter 5. If mRNA and proteins are isolated from the different cell lineages in
3D co-culture, further functional changes may be observed as a result of cell-cell interaction. Particularly, interesting changes may result from the addition of M1 or
M2 macrophages to the apical chamber of the system, as if they had been recruited by pro-inflammatory signals produced by Leydig and germ cells. Antibiotics or anti- chlamydial antibodies could also be added to the apical chamber to determine if further cellular functional changes are induced or if normal function can be restored in vitro. These types of experiments will inform the direction of in vivo model development.
An example of an animal model that could be developed in the future to complement
Chapter 6, would explore whether the negative effects of infection in the testis can be attenuated or reversed. Vaccination and antibiotic therapy should be investigated as methods for preventing and treating male chlamydial infertility. The effects of these treatments could be investigated by measuring whether infection can be prevented from reaching, and cleared from, the testes but also by whether sperm quality can be recovered. As sperm quality is the important determinant of testicular function and male fertility, this would be a requirement in understanding how vaccination and antibiotic therapy effect testicular health.
Chapter 9: General Discussion 231
As a downstream measure of how testicular health is affected by vaccination and antibiotic therapy, the future direction of Chapter 7 would include breeding treated sires with healthy dams. If vaccination can prevent testicular infection, it would be important to establish that no ill-effects on sperm health are produced if a vaccine that is developed is required to target the testes or upper MRT. If infection can be cleared from the testes using antibiotics, understanding whether pup vitality and development return to normal would have clinical relevance. If parameters are not recovered, this represents another important avenue of investigation that needs to be addressed, and the multigeneration effects of infection should be examined in greater depth.
In the vein of modelling the effects from infected fathers on offspring, the future direction of Chapter 8 also includes gaining a deeper understanding of the relationship between testicular infection and IVF outcomes. Ideally, collection of fine-needle biopsy samples would continue in order to expand the study and increase the power. The expansion of this study could include matched urine, swab, semen, and biopsy samples (at multiple time-points where possible) to establish whether asymptomatic infections can be found in the urethra, bladder, upper MRT and the testis, or whether sites are mutually exclusive. Where positive samples are identified, treatment with antibiotics could be recommended by the patient’s physician, and then applicable sites may be able to be resampled. For example, the semen could be resampled. Currently, the semen from infected patients is aspermic, so it would be interesting to discover whether sperm can be found in semen after treatment and if this sperm is now able to be successfully used in IVF.
232 Chapter 9: General Discussion
9.3 Significance and Conclusion
The significance of this study reaches predominantly into the understanding of male chlamydial infertility. A clinically relevant form of unrecognized male infertility has been identified. This highlights the importance of chlamydial screening programs for both men and women, but also the importance in fertility clinics of testing patients for asymptomatic infections. A patient’s clinical history being clear of STIs like
Chlamydia, in an age where asymptomatic infections are becoming increasingly prominent, may not be a high enough standard.
Furthermore, currently screening of urine specimens is the standard for chlamydial detection. Research is now accumulating that shows urine specimens cannot accurately detect 100% of MRT infections, particularly those in the upper reproductive tract that are most damaging to fertility [35, 36]. Differences in detection rates between urine, urethral swab, semen or other samples underscores the inadequacy of current screening methods and emphasizes the requirement for improved and standardized testing protocols [35, 36, 437].
To protect the reproductive potential of the population in the future, development of prophylactic vaccination and more efficient therapeutics against silent STIs will be key. This project accentuates the current requirement for development of a male anti- chlamydial vaccine but also the difficulties that must be overcome in combatting the unique infection type identified.
The damage these infections can cause to spermatogenesis and fertility is severe, with changes induced in all testicular cell populations and structures. Equally, if not more alarming, are the harmful effects to normal development that offspring can experience when born to chronically infected male mice. If translatable to a human
Chapter 9: General Discussion 233
setting, these results suggest that generations of children could have compromised developmental outcomes from an infection contracted by their fathers, possibly years before their birth. Importantly, this project shows that the seriousness of male chlamydial infections has been dramatically underestimated.
234 Chapter 9: General Discussion
Appendices
Appendix A
Appendix A shows the in vivo model of macrophage-mediated transmission discussed in Chapter 4. This data was primarily gathered by Dr Charles Armitage, Dr
Avinash Kollipara, and Logan Trim. This data revealed that Chlamydia positive macrophages were detectable in mouse blood three days post intra-penile infection.
Whole blood underwent flow cytometric analysis to differentiate macrophages from other immune cells, and then to isolate those that contained infection. A small population of the total number of macrophages present in the blood sample were shown to be infected.
Regardless of the small size of the blood-borne infected macrophage population, infected macrophages were also detected in the testes of mice 2 week post intra- penile infection. Whole testes were reduced to a single cell suspension, which was analysed by flow cytometry (methods contained in Section 3.3.10).
Appendices 235
Figure A1 Detection of C. muridarum infected macrophages in blood and testes. Figure A1 A shows that C. muridarum can be detected within circulating macrophages, by flow cytometry, three-days after intra-penile infection of male C57BL/6 mice. Figure A1 B shows that infected macrophages can also be isolated from the testes of male C57BL/6 mice, three-days after intra-penile infection.
236 Appendices
Appendix B
1 5 0 0 0
0.9920 s
e 1 0 0 0 0 0.9999
t
s
e
T
/
U F
I 5 0 0 0 0.9986
0 2 wk s 4 wk s 8 wk s
T im e P o s t I n fe c tio n
I n ta c t V a s e c to m y
Figure A2 Detection of testicular C. muridarum from vasectomised versus intact mice. Figure A2 shows the vasectomy experiment discussed in Chapter 4. By comparing the titre of C. muridarum that was cultured from the testes of vasectomised mice versus mice with intact reproductive tracts, it was established that Chlamydia travels around the MRT via the circulatory system. No statistically significant differences were found in the titres of testicular Chlamydia two-, four-, or eight-weeks post intra- penile infection.
Appendices 237
Appendix C
A
Table 14: Full DEG list for C. muridarum infected vs non-infected TM3 cells. Gene Symbol log2 Fold Change Expression P value 0610010B08Rik 4.347081785 Up 5.37E-20 2610528A11Rik 1.38180653 Up 0.000397 adar 1.470775308 Up 3.75E-83 aI607873 3.627925962 Up 5.99E-25 apod 3.478519514 Up 2.17E-13 apol9a 3.449141404 Up 1.37E-115 apol9b 3.917547988 Up 4.98E-144 batf2 2.423200262 Up 2.06E-07 bC147527 2.524120585 Up 8.93E-07 bcl3 1.005863933 Up 4.46E-17 bst2 2.539455291 Up 2.05E-125 c4b 1.150753701 Up 6.35E-17 casp4 1.495411838 Up 5.34E-27 ccl2 3.842176381 Up 2.26E-249 ccl20 2.674660668 Up 8.66E-08 ccl4 4.122945008 Up 1.94E-18 ccl5 4.620102305 Up 9.32E-109 ccl7 3.643941056 Up 8.50E-143 ccrl2 3.659450702 Up 2.79E-31 cd274 1.35608839 Up 1.38E-08 cfb 2.807417981 Up 1.73E-19 cmpk2 2.796616837 Up 4.50E-08 cpz -1.092294328 Down 3.85E-05 cxcl1 1.997488243 Up 7.62E-155 cxcl10 7.021498397 Up 2.11E-128 cxcl5 1.988745603 Up 2.72E-105 cxcl9 4.701719343 Up 8.39E-28 daxx 1.204249144 Up 2.04E-28 ddx58 2.126371959 Up 4.28E-165 ddx60 5.365551919 Up 7.30E-34 dhx58 4.292525409 Up 4.64E-180 dpt 1.41987071 Up 0.001996 dtx3l 2.07223413 Up 7.84E-110 eif2ak2 1.608016735 Up 1.03E-70 F830016B08Rik 1.896511411 Up 3.93E-08 fos -1.798456426 Down 2.82E-83 fosb -1.272475388 Down 4.19E-06 fosl1 1.775940737 Up 3.09E-20 gbp10 4.255492752 Up 1.05E-20
238 Appendices
gbp2 4.827674102 Up 8.69E-108 gbp2b 5.299633192 Up 1.44E-106 gbp3 6.726774926 Up 7.40E-138 gbp4 3.210273364 Up 4.51E-13 gbp5 6.415022143 Up 1.20E-54 gbp6 4.102190372 Up 1.26E-108 gbp7 4.663019369 Up 9.72E-92 gbp8 1.733103795 Up 0.000716 gbp9 2.710518638 Up 6.63E-19 gch1 1.09157355 Up 0.001894 gm12185 1.004308443 Up 2.22E-09 gm12250 6.021052583 Up 5.31E-46 gm15433 2.636986312 Up 1.18E-16 gm1966 1.76150746 Up 4.92E-09 gm2666 3.105179672 Up 3.55E-14 gm35498 1.009443185 Up 3.27E-07 gm38510 2.626237885 Up 8.57E-10 gm4070 2.24302845 Up 7.37E-105 gm4841 5.642815609 Up 5.29E-43 gm4951 7.716826241 Up 9.28E-91 gm5431 3.011649464 Up 5.16E-32 gm7609 2.020044947 Up 8.14E-37 gvin1 1.399596906 Up 4.57E-05 h2-D1 2.100394666 Up 1.22E-07 h2-K1 1.283825128 Up 7.49E-52 h2-T22 1.111755304 Up 3.03E-42 h2-T23 1.088969693 Up 1.46E-11 h2-T24 2.565578765 Up 2.64E-31 hap1 1.202846271 Up 1.39E-21 heatr9 1.659397103 Up 0.001094 helz2 1.806690376 Up 2.89E-121 herc6 3.478875306 Up 5.43E-89 hsh2d 2.345493292 Up 2.23E-07 icam1 1.695712184 Up 0.000865 ifi202b 1.506900384 Up 3.89E-11 ifi204 2.298056901 Up 1.96E-72 ifi205 1.909787704 Up 0.000178 ifi27 1.381391767 Up 4.53E-28 ifi27l2a 1.791877656 Up 3.81E-25 ifi35 2.531636714 Up 2.78E-64 ifi44l 5.466589276 Up 1.56E-37 ifih1 2.803976184 Up 8.99E-24 ifit1 6.629692434 Up 1.39E-112 ifit1bl1 2.42888724 Up 1.87E-06 ifit1bl2 5.626561144 Up 2.36E-51
Appendices 239
ifit2 1.967612055 Up 2.67E-69 ifit3b 7.928455396 Up 1.56E-187 ifitm3 1.242592468 Up 2.13E-55 ifnb1 2.234099289 Up 1.37E-05 iigp1 6.916167633 Up 7.51E-170 il12b 1.559965655 Up 0.001725 il18bp 3.371302019 Up 3.95E-17 il6 2.369902865 Up 2.16E-10 irf5 1.137233583 Up 1.41E-05 irf9 2.920040073 Up 2.75E-133 irgm1 2.319601057 Up 1.78E-197 irgm2 5.375116452 Up 4.30E-272 isg15 5.518789097 Up 8.88E-243 krt16 1.569017971 Up 0.002101 lgals3bp 1.952122793 Up 3.69E-121 lipg 1.552772628 Up 1.83E-36 LOC100039029 2.088867344 Up 3.81E-07 LOC100044068 1.475903218 Up 2.55E-34 LOC101055663 2.041630555 Up 5.36E-06 LOC101055758 2.593674162 Up 1.72E-29 LOC102641031 2.595474776 Up 6.81E-33 LOC108167347 1.774386785 Up 0.000499 LOC108168003 1.772705441 Up 0.000436 ly6a 3.771261578 Up 2.95E-38 ly6e 1.429238576 Up 1.15E-65 misp 2.191602532 Up 3.74E-06 mitd1 1.240779333 Up 1.60E-19 mnda 2.160259433 Up 1.48E-55 mndal 1.851774186 Up 3.70E-19 mpeg1 3.339662402 Up 2.64E-16 neurl3 1.490935079 Up 0.001244 nfkbie 1.129638803 Up 8.30E-16 nfkbiz 1.120189283 Up 1.45E-27 nlrc5 1.843202025 Up 1.27E-06 nmi 1.109272988 Up 2.02E-13 nos2 2.33193031 Up 3.78E-06 oas1a 5.629997183 Up 4.90E-120 oas1c 1.451036098 Up 5.73E-18 oas1g 5.753541071 Up 4.55E-45 oas2 7.181314656 Up 8.60E-187 oas3 4.047897447 Up 8.98E-84 oasl1 5.748263296 Up 1.14E-167 parp10 1.034844847 Up 1.77E-27 parp11 1.049378897 Up 1.23E-24 parp12 1.447656331 Up 9.66E-40
240 Appendices
parp14 3.127035391 Up 3.68E-200 parp9 2.016316874 Up 1.86E-103 perm1 1.495294812 Up 8.21E-05 pglyrp3 1.569281469 Up 2.07E-06 phf11a 2.444692689 Up 1.25E-14 phf11b 2.429003336 Up 1.00E-50 phf11d 4.906820759 Up 2.96E-84 plac8 1.746195155 Up 0.000561 prl2c2 1.254068163 Up 3.70E-05 prl2c3 1.282728667 Up 0.000109 psmb10 1.271165085 Up 1.40E-23 pydc3 4.245947813 Up 1.02E-19 relb 1.068716598 Up 5.10E-20 rsad2 7.696175786 Up 6.52E-127 rtp4 6.370671972 Up 3.54E-237 saa3 4.963364032 Up 6.73E-92 samd9l 2.691657 Up 3.16E-128 sectm1a 1.491520574 Up 0.000883 slc4a8 -1.525893147 Down 0.000275 slfn2 4.497678741 Up 1.05E-75 slfn8 5.254114687 Up 7.24E-194 slfn9 1.270034134 Up 1.71E-47 slpi 1.416397657 Up 1.32E-05 socs1 1.048882464 Up 2.47E-09 sp100 2.138274376 Up 3.05E-80 sp110 1.735982415 Up 1.02E-16 stat2 2.59215777 Up 1.64E-237 stra6 -1.075160808 Down 5.77E-08 tap1 1.613993246 Up 8.34E-49 tdrd7 1.370619374 Up 5.43E-38 tgtp1 4.055035765 Up 2.37E-124 tlr2 1.138674486 Up 5.88E-16 tlr3 2.438513617 Up 2.79E-85 tmem140 1.553284541 Up 2.54E-10 tnfaip3 1.599503996 Up 6.11E-55 tnfsf10 4.839027732 Up 1.31E-28 tnip3 1.442189474 Up 0.000157 tor3a 2.341765697 Up 2.51E-160 trex1 1.902901649 Up 5.45E-33 trim12c 1.699300915 Up 7.38E-30 trim21 1.917604307 Up 1.48E-29 trim25 1.526924833 Up 3.41E-33 trim30a 4.433592431 Up 1.18E-275 trim30d 2.965554087 Up 3.37E-20 trim34a 2.570866558 Up 6.23E-36
Appendices 241
trim34b 2.753164062 Up 6.18E-17 uba7 1.431205129 Up 7.04E-29 ube2l6 1.422831591 Up 1.44E-43 usp18 7.544886567 Up 1.10E-290 zbp1 6.555315 Up 5.11E-188 znfx1 1.516844864 Up 7.12E-83
B
Table 15: Full DEG list for C. muridarum infected vs non-infected TM4 cells. Gene Symbol log2 Fold Change Expression P value 1700013F07Rik -2.91556 Down 1.94E-16 1700020L24Rik -1.41913 Down 0.001052 1700057G04Rik -1.88151 Down 0.000119 1810010H24Rik -1.46974 Down 9.04E-07 2010315B03Rik -1.29261 Down 1.86E-08 3110043O21Rik -1.03732 Down 2.39E-43 4930438A08Rik -3.27124 Down 7.13E-37 4930467E23Rik -1.05296 Down 0.002131 6330416G13Rik -1.00191 Down 3.85E-17 abca12 -1.35494 Down 0.009057 abca4 -1.32523 Down 2.23E-06 abca8b -1.68236 Down 1.55E-22 abcb1a -1.88437 Down 4.18E-08 abcc4 -1.26953 Down 1.05E-110 ablim3 -1.34444 Down 0.012143 acot2 -1.84914 Down 3.22E-135 acox2 -2.19076 Down 6.17E-06 actg1 1.058474 Up 1.69E-288 adck3 -2.09883 Down 4.65E-12 adm2 -1.09561 Down 9.53E-36 adtrp -1.65699 Down 2.33E-05 AF529169 -2.61939 Down 9.22E-07 afap1l1 -1.38931 Down 0.008842 afap1l2 -1.09663 Down 9.71E-09 aig1 -1.34134 Down 9.58E-18 akap3 -1.81105 Down 9.93E-10 akap8l -1.17373 Down 9.31E-56 akna -1.28746 Down 4.98E-44 aknad1 -1.25991 Down 0.01295 akr1b7 -3.01289 Down 6.54E-18 amz1 -1.77881 Down 4.02E-10 angptl6 -3.55274 Down 5.38E-221
242 Appendices
angptl7 -1.70814 Down 9.36E-11 ank2 -1.27143 Down 1.62E-89 ankle1 1.001708 Up 3.73E-06 ankrd37 1.219639 Up 3.94E-09 anxa8 1.366463 Up 0.011493 apitd1 1.082679 Up 0.004815 apln 1.52217 Up 4.86E-49 apobec1 -2.09368 Down 3.44E-106 aqp9 -2.15561 Down 5.22E-05 arap3 -2.14704 Down 3.20E-16 arhgap33 -2.33831 Down 1.80E-99 arhgef6 -1.40086 Down 2.12E-05 arl14ep -1.13622 Down 1.60E-91 atf3 -2.06888 Down 1.27E-94 atp8a1 -1.19465 Down 2.37E-09 avil -3.66021 Down 1.25E-193 b4galnt2 -2.58209 Down 3.77E-09 bcl2l13 1.121239 Up 0.006933 bcl6b 1.060728 Up 8.06E-42 bend6 -1.45121 Down 1.96E-28 bmp6 -1.45561 Down 0.002734 bnip3 1.093333 Up 9.22E-106 cabp1 -2.06452 Down 4.19E-11 cage1 -1.34991 Down 5.85E-06 calcrl -1.33076 Down 1.28E-32 car6 -1.52944 Down 0.002661 car7 -1.50255 Down 0.004098 carns1 -1.58432 Down 0.001655 casp4 -1.56535 Down 3.76E-17 cass4 -3.10297 Down 2.27E-23 ccdc169 -1.96654 Down 1.29E-05 ccdc174 -1.03258 Down 4.40E-33 ccdc28a -1.14461 Down 9.93E-08 ccl5 -1.03491 Down 6.43E-27 ccne2 1.412361 Up 1.37E-47 ccrl2 -1.43461 Down 0.000186 cd274 -1.4246 Down 5.30E-09 cd300lb 1.495033 Up 1.25E-29 cd46 -1.14482 Down 0.007662 cd74 -1.9761 Down 2.14E-08 cdc6 1.203843 Up 7.02E-72 cdnf -1.04486 Down 0.002251 cdsn -3.13445 Down 3.12E-278 ces2g -1.32601 Down 0.001546 chac1 -1.34591 Down 4.87E-158
Appendices 243
chd5 -2.03976 Down 2.52E-51 chka -1.56236 Down 8.73E-188 ciart -1.07222 Down 1.82E-28 clca1 -1.00324 Down 8.75E-06 cnppd1 -1.1705 Down 1.01E-46 col14a1 -2.71779 Down 2.34E-14 cox6a2 -2.77394 Down 2.08E-34 cp -2.02291 Down 2.29E-13 cphx1 -2.2871 Down 1.90E-05 cps1 -1.47278 Down 0.004869 cr1l -1.13714 Down 8.71E-66 crispld1 1.073562 Up 0.012423 cry2 -1.0575 Down 1.70E-26 cth -1.02734 Down 8.06E-77 cx3cl1 1.071502 Up 4.24E-88 cxadr -1.3936 Down 3.72E-45 cxcl1 1.863904 Up 1.10E-55 cxcl2 1.756688 Up 0.001064 cyb561 -1.24783 Down 0.01427 cyp11b1 1.178014 Up 6.68E-16 cyp4f18 -1.31827 Down 0.00493 cyp4f40 -2.79284 Down 5.83E-26 cytip 1.062568 Up 0.0122 daam2 -1.02819 Down 6.17E-14 dach2 -1.42723 Down 6.51E-18 ddit3 -1.34226 Down 1.15E-123 ddx26b -1.05048 Down 1.10E-14 ddx60 -1.66159 Down 6.64E-11 dennd2d -2.67474 Down 3.89E-35 dennd4a -1.13342 Down 5.45E-93 dgat2 -2.11136 Down 1.27E-65 dhrs3 -1.67272 Down 2.08E-31 dhrs7 -1.42164 Down 1.46E-54 dna2 1.246208 Up 1.04E-16 dnmt3l -2.58898 Down 2.23E-14 doc2g -1.1195 Down 1.16E-24 dock3 -2.22534 Down 5.84E-13 doxl2 -1.3748 Down 0.010444 dpf2 -1.03584 Down 2.04E-120 dppa2 -1.68242 Down 0.001946 dscc1 1.042547 Up 7.41E-19 dthd1 -2.99116 Down 3.59E-42 dtna -1.18235 Down 3.43E-10 dtx4 -1.20896 Down 1.10E-40 dus4l -1.11965 Down 4.36E-22
244 Appendices
dusp5 1.146061 Up 6.77E-17 dynap -1.00244 Down 1.02E-10 dyrk4 -3.20787 Down 6.73E-25 e2f2 1.267307 Up 1.35E-15 e2f8 1.111581 Up 6.78E-42 ecm2 -1.19502 Down 0.011193 eda2r -2.10924 Down 1.10E-53 egr3 1.36433 Up 8.01E-15 elavl2 -1.80426 Down 1.52E-38 elfn2 -1.59068 Down 0.000283 eml1 1.021348 Up 5.67E-64 epg5 -1.05405 Down 2.69E-43 exph5 -1.26893 Down 0.013099 extl1 -3.1658 Down 9.53E-13 fam171b -2.05964 Down 1.91E-213 fam20a -1.03627 Down 9.06E-47 fam71f1 -1.09999 Down 1.28E-12 fbxo32 -1.30752 Down 1.63E-30 fcamr -1.6016 Down 0.003216 fcrlb -1.36946 Down 7.71E-09 fgf21 -3.05311 Down 5.55E-32 fibin -2.13524 Down 2.07E-05 flrt1 -4.69123 Down 5.43E-47 flt3l -1.60539 Down 1.35E-10 foxs1 1.238914 Up 3.28E-09 fras1 -1.17901 Down 0.002591 fst 1.255703 Up 3.79E-16 fut1 -1.63335 Down 0.002105 fyn -1.0716 Down 1.72E-66 gbp2b -1.03366 Down 0.00188 gbp6 -1.78322 Down 8.73E-11 gbp8 -1.42545 Down 0.000383 gbp9 -1.23267 Down 5.48E-06 gbx1 -1.95566 Down 9.85E-20 gch1 -1.4128 Down 3.54E-28 gdap1l1 -2.97771 Down 1.64E-14 gdf15 -1.04928 Down 0.000759 gfpt2 -2.08424 Down 5.75E-05 gfra1 -1.02582 Down 0.011502 ggt5 -1.25635 Down 5.04E-15 gins2 1.006468 Up 7.83E-35 gjb3 1.573125 Up 1.18E-06 gjb4 1.115997 Up 5.04E-05 gjb6 -1.25455 Down 0.016652 glce -1.03263 Down 1.06E-93
Appendices 245
glrb -1.10535 Down 0.000242 glrp1 -1.00529 Down 0.0074 gm10486 -1.61721 Down 0.000114 gm10487 -1.64157 Down 0.000394 gm14308 -1.98477 Down 1.72E-16 gm15433 -1.4494 Down 0.000262 gm20939 -1.01813 Down 6.23E-10 gm32856 -1.10041 Down 7.35E-51 gm4070 -1.28998 Down 1.53E-09 gm6445 -1.39573 Down 3.47E-11 gml 1.408079 Up 0.000644 gnpnat1 -1.14062 Down 7.28E-91 gpr137b -1.11703 Down 3.55E-35 gpr141 -2.12005 Down 2.30E-07 gpr179 -2.07091 Down 1.89E-05 gpt2 -1.41773 Down 3.98E-298 grid1 1.395423 Up 8.12E-05 gtpbp2 -1.43377 Down 1.45E-103 gypa -2.23363 Down 3.95E-05 h2-DMb1 1.636124 Up 0.002623 hdac11 -1.03388 Down 4.78E-08 hdac4 -1.31413 Down 1.85E-53 hecw2 -1.48714 Down 0.003664 hfe -1.31052 Down 7.94E-05 hhipl1 -1.07948 Down 1.89E-18 hlf -1.45654 Down 3.47E-11 hmga2 1.105822 Up 1.90E-163 htatip2 -1.24952 Down 0.000367 icam1 1.491249 Up 0.005865 id1 1.406636 Up 2.56E-39 id2 1.029048 Up 9.58E-07 id3 1.011229 Up 9.94E-15 ifi44 -1.72539 Down 3.34E-73 ifit1 -1.49667 Down 2.01E-115 ifit1bl2 -1.18761 Down 1.12E-06 ifit3b -1.01182 Down 2.67E-09 ifitm6 -2.18124 Down 2.20E-05 igfbp4 -1.59124 Down 2.58E-203 inca1 -1.12416 Down 7.89E-05 irgm2 -1.10492 Down 1.22E-08 irx1 -1.02625 Down 3.14E-17 isoc1 -1.03434 Down 1.50E-81 itgad -1.50529 Down 0.005347 itgb2 -1.18437 Down 3.96E-05 itih2 -1.43574 Down 3.97E-05
246 Appendices
itk -3.53424 Down 6.44E-26 kcnip4 -1.09918 Down 0.010298 kcnma1 -2.51806 Down 4.89E-89 kctd11 1.220174 Up 2.01E-39 kdm7a -1.81728 Down 1.45E-55 kif21b -1.3062 Down 1.07E-114 kiss1r -1.06143 Down 0.011527 klf10 -1.12485 Down 9.75E-68 klf11 -1.48478 Down 2.20E-34 klf15 -1.97761 Down 3.77E-05 klf4 -1.28139 Down 2.69E-51 klhl24 -1.10437 Down 2.16E-24 klhl33 -1.58939 Down 6.71E-06 kprp 1.107084 Up 2.74E-05 kyat1 -1.15246 Down 1.26E-10 lce1f 1.340591 Up 0.005749 ldha 1.309151 Up 1.45E-250 ldlrad3 -1.14546 Down 7.52E-51 letm2 -1.27189 Down 3.12E-07 lhfpl2 -1.12978 Down 1.81E-90 lins1 -1.12623 Down 1.09E-27 LOC101055758 -2.34541 Down 1.85E-12 LOC105242472 -1.22139 Down 0.015787 LOC108167848 -1.43817 Down 3.43E-17 lrp2 -2.59258 Down 8.62E-17 lrrc17 -1.39378 Down 0.000954 lrrc51 -1.15939 Down 6.71E-09 lum -1.06346 Down 0.008184 maats1 -2.6761 Down 1.40E-43 mamdc2 -3.92543 Down 1.14E-167 map3k19 -1.97342 Down 1.51E-10 map6d1 -1.68249 Down 0.000931 mcm5 1.113843 Up 2.18E-165 mettl7b -2.14664 Down 2.37E-09 mib2 -1.45378 Down 2.16E-81 mknk1 -1.10397 Down 1.45E-48 mmp11 -1.42906 Down 1.21E-85 mmp28 -1.76249 Down 3.20E-18 mpp2 1.425238 Up 5.52E-68 msx3 -1.59038 Down 0.00266 mtm1 -1.90106 Down 1.45E-74 mtss1l -1.08963 Down 7.56E-49 mxd3 1.160027 Up 3.58E-17 mybl2 1.094977 Up 7.47E-64 mycn 1.073155 Up 9.90E-07
Appendices 247
myo5c -1.93787 Down 4.26E-05 myom2 -1.90573 Down 5.02E-06 naaa -1.09334 Down 4.53E-35 nab2 1.102062 Up 8.04E-104 nanos1 1.106616 Up 9.42E-10 napb -2.46585 Down 4.75E-102 ndrg1 -1.30415 Down 1.26E-263 ndufa4l2 -1.10781 Down 0.014763 ngef 1.013357 Up 0.001593 nipal3 -1.16965 Down 1.60E-06 nnmt -1.3874 Down 3.28E-22 nrap -2.52092 Down 1.50E-08 nrg4 -1.17686 Down 0.000653 nudt8 -1.22814 Down 1.12E-23 nup210 -1.99846 Down 0.000134 nupr1 -1.2998 Down 2.49E-153 oasl1 -1.31855 Down 2.67E-25 oasl2 -1.53142 Down 8.13E-62 oit3 1.016197 Up 0.004421 orm2 -1.50858 Down 6.96E-05 ostn -3.64693 Down 9.62E-70 otub2 -1.08194 Down 3.88E-28 p2rx3 -2.55608 Down 1.84E-229 paqr3 -1.9465 Down 2.56E-122 pax8 -1.05686 Down 0.000103 pbld2 -1.39631 Down 9.08E-05 pcdhb5 -1.0387 Down 7.93E-11 pcdhga8 -1.64825 Down 0.002144 pde1a -1.09388 Down 1.61E-32 pde3b -2.94555 Down 2.18E-39 pdzd7 -2.1304 Down 3.31E-21 phyhd1 -1.45568 Down 6.06E-15 pi16 -1.88704 Down 7.57E-05 pik3ip1 -1.36304 Down 9.69E-22 pla2g2e -2.13728 Down 5.56E-05 plce1 -2.44484 Down 1.71E-08 plcl1 -1.50319 Down 0.005719 plcl2 -1.51121 Down 9.13E-71 plek -1.41094 Down 0.003658 pnrc2 -1.13937 Down 1.05E-112 ppef1 -1.79261 Down 0.000107 ppef2 -2.06536 Down 2.32E-05 prdm9 -1.06288 Down 1.95E-08 prl2c2 1.592082 Up 6.91E-06 prl2c3 1.589504 Up 0.000243
248 Appendices
prr32 -2.3775 Down 1.01E-05 ptger4 1.046027 Up 5.15E-11 ptprt -2.42252 Down 3.88E-10 ptpru -1.13589 Down 0.014143 ptx3 -1.49236 Down 6.53E-22 pycr1 -1.72265 Down 8.63E-183 rab39b -1.4512 Down 7.01E-63 rcan2 1.014113 Up 1.27E-17 reep6 -1.84012 Down 2.43E-103 rgs2 -1.69892 Down 4.77E-41 rhbdd1 -1.26422 Down 1.55E-158 rmnd5a -1.03869 Down 2.07E-107 rorc -1.41995 Down 0.002699 rps6ka2 -1.40212 Down 2.00E-152 rrm2 1.386767 Up 2.89E-208 rsad2 -1.2388 Down 8.21E-19 rtp3 -1.38001 Down 0.0003 rtp4 -1.02884 Down 3.06E-14 rwdd3 -1.20716 Down 9.29E-08 s100a5 1.408558 Up 0.009368 samd12 -2.56534 Down 2.31E-06 samt1 1.350828 Up 0.012567 scn1a -1.92036 Down 0.000374 sel1l -1.14782 Down 9.36E-136 sema4d -2.1567 Down 2.24E-05 serpinb1a -1.17683 Down 0.001565 serpinb1b -1.18299 Down 0.012935 sh2d1b1 1.389536 Up 2.07E-12 sh2d4a -1.22734 Down 0.004934 sh2d6 -4.32538 Down 4.79E-153 sh3tc2 -1.16769 Down 0.004041 shcbp1 1.011986 Up 6.61E-53 slamf9 -1.3117 Down 0.000581 slc19a3 -3.06742 Down 2.39E-10 slc1a1 -2.28859 Down 5.15E-08 slc1a4 -1.00955 Down 3.73E-121 slc24a3 -1.69854 Down 5.79E-23 slc25a27 -1.56807 Down 2.87E-14 slc2a1 1.243117 Up 6.26E-169 slc30a1 -1.04439 Down 2.86E-11 slc38a7 -1.4077 Down 1.61E-60 slc7a11 -1.5744 Down 8.77E-154 slc7a3 -1.55741 Down 3.49E-95 slc8a1 -1.04411 Down 5.69E-24 slc9a9 -2.43895 Down 5.35E-77
Appendices 249
sned1 -2.02315 Down 7.39E-08 snx33 -1.0167 Down 4.97E-30 soat2 -3.40071 Down 2.42E-39 sp110 -1.24508 Down 0.01225 sspo -3.80874 Down 2.42E-47 stard5 -1.3657 Down 3.69E-78 stard6 -1.13612 Down 0.012658 stbd1 -1.30113 Down 1.70E-90 steap1 -1.02205 Down 1.14E-82 sun5 -1.437 Down 0.007677 taf7l -3.35934 Down 8.15E-77 tcf19 1.052586 Up 1.41E-38 tcp11l2 -1.03049 Down 1.01E-07 tfcp2l1 -2.2339 Down 1.91E-08 tfrc 1.329403 Up 1.97E-127 tg -2.11717 Down 1.55E-06 them4 -1.52888 Down 7.01E-36 tk1 1.231232 Up 4.24E-125 tmem116 -1.22535 Down 0.002582 tmem140 -1.59542 Down 2.55E-08 tmem154 -1.00011 Down 2.93E-08 tmem240 -1.21982 Down 4.15E-09 tmem74 -1.25827 Down 0.000204 tmtc2 -2.22656 Down 3.19E-08 tnfrsf4 1.502523 Up 0.004313 tnfsf9 1.238719 Up 1.25E-18 trem3 -2.38947 Down 3.01E-12 trib3 -1.56087 Down 9.96E-257 trim66 -1.30596 Down 1.01E-07 trp53inp1 -1.576 Down 6.30E-14 tsc22d3 -1.34417 Down 2.00E-42 tspyl4 -1.41556 Down 7.17E-29 ugt1a6a -1.44177 Down 2.57E-53 ugt1a6b -1.22152 Down 1.06E-18 ung 1.425617 Up 4.44E-45 upp1 1.092856 Up 7.57E-07 vmn2r3 -2.00848 Down 0.00011 vrtn -1.40357 Down 0.008261 vwa1 1.150873 Up 1.06E-11 wars -1.14663 Down 2.49E-143 ypel1 -1.19632 Down 0.006574 ypel3 -1.38533 Down 1.06E-29 ypel4 -1.18135 Down 2.29E-05 zbp1 -1.01879 Down 4.33E-05 zbtb18 -1.31761 Down 3.74E-73
250 Appendices
zcchc5 -1.47321 Down 0.002964 zfp362 -1.00853 Down 1.20E-17 zfp385b -1.10985 Down 1.91E-08 zfp493 -1.44341 Down 1.41E-06 zfp661 -1.09982 Down 6.94E-11 zfp759 -1.07658 Down 2.15E-05 zfp760 -1.204 Down 1.50E-27 zfp874a -1.07259 Down 2.17E-06 zfp874b -1.07077 Down 6.18E-11 zfp945 -2.0687 Down 7.05E-78 zfr2 -1.1674 Down 6.02E-06 zyg11a -1.66376 Down 0.002159 zyg11b -1.16679 Down 3.53E-117
C
Table 16: Full DEG list for C. muridarum infected vs non-infected GC-1 cells. Gene Symbol log2 Fold Change Expression P value acod1 3.235258935 Up 7.70E-12 AI607873 2.753480745 Up 1.03E-17 arhgap8 1.098709054 Up 6.85E-05 batf2 1.505699799 Up 0.002094 bcl2l15 1.043036934 Up 6.52E-07 birc3 1.050406159 Up 7.26E-09 bst2 2.335883338 Up 6.49E-175 casp4 1.187889391 Up 6.07E-44 ccl20 3.018012292 Up 2.86E-13 ccl5 1.964245109 Up 1.52E-47 ccl7 4.058668801 Up 9.03E-84 ccrl2 1.644799889 Up 2.32E-13 cd274 2.066544368 Up 6.49E-64 cdc45 -1.066442992 Down 0.000535 cebpd 1.046560993 Up 4.90E-35 cfb 2.344851282 Up 3.06E-09 ch25h 2.142069913 Up 4.99E-06 chil1 1.873845872 Up 2.28E-17 cmpk2 3.908018866 Up 1.58E-160 csprs 1.771852758 Up 0.0002723 cxcl16 1.212527598 Up 1.04E-114 cxcl2 1.504755039 Up 0.001444 cxcl5 3.031161538 Up 2.78E-182 cxcr4 -1.188805413 Down 1.65E-08 ddx60 3.206834897 Up 2.09E-56
Appendices 251
dhx58 3.379232639 Up 1.19E-98 dtx3l 1.907229261 Up 1.40E-161 eif2ak2 1.462210904 Up 2.48E-130 fam180a -1.492492092 Down 5.22E-50 fam46a 1.812881192 Up 9.41E-26 fas 1.617936774 Up 1.91E-30 gbp10 2.30141999 Up 2.18E-11 gbp2 2.587556011 Up 2.57E-248 gbp2b 2.926736747 Up 6.00E-267 gbp3 3.498566211 Up 1.62E-273 gbp4 2.910412136 Up 1.19E-65 gbp5 3.847163469 Up 2.74E-33 gbp6 3.05826977 Up 3.41E-67 gbp7 2.880717153 Up 1.85E-73 gbp9 2.492071721 Up 8.93E-100 gm10705 -1.665004955 Down 0.0003015 gm12250 2.65670885 Up 5.21E-08 gm2573 3.112491444 Up 1.24E-10 gm35498 1.118603416 Up 1.43E-05 gm39701 1.487777453 Up 0.0010589 gm4070 2.746623753 Up 7.87E-81 gm4841 2.03436208 Up 3.21E-05 gm4951 5.134558875 Up 1.15E-48 gm5431 2.534490213 Up 2.87E-20 gm7609 2.529033982 Up 1.27E-08 gm8909 1.012715352 Up 0.0026202 gm9840 -1.455559647 Down 0.0023543 gpr68 1.193804584 Up 2.52E-10 gpr84 1.56539212 Up 0.0001933 h2-D1 1.234664479 Up 2.38E-90 h2-K1 1.032625898 Up 2.46E-178 h2-Q4 1.418611498 Up 0.0001705 h2-T24 1.552550874 Up 1.22E-05 herc6 1.717479578 Up 1.21E-92 hp 2.50818036 Up 1.97E-12 icam1 2.029494372 Up 1.45E-19 ifi203 2.032485249 Up 5.38E-07 ifi27 1.321944605 Up 4.53E-68 ifi27l2a 1.603054584 Up 8.44E-09 ifi35 1.619179883 Up 9.10E-87 ifi44 4.474966572 Up 5.70E-263 ifi47 2.46736813 Up 6.34E-72 ifih1 2.00125582 Up 1.75E-141 ifit1bl1 3.399745216 Up 1.67E-22 ifit1bl2 3.531534905 Up 2.86E-47
252 Appendices
ifit2 1.694185372 Up 9.73E-130 ifit3 4.499855486 Up 1.12E-220 ifitm3 1.590277218 Up 4.97E-194 igtp 3.122342081 Up 2.49E-285 iigp1 2.520393918 Up 5.37E-16 il23a 1.869852885 Up 9.61E-10 irf5 1.277310853 Up 1.25E-26 irf9 2.169644077 Up 2.45E-146 irgm2 3.665567686 Up 1.36E-247 krt16 1.353841279 Up 3.50E-07 lcn2 1.221047534 Up 3.67E-34 lgals3bp 1.847124399 Up 8.21E-260 lgals9 1.563186149 Up 3.46E-211 LOC100044068 1.073741109 Up 5.05E-49 LOC100862473 2.172981045 Up 7.24E-17 LOC101055663 1.745819863 Up 0.0002895 LOC101055758 1.338992025 Up 0.0015005 LOC102641031 2.174776212 Up 8.52E-120 ly6a 1.180604613 Up 4.76E-46 ly6e 2.050275421 Up 2.70E-209 madcam1 1.863724364 Up 1.34E-06 misp 1.580476876 Up 8.53E-05 mitd1 1.042720459 Up 4.22E-30 mndal 1.483110884 Up 4.97E-06 mpeg1 2.458662822 Up 1.05E-22 neurl3 2.934667817 Up 3.19E-24 nfkbia 1.513033645 Up 3.58E-132 nfkbie 1.197430621 Up 2.93E-53 nfkbiz 2.089347871 Up 2.61E-54 nos2 4.696352896 Up 1.25E-77 nrep -1.107022806 Down 0.0001279 nrg1 1.045241049 Up 5.00E-27 oas1a 3.170882367 Up 1.63E-239 oas1c 1.509350091 Up 3.37E-17 oas1g 3.088493963 Up 2.90E-106 oas2 3.907996364 Up 5.63E-150 oasl1 1.817694146 Up 9.56E-168 olfr56 1.57859912 Up 0.0012442 parp10 1.316803868 Up 1.49E-71 parp12 1.785889786 Up 4.15E-158 parp14 3.065011522 Up 1.29E-264 parp9 1.64657095 Up 7.64E-116 pglyrp3 1.751558095 Up 3.35E-64 phf11a 2.010367485 Up 2.56E-08 phf11b 2.195613501 Up 3.52E-26
Appendices 253
phf11d 2.001977896 Up 2.37E-101 plekha4 1.146842644 Up 7.28E-07 pou3f1 1.031499626 Up 1.21E-45 prnd 1.618138968 Up 0.0007016 psmb10 1.291312323 Up 9.02E-28 psmb8 1.712120101 Up 1.29E-11 ptchd4 1.006962407 Up 0.000224 rnf213 1.117219101 Up 5.90E-15 rpl10 -1.747423583 Down 4.18E-06 rpl34-ps1 -1.312266556 Down 0.0032458 rtp4 3.674101285 Up 1.71E-302 saa3 4.655116571 Up 7.15E-129 samd9l 2.744816171 Up 4.07E-241 serpina3f 1.226917432 Up 3.14E-07 serpina3g 1.09324432 Up 6.37E-12 serpina3i 1.272102791 Up 0.0002326 slfn2 2.333145642 Up 3.47E-234 slfn8 2.428724129 Up 6.93E-11 slpi 1.300686922 Up 1.92E-25 socs1 1.39442608 Up 2.90E-07 sp100 2.86670934 Up 1.04E-108 sp110 2.136721212 Up 1.07E-23 spib 1.249626415 Up 1.93E-06 sprr2e 1.649991005 Up 0.0006256 stat2 1.77709284 Up 4.47E-166 tap1 1.707761641 Up 3.92E-50 tapbp 1.149395159 Up 2.59E-216 tdrd7 1.011185559 Up 8.61E-48 tgtp1 2.623876103 Up 1.63E-64 tgtp2 3.615045154 Up 3.62E-51 tlr2 1.392703882 Up 2.08E-18 tlr3 1.200438817 Up 2.06E-22 tmem130 1.164845816 Up 0.0005328 tnf 1.728744424 Up 0.00026 tnfaip3 1.369730202 Up 1.98E-79 tnfsf10 2.41225558 Up 4.56E-07 tnfsf15 1.028240062 Up 5.48E-135 tor3a 1.169029002 Up 7.04E-92 traf1 1.668444212 Up 5.89E-05 trex1 1.367962793 Up 2.15E-21 trim12c 1.616761348 Up 1.77E-42 trim14 1.206490206 Up 1.20E-40 trim21 1.927975252 Up 9.24E-35 trim25 1.356011924 Up 4.07E-109 trim30a 3.453550587 Up 7.64E-71
254 Appendices
trim34a 2.190293751 Up 2.41E-27 trim34b 2.155576015 Up 8.66E-13 trim45 1.901626532 Up 2.22E-05 uba7 1.81118555 Up 3.25E-34 ube2l6 2.327955199 Up 1.05E-135 vcam1 1.441091831 Up 4.10E-186 vnn3 2.338221685 Up 7.79E-17 xaf1 2.88783217 Up 1.89E-225 xirp2 1.143215833 Up 0.0003249 zbp1 3.39572816 Up 2.19E-93 zc3h12a 1.690524313 Up 2.34E-28 znfx1 1.059825076 Up 3.79E-66
D
Table 17: Common DEGs between infected TM3, TM4, and GC-1 cells. Gene Symbols TM3 TM4 GC-1 aI607873 Up Up batf2 Up Up bst2 Up Up casp4 Up Down Up ccl20 Up Up ccl5 Up Down Up ccl7 Up Up ccrl2 Up Down Up cd274 Up Down Up cfb Up Up cmpk2 Up Up cxcl1 Up Up cxcl2 Up Up cxcl5 Up Up ddx60 Up Down Up dhx58 Up Up dtx3l Up Up eif2ak2 Up Up gbp2 Up Up gbp2b Up Down Up gbp6 Up Down Up gbp8 Up Down gbp9 Up Down Up gch1 Up Down gm12250 Up Up gm15433 Up Down
Appendices 255
gm35498 Up Up gm4070 Up Down Up gm4841 Up Up gm4951 Up Up gm5431 Up Up gm7609 Up Up h2-D1 Up Up h2-K1 Up Up h2-T24 Up Up herc6 Up Up icam1 Up Up Up ifi27 Up Up ifi27l2a Up Up ifi35 Up Up ifih1 Up Up ifit1 Up Down ifit1bl1 Up Up ifit1bl2 Up Down Up ifit2 Up Up ifit3b Up Down ifitm3 Up Up iigp1 Up Up irf5 Up Up irf9 Up Up irgm2 Up Down Up krt16 Up Up lgals3bp Up Up LOC100044068 Up Up LOC101055663 Up Up LOC101055758 Up Down Up LOC102641031 Up Up ly6a Up Up ly6e Up Up misp Up Up mitd1 Up Up mndal Up Up mpeg1 Up Up neurl3 Up Up nfkbie Up Up nfkbiz Up Up nos2 Up Up oas1a Up Up oas1c Up Up oas1g Up Up oas2 Up Up
256 Appendices
oasl1 Up Down Up parp10 Up Up parp12 Up Up parp14 Up Up parp9 Up Up pglyrp3 Up Up phf11a Up Up phf11b Up Up phf11d Up Up prl2c2 Up Up prl2c3 Up Up psmb10 Up Up rsad2 Up Down rtp4 Up Down Up saa3 Up Up samd9l Up Up slfn2 Up Up slfn8 Up Up slpi Up Up socs1 Up Up sp100 Up Up sp110 Up Down Up stat2 Up Up tap1 Up Up tdrd7 Up Up tgtp1 Up Up tlr2 Up Up tlr3 Up Up tmem140 Up Down tnfaip3 Up Up tnfsf10 Up Up tor3a Up Up trex1 Up Up trim12c Up Up trim21 Up Up trim25 Up Up trim30a Up Up trim34a Up Up trim34b Up Up uba7 Up Up ube2l6 Up Up zbp1 Up Down Up znfx1 Up Up Up: over-expressed; Down: under-expressed; Red: common between TM3/TM4/GC- 1; Green: common between TM3/TM4, Blue: common between TM4/GC-1.
Appendices 257
Appendix D
A
258 Appendices
B
Appendices 259
C
260 Appendices
D
Appendices 261
E
262 Appendices
F
Appendices 263
G
264 Appendices
H
Appendices 265
I
266 Appendices
J
Appendices 267
K
268 Appendices
L
Appendices 269
Figure A4 Differentially expressed KEGG pathways for C. muridarum infected vs non-infected TM3, TM4, and GC-1 cells. Figure A4 shows the remaining KEGG pathways discussed in Chapter 5. For TM3 cells, the four remaining pathways included NOD-like receptor signalling (A), Influenza A infection (B), Measles infection (C), and TNF signalling (D). For TM4 cells. The four remaining pathways included Phototransduction-fly (E), Legionellosis (F), Rap1 signalling (G), and Renin secretion (H). The remaining pathways for GC-1 cells included, Herpes Simplex infection (I), TNF signalling (J), Influenza A infection (K), and finally Measles infection (L).
270 Appendices
Appendix E
Figure A5 Active replication of C. muridarum in mouse testes shown by TC0500. Figure A5 shows the TC0500 staining discussed in Chapter 6. Chlamydial TC0500 (green) was detected by immunohistochemistry in the testes of C57BL/6 mice, six- months after intra-penile infection. A DAPI nuclear counterstain (blue) was also used. Images are representative of n = 5 replicates.
Appendices 271
Appendix F
A S e m in ife r o u s T u b u le L u m e n B P C N A
3 0 0 0 0 3
**** *
2
m
m
2
2 0 0 0 0 / 2
m
s
l
l
m
e
a
c
e
e
r
v A
1 0 0 0 0 i 1
t
i
s
o P
0 0 N o n -I n fe c te d I n fe c te d N o n -in fe c te d I n fe c te d
Figure A6 Quantification of seminiferous tubule lumen size and PCNA+ cells in C. muridarum infected mouse testes vs non-infected mice. Figure A6 shows the area/mm2 of seminiferous tubule lumen (A) and PCNA+ germ cells (B) present in the testes of infected (black bars) and non-infected (white bars) mice, six-months post intra-penile infection with C. muridarum or mock infection with SPG. Graphs were generated with GraphPad Prism (version 7, mean and SD) and a Students T-test was applied, differences were considered significant when P < 0.05 (*P < 0.05, ****P < 0.0001).
272 Appendices
Appendix G
S p e r m C a p a c ita tio n ns ns
n 1 0 0 o
i *
t a
l *
y r
o 8 0
h
p
s
o h
P 6 0
e
n
i
s
o r
y 4 0
T
g
n i
w 2 0
o
h
S
% 0 N o n-c a pa c ita ting C a pa c ita ting
B W W M e d ia T y p e
N o n -In f e c te d
In fe c te d
Figure A7 Differential capacitation observed in sperm isolated from C. muridarum infected vs non-infected mice. Figure A7 shows the percentage of sperm displaying capacitation (measured by tyrosine phosphorylation staining) isolated from the vas deferens of infected (black bars) and non-infected (white bars) mice, six-months post intra-penile infection with C. muridarum or mock infection with SPG. Graphs were generated with GraphPad Prism (version 7, mean and SD) and a Students T-test was applied, differences were considered significant when P < 0.05 (*P < 0.05).
Appendices 273
Appendix H
Figure A8 Detection of active chlamydial replication in human testis by TC0500 IHC. Figure A8 shows immunohistochemical detection of chlamydial TC0500 (dark brown) in human testicular biopsy sections. The inclusions were present in different structures within the tissue; (b) shows inclusions in the interstitum and basement membrane, (c) shows intra-tubular inclusions, and (a) shows non-infected control tissue.
274 Appendices
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