The Pennsylvania State University
The Graduate School
Department of Animal Science
CHARACTERIZATION OF THE PRAME/PRAMEY GENE FAMILY DURING
SPERMATOGENESIS
A Thesis in
Animal Science
by
Yaqi Zhao
2013 Yaqi Zhao
Submitted in Partial Fulfillment of the Requirements for the Degree of
Master of Science
December 2013
The thesis of Yaqi Zhao was reviewed and approved* by the following:
Wansheng Liu Associate Professor of Animal Genomics Thesis Advisor
Francisco J. Diaz Assistant Professor of Reproductive Biology
Joy L. Pate Professor of Reproductive Physiology
Terry D. Etherton Distinguished Professor of Animal Nutrition Head of the Department of Animal Science
*Signatures are on file in the Graduate School
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ABSTRACT
Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis (CT) antigen that is predominantly expressed in normal gametogenic tissues and a variety of tumors. Like other CT antigens, PRAME is a multi-copy gene family representing one of the most amplified gene families in eutherian mammals. Members of the PRAME gene family encode leucine-rich repeat (LRR) proteins that fold into a horseshoe shape and provide a versatile structural framework for the formation of protein-protein interactions.
PRAME has been extensively studied in cancer biology and is believed to play a regulatory role in cancer cells. The function of PRAME during testicular development and spermatogenesis remains unknown. The objective of this study is to characterize the expression of PRAME during spermatogenesis, to identify protein(s) that interact with
PRAME and to explore the possible functions of PRAME. We chose the bovine
PRAMEY (on chromosome Y) and mouse Pramel1 (on chromosome 4) as representatives of the PRAME gene family in this study. Using a custom peptide-specific antibody,
PRAMEY was characterized as a testis- and spermatozoa-specific protein in bovine.
PRAMEY was expressed in the acrosome of spermatids, as well as the acrosome and flagellum of spermatozoa. Immunogold electron microscopy revealed the subcellular localization of PRAMEY in spermatids and spermatozoa: PRAMEY was firstly localized in the acrosomal granule of step 4 spermatids, migrated with the content of acrosomal granule during spermiogenesis, and was finally present in the acrosome lumen of mature spermatozoa. To identify the PRAME interactive protein(s), we performed co- immunoprecipitation (co-IP) using the PP1γ2 and PRAMEY antibodies in bovine
iv spermatozoa. The results showed that PRAMEY was co-immunoprecipitated with PP1γ2 in caput and caudal epididymal sperm. By blocking PRAMEY during in vitro fertilization
(IVF), fewer embryos were formed while the incidence of polyspermy increased, suggesting a role of PRAMEY in fertilization and blocking of polyspermy. In mice, the expression level of the PRAMEL1 protein was very low in newborn testes, then was increased gradually from 1- to 3-week-old testes, and remained constant after three weeks of age. Immunofluorescent staining on testis cross sections revealed that PRAMEL1 was localized in the cytoplasm of spermatocytes in 2-week-old testis, and translocated to the acrosomal region of round spermatids in 3-week-old testis. Taken together, these results suggest that PRAME is involved in acrosome formation during spermatogenesis, functions in block to polyspermy during fertilization, may interact with PP1γ2 and play a role in the regulation of sperm motility.
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TABLE OF CONTENTS
List of Figures ...... vii
List of Tables ...... viii
Acknowledgements ...... ix
Chapter 1 Review of the Literature ...... 1
1. Cancer/testis (CT) antigen ...... 1 1.1 CT antigens in cancer ...... 1 1.2 CT antigens in testis ...... 4 2. Preferentially expressed antigen in melanoma (PRAME) ...... 5 2.1 The discovery of PRAME ...... 5 2.2 PRAME is a largely expanded gene family in eutheria ...... 6 2.3 PRAME is a leucine rich repeat (LRR) protein ...... 7 2.4 Expression and function of PRAME in cancer ...... 10 2.5 Expression and Function of PRAME in reproduction ...... 12 3. Spermatogenesis ...... 14 3.1 Phases of spermatogenesis ...... 15 3.2 The first wave of spermatogenesis ...... 19 3.3 Acrosome biogenesis ...... 21 3.4 Structure of the mature acrosome ...... 22 3.5 Molecular mechanism of acrosome biogenesis ...... 24 3.6 Modulation of sperm motility by PP1γ2 complexes ...... 25 4. Fertilization and block of polyspermy ...... 27 5. Summary and the objectives of this study ...... 28
Chapter 2 Characterization of the Bovine PRAMEY During Spermiogenesis, and its Potential Role in the Block to Polyspermy During Fertilization ...... 30
1. Introduction ...... 30 2. Materials and methods ...... 33 3. Results...... 42 4. Discussion ...... 75
Chapter 3 The Expression and Localization of the Mouse PRAMEL1 during the First Wave of Spermatogenesis ...... 81
1. Introduction ...... 81 2. Materials and Methods ...... 83 3. Results...... 86
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4. Discussion ...... 92
Summary ...... 94
References ...... 95
vii
LIST OF FIGURES
Figure 1-1. The 3D structure of PRAME protein showing its horseshoe shape (Chang et al., 2011)...... 9
Figure 1-2. Mouse stages in the cycle of the seminiferous epithelium (Hess and Renato de Franca, 2008)...... 17
Figure 1-3. Bovine stages in the cycle of the seminiferous epithelium (Berndston and Desjardins, 1974)...... 18
Figure 2-1. Identification of the bovine PRAMEY protein in caudal spermatozoa by western blot with the custom-made PRAMEY antibody...... 45
Figure 2-2. Comparison of the bovine sperm PRAMEY proteins extracted either by different buffers or at different freeze-thaw times...... 46
Figure 2-3. Temporal expression of the bovine PRAMEY protein during testis development...... 48
Figure 2-4. Localization of the bovine PRAMEY during spermiogenesis...... 50
Figure 2-5. Localization of the bovine PRAMEY in testicular spermatozoa, caput and caudal epididymal spermatozoa ...... 52
Figure 2-6. The subcellular localization of the bovine PRAMEY in spermatids and spermatozoa revealed by immunogold electron microscopy...... 57
Figure 2-7. Co-immunoprecipitation analysis of PP1γ2 with PRAMEY in bovine. ... 66
Figure 2-8. 2-D electrophoresis of the bovine caudal sperm proteins...... 68
Figure 2-9. Enrichment of the bovine sperm PRAMEY protein by immunoprecipitation...... 70
Figure 2-10. Sperm-oocyte binding and IVF using caudal sperm incubated with the PRAMEY antibody...... 73
Figure 3-1. Temporal expression analysis of Pramel1 by RT-PCR during the mouse testis development...... 88
Figure 3-2. Expression analysis of the mouse PRAMEL1 protein in testes...... 89
Figure 3-3. Immunofluorescent localization of the mouse PRAMEL1 during early testis development...... 91
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LIST OF TABLES
Table 2-1. The bovine PRAMEY antibody staining in spermatozoa isolated from testis, caput and caudal epididymis ...... 54
Table 2-2. Proteins identified in the IP eluate by mass spectrometry ...... 71
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ACKNOWLEDGEMENTS
My studying at Penn State University has been a great exploration, in which I gained knowledge, strength and courage. There have been so many fantastic individuals that helped me along the way. Firstly, I would like to express my sincere appreciation to
Dr. Wansheng Liu, my advisor, for giving me the opportunity to work under your instruction. You have inspired me throughout my study and your dedication to research is truly remarkable.
I would like to thank my committee members, Dr. Joy Pate and Dr. Francisco
Diaz, for your guidance and diverse perspectives. I could always get your help in time whenever I walked into your offices. Dr. Pate, you are a great role model in not only research but also being an instructor. It’s always encouraging to talk with you. Dr. Diaz, I appreciate the time you took out from your busy schedule to help me on the bovine in vitro fertilization and mouse PRAMEL1 experiments. It was a great pleasure to work with you and I really learned a lot from you.
I would like to thank Dr. Gang Ning, for helping me to study the subcellular localization of PRAMEY using the powerful electron microscope. I would like to thank
Dr. Tatiana Laremore, for leading me to explore the protein world using 2-D electrophoresis and mass spectrometry. Your knowledge and expertise greatly facilitated my research.
I would like to thank Dr. Cooduvalli Shashikant and Dr. K. Sandeep Prabhu for the suggestions on protein sample process. I would like to thank Dr. Peter Sutovsky and
Dr. Richard Saacke for the help on iEM data interpretation. I would like to thank Dr.
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Srinivasan Vijayaraghavan (Kent State University) for providing the PP1γ2 and sds22 antibodies. I would like to thank Dr. Olga Maria Ocon-Grove for helping me set up and carry out the bovine IVF experiments. I would like to thank Kate Antony for taking care of the mice for me.
Furthermore, I would like to thank my current and former lab mates, Dr. Bhavesh
Mistry, Dr. Ti-Cheng Chang, Xiangpeng Yue and all the undergraduate students in our lab. We are a fantastic group and I am incredibly grateful for all your support. I would like to thank my dear friends and colleague, Jackie Yun Ying, Dr. Liying Ma, Dr. Xi
Tian, Tianyanxin Sun and John Cantolina. I could always ask you for advice whether related to life or research projects. I would also like to thank the faculty and staff in the
Department of Animal Science, who sustain a collaborative environment that has augmented my education.
Finally, I would express my deepest thanks to my family for always being there for me. I dedicate this work to my parents and I am grateful for all of your love and support.
Chapter 1
Review of the Literature
1. Cancer/testis (CT) antigen
Cancer/testis (CT) antigen genes refers to a special set of genes which are normally expressed only in male testis and abnormally activated in various cancers (Chen et al., 1997). Later studies revealed that some CT antigens are also present in ovary and trophoblasts (Gjerstorff et al., 2007; Koslowski et al., 2004; Simpson et al., 2005).
Therefore, an alternative name, “cancer/germline antigen” was proposed (Akers et al.,
2010), though most people still use the term “cancer/testis antigen”. CT antigens are absent or expressed at a very low level in normal somatic tissues (Simpson et al., 2005).
There are 158 CT antigen families identified, with more than 277 members known to date
(CTDatabase, http://www.cta.lncc.br/). This section gives a general review of CT antigens, while research findings on PRAME are reviewed in section 2.
1.1 CT antigens in cancer
CT antigens are frequently expressed in a wide variety of cancer types, while rarely observed in a few cancers including colon cancer and renal cancer (Hofmann et al.,
2008; Scanlan et al., 2002; Simpson et al., 2005). The expression of CT antigens also varied within a certain cancer type and were correlated with the stages of cancer
2 progression (Cheng et al., 2009; Kim et al., 2010; Koop et al., 2013). In previous expression studies, 53% of breast cancer, 74% of melanoma and 67% of lung cancer patients expressed the tested CT antigens in the tumor cells; while the other cancer patients lacked expression of those CT antigens (Sahin et al., 1998; Tajima et al., 2003).
More interestingly, cancer patients tended to co-express several CT antigens simultaneously (Glazer et al., 2009; Sahin et al., 1998; Smith et al., 2009). The expression of different CT antigens seems to be coordinated, which suggests a common molecular mechanism of gene regulation (Akers et al., 2010).
Epigenetic modification was proposed as the key mechanism to regulate CT antigen expression in cancer cells (Karpf, 2006). Both global and promoter specific DNA hypomethylation were associated with CT antigen gene expression (De Smet et al., 1996,
1999). Global DNA hypomethylation was observed in various tumors, which usually affects repetitive DNA sequences. Global DNA hypomethylation can lead to the hypomethylation of CpG-rich promoters (Ehrlich, 2002; De Smet et al., 1996;
Woloszynska-Read et al., 2008). The promoters of most CT antigen genes contained CpG islands or CpG-rich regions (Illingworth and Bird, 2009), which were methylated under normal conditions. During tumorigenesis, some CpG sites of the CT antigen gene promoter were hypomethylated, which was correlated with CT antigen expression (Karpf et al., 2009).
The abnormal expression of CT antigens in cancer may play a fundamental role in tumorigenesis. For example, the extensively studied MAGEA (melanoma antigen family
A) members are involved in several signaling pathways that have multiple regulatory roles in cancer. MAGEA1 was the transcription suppressor of NOTCH1 pathway, in
3 which MAGEA1 bound to SKI-interacting protein (SKIP) and recruited histone deacetylase (Laduron et al., 2004). MAGEA11 was involved in the regulation of androgen-receptor function by modulating its internal domain interactions (Bai et al.,
2005). The expression of MAGEA1, MAGEA2 or MAGEA3 in human cell lines resulted in the resistance to tumor necrosis factor (TNF)-mediated cytotoxicity (Park et al., 2002).
More importantly, the overexpression of MAGE2 and MAGE6 rendered human cancer cells resistant to chemotherapeutic drug (PACLITAXEL and DOXORUBICIN) (Simpson et al., 2005), which was considered as a super invasive phenotype during malignant transformation (Glynn et al., 2004). There were also functional studies in other CT antigens. G antigen (GAGE) might directly contribute to the malignant phenotype in tumorigenesis by reducing apoptosis. GAGE-7C rendered the transfected Hela cells resistant to interferon-γ (IFNG) induced apoptosis or death receptor Fas/CD95/APO1- induced apoptosis (Cilensek et al., 2002). Some CT antigens might be involved in controlling gene expression, e.g., synovial sarcoma X [SSX] appeared to localize in nucleus and function as a transcriptional repressor (Thaete et al., 1999). The expression of meiotic-related CT antigens (such as synaptonemal complex protein 1 [SCP1] and
SPO11) in cancer was thought to have a role in abnormal chromosome segregation during cancer development (Simpson et al., 2005).
CT antigens are highly antigenic, as many CT antigens were discovered by screening the cDNA expression libraries with antibodies from the cancer patients (Old and Chen, 1998; Sahin et al., 1995). Meanwhile, CT antigens were restrictively expressed in tumor and crucial for tumorigenesis. These features enlightened researchers to use CT antigens as targets for cancer immunotherapy (Simpson et al., 2005). However, due to the
4 complexity of tumorigenesis, one cancer patient usually expressed multiple CT antigens, which increased the difficulty of designing effective cancer vaccines. In clinical trials, CT antigen based immunotherapy was shown to shrink the tumor, but didn’t significantly increase the cancer patient survival rate (Fratta et al., 2011; Kawada et al., 2012). More efforts are needed to understand the role of CT antigens in tumorigenesis and to use them in cancer treatment.
1.2 CT antigens in testis
The testis is not a tumor, but it specifically expresses CT antigens. Although CT antigens can induce an immune response in cancer patients, they are not exposed to the host immune system in the testis (Wong and Cheng, 2005). The specialized junctions that form blood-testis barrier (BTB) separate the apical compartments of seminiferous epithelium from the host immune system. In addition, some secretory factors from Sertoli cells, e.g. interferons and cytokines, also help to maintain the immune privilege status in testis (Jégou et al., 1995; Mruk and Cheng, 2004).
Many CT antigens are expressed at specific stages of spermatogenesis. For example, SPA17 (sperm autoantigenic protein 17, also known as SP17) is selectively expressed in spermatozoa (Chiriva-Internati et al., 2009; Kong et al., 1995; Lea et al.,
1996). SCP1 (synaptonemal complex protein 1) is expressed from zygotene to diplotene spermatocytes, during which meiosis occurs (Meuwissen et al., 1992). The two alternatively spliced forms of ACRBP (acrosin binding protein), ACRBP-W and
ACRBP-V5, are expressed at different stages: ACRBP-W was found to be expressed
5 from pachytene spermatocyte until mature spermatozoa (Baba et al., 1994; Kanemori et al., 2013); the expression of ACRBP-V5 was restricted from pachytene spermatocytes to round spermatids (Kanemori et al., 2013). The diverse expression patterns of CT antigens suggest they are involved in different stages of spermatogenesis.
Although the biological functions of CT antigens remain largely unknown, CT antigens may play a wide range of different roles, including, spermatogonia stem cell self-renewal, e.g., piwi-like RNA-mediated gene silencing 2(PIWIL2) (Cox et al., 2000;
Saxe and Lin, 2011), pairing of the homologous chromosomes during meiosis, e.g., Scp1
(Meuwissen et al., 1992; Türeci et al., 1998), proacrosin regulation and acrosomal matrix disassembly, e.g., ACRBP (Baba et al., 1994; Kanemori et al., 2013) and sperm-egg interaction, e.g., SPA17 (McLeskey et al., 1997; Primakoff and Myles, 2002). With the development of technology/experimental methods, more CT antigens will be studied and we expect to have a comprehensive understanding of their roles during spermatogenesis.
2. Preferentially expressed antigen in melanoma (PRAME)
2.1 The discovery of PRAME
Human PRAME was first discovered in the melanoma cell lines MEL.B, which expressed an antigen recognized by a HLA-A24-restricted autologous cytolytic T lymphocytes (CTL) clone (Ikeda et al., 1997). PRAME was also known as OPA interacting protein 4 (OIP4), as the C-terminal of PRAME was identified to bind with
OPAP proteins by yeast two-hybrid assay (Williams et al., 1998). The human PRAME
6 gene (Gene ID: 23532) was located on the reverse strand of chromosome 22 (22q11.22).
PRAME had five alternative transcript variants (NM_006115.3, NM_206953.1,
NM_206954.1, NM_206955.1, and NM_206956.1). Those transcript variants mainly differed on the 5' UTR region (NM_206955.1 also contains an alternative first exon).
However, all five variants encoded the same protein, which was 509 aa in length and the predicted molecular weight was ~ 58 kDa.
2.2 PRAME is a largely expanded gene family in eutheria
Similar to many other CT antigen genes, PRAME has expanded in the genome.
Bioinformatic analysis suggested that the PRAME gene family may have originated between approximately 95 and 85 million years ago (MYA, Birtle et al., 2005; Springer et al., 2003). Researchers also found that members in the PRAME gene family expanded independently within the primate, bovid and rodent lineage (Birtle et al., 2005; Chang et al., 2011). After the divergence from the last common ancestor, PRAME genes in each lineage expanded by both rapid duplication and pseudogene creation. Two large segmental duplications in human PRAME genes were estimated to occur recently within the past 3 MY (Birtle et al., 2005). There are at least 22 copies of PRAME/PRAME-like genes and 10 pseudogenes in the human genome (Birtle et al., 2005; Chang et al., 2011).
The Prame gene family is the third largest gene family in the mouse genome, with about
90 copies of genes and pseudogenes (Church et al., 2009). There are roughly 30 copies of
PRAME family members in the bovine autosomes (based on the bovine genome draft assembly by the University of Maryland), and 2~30 copies on the Y chromosome (Yue et
7 al., 2013). However, no PRAME ortholog was identified in the opossum, platypus, chicken, frog and zebrafish, suggesting that the PRAME gene family was eutheria- specific (Chang et al., 2011).
Paralogs for PRAME genes in human were named as PRAME family member 1
(PRAMEF1), PRAME family member 2 (PRAMEF2)… until PRAME family member 23
(PRAMEF23) (http://www.genenames.org/genefamilies/PRAME). PRAME-Like
(PRAMEL) was later approved to be a pseudogene and renamed as PRAME family member 24, pseudogene (PRAMEF24P). Among the 10 copies of pseudogenes identified in the human genome (Birtle et al., 2005), only PRAMEF24P was named. All the above
PRAME paralogs (except the pseudogene PRAME24P) was expressed in melanoma. The nomenclature of the PRAME paralogs in mouse, bovine and other eutherian mammals is not standardized, for example, the mouse PRAME paralogs were named as Prame-like
(Pramel1-7), Prame-family member (Pramef1-17), and Oogenesin (Oog1-4), etc.
(http://www.ncbi.nlm.nih.gov/). The lack of a standard nomenclature for this gene family lead to different names and gene symbols in literature and databases.
2.3 PRAME is a leucine rich repeat (LRR) protein
Leucine-rich repeat (LRR) is a protein structural motif, which usually contains a conserved sequence LxxLxLxxN/CxL (L can be leucine, valine, isoleucine and phenylalanine, Kajava, 1998; Kajava et al., 1995). Over 2000 LRR proteins were identified in viruses, bacteria, arcaea and eukaryotes (Enkhbayar et al., 2004a). Protein crystal structure and 3D structure modeling studies revealed that LRR proteins tended to
8 fold into a horseshoe structure, which provided the structural framework for protein- protein interactions (Enkhbayar et al., 2004a; Kobe and Deisenhofer, 1994). It is believed that LRR proteins are involved in a variety of molecular recognition processes such as signal transduction, cell adhesion, DNA repair, recombination, transcription, RNA processing and immune response (Enkhbayar et al., 2004b).
PRAME belongs to the LRR protein family. The human PRAME contains 13 predicted LRR domains (Birtle et al., 2005; Kajava, 1998; Wadelin et al., 2010) and
21.8% of PRAME residues were leucine or isoleucine (Wadelin et al., 2010). The predicted bovine PRAME protein horseshoe structure is shown in Figure 1-1 (Chang et al., 2011). In human cancer cell lines, Cul2 (Cullin family member 2), Elongin C, O- sialoglycoprotein endopeptidase (OSGEP) and L antigen family member 3 (LAGE3) were identified as the interaction partners of PRAME (Costessi et al., 2011, 2012). It was proposed that PRAME functioned as the bridge to link Cullin2 Ligases (through Cul2 and
Elongin C) and the EKC Complex (through OSGEP and LAGE3, Costessi et al., 2012).
However, the interaction partner(s) of PRAME in testis have not been reported to date.
9
Figure 1-1. The 3D structure of PRAME protein showing its horseshoe shape (Chang et al., 2011). The outer layer of PRAME protein is composed of α-helices and the inner layer of parallel β-strands. The model was built based on the PRAME gene (GenBank acc. no. XM_001256020.1) on bovine chromosome16. The predicted DNA binding site is highlighted in orange. The LXXLL motifs are highlighted in pink.
10 2.4 Expression and function of PRAME in cancer
After the identification of PRAME in melanoma, leukemia and lung cancer (Ikeda et al., 1997), the expression of PRAME and many other CT antigens in tumors was studied by large-scale microarray analysis. Consequently, the PRAME transcripts were over expressed in a large variety of cancers, including sarcoma, cervical squamous cell carcinoma, neuroblastoma, and adrenal tumors (Allander et al., 2002; Kilpinen et al.,
2008). Also, several studies showed that the expression of PRAME was associated with progression and/or poor outcome in melanoma (Haqq et al., 2005), neuroblastoma
(Oberthuer et al., 2004), serous ovarian adenocarcinomas (Partheen et al., 2008) and chronic myeloid leukemia patients (Radich et al., 2006).
Custom and commercial antibodies against human PRAME were developed to study the expression of the PRAME protein in cancer cells. Using a custom polyclonal antibody against the internal region of PRAME (peptide sequence
FPEPEAAQPMTKKRKVDG), a 58 kDa protein was detected in human melanoma cell lines (Epping et al., 2005) and K562 cells (human immortalised myelogenous leukemia line, Costessi et al., 2011). Commercial antibodies (Abcam 32185; Novus Biologicals
H00023532-B01P) also detected proteins of the expected size (58 kDa) in leukemic cells or carcinoma cells (Brenne et al., 2012; Partheen et al., 2008; Tanaka et al., 2011).
However, in another study, two bands (75 kDa and 58 kDa) were detected using the same antibody (Abcam 32185) in cells transfected with PRAME constructs (Quintarelli et al.,
2008). There was a report that a single 33 kDa band was detected using a monoclonal
PRAME antibody in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma
11 (MCL) cell lines (Proto-Siqueira et al., 2006). It is not clear why those detected proteins were different from the expected size, though it could be due to post-translational modifications, e.g., phosphorylation, or protein cleavage.
The subcellular localization of PRAME was studied in cancer cell lines and cell lines that expressed recombinant PRAME. The native PRAME protein was localized in both nuclear and cytoplasmic regions in several human cancer cell lines, including HL60
(promyelocytic leukemia cells), K562 (myelogenous leukemia), JURKAT (T cell leukemia) and U937 (histiocytic lymphoma) cells (Wadelin et al., 2010). In PRAME-
GFP or PRAME-FLAG transfected cell lines, the recombinant PRAME localized in nucleus and/or cytoplasm, depending on the cell types: PRAME-GFP was detected in the nuclear region of CHO cells (Tajeddine et al., 2005), in both nuclear and cytoplasm of
Hela cells, while mainly localized in the cytoplasm in U2OS cells (Wadelin et al., 2010).
The high expression of PRAME was correlated with chromosomal translocation in acute myeloblastic leukaemias, in which the genes for AML1 (also known as RUNX1, runt-related transcription factor 1) and ETO (also known as RUNX1T1, runt-related transcription factor 1) transcription factors are fused (Baren et al., 1998). In chronic myeloid leukemia (CML) cell models, PRAME was induced by the over-expression of
BCR/ABL, which was a fusion gene resulted from Philadelphia chromosome (Watari et al., 2000). How the AML1-ETO and BCR-ABL fusion proteins contributed to PRAME up regulation remains unknown. In melanoma cells, PRAME was shown to act as a dominant repressor of retinoic acid receptor (RAR) signaling (Epping et al., 2005).
PRAME prevented RAR activation and gene transcription by binding to the RAR and recruiting polycomb proteins, which resulted in the inhibition of RAR-related cell
12 proliferation arrest, differentiation and apoptosis (Epping et al., 2005). In another study, over expression of SOX9 in melanoma cells down regulated PRAME expression and restored RAR signaling (Passeron et al., 2009). In contrast, PRAME was not associated with RAR signaling in primary acute myeloid leukemia (Steinbach et al., 2007). Recent studies showed that PRAME was a subunit of a Cullin2-based E3 ubiquitin ligase in leukemia cells, which was involved in nuclear transcription factor Y (NFY)-mediated transcriptional regulation (Costessi et al., 2011, 2012). These studies indicated the functional diversity and complicated regulation mechanisms of PRAME in different cancer types.
2.5 Expression and Function of PRAME in reproduction
There are relatively few studies examining the role of PRAME gene family in reproduction. The human PRAME mRNA is highly expressed in testis, with a very low level of expression in ovaries, endometrium and adrenals (Ikeda et al., 1997; Kilpinen et al., 2008). Mouse is the most commonly used model to study the PRAME gene family.
PRAME genes can be divided into four groups depending on where they are expressed in mouse: testis (Prame, Pramel1 et al.) (Mistry et al., 2013; Wang et al., 2010), ovary
(Oogenesin-1, Oogenesin-2, Oogenesin-3 and Oogenesin-4 et al.) (Minami et al., 2003;
Monti and Redi, 2009), embryo (Pramel4, Pramel5, Pramel6, Pramel7 et al.) (Bortvin et al., 2003; Casanova et al., 2011) and those expressed in both male and female germlines and some of the somatic tissues (Pramef8, Pramef12, Pramel et al.) (Mistry et al., 2013).
13 As the representatives of ovary-specific/predominant PRAME family members,
Oogenesin-1, Oogenesin-2, Oogenesin-3 and Oogenesin-4 were first identified as a cluster of genes on mouse chromosome 4 (Minami et al., 2003; Monti and Redi, 2009).
Oogenesin-1 is expressed in the oocytes of all stages (from primordial to antral follicles); while Oogenesin-2, -3 and -4 are only expressed from primary to preovulatory follicles
(no expression detected in primordial follicles). Interestingly, Oogenesin-1 is also expressed in the early embryos (from one-cell to four-cell stage), with the protein localized in the nuclear region. The predicted molecular weight of OOGENESIN-1 is 58 kDa (predicted from ExPASy http://web.expasy.org/compute_pi/). However, by western blot analysis, OOGENESIN-1 was detected as a single 46 kDa band in oocyte, one-cell and two-cell embryos, while two bands (46 kDa and 58 kDa) were detected in four-cell embryos. It was suggested that OOGENESIN-1 played a role in oogenesis and zygotic transcription of early preimplantation embryos (Minami et al., 2003).
Pramel4, Pramel5, Pramel6, Pramel7 represented the embryo-specific PRAME family members. Pramel6 and Pramel7 are both restrictively expressed to the preimplantation embryo with no expression at later stages (Casanova et al., 2011).
Pramel6 mRNA is homogeneously expressed in all cells of the morula and blastocyst.
Pramel7 mRNA is exclusively expressed in the interior part of the morula and the inner cell mass of the blastocyst. The overexpression of Pramel7 in embryo stem cells (ESCs), but not Pramel6, could induce the up regulation of pluripotency genes, which resulted in the suppression of ESCs differentiation. Pramel7 was recognized as an essential factor in
LIF/STAT3-dependent self-renewal in ESCs. It was proposed that Pramel7 functioned via binding to other proteins instead of directly acting as a transcription factor during
14 embryo development; the protein interaction partners of Pramel7 were not identified
(Casanova et al., 2011).
Wang et al. identified expression of mouse Pramel1 in testis and spermatogonia, which suggested a role of Pramel1 in early spermatogenesis (Wang et al., 2001). Our recent data indicated that PRAMLE1 was expressed in the acrosome and flagellum of sperm (Mistry et al., 2013). In bovine, PRAME comprised multiple copies on chromosome 16, and a single copy on chromosome 17. The chromosome 17 copy
(PRAME17) underwent an autosome-to-Y transposition during evolution, resulting in the
PRAMEY subfamily (Chang et al., 2011). PRAMEY is exclusively expressed in testis
(Chang et al., 2011). PRAMEY is unique to the bovid lineage, and the transposition of this gene family to the Y chromosome suggests an important role of PRAMEY in spermatogenesis and male fertility. By in situ hybridization, the PRAMEY RNA was specifically localized in spermatids; interestingly, the antisense RNA was broadly expressed across the seminiferous tubules of adult testis (Chang et al., 2011). The subcellular localization and functions of testis-specific PRAME members have not been studied, and thus, became one of the main objectives of this research project.
3. Spermatogenesis
Spermatogenesis is a complex cellular transformation process within the seminiferous tubule of testis, in which the diploid spermatogonial stem cells ultimately develop into haploid spermatozoa (Hess and Renato de Franca, 2008). Spermatogenesis
15 has been widely studied in different species since 1950s, with focus on several model animals including mouse, rat and bovine.
3.1 Phases of spermatogenesis
Spermatogenesis is characterized by three functional phases: mitosis, meiosis and spermiogenesis (Russell et al., 1993). In the proliferation phase, three types of spermatogonia are present: type A (undifferentiated spermatogonia), intermediate and type B. Some undifferentiated type A spermatogonia can self-renew to ensure maintenance of the stem cell pool, while others differentiate into type B spermatogonia
(de Rooij, 1998). Type B spermatogonia divide by mitosis to form two primary spermatocytes, which then enter the meiosis phase. Primary spermatocytes underwent the first meiotic division (Meiosis-I) to generate secondary spermatocytes. Prophase of meiosis-I is composed of a prolonged period, and there were four stages: preleptotene, leptotene, zygotene and pachytene. After that, the second meiotic division (Meiosis-II) happens rapidly and results in the production of haploid round spermatids (Handel and
Schimenti, 2010). In the spermiogenesis phase, round spermatids are transformed into elongated spermatids and eventually highly differentiated spermatozoa (O’Donnell et al.,
2011).
Spermiogenesis in mouse is divided into 16 steps (Figure 1-2) based on the morphological changes of spermatids: step 1-8 spermatids were rounds spermatids; step
9-16 spermatids were elongating spermatids (Ahmed and de Rooij, 2009).
Spermatogenesis occurrs in a time regulated manner, therefore the fixed association of
16 different germ cell types exists within the seminiferous tubules. “Stages” were used to define the various cellular associations. For example, in mouse, the stage 1 seminiferous tubule includes four types of germ cells (type A spermatogonia, pachytene spermatocyte, step 1 spermatids and step 13 spermatids); while stage 10 includes three types of germ cells (leptotene spermatocyte, pachytene spermatocyte and step 10 spermatids). The number of stages varies among species: there were 6 stages defined in human (Clermont,
1963), 12 stages in bull (Figure 1-3) (Berndston and Desjardins, 1974), 12 stages in mouse (Figure 1-2) (Oakberg, 1957) and 14 stages in rat (Perey et al., 1961).
17
Figure 1-2. Mouse stages in the cycle of the seminiferous epithelium (Hess and Renato de Franca, 2008). State I-XII was characterized during spermatogenesis. Spermatogonia (A, In, B); spermatocytes (PI: preleptotene, L: leptotene, Z: zygotene, P: pachytene, D: diakinesis, Mi: meiotic division); round spermatids (1-8); elongated spermatids (9-16). Image reproduced with kind permission of Springer in the format Journal via Copyright Clearance Center.
18
Figure 1-3. Bovine stages in the cycle of the seminiferous epithelium (Berndston and Desjardins, 1974).
State I-XII was characterized during spermatogenesis. Spermatogonia (A, In, B1, B2); spermatocytes (PI: preleptotene, L: leptotene, Z: zygotene, P: pachytene, II: secondary spermatocytes); round spermatids (1-7); elongated spermatids (8-14). The lateral profile of steps 10-14 spermatids were also included.
19 As just mentioned above, spermatogenesis is a timely ordered event; in other words, in any one area of seminiferous tubules, stage 1 would be followed by stage 2, state 3…until completed. The period to finish one round of all the stages is called a
“cycle”. For example, the cycle of spermatogenesis is ~ 12 days in rat (Clermont,
Leblond and Messier, 1959). Interestingly, a successive order of the stages was also observed along the length of seminiferous tubules. The length of seminiferous tubules which covered a complete series of stages is called a “wave” (Perey et al., 1961). For example, the average length of one wave of spermatogenesis was ~ 2.6 cm in mouse and
3.2 cm in rat (Perey et al., 1961).
3.2 The first wave of spermatogenesis
In adult males, spermatozoa are produced asynchronously, which means different area of seminiferous tubules could be at different stages of spermatogenesis (Desjardins and Ewing, 1993). The adult male testis contains numerous cell types and it is challenging to identify genes at a specific cell type. However, during the first wave of spermatogenesis in prepubertal male, germ cells multiply and differentiate synchronously
(NEBEL et al., 1961). Therefore, prepubertal testis of a given age have relatively homogenous cell types, which provides a powerful tool to study spermatogenesis. The analysis of gene expression based on stages of testis development also reveals the simultaneous progression of spermatogenesis. Stage-specific transcripts were identified by comparing the transcriptome of prepubertal mouse testes at a given age (Schultz et al.,
2003; Shima et al., 2004).
20 Before birth, primordial germ cells (PGCs) are formed from epiblast cells
(Richardson and Lehmann, 2010; Saitou, 2009). Primordial germ cells (PGCs), which are the precursors to sperm, migrate during embryonic development (Richardson and
Lehmann, 2010). PGCs became gonocytes once they reach the somatic gonadal (Sertoli cells) precursors. After birth, the gonocytes move to the basement membrane of the tubule and form type A spermatogonia (Vergouwen et al., 1991). The germ cell types were characterized in each stage of postnatal testis development in mice (Auharek and de
França, 2010; Vergouwen et al., 1993). In C57BL/6J mice, at 1, 5, 10 and 15 days of age, the most advanced type of germ cell were gonocytes, differentiated spermatogonia, pre- leptotene spermatocytes and pachytene spermatocytes, respectively. Round spermatids were seen at day 20 and were followed by step 9/10 spermatids at day 28. The full establishment of spermatogenesis occurred at about 5 weeks of age in mice (Vergouwen et al., 1993). At completion of the first wave of spermatogenesis, the systematically arranged germ cells (spermatogonia, spermatocytes and spermatids) were observed in all seminiferous tubules.
In bovine, the testicular development is according to when sperm first appear
(“first sperm”) and when the ejaculate contains 50 million s perm. The mean age of “first sperm” was 258 days in the above study evaluating 6 breeds of beef bulls, which ranged from 236 days (Brown Swiss) to 268 days (Hereford-Angus crossbreed). The first production of an ejaculate containing at least 50 million sperm with minimum 10% motility was defined as the age of puberty. The mean age of puberty was 294 days with a range from 264 days to 326 days in different breeds.
21 3.3 Acrosome biogenesis
The acrosome is a unique granular vesicle of mature spermatozoon, which contains hydrolytic enzymes and plays an important role during fertilization. The acrosome is formed through an integrated process of vesicle production, trafficking and fusion (Abou-Haila and Tulsiani, 2000). Acrosome biogenesis started from the late pachytene spermatocyte phase of meiosis and continues until the spermiogenesis
(Anakwe and Gerton, 1990; Escalier et al., 1991; Kashiwabara et al., 1990). At the beginning, proacrosomal vesicles were formed from Golgi apparatus of pachytene spermatocytes. These vesicles are filled with dense, granular components and are called proacrosomal granules. These small proacrosomal granules fused into a large single acrosomal granule within a large vesicle during the Golgi phase of spermiogenesis.
During the subsequent capping phase, the acrosome granule grow in size by transporting newly synthesized protein from Golgi apparatus. The acrosomal granule and the sperm head cap composed of the acrosomic system. The acrosomal granule condensed and flattened, and began to elongate in the ends of capping phase. During acrosomal phase, the acrosomal system migrated over the nuclear surface of elongated spermatids and started morphological changes. The acrosome finally matured into a thin Periodic acid-
Schiff (PAS)-positive structure during the maturation phase of spermiogenesis (Anakwe and Gerton, 1990; Escalier et al., 1991; Kashiwabara et al., 1990). The morphological changes of acrosome biogenesis have been well documented; however, the molecular basis still remains to be studied.
22 The morphology changes of acrosome during spermatogenesis were carefully characterized in several species. In the mouse, proacrosomal vesicles and granules were formed in step 2/3 spermatids. The acrosome vesicle began to flatten since step 4/5 spermatids, until covered about 1/3 of the nuclear surface in step 8 spermatids. With the beginning of spermatid elongation, acrosome migrated and underwent morphological changes. The acrosome finally matured into a hook-shaped structure with protrudes at the apex (Hess and Renato de Franca, 2008). In bovine, the acrosome biogenesis started from step 2 round spermatids, in which two or more small PAS+ proacrosomal granules were present within the Golgi zone. The fusion of those proacrosomal granules resulted in a single, centrally located acrosomal granule in step 3 round spermatids. The head cap of spermatids developed in step 5 round spermatids, which formed the acrosomal system with the acrosomal granule. The acrosomal granule and head cap were distinguishable in step 5 to step 10 spermatids. Since step 11, the acrosomal granule and head cap fused together. In the later stages, the acrosome continued to develop and mature, with less change in morphology and more changes in content (Berndston and Desjardins, 1974).
3.4 Structure of the mature acrosome
The membranes surrounding the acrosome lumen can are outer acrosomal membrane (OAM) and inner acrosomal membrane (IAM). OAM is close to the plasma membrane, while IAM is associated with nuclear membrane. Proteins in the acrosome are either soluble constitute or component of the acrosomal matrix, depending on whether the protein can be solubilized by non-ionic detergent such as Triton X-100 (Huang et al.,
23 1985). In other words, soluble constitute generally included soluble proteins and membrane extracts, while the acrosomal matrix was the membrane-free component of acrosome after Triton X-100 extraction. Alternatively, acrosomal matix could be defined as the electron dense material within the lumen of acrosome when examined by electron microscope (Buffone et al., 2008). Moreover, distinct domains within the acrosomal matrix could be defined using differential solubilization procedure or electron microscope. For example, the acrosomal matix of guinea pig sperm could be separated into three domains named M1, M2 and M3 (Foster et al., 1997; Olson et al., 1987).
Defining soluble constitute and acrosomal matrix could help to understand the process of organization of the acrosomal contents, and the controlled disassembly mechanism during acrosomal exocytosis (Buffone et al., 2008).
A significant feature of the acrosome is the presence of a variety of enzymes, such as protease, phosphatases, glycohydrolases, esterases and sulfatases (Tulsiani et al.,
1998a; Zaneveld and Jonge, 1991). Many enzymes are found in the acrosomal matrix
(Buffone et al., 2008), as well as membrane-bound (Honda et al., 2002).
Acrosin/proacrosin is the most widely studied acrosome enzyme, which is a trypsin-like serine protease. The enzymatically inactive form, proacrosin, is localized in the acrosomal matrix. The 55 kDa proacrosin is converted to 35 kDa mature acrosin by cleavage, which has protease activity (Hardy et al., 1991; Noland et al., 1989;
Westbrook-Case et al., 1994). Acrosin was thought to be essential for zona pellucida penetration during fertilization; however, the acrosin-null mice was found to be fertile
(Baba et al., 1994). Therefore, acrosin was proposed to play a role during acrosomal exocytosis by regulating or interacting with other acrosomal component proteins (Honda
24 et al., 2002). In addition to the enzymatic protease activity, acrosin also exhibited carbohydrate-binding activity and was characterized as a ZP-binding protein (Jones and
Brown, 1987; Jones et al., 1988; Töpfer-Petersen and Henschen, 1987; Urch and Patel,
1991).
3.5 Molecular mechanism of acrosome biogenesis
HRB (human immunodeficiency virus Rev-binding protein, also known as Rab or hRip), GOPC (Golgi-associated PDZ- and coiled-coil motif-containing protein) and
VSP54 (vacuolar protein sorting-associated protein 54) were defined as essential proteins for acrosome biogenesis by contributing to vesicle formation (Kang-Decker et al., 2001;
Paiardi et al., 2011; Yao et al., 2002). HRB was required for the docking and/or fusion of proacrosomal vesicles to form the single large acrosomal vesicle (Kang-Decker et al.,
2001). In the mouse Hrb-deficient sperm, the proacrosomal vesicles failed to fuse to a single acrosomal vesicle, which blocked acrosome development (Kang-Decker et al.,
2001). GOPC was identified as a Golgi-associated protein which might play a role in vesicle transport from the Golgi apparatus (Yao et al., 2001). Lack of GOPC lead to the fragmentation of acrosomes in early round spermatids, which resulted in the malformation of acrosome (Yao et al., 2002). VSP54 was a vesicle protein for sorting, which was a subunit of Golgi-associated retrograde protein (GARP) complex and required for retrograde traffic from endosomes to the trans-Golgi network (TGN)
(Liewen et al., 2005; Quenneville et al., 2006). The Vsp54 mutant mouse could not form a single acrosomal vesicle during spermatogenesis and was infertile (Paiardi et al., 2011).
25 Though HRB, GOPC and VSP54 were involved in vesicle fusion, transport and sorting, respectively, knock out or mutant of these proteins all finally resulted in globozoospermia (Kang-Decker et al., 2001; Paiardi et al., 2011; Yao et al., 2002).
Globozoospermia (also known as round-headed spermatozoa) is an infertility syndrome, the most prominent feature of which is the malformation or absence of the acrosome
(Kullander and Rausing, 1975; Lalonde et al., 1988; Singh, 1992). Interestingly, the absence of mitochondrial shealth (Kang-Decker et al., 2001) or abnormal arrangement of mitochondria (Paiardi et al., 2011; Yao et al., 2002) were observed in these mutants. The mitochondrial sheath was formed by migration of the mitochondria during spermatogenesis, which formed a sheath wrap around the midpiece of sperm flagellum.
Mitochondrial sheath was the power supply of sperm flagellum and played an important role in sperm motility (Sun, 2010). It was proposed that acrosome biogenesis and mitochondrial sheath formation shared similar proteins/pathways (Jan et al., 2012).
Interestingly, our study has shown that PRAME/PRAMEY was localized in both acrosome and flagellum of sperm, indicating a role of PRAME/PRAMEY in both acrosome biogenesis and sperm motility.
3.6 Modulation of sperm motility by PP1γ2 complexes
The spermatozoa is a highly differentiated and compartmentalized cell. The transcription and translation were almost completely stopped, therefore, post-translational modification plays an important role in sperm. Several phosphoprotein phosphatases
(PPPs) are expressed in testis, including PP1α, PP1β, PP1γ1, , PP1γ2, PP2A, PP2B, PP4,
26 PP5, PP6 and PP7 (Fardilha et al., 2011), indicating an important role of phosphorylation in testis/spermatogenesis. PP1 (protein phosphatase 1) was a major serine/threonine- phosphatase, which has more than 200 PP1 interacting proteins (PIPs) identified (Cohen,
2004; Johnson and Hunter, 2005). PP1 has three isoforms (α, β and γ) which were 90% identical (Cohen, 2002). Among them, PP1γ2 was identified as a testis/sperm specific isoform, which was specific expressed in sperm; in contrast, PP1γ1 was not present in sperm. PP1γ2 was expressed in the sperm flagellum including the midpiece, which was the power supply of sperm motility (Huang et al., 2002). PP1γ2 was also present in sperm head, suggesting a role in acrosome biogenesis/acrosome reaction (Huang et al., 2002).
It has been suggested that the sperm acquiring motility by the inhibition of PP1γ2 phosphatase activity. The immotile caput sperm become motile after transition to the caudal epididymis. Accordingly, PP1γ2 was active in caput epididymis and the phosphatase activity was inhibited in caudal epididymis (Mishra et al., 2003). Adding
PP1 inhibitors to immotile caput sperm resulted in the acquiring of motility, demonstrating PP1γ2 as a direct regulator of sperm motility (Vijayaraghavan et al.,
1996). Two of the LRR proteins, sds22 (also known as PPP1R7, protein phosphatase 1, regulatory (inhibitor) subunit 7) (Huang et al., 2002; Mishra et al., 2003) and TLRR (also known as PPP1R42, protein phosphatase 1, regulatory subunit 42) (Fawcett et al., 1971;
Wang et al., 2010), were identified as interaction partners of PP1γ2. Sds22 was mainly expressed in the sperm head, connecting piece and principle piece of flagellum, with relative weak expression in midpiece of flagellum (Huang et al., 2002). Sds22 was shown to bind to and inhibit the phosphatase activity of PP1γ2 in caudal sperm (Mishra et al.,
2003). In contrast, sds22 did not bind to PP1γ2 in caput sperm (Mishra et al., 2003). It
27 was suggested that the decrease of phosphatase activity by sds22 induced the sperm mobility initiation. TLRR was proposed to regulate the phosphorylation of proteins by localization PP1γ2 through spermatid manchette microtubules (Wang et al., 2010).
4. Fertilization and block of polyspermy
Fertilization is a process that includes the penetration of sperm into the oocyte, formation of the male and female pronuclear and the union of the two haploid genomes
(Chang, 1968). Normally, one oocyte is fertilized by one sperm, to ensure that the zygote contains exactly two copies of each chromosome. Polyspermy describes the event that an oocytes is fertilized by more than one sperm (Sato, 1979). Polyspermy generally results in an non-viable zygote. The polyploidy embryos were detected in 10~20% of spontaneous abortion in human (Hassold et al., 1980; Jacobs et al., 1978; Michelmann et al., 1986). Therefore, mammals have developed a variety of mechanisms to reduce polyspermy. Blocks of polyspermy occur at two levels on the mammalian oocyte: the zona pellucida (ZP) and the oocyte plasma membrane. The ZP block involves the exocytosis of cortical granules (CGs) of the oocytes, which renders the ZP to a form that cannot support sperm binding (Abbott and Ducibella, 2001). The mechanism of the membrane block in mammalian egg is not clear, but there are several evidences to support the existence of membrane block. Zona-free fertilized oocytes were challenged with a second insemination during in vitro fertilization (IVF), which eliminates the ZP block of polyspermy. The results showed that the very few sperm penetrated the cytoplasm membrane of the zona-free fertilized oocytes, indicating the zygote membrane
28 was not supportive for sperm interaction and function as a second block for polyspermy
(Horvath et al., 1993; Sengoku et al., 1995; Wolf, 1978; Zuccotti et al., 1991). The calcium signaling was believed to trigger the CG exocytosis and ZP modification. During fertilization and egg activation, a rise in cytosolic Ca2+ activates the effector protein
CaMKII and calcineurin, and possible other pathways (Krauchunas and Wolfner, 2013).
The detailed molecular basis of the block of polyspermy needs further study.
5. Summary and the objectives of this study
The PRAME protein belongs to a group of cancer/testis antigens that are predominantly expressed in normal testis and a variety of tumors, and involved in immunity and reproduction (Ikeda et al., 1997; Simpson et al., 2005). The PRAME gene has been amplified during evolution and constitutes a large gene family in eutherian mammals (Birtle et al., 2005; Chang et al., 2011; Church et al., 2009). PRAME has been transposed to and amplified on the bovid Y-chromosome, indicating an important role in male fertility (Chang et al., 2011). Members of the PRAME gene family encode leucine- rich repeat (LRR) proteins that fold into a horseshoe shape, which provides a versatile structural framework for the formation of protein–protein interactions in diverse molecular recognition processes (Epping et al., 2005; Kobe and Kajava, 2001; Wadelin et al., 2010). Although PRAME is known to play a regulatory role in cancer cells (Costessi et al., 2011, 2012; Epping et al., 2005), the function of PRAME in germ cells remains unknown. This thesis aims study the functional role of PRAME in spermatogenesis by focusing on two representative members of PRAME family, PRAMEY in cattle and
29 Pramel1 in mice. Understanding how PRAME function during spermatogenesis will provide novel insights into the molecular mechanisms that regulate germ cell morphogenesis.
30 Chapter 2
Characterization of the Bovine PRAMEY During Spermiogenesis, and its Potential Role in the Block to Polyspermy During Fertilization
1. Introduction
Throughout postpubertal male reproductive life, spermatozoa are formed from spermatogonial stem cells (SSCs). Spermatogenesis is a complex cellular transformation process within the seminiferous tubules of the testis, which can be divided into three functional phases: mitosis, meiosis, and spermiogenesis. During the last phase, spermiogenesis, haploid round spermatids are transformed into highly differentiated mature spermatozoa that are released into the seminiferous tubule lumen (Hess and
Renato de Franca, 2008; Leblond and Clermont, 1952).
Spermiogenesis begins with formation of the acrosome from Golgi-derived vesicles (Kawa et al., 2006). The acrosome is a Golgi-derived organelle overlying the anterior region of the sperm head. The acrosome is one of the defining features of sperm development (Martínez-Menárguez et al., 1996; Tang et al., 1982), and plays an important role during fertilization, including zona pellucida (ZP) binding/penetration and sperm-egg adhesion/fusion (Ikawa et al., 2010). The biogenesis of the acrosome starts from the late pachytene spermatocyte phase of meiosis and the detailed process has been described in several reviews (Anakwe and Gerton, 1990; Escalier et al., 1991;
Kashiwabara et al., 1990). At the beginning, proacrosomal granules developed from the
Golgi apparatus of pachytene spermatocytes. These small granules fuse into a large single acrosomal granule within the Golgi zone. The acrosomal granule and head cap form the
31 acrosomal system. The acrosomal granule enlarges during development by accumulation incoming proteins from the Golgi apparatus. In the following steps, the dense acrosomal granule flattens and extended over the sperm head. During the last half of spermiogenesis, the acrosome undergoes extensive morphological changes, which results in different shapes of acrosomes in different species. Also, additional modifications of the acrosome can occur during the transition in the epididymis (Toshimori, 1998; Tulsiani et al., 1998b).
During spermatogenesis, numerous germ-cell specific antigens are expressed in testicular cells. Interestingly, many of these antigens are absent or expressed at a very low level in normal somatic tissues, however, they are detected at a high level in various kinds of tumors (Cheng et al., 2011). Due to the restricted expression pattern in testis and cancer, these antigens are designated as cancer/testis antigens (CT antigens). Some CT antigens are also expressed in the ovary and trophoblast cells of the early embryo (Old,
2001; Simpson et al., 2005). During spermatogenesis, many CT antigens are expressed transiently at a specific stage (e.g. SCP1, Meuwissen et al., 1992); while some are expressed in several stages of spermatogenesis (e.g. Trophinin and PRAMEL1) (Mistry et al., 2013; Saburi et al., 2001). The stage-specific appearance of these antigens on male germ cells suggests that they play a role in spermatogenesis (Tulsiani et al., 1998a).
Preferentially expressed antigen of melanoma (PRAME) is a CT antigen discovered initially in melanoma cell lines MEL.B (Ikeda et al., 1997). Early studies found that PRAME was a dominant repressor involved in retinoic acid receptor (RAR) signaling in melanoma cells (Epping et al., 2005, 2007). Recent studies indicate that
PRAME is involved in NFY-mediated transcriptional regulation as a subunit of a
32 Cullin2-based E3 ubiquitin ligase in leukemia cells (Costessi et al., 2011, 2012). PRAME contains leucine-rich repeats (LRRs) motif, which primarily provides a structural framework for the formation of protein-protein interaction (Chang et al., 2011; Kobe and
Kajava, 2001; Wadelin et al., 2010). It has been reported that another LRR protein,
PPP1R7 (protein phosphatase 1, regulatory (inhibitor) subunit 7, also known as sds22) interacts with PP1γ2 in bovine caudal epididymal spermatozoa (Huang et al., 2002;
Mishra et al., 2003). PP1γ2 is a testis/spermatozoa specific phosphatase, which is a key component for regulatation of spermatozoa motility and male fertility (Fardilha et al.,
2011; Smith et al., 1996, 1999; Vijayaraghavan et al., 1996).
To date, few studies pertaining to the role of PRAME during spermatogenesis have been performed. Our recent study demonstrated that the mouse PRAMEL1, a member of the Prame gene family, was first expressed in the cytoplasm of pachytene spermatocytes in the 2-week-old mouse testis, and then translocated to the acrosomal head cap of round spermatids in the 3-week-old testis. In mature spermatozoa,
PRAMEL1 localized in the anterior acrosome region of spermatozoa, and was differentially expressed in the flagellum and cytoplasmic droplets (Mistry et al., 2013).
Like many of the other multiple copy CT genes, PRAME was amplified in Eutheria during evolution (Birtle et al., 2005; Chang et al., 2011). In bovine, there are multiple copies of PRAME on chromosome 16, and a single copy on chromosome 17. The latter underwent an autosome-to-Y transposition and amplification thereafter resulting in a Y- linked PRAME gene (PRAMEY) sub-family (Chang et al., 2011). Phylogenetic analysis indicated that this autosome-to-Y transposition event occurred during evolution in the bovid lineage only (Chang et al., 2011), suggesting an enhanced role of PRAMEY in
33 bovid spermatogenesis and male fertility. The copy number variation of PRAMEY was found to correlate with male reproductive traits and may be used as a valuable selection marker for male fertility (Yue et al., 2013). The objectives of this study were to characterize the detailed expression and subcellular localization pattern of the PRAMEY protein in bovine spermatids/spermatozoa, identify its protein interaction partner, and to assess the functional role of PRAMEY during spermatogenesis and fertilization.
2. Materials and methods
Testis collection, sperm isolation and protein extraction
Testes with intact tunica and attached epididymis were obtained from mature bulls at a local slaughter house (Nicholas Meat Packing Co., Loganton, PA). The testes were transported to the laboratory in ice-filled coolers and processed within two hours.
Testes of >10 bulls were collected for this project. The epididymi were briefly washed in ice-cold washing buffer (120 mM NaCl, 10 mM KCl, 10 mM Tris-HCl and supplied with
1 mM PMSF, 1 mM EDTA, 1 mM DTT, pH 7.4) to remove blood. The caput and caudal epididymal spermatozoa were isolated in modified phosphate buffered saline (PBS)
(containing 137 mM NaCl, 2.7 mM KCl, 10 mM Phosphate Buffer, supplied with 1 mM
PMSF, 1 mM EDTA, 1 mM DTT, pH 7.4). The isolated spermatozoa were washed twice in the modified PBS, aliquoted and pelleted. The sperm pellets were immediately frozen in liquid nitrogen.
34 The sperm pellets were extracted using different protein extraction buffers, including CelLytic Buffer (Sigma C3228), Pierce IP Lysis Buffer (Pierce 87787),
ReadyPrep Rehydration/Sample Buffer (BioRad 163-2106) and Laemmli Sample Buffer
(BioRad 161-0737). Protease Inhibitor Cocktail (Thermo 87785, containing AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin A and EDTA) and Phosphatase Inhibitor
Cocktail (Thermo 78420, containing sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate) were used as needed. The protein extraction method using CelLytic Buffer, IP lysis buffer and ReadyPrep Rehydration/Sample Buffer was as following: the sperm pellets were taken out of -80 °C freezer, added ice-cold extraction buffer (supplemented with protease inhibitor and phosphatase inhibitor cocktail) was immediately added and the pellet was re-suspended. After incubation on ice for 10 min, the lysates were centrifuged for 10 min at 13,000 g, at 4 °C. The supernatant were referred as sperm extracts in this study. Alternatively, when the Laemmli Sample
Buffer was used for the protein extraction (referred as Laemmli extracts), the sperm pellets were directly boiled at 95°C for 5 min in the Laemmli Sample Buffer supplemented with 5% β-mercaptoethanol.
Antibody production
A PRAMEY-specific custom antibody was produced by New England Peptide,
LLC (Gardner, MA). Two rabbits (New Zealand White - SPF) were injected with a synthetic peptide (CAQAGLKPEQA) as an antigen. The peptide sequence
“AQAGLKPEQA” was a unique sequence within the internal region of PRAMEY; the
35 terminal Cysteine residue (C) was added in order to facilitate coupling of the peptide to the carrier protein and affinity matrix. The antibody titers were determined by ELISA and the IgG fraction of the antibody was affinity purified. The PP1γ2 and sds22 antibodies were kindly provided by Dr. Srinivasan Vijayaraghavan (Kent State University) and described previously (Huang et al., 2002).
Gel electrophoresis and western blot
For one dimensional SDS-PAGE, the protein extracts were separated by 8%-16%
Precise Tris-Glycine gel (Thermo 25268). The gel was electronically transferred to PVDF membrane (Thermo 88518), blocked in 5% non fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBST). After being briefly washed in TBST, the membrane was incubated in primary antibody in blocking buffer (1.5 µg/ml) at 4 °C for overnight.
The membrane was washed three times for 5 min each and incubated in donkey anti- rabbit IgG-HRP (Santa Cruz, sc-2313, 1:5000) for 1 h. The reactive proteins were detected by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo 34094).
To prove the antibody specificity, pre-absorption of the anti-PRAMEY was conducted by incubating the antibody with 200-fold molar excess of peptide for 1 h at RT. The pre- absorbed antibody or pre-immune rabbit IgG (Thermo NC-100-P0) was used to replace the primary antibody as negative control.
The two-dimensional (2-D) electrophoresis was performed following the instruction of ReadyPrepTM 2-D Starter Kit (BioRad, 163-2105). The sperm protein was extracted using ReadyPrep Rehydration/Sample Buffer (Biorad 163-2106) supplied with
36 10% isopropanol, 12.5% (v/v) water-saturated isobutanol and 5% (v/v) glycerol
(Leimgruber, 2005). The caudal sperm pellet was suspended in the sample buffer and incubated on a shaker for 10 min. The supernatant was used as the protein sample for 2-D electrophoresis. Two parallel gels were run each time. The protein sample was applied to the IPG Strips (11cm, pH 3-10, BioRad 163-2014) by negative rehydration at 4 °C for overnight. Isoelectric focusing (first dimension separation) was performed in PROTEIN
IEF cell (BioRad 165-4000) for 5.3 h. Upon the completion of isoelectric focusing, the
IPG strips were equilibrated in equilibration buffer I (G-Biosciences 786-224, supplied with 20 mg/ml DTT) for 10 min and equilibration buffer II (G-Biosciences 786-224, supplied with 250 mg/ml iodoacetamide) for 10 min. The second dimension separation was performed using 8–16% Criterion™ TGX™ Precast Gel (BioRad 567-1101). After finishing electrophoresis, one gel was stained using coomassie blue (Thermo Scientific
24615), and the other gel was transferred to PVDF membrane and blotted using anti-
PRAMEY antibody as the procedure described above.
Indirect immunofluorescence
The testicular tissues were cut to small cubes (3 mm × 3 mm × 3 mm) and fixed in Bouin’s solution (RICCA chemical company R1120000-1A 1120-32) for 20 h in 4 °C.
The fixed tissues were dehydrated in 30% ethanol for 2 h, 50% ethanol for 2 h and 70% ethanol overnight. After several changes of 70% ethanol, the tissues were embedded in paraffin and cut to 4 µm fine sections. The staining procedure was described previously
(Mistry et al., 2013). Following deparaffinization and rehydration, the sections
37 underwent heat-induced antigen retrieval by boiling in sodium citrate buffer (10 mM
Sodium Citrate, 0.05% Tween 20, pH 6.0). After washing in PBS, the sections were blocked in 10% donkey serum in PBS for 1 h at RT, and then incubated with anti-
PRAMEY (1.5µg/ml in 0.1% Triton X-100) overnight at 4 °C. Following a wash step (3
× 5 min in PBS), sections were incubated with FITC-conjugated donkey anti-rabbit IgG
(sc-2090) (1:300) for 1 h at RT, washed again, and mounted with VECTASHIELD
Mounting Medium with DAPI (Vector, H-1200).
The testicular and epdidymal spermatozoa were isolated by cutting the testis and epididymis in PBS. The testicular and epdidymal spermatozoa were fixed in 4% paraformaldehyde on ice for 30 min. The fixed spermatozoa were washed three times in
PBS, placed on slides and allowed to air dry. For immunofluorescence staining, the sperm slides were rehydrated by PBS, blocked in 10% donkey serum for 1 h at RT. After washing with PBS, the slides were incubated with the anti-PRAMEY (1.5 µg/ml in 0.1%
Triton X-100) overnight at 4 °C, washed twice in PBS, and incubated in donkey anti- rabbit IgG-FITC (sc-2090) (1:300) for 1 h at RT. The slides were washed twice in PBS, mounted with VECTASHIELD Mounting Medium with DAPI (Vector, H-1200), and examined on an Olympus BX51 fluorescence microscope with DP Controller image software (Olympus America Inc., Melville, NY).
Immunogold electron microscopy
The testis tissue and caudal epididymal sperm were processed by the method of cryofixation and Lowicryl embedding (Maunsbach and Afzelius, 1998). The testis tissue
38 and sperm pellet were fixed with 4% paraformaldehyde plus 0.1% glutaradehyde for 1 h at RT, changed to fresh fixative and fixed for another 1 h at 4 °C. The sperm pellets were pre-embedded in agarose gel. The tissue and sperm pellets were cut to small blocks (< 0.5 mm in any direction), completely infiltrated with 2.3 M sucrose at 4 °C for overnight, and cryofixed in liquid nitrogen. Great care was taken not to warm the specimen since this step. The freeze-substitution was performed in Automatic Freeze Substitution System.
The frozen samples were transferred to 0.5% uranyl acetate in methonal which was kept at -90 °C in the freeze-substitution unit. Five hours later, most of the solution was withdrawn and fresh methanol/0.5% uranyl acetate solution was added. The temperature was raised to -80 °C for 24 hr. The specimens were then rinsed three times with pure methanol at -70 °C over a period of 8 h, and three times with pure methanol at -45 °C over a period of 20 h. The samples were infiltrated at 45 °C, with 1:1 mixture of methanol and Lowicryl HM20 for 6 h, 1:2 mixture of methanol and Lowicryl HM20 for
14 h, pure Lowicryl HM20 for 8 h with three changes, and pure Lowicryl HM20 for 24 h.
The samples were changed to fresh Lowicryl HM20 and polymerized with indirect UV light at -45 °C for 48 h and 0 °C for another 24 h. The specimen were taken to RT after completely polymerization and sectioned to 90 nm ultrathin sections on nickel grid by conventional ultramicrotomy. To check the morphology of the spermatids and spermatozoa, the sections were stained 6 min at 60 °C with 2% UA and 3 min at RT with
0.3% lead, and examined by transmission electron microscope (FEI Tecnai G2 Spirit
BioTwin, at Microscopy and Cytometry Facility, Huck Institutes of the Life Sciences).
For immunogold labeling, the sections were blocked by 1% BSA in PBS for 30 min, labeled with anti-PRAMEY (1.5 µg/ml) and 10 nm- or 5 nm-gold-conjugated goat anti-
39 rabbit IgG (Ted Pella, 15726) (1:50). The sections were stained with 2% uranyl acetate and examined in transmission electron microscope (FEI Tecnai G2 Spirit BioTwin, at
Microscopy and Cytometry Facility, Huck Institutes of the Life Sciences).
Immunoprecipitation(IP)/co-immunoprecipitation(co-IP)
The IP/co-IP was performed using Pierce Crosslink Immunoprecipitation Kit
(Thermo 26147) per the manufactory’s instruction. Briefly, the anti-PP1γ2 or anti-
PRAMEY antibody was bound to protein A/G plus agarose bead; the bonds between antibody and protein A/G were cross-linked by DSS (disuccinimidyl suberate). The sperm pellets were extracted using IP lysis buffer as described above. The protein extracts were pre-cleared by Pierce control agarose resin, and then incubated with anti-
PP1γ2 or anti-PRAMEY immobilized agarose beads in mini-spin columns overnight at 4
°C. The antigen (protein complex) was eluted from the beads by the elution buffer
(Thermo 21004). The input sample (total protein extracts) and the IP/Co-IP eluate were separated by SDS-PAGE and immunoblotted by anti-PP1γ2, anti-PRAMEY or anti- sds22. For protein identification purpose, the IP/co-IP eluate was separated by SDS-
PAGE and the corresponding gel slices were cut out and analyzed by mass spectrometry using Thermo LTQ Orbitrap (Thermo Fisher Scientific, Inc.).
40 Oocyte collection and maturation
The ovaries were collected from the reproductive tract of mature cows immediately after the sacrifice of the animals at the same slaughter house (Nicholas Meat Packing Co.,
Logaton, PA) where the testes were collected. The fresh ovaries were placed in 0.9% saline (38 °C) and transported to the laboratory within 2 h. Upon arrival in the laboratory, the ovaries were immediately rinsed in 0.9% saline (38 °C) to remove excess blood, and the oocytes were aspirated from visible ovarian follicles. The oocytes were matured in vitro in medium 199 (Invitrogen 31100-035) supplied with 10% bovine steer serum (v/v), sodium pyruvate (22 µg/ml), Glutamax (Life Technology 35050-061), folltropin (25
µg/ml), estradiol (2 µg/ml), and gentamicin (50 µg/ml) for 22-24 h at 38.5 °C in 5% CO2.
Sperm preparation for in vitro fertilization (IVF)
The freshly collected testes with attached epididymis were transported to the laboratory within 2 h in a Styrofoam box at a temperature that ranged between 25 °C to 28 °C. The caudal epididymis were dissected and rinsed with 0.9% saline to remove blood.
Epididymal sperm were harvested by making 5-6 incisions in the caudal epididymis, rinsed with sperm/oocyte washing medium (Minitube 19982-1200, supplied with 22
µg/ml sodium pyruvate and 7.5 µg/ml gentamicin) and placed in the 60 mm petri dish for
10 min to allow sperm to swim out into the medium. Approximately equal number of sperm from 3 bulls were pooled together and adjusted to the final concentration of 5 ×
107/ml. Sperm were incubated in sperm/oocyte washing medium containing: (a) no anti-
41 PRAMEY (control) and (b) 5 µg/ml anti-PRAMEY (treatment) for 1.5 h at 38.5 °C incubator.
Sperm-oocyte binding
The cumulus cells of matured oocytes were removed by pipetting in 200 µg/ml hyaluronidase. Thirty to thirty-five cumulus cell-free oocytes were inseminated with sperm from control or antibody-treated sperm in BoviPRO™ In vitro Fertilization
Medium (Minitube 19982/4115) supplemented with 10 ug/ml heparin, 1 mM of caffeine,
10 µM hypotaurine, 20 µM penicillamine and 1µM epinephrine. The final sperm concentration was 6 × 106/ml. The antibody concentration in the anti-PRAMEY antibody-treated group was 5 µg/ml. Oocytes and sperm were co-incubated for 6 h at
38.5 °C in 5% CO2. After co-incubation, oocytes were fixed in 4% paraformaldehyde and stained with phalloidin and DAPI. The number bound to each zona pellucida (ZP) was evaluated by fluorescence Olympus BX51 fluorescence microscope.
In vitro fertilization (IVF)
The mature oocytes with intact cumulus cells were inseminated as described above.
Oocytes and sperm were co-incubated in fertilization medium for 20-22 h at 38.5 °C in
5% CO2. After incubation, cumulus cells and accessory sperm were removed by pipetting, washed in sperm/oocyte washing medium and transferred to culture medium
(Minitube 19982/4115, supplied with 0.5 mg/ml myo-inositol, non-essential amino acids
42 and essential amino acids). After 24 h, the oocytes/embryos were fixed in 4% paraformaldehyde and stained with phalloidin and DAPI. The 2-cell, 4-cell embryos and other stages of embryos were evaluated by fluorescence microscopy as described above.
Statistical analysis
The IVF experiment was repeated twice and data from each experiment were pooled. To compare the differences between the control and antibody-treated groups, T-test was performed on the sperm-oocyte binding data using mean number of spermatozoa bound per ZP for each treatment, while χ2 test was performed on the data of polyspermy fertilization and embryo rate. The significance level for these tests was P < 0.05.
3. Results
The PRAMEY is identified as a 30 kDa protein in bovine spermatozoa
The peptide-specific antibody was produced for the analysis of PRAMEY by immunoblotting. Caudal epididymal spermatozoa were collected immediately after the sacrifice of animals, to minimize protein degradation. As shown in Figure 2-1, the anti-
PRAMEY antibody identified an immunoreactive protein with molecular weight of ~30 kDa in caudal epididymal spermatozoa. The specificity of the antibody was proved by peptide pre-absorption. After pre-incubating with the corresponding PYAMEY peptide, the immunoreactivity of the antibody was blocked and no band was detected (Figure 2-1).
In the other negative control experiment, anti-PRAMEY was replaced by the pre-immune
43 rabbit IgG, which also detected no band, confirming the specificity of this PRAMEY antibody (Figure 2-1).
We noticed that, in addition to the ~30 kDa major band, two minor bands, ~26 kDa and ~13 kDa in size, were also detected by the PRAMEY antibody under certain protein extraction conditions. In order to find out the reason(s), we performed western blot analyses with proteins extracted under different conditions, including different buffer compositions with or without protease inhibitors (see details in Methods). When the sperm proteins were extracted using the ReadyPrep Rehydration/Sample Buffer (BioRad
163-2106), or directly boiled in Laemmli Sample Buffer (BioRad 161-0737), a single 30 kDa protein was identified (Figure 2-2 A). However, when the moderate-strength buffer
(CelLytic buffer and IP Lysis buffer) was applied to protein extraction, which was formulated for co-IP analysis, the two minor bands (26 kDa and 13 kDa) were observed
(Figure 2-2 A). Despite this, moderate-strength buffers were used for co- immunoprecipitation, to minimize the destruction of protein-protein interaction.
The freeze-thaw method is another common method to lyse mammalian cells
(Bodzon-Kulakowska et al., 2007; Brasier and Fortin, 2001). To further investigate the possible reason for the presence of the minor bands, a carefully-designed freeze-thaw stability test was carried out. The fresh sperm pellets were snap-frozen, and then kept in
RT for different times (0 min, 5 min and 30 min) before boiling in Laemmli Buffer and loading to the gel for western blotting. We noticed that the appearance and the intensity of the minor bands related to the thawing time of the sperm pellets (Figure 2-2 B). The western blot result with the freeze-thaw sperm protein at 0 min (Figure 2-2 B) was almost identical to the results with extraction using the ReadyPrep Rehydration/Sample Buffer
44 (Figure 2-2 A). When the sperm pellets thawed at RT for 5 min, the two minor bands were clearly visible, although the 30 kDa band was still the major band. However, when the sperm pellets were thawed for 30 min, the 30 kDa band almost disappeared, and the two minor bands became much stronger (Figure 2-2 B). These results indicated that the
26 kDa and 13 kDa bands were most likely the degraded products of the 30 kDa peptide.
45
Figure 2-1. Identification of the bovine PRAMEY protein in caudal spermatozoa by western blot with the custom-made PRAMEY antibody. Protein extracts from the bovine caudal epeididymal spermatozoa (7 µg per lane) were separated by SDS-PAGE, transferred to a PVDF membrane and blotted by the affinity purified anti-PRAMEY antibody. PRAMEY was detected as a 30 kDa protein. As negative controls, the anti-PRAMEY antibody was pre-absorbed using the corresponding peptide, or replaced by the pre-immune rabbit IgG. No band was detected in negative controls. Numbers on the left indicated the molecular weight of standard proteins.
46
Figure 2-2. Comparison of the bovine sperm PRAMEY proteins extracted either by different buffers or at different freeze-thaw times. A. Protein extraction buffer effect. Caudal epididymal sperm were extracted using ReadyPrep Rehydration/Sample Buffer or CelLytic buffer and analyzed by western blot. A single 30 kDa band was shown in the protein extracted by ReadyPrep Rehydration/Sample Buffer, while three bands (30 kDa, 26 kDa and 13 kDa) were observed in protein sample extracted using CelLytic buffer. B. Sperm freeze-thaw stability test. The fresh caudal epididymal sperm pellets were frozen and kept in RT for different time (0 min, 5 min or 30 min) before protein extraction to test the freeze-thaw stability. A single 30 kDa band was detected in immediately extracted sample (0 min), while the two minor bands (26 kDa and 13 kDa) appeared in the sample thawed for 5 or 30 mins. Obviously, the relative proportion of the three bands varied: the two minor bands were relatively weak in the slightly thawed sample (thawed in RT for 5 min), and became stronger than the 30 kDa band in the completely thawed sample (kept in RT for 30 min).
47 The PRAMEY is expressed in pubertal and mature bull testes
The expressions of the bovine PRAMEY in testes of different developmental stages were examined by western blot. PRAMEY was detected in 8-m and mature testes, while no expression was detected in 20-d and 4-m-old testes (Figure 2-3). As bulls entered puberty at about 8 m of age, the initiation of PRAMEY expression in testis from the age of puberty and into maturity indicated that PRAMEY was involved in the late stages in spermatogenesis.
48
Figure 2-3. Temporal expression of the bovine PRAMEY protein during testis development. The protein was extracted from bull testes of different ages and analyzed by western blot. PRAMEY was expressed in 8-month and mature bull testes, while there was no protein detected in 20 day- and 4 month-old testes. Beta-ACTIN was used as a loading control. 20d: 20 day, 4m: 4 month, 8 m: 8 month.
49 The bovine PRAMEY protein is localized in the acrosomal region of spermatids and spermatozoa during spermiogenesis
The localization of PRAMEY was examined in the sections of the mature testis by indirect immunofluorescence staining. As shown in Figure 2-4 A, the PRAMEY staining covered about half of the nuclear surface in round spermatids (step 7), and formed a cap-like structure (called “head cap”, Berndston and Desjardins, 1974) in the peri-nuclear region; specifically, the acrosomal granule was strongly stained (Figure 2-4
A, a-c). In elongated spermatids (step 10), the PRAMEY staining covered two-thirds of the anterior portion of the nucleus, and still strongly stained the acrosomal granule
(Figure 2-4 A, e-g). Starting from step 10 of elongated spermatids, the nucleus began to appear flattened when viewed laterally (Berndston and Desjardins, 1974); Figure 2-4 A, i-k shows a lateral view of the elongated spermatids. To obtain a more detailed staining pattern, spermatids were isolated from squashed seminiferous tubules, which provided a better view of step 6 (Figure 2-4 B, a), step 8 (Figure 2-4 B, b) and step 10 (Figure 2-4 B, c) spermatids. PRAMEY evenly covered the entire acrosomal region in step 14 spermatids (Figure 2-4 A, m-o). There was no fluorescence observed when the anti-
PRAMEY antibody was replaced with pre-immune rabbit IgG (Figure 2-4 A, d, h, l, p), indicating that non-specific staining was minimal.
50
Figure 2-4. Localization of the bovine PRAMEY during spermiogenesis. A. Cross-sections of mature bull testis were stained with anti-PRAMEY (green). The bovine PRAMEY expression was observed mainly in the acrosomal region of all stages of spermatids from round spermatids (c) to elongated spermatids (g, k) to step 14 spermatids (o). The staining in the acrosomal granule (c, g) was relatively strong. The nuclear was stained with DAPI. No staining was observed in the pre-immune rabbit IgG negative control (d, h, l, p). Scale bar = 20 µm. B. Testicular spermatids were isolated and stained. PRAMEY was expressed in the acrosomal granule and head cap of step 6 (a), step 8 (b) and step 10 (c) spermatids. Scale bar = 10 µm.
51 The localization of PRAMEY in acrosome/post-acrosomal sheath junction and flagellum of testicular and epididymal spermatozoa
The localization of PRAMEY in testicular/epididymal spermatozoa was explored using indirect immunofluorescence staining. The similar staining pattern was observed in testicular (Figure 2-5, a-c), caput (Figure 2-5, e-g) and caudal (Figure 2-5, i-k) spermatozoa: in the sperm head, PRAMEY was localized in the acrosome (Ac), and/or postacrosomal sheath junction (J); PRAMEY was also stained in the flagellum (Figure 2-
5). No signal was observed in the pre-immune rabbit IgG or secondary antibody only negative control, indicating non-specific staining was minimal. To further study the three localization patterns in the sperm head (“Ac”, “Ac+J”, “J”), over 200 spermatozoa were examined from each group (Table 2-1). The results revealed that the percentage of the three localization patterns differed in testicular, caput and caudal spermatozoa. The percentage of “Ac” stained only sperm decreased with the maturation of spermatozoa
(87.0% in testicular spermatozoa, 25.9% and 7.3% in caput and caudal epididymal spermatozoa); while the percentages of spermatozoa with “Ac+J” and “J” staining was increased (Table 2-1).
52
Figure 2-5. Localization of the bovine PRAMEY in testicular spermatozoa, caput and caudal epididymal spermatozoa
53 Testicular (A), caput (B) and caudal (C) epididymal spermatids were isolated and stained with anti-PRMAEY antibody. PRAMEY was localized in the acrosome (a, e, i), both acrosome and postacrosomal sheath junction (b, f, g), or postacrosomal sheath junction (c, g, k) in the sperm head. The staining was also observed in the flagellum (a-c, e-g, i-k). No staining was observed in the pre-immune rabbit IgG negative control (d, h, l). The nuclear was stained with DAPI. Scale bar = 10 µm.
54 Table 2-1. The bovine PRAMEY antibody staining in spermatozoa isolated from testis, caput and caudal epididymis
Acrosome and Postacrosomal Number of Acrosome postacrosomal sheath sheath junction No Sperm origin sperm (Ac) junction (Ac + J) (J) staining Testicular 123 82.9% 13.0% 4.1% 0.0% Caput epididymis 264 25.9% 49.4% 20.0% 4.7% Caudal epididymis 259 7.3% 60.6% 27.4% 4.7%
55 PRAMEY is primarily associated with the acrosomal granule and acrosomal matrix by Immunoelectron microscopy (iEM)
Immunoelectron microscopy was used to study the detailed subcellular localization of PRAMEY in spermatids and spermatozoa. Firstly, the ultrastructure
(without immunogold labelling) of elongated spermatids and caudal epdidymal spermatozoa are shown in Figure 2-6 A1 and A2, B1 and B2. The membrane structure of the acrosome was well preserved. The outer acrsomal membrane (OAM) was distinguishable from the plasma membrane (P), and the inner acrosomal membrane
(IAM) was also clearly observed. There was a structure between IAM and outer nuclear membrane (ON), which is called subacrosomal layer (S). For immunogold staining, the earliest stage of immunogold labeling we observed was in step 4 spermatids. A single, prominent, centrally located acrosomal granule was shown, with intense immunogold labelling indicating the high level of PRAMEY expression (Figure 2-6 C1 and C2). The head cap of the acrosomal system had not developed in this stage. The PRAMEY continued to be expressed in the acrosomal granule in the step 7 spermatids (Figure 2-6
D1 and D2), which had already formed the acrosomal head cap. Upon elongation of spermatids, PRAMEY still was localized in the acrosomal granule of step 10 spermatids and now appeared in the lumen of the acrosome (Figure 2-6 E1 and E2). Importantly,
PRAMEY was localized in the electron dense region within the acrosome, indicating it was part of the acrosomal matrix. In the mature caudal epididymal spermatozoa,
PRAMEY was localized in the lumen of the acrosome, particular in the peri-IAM region
(but did not actually label the IAM) (Figure 2-6 F1, F2 and F3, G1 and G2). Comparing with step 10 spermatids (Figure 2-6 E2), the electron dense region of the acrosome was
56 thinner in cauda spermatozoa, but PRAMEY was still localized in the electron dense region within the acrosome (Figure 2-6 G1 and G2).
57 Figure 2-6. The subcellular localization of the bovine PRAMEY in spermatids and spermatozoa revealed by immunogold electron microscopy.
P: Plasma membrane, OAM: outer acrosomal membrane, A: lumen of the acrosome, IAM: inner acrosomal membrane, S: subacrosomal layer, PS: postacrosomal sheath, ON: outer nuclear membrane, ES: equatorial segment, J: postacrosomal sheath junction, G: acrosomal granule, N: nuclear, HC: head cap.
58
A1 A2
A1. The ultrastructure of elongated spermatids. The sagittal section showed clear membrane structure of the acrosome. Scale bar = 200 nm. A2, an enlarged picture of A1. Scale bar = 100 nm.
59
B1 B2
B1. The ultrastructure of caudal epididymal spermatozoa. The sagittal section of frontal part of the sperm head showed the acrosome structure and equatorial segment (ES). Scale bar = 1 µm. B2, an enlarged picture of B1. Scale bar = 100 nm.
60
C1
C2
C1. The immunogold labeling of step 4 spermatids. A clear single acrosomal granule (G) was shown, while the acrosomal head cap was not yet developed at this stage. Scale bar = 1 µm. C2, an enlarged picture of C1. The gold particle (10 nm) labeling was very clear and intense in the acrosomal granule. Scale bar = 500 nm.
61
D1
D2
D1. The immunogold labeling of step 7 spermatids. The acrosmic granule (G) and head cap (HC) formed the acrosomal system. Scale bar = 1 µm. D2, an enlarged picture of D1. The gold particle (10 nm) labeling was very clear and intense in the acrosomal granule. Scale bar = 500 nm.
62
E1 E2
E1. The immunogold labeling of step 10 spermatids. The sagittal section cut through the acrosomal region. Scale bar = 1 µm. E2, an enlarged picture of E1. The gold particle (10 nm) labeling was shown in the acrosomal granule, and the electron dense region of the acrosome lumen. Scale bar = 500 nm.
63
F1 F2 F3
F1. The immunogold labeling of caudal epididymal spermatozoa. The sagittal section cut through the acrosome and part of the post-acrosomal region. Scale bar = 1 µm. F2 and F3 enlarged pictures of F1. The gold particle (10 nm) labeling was shown in the lumen of the acrosome. Scale bar = 500 nm.
64
G1 G2
G1 and G2. The immunogold labeling of caudal epididymal spermatozoa. The sagittal section cut through the head part (G1) and the middle part (G2) of the acrosome. The gold particle (5 nm) labeling was shown in the electron dense region of the acrosome lumen, particular in the peri-IAM region. Scale bar = 200 nm.
65 PRAMEY interacts with PP1γ2 in epididymal spermatozoa
We hypothesized that PP1γ2 was the potential interaction partner of PRAMEY, because there is considerable structural similarity between PRAMEY and sds22 (see introduction). To study the interaction of PP1γ2 and PRAMEY in caput and caudal epididymal spermatozoa, co-IP analysis was performed with anti-PP1γ2 or anti-
PRAMEY antibody to enrich the PP1γ2 or PRAMEY complex from sperm extracts. The caput or caudal sperm extracts were used as input protein, in which both PP1γ2 and
PRAMEY were detected (Figure 2-7 A and B). In the co-IP performed using anti-PP1γ2, both PP1γ2 and PRAMEY were detected by western blot using the corresponding antibody, indicating PP1γ2 and PRAMEY formed a complex in caput and caudal epididymal spermatozoa. However, in the co-IP performed using anti-PRAMEY, only
PRAMEY were detected. In the co-IP of caput sperm, anti-sds22 antibody was used as a negative control, as sds22 did not interact with PP1γ2 in caput sperm. As expected, no sds22 was observed in the co-IP performed using anti-PP1γ2 (Figure 2-7 A).
66
Figure 2-7. Co-immunoprecipitation analysis of PP1γ2 with PRAMEY in bovine. Caput (A) or caudal (B) sperm extracts were incubated with anti-PP1γ2 or anti-PRAMEY immobilized on Protein A/G plus agarose bead. The sperm extracts and the immuno- precipitates were separated by SDS-PAGE and immunoblotted by anti-PP1γ2, anti- PRAMEY and anti-sds22 antibodies. Both PP1γ2 and PRAMEY were detected in the coIP eluate of anti-PP1γ2. Only PRAMEY was detected in the coIP eluate of anti- PRAMEY. No sds22 was detected in the co-IP eluate of anti-PRAMEY or anti-PP1γ2 in the caput sperm (A). Arrows on the right mark the bands for the IgG heavy chain (upper) and light chain (lower).
67 Two-dimensional (2-D) electrophoresis identifies the PRAMEY protein
To characterize the sequence of the bovine PRAMEY protein, we purified/isolated the PRAMEY by 2-D electrophoresis, and sequenced the isolated protein by mass spectrometry (MS). The caudal sperm protein were extracted using a low solubilizing power sample buffer (see Methods), which minimized the release of nuclear protein to reduce the interference of the abundant nuclear proteins. Two parallel gels were prepared and the crude protein extracts were separated by 2-D electrophoresis. The commassie blue stained gel revealed well resolved protein spots (Figure 2-8 A). To locate the PREAMEY protein, the parallel gel was transferred to PVDF membrane and blotted by the anti-PRAMEY. The PRAMEY was identified as a ~30 kDa protein spot in the pH range of 5-7. However, in the corresponding region of the Commassie blue stained gel, no visible protein spot was identified (Figure 2-8, red rectangles). The 2-D electrophoresis was repeated twice, which showed similar staining patterns and western blot results.
68
Figure 2-8. 2-D electrophoresis of the bovine caudal sperm proteins. Cauda sperm extracts were separated by 2-D electrophoresis. One gel was stained with commassie blue (A), which showed well-resolved protein spots. The other parallel gel was transferred to PVDF membrane and blotted by anti-PRAMEY (B), which identified the PRAMEY protein as ~30 kDa in size and within the 5-7 pH range. The corresponding region was marked with a red rectangle.
69 Immunoprecipitation to enrich the PRAMEY protein for mass spectrometry
IP was performed using anti-PRAMEY antibody immobilized on protein A/G agarose beads. From the western blot results (Figure 2-9), PRAMEY was detected in the input cauda sperm extracts and enriched in the IP eluate. Three major bands (band 1, 3 and 4) were revealed by coomassie blue staining, while band 2 was a very faint band in- between band 1 and 3 (Figure 2-9). Band 1 and 3 were the same sizes as the PRAMEY proteins identified by western blot (30 kDa and 26 kDa), while band 4 was ~40 kDa in size. The band 1, 2, 3 and 4 were cut out and analyzed by mass spectrometry for protein identification. As shown in Table 2-2, acrosin and acrosin-binding protein were identified in all four bands, while no PRAMEY protein was identified.
70
Figure 2-9. Enrichment of the bovine sperm PRAMEY protein by immunoprecipitation. A. The caudal epididymal sperm extracts (14 µg), the flow-through after IP (14 µg), and 16 µl of the IP eluted complex were analyzed by western blot probed with PRAMEY antibody. B. In a parallel lane of the same gel, 24 µl of the IP eluted complex were resolved by SDS-PAGE and stained by coomassie blue. The band 1, 2, 3 and 4 were indicated by the arrow heads on the right. The four bands were cut out and analyzed by mass spectrometry.
71 Table 2-2. Proteins identified in the IP eluate by mass spectrometry
Number MW Band Accession Description Score Coverage of [kDa] No. Peptides PREDICTED: acrosin-binding 1 297475301 protein 419.12 21.77 7 61.2 334285121 acrosin precursor 135.91 17.63 5 45.4 PREDICTED: acrosin-binding 297475301 protein 558.51 17.71 6 61.2 334285121 acrosin precursor 219.90 17.87 6 45.4 immunoglobulin lambda-like 2 139948632 polypeptide 1 precursor 43.66 21.28 3 24.7 156120505 izumo sperm-egg fusion protein 4 12.96 14.29 2 17.8 41386699 heat shock-related 70 kDa protein 2 10.28 6.97 3 69.2 32189336 ADP/ATP translocase 3 6.32 8.39 2 32.9 PREDICTED: acrosin-binding 297475301 protein 655.32 17.71 6 61.2 334285121 acrosin precursor 144.51 17.63 5 45.4 3 immunoglobulin lambda-like 139948632 polypeptide 1 precursor 85.65 22.13 4 24.7 27807407 ferritin light chain 7.26 14.86 2 20.0 334285121 acrosin precursor 789.82 48.31 15 45.4 PREDICTED: acrosin-binding 4 297475301 protein 18.26 6.27 2 61.2 saccharopine dehydrogenase-like 77735529 oxidoreductase 14.39 9.79 3 47.3
72 Incubating sperm with the bovine PRAMEY antibody increased the polyspermy rate and decreased the embryo rate in IVF
The sperm-oocyte binding and IVF experiments were performed using caudal epididymal sperm incubated with/without the PRAMEY antibody. There were more sperm cells bound per oocyte in the antibody treated group than the control group (Figure
2-10 A and B, P = 0.0001, t-test). The average number of sperm cells per oocyte in control group was 22, and 97 sperms per oocyte in the antibody treated group (Figure 2-
10 B). During IVF, normally fertilized zygotes contained two pronuclei, while more than two pronuclei were observed in polyspermic fertilization (Figure 2-10 C). A high level of polyspermic fertilization was observed in the antibody-treated group during IVF (Figure
2-10 D, P = 0.02, χ2 test). Fertilization rates of oocytes with control or antibody-treated sperm were presented as a percentage of embryos (including 2-cell and 4-cell stages)
(Figure 2-10 E). Fewer embryos were observed in the antibody-treated group (Figure 2-
10 F, P = 0.007, χ2 test), indicating a decreased fertilization rate after the PRAMEY antibody treatment.
73
Figure 2-10. Sperm-oocyte binding and IVF using caudal sperm incubated with the PRAMEY antibody. A. The representative pictures of sperm-oocyte binding between the control (without antibody) and PRAMEY antibody treated sperm. B. Number of sperm bound per zona pellucida (Mean number ± S.E.M.). Sperm were incubated with 0 (control) or 5 µg/ml (antibody-treated) anti-PRAMEY prior and during insemination. The asterisk indicated P < 0.05. C. Polyspermic fertilization and normal fertilization. Six pronuclei were observed in this polysermic fertilized oocyte, while two pronuclei were present in normally fertilized oocyte. D. Percentage of polyspermic fertilization. Oocytes were incubated with 0 (control) or 5 µg/ml (antibody-treated) anti-PRAMEY prior and during fertilization. The experiments were repeated twice and the error bar represents S.E.M. The asterisk indicated P < 0.05. E. The representative pictures of 2-cell and 4-cell embryos.
74 F. Percentage of embryos (including 2-cell and 4-cell embryos). Sperm were incubated with 0 (control) or 5 µg/ml (antibody-treated) anti-PRAMEY prior and during fertilization. The experiments were repeated twice and the error bar represents S.E.M. The asterisk indicated P < 0.05.
75 4. Discussion
Like many of the other CT antigen genes, PRAME was largely expanded in the eutherian genome (Chang et al., 2011). One way to study the multicopy gene families was to develop an isoform-specific antibody. In the present study, we produced a custom anti-PRAMEY antibody using a PRAMEY-specific short peptide (11-aa). The specificity of this antibody was validated by the peptide pre-absorption. The western blot results confirmed the presence of PRAMEY in the bovine testis and epididymal spermatozoa, which is 30 kDa in size. We found that the PRAMEY in epididymal spermatozoa underwent specific cleavage/degradation under some extraction conditions, which resulted in two minor bands (26 kDa and 13 kDa) in addition to the 30 kDa major bands.
The ratio of these three bands varied depending on the buffer compositions (such as pH, buffer strength, detergent used, etc.), the presence of protease inhibitors, the freshness and the freeze-thaw cycle of the samples. It has been reported that another sperm acrosomal protein, sp56 (Sperm fertilization protein 56, also known as Zona pellucida sperm-binding protein 3 receptor), also underwent similar specific cleavage/degradation during sample process (Kim et al., 2001) though the underlying molecular mechanism is unknown.
The predicted molecular weight for the full length PRAME protein is ~58 kDa, including the bovine PRAMEY, and its orthologs the human PRAME, the mouse
PRAMEL1 and OOG1 (http://web.expasy.org/compute_pi/). However, in the case of
PRAMEY, the 30 kDa band was recognized as the major PRAMEY protein in bovine, which was different from the predicted size. In humans, we detected a ~70 kDa protein in
76 semen and sperm, using two antibodies targeting different regions of the human PRAME sequence (unpublished data). One of the human antibodies reacts with the expected 58 kDa protein in melanoma cell lines (Epping et al., 2005) and K562 cells (Costessi et al.,
2011). In mice, the predicted 58 kDa protein was detected by the PRAMEL1 antibody in testis, while a minor band of 42 kDa was also observed (Mistry et al., 2013). Mouse
OOG1 was detected as a single 46 kDa protein in oocyte, one-cell and two-cell embryos, while two bands (46 kDa and 58 kDa) were detected in four-cell embryos (Minami et al.,
2003). These data suggest that members of the PRAME family might undergo post- translational modification during spermatogenesis, oogenesis and embryo development.
We believe that these post-translational modifications could be important for the protein to function and should be investigated in depth in the future.
The localization pattern of PRAMEY during spermatogenesis strongly suggests that PRAMEY is involved in acrosome biogenesis. The earliest stage of PRAMEY expression we observed was in step 4 spermatids, in which PRAMEY was localized in the newly-formed acrosomal granule. With the elongating of spermatids, PRAMEY was finally relocated to the lumen of acrosome. PRAMEY was particularly localized in the electron dense region, which can be designated as “acrosmal matrix” (Buffone et al.,
2008). Another PRAME family member, the mouse PRAMEL1, was also predominantly expressed in the acrosomal region in round spermatids; however, the expression was not detected in the acrosomal granule (Mistry et al., 2013). This difference indicated that the two proteins may be involved in different pathways during acrosome formation. As
PRAMEY redistributed with the content of the acrosomal granule during the process of acrosome biogenesis, the pathway of PRAMEY transport could be part of the so-called
77 “Golgi-acrosomal granule tract” proposed by Toshimori et al. (Toshimori et al., 1992).
Generally, the proteins involved in this pathway are initially accumulated in the proacrosomal/acrosomal granule and further transported to the head cap matrix. In contrast, the mouse PRAMEL1 may participate in another protein transport pathway called “Golgi-head cap tract” (Toshimori et al., 1992), in which proteins are not associated with the (pro)acrosomal granule. It is very interesting that the two orthologs of
PRAME, PRAMEY and PRAMEL1, may participate in different pathways during acrosome formation.
Intra-acrosomal migration is one of the steps in the organization of acrosomal contents. Interestingly, several acrosomal antigens have been reported to migrate and redistribute within the acrosome during acrosome biogenesis. In the human, ACRV1
(acrosomal vesicle protein 1, also known as SP-10) localized in the entire acrosomic system in early stages and then primarily localized in the acrosomal membrane in late stages of spermatogenesis (Kurth et al., 1991). The porcine proacrosin distributed from the entire acosomic system to mainly the acrosomal granule, and finally localized to the anterior acrosome (Bozzola et al., 1991). MN7 showed the most significant redistribution during rat spermatogenesis, MN7 first appeared in the proacrosomal granules of step 1/2 spermatids and the entire acrosomal region in step 3 spermatids, then continued to be expressed in the head cap but diminished from the acrosomal granule during the cap phase, finally presented on the anterior acrosome and absent from the equatorial segment
(Tanii et al., 1994). In our study, PRAMEY was distributed within the acrosome during acrosome biogenesis, which was from the acrosmal granule to the anterior part of the acrosome matrix. No PRAMEY was detected in the equatorial segment region.
78 In the present study, we successfully located the PRAMEY protein in the 2-D gel using antibody. However, we did not locate the corresponding spot by coomassie staining, suggesting the amount of protein was not enough to be detected by coomassie blue staining or analyzed by mass spectrometry. Considering the PRAMEY protein was easy to degrade/cleave (as discussed above), a great proportion of PRAMEY protein might have degraded during the rehydration and isoelectric focusing (~22 h).
Alternatively, IP was performed to enrich the PRAMEY protein using antibody immobilized on the agarose bead. The mass spectrometry analysis revealed that acrosin and acrosin-binding protein were detected in all four gel slices, while no PRAMEY was detected. Acrosin and proacrosin exhibit lectin-like carbohydrate-binding properties
(Jones and Brown, 1987; Urch and Patel, 1991), which could bind to saccharide including the agarose bead we used for IP. This might explain why we have detected a large amount of acrosin and acrosin-binding protein in the IP eluate. For future experiment, other types of bead (such as polymer-coated magnetic beads) could be used to eliminate acrosin binding when enriching the PRAMEY proteins.
Previous studies revealed that sds22 interacted with PP1γ2 in caudal sperm and functioned as a regulator of PP1γ2 catalytic activity. In contrast, sds22 did not interact with PP1γ2 in caput sperm (Huang et al., 2002; Mishra et al., 2003). The structural similarity shared by PRAMEY and sds22 inspired us to consider PP1γ2 as a potential interaction partner of PRAMEY. The immunofluorescence staining of PRAMEY (Figure
2-5) and a previous result of PP1γ2 in bovine (Huang et al., 2002) suggested the two proteins appear to colocalize in anterior region of sperm head and the sperm flagellum in cattle. Our co-IP results revealed that PRAMEY interacted with PP1γ2 in epididymal
79 spermatozoa, and formed a protein complex which can be immunoprecipitated by anti-
PP1γ2 antibody. As PP1γ2 is the major phosphatase in spermatids/spermatozoa and plays an important role in male fertility (Fardilha et al., 2011), the interaction between
PRAMEY and PP1γ2 indicated PRAMEY might be involved in the PP1γ2-related regulation of spermatogenesis, as well as the modulation of sperm motility. Surprisingly, in the co-IP eluate of anti-PRAMEY, PP1γ2 was not detected. A possible explanation was that the antibody binding site overlapped with the interaction site between PRAMEY and PP1γ2. Under this circumstance, an alternative anti-PRAMEY antibody which differs in epitope specificity could be produced and used for co-IP verification in the future.
Structurally, the acrosomal region consists of the anterior acrosome and the equatorial segment (posterior acrosome). Functionally, the anterior acrosome participates acrosome reaction and sperm-zona pellucida (ZP) interaction, while the equatorial segment may play a role in sperm-oocyte membrane fusion (Yoshinaga and Toshimori,
2003). Our IVF data suggested that PRAMEY may play a role in the block to polyspermy during fertilization. Mammalian oocytes have two levels of block to the polyspermy: zona pellucida and the plasma membrane (Gardner and Evans, 2006). The ZP block involves the exocytosis of cortical granules (CGs) upon the binding of sperm to ZP, which causes the conversion of ZP to a form that cannot support sperm binding (Abbott and Ducibella, 2001). Sperm treated with PRAMEY antibody bound to oocytes in higher numbers and the polyspermy rate was also increased (Figure 2-10), suggesting a delayed or disturbed induction of ZP block. The subcellular localization of PRAMEY, which was in the anterior region of acrosomal matrix but not in the equatorial segment, also
80 supported the hypothesis that PRAMEY may involve the block to polyspermy at the ZP level instead of membrane level.
This study shows that the PRAMEY is a testis- and spermatozoa- specific protein, expressed in spermatids and spermatozoa during spermatogenesis, localized in the acrosome and flagellum of sperm, and may be involved in the PP1γ2-related regulation of sperm motility, as well as play a role in the block of polyspermy during fertilization. Our results strongly suggest that PRAMEY play an important role in spermatogenesis and male fertility.
Chapter 3
The Expression and Localization of the Mouse PRAMEL1 during the First Wave of Spermatogenesis
[Published in part as an article entitled “Differential expression of PRAMEL1, a cancer/testis antigen, during spermatogenesis in the mouse” by Bhavesh V. Mistry, Yaqi Zhao, Ti-Cheng Chang, Hiroshi Yasue,
Mitsuru Chiba, Jon Oatley, Francisco Diaz, and Wan-Sheng Liu, in PloS One 8, e60611, 2013]
1. Introduction
The adult male testis is composed of spermatogenic cells of different stages
(Desjardins and Ewing, 1993), which makes it a challenge to study the gene expression in a given cell type. Several methods have been developed to isolate testicular cell populations, such as fluorescence-activated cell sorting (FACS) and velocity sedimentation separation (Han et al., 2001; Lassalle et al., 1999; Mays-Hoopes et al.,
1995). However, those methods require specialized equipment/reagent, large numbers of animals, and are usually time consuming. Alternatively, as the first wave of spermatogenesis is synchronized (NEBEL et al., 1961), analyzing changes of gene expression based on stages of development is used as an alternative strategy to determine the cellular process of spermatogenesis (Shima et al., 2004). The analysis of gene expression based on stages of testis development also reveals the simultaneous progression of spermatogenesis.
82 The germ cell types have been characterized in each stage of postnatal testis development in mice (Auharek and de França, 2010; Vergouwen et al., 1993). In
C57BL/6J mice, the most advanced type of germ cells were gonocytes, differentiated A3 spermatogonia, pre-leptotene spermatocytes and pachytene spermatocytes, respectively, at 1, 5, 10 and 15 days (d) of age. Round spermatids were seen at 20 d and were followed by step 9/10 spermatids on 28 d. After completion of the first wave of spermatogenesis, the systematically arranged germ cells (spermatogonia, spermatocytes and spermatids) are observed in all seminiferous tubules (Auharek and de França, 2010; Vergouwen et al.,
1993).
Preferentially expressed antigen in melanoma (PRAME) is a member of the cancer/testis (CT) antigens, and was originally identified as a tumor antigen expressed in melanoma (Ikeda et al., 1997). The mouse Prame gene family is the third largest gene family in the mouse genome, with about 90 copies of genes and pseudogenes (Church et al., 2009). The previous work in our laboratory suggested that the Prame-like 1
(Pramel1) was a male-specific Prame family member, which localized in the acrosomal region and flagellum of spermatozoa in adult mice (Mistry et al., 2013). The current study aimed to analyze the time course (from newborn to mature) expression of the mouse Pramel1 and to localize the PRAMEL1 protein during the first wave of spermatogenesis and in the early testis development.
83 2. Materials and Methods
Animals
C57BL/6J mice were bred in our colony at the Pennsylvania State University mouse facility, and a total of six pairs of breeders were set up. Testes were removed from mice at 0 day (newborn), 1 week (w), 2 w, 3 w, 4 w and 8 w of age. At least three animals (biological replicates) were used for each age. All animal procedures were approved by the Pennsylvania State University Institutional Animal Care and Use
Committee (IACUC).
Collection and preparation of testicular tissues
Testes were removed immediately after the sacrifice of animals and snap frozen in liquid nitrogen for RNA and protein extraction. For histological analysis, testes were removed carefully from the animals to minimize trauma to the delicate seminiferous tubules. Prior to placement of each testicle into fixative, the tunica albuginea was shallowly pierced at each pole 5 times with a 21-gauge needle to aid in the penetration of the fixative. The testes were fixed in Bouin’s solution (RICCA Chemical Company
#1120) (at least 10 fold of volume of the tissue) in 4 °C, for 6~8 hours (h) for 1 w- and 2 w-old testes, and overnight for 3 w-old testes. After briefly washed by water, the testes were washed in 70% ethanol, dehydrated, and embedded in paraffin blocks (Latendresse et al., 2002). The paraffin-embedded tissues were sectioned at 4 µm using a microtome and mounted on glass slides.
84 RNA extraction and RT-PCR
Total RNA of testes was extracted using RNeasy Mini Kit (Qiagen, Valencia,
CA). The RNA samples were treated with DNase I (Ambion, Austin, TX) and reverse transcribed using the SuperscriptTM III First-Strand Synthesis System (Invitrogen,
Carlsbad, CA) as described by the manufacturer. RT-PCR was performed in 20 µl with
10 ng cDNA, 200 µM dNTPs, 1.5 mM MgCl2, 2.5 µM of each primer, 1 unit hot-star Taq
DNA polymerase (Qiagen, Valencia, CA). The PCR conditions were: 95 °C for 15 min followed by 35 cycles each of 95 °C for 50 s, 55-65 °C for 50 s and 72 °C for 50 s, with a final extension at 72 °C for 10 min. Products were resolved on 1.5% agarose gels with ethidium bromide in 1×TAE buffer. Sequence-specific primers used for PCR amplification of the mouse Pramel1 (NM_031377) was: sense, 5’-
TCTGCTCTGGATGACATACC-3’; antisense, 5’-GGCAACCTGTTCCACAGCTT-3’.
PRAMEL1 antibody
A PRAMEL1 antibody (termed as PRAME like-1 (S-16), Catalog # sc-34513) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). This antibody is an affinity-purified goat polyclonal antibody raised against a peptide of PRAME like-1 of the mouse protein (accession no. NP_113554), which was chosen from the C-terminal region between 350-400 amino acid (aa) with a protein sequence similarity ≤ 58% to all other members of the mouse Prame gene family (NCBI Blastp analysis).
85 Protein extraction and western blot
The testes were homogenized in ice-cold CelLytic buffer (Sigma C3228) supplied with Protease Inhibitor Cocktail (Thermo 87785, containing AEBSF, aprotinin, bestatin,
E-64, leupeptin, pepstatin A and EDTA) and Phosphatase Inhibitor Cocktail (Thermo
78420, containing sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β- glycerophosphate). The lysed samples were centrifuged at 12,000 g，4 °C to pellet the tissue debris. The protein-containing supernatant were transferred to a new tube and used for gel electrophoresis, or saved in -80 °C for future work.
The testicular proteins were mixed with loading buffer (Thermo 39001) supplied with 100 mM DTT, boiled at 95 °C for 5 min and cooled to RT before applied to gel. The proteins were separated using 8%-16% Precise Tris-Glycine gel (Thermo 25268), transferred to PVDF membrane and blocked in 5% milk in TBST (25mM Tris, 0.15M
NaCl, 0.05% Tween-20, pH 7.5) at RT for 1 h. The membrane was incubated with anti-
PRAMEL1 (1:200) at 4 °C with gentle shaking overnight. After washing three times in
TBST, the membrane was incubated with a donkey anti-goat IgG-HRP (Santa Cruz, sc-
2020) (1:5000) for 1 h. The reactive proteins were detected by SuperSignal West Femto
Maximum Sensitivity Substrate (Thermo 34094). The peptide (sc-34513 P) used for making the PRAMEL1 antibody was applied to confirm the antibody specificity. The antibody and a five-fold (by weight) excess of peptide were incubated together for 2 h at
RT, which was analyzed by western blotting.
86 Indirect immunofluorescence staining
Testis cross-sections underwent deparaffinization in Xylene, followed by rehydration in a series of ethanol, and underwent heat-induced antigen retrieval by boiling in sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) during a period of 20 min. After washing in PBS, the sections were blocked in 10% donkey serum for 1 h at RT, and then incubated with anti-PRAMEL1 (1:50) overnight at
4 °C. Following a wash step (3 × 5 min in PBS), sections were incubated with FITC- conjugated donkey anti-goat IgG (sc-2024, Santa Cruz Biotechnology) (1:100) for 1 h at
RT, washed again, and mounted with SlowFade® Gold Antifade Reagent with DAPI
(Life technologies S36938). Sections incubated with peptide-preabsorbed PRAMEL1 antibody or secondary antibodies only were used as negative controls.
3. Results
Temporal expression analysis of the mouse Pramel1 in testis
To examine the temporal expression of the mouse Pramel1 gene (NM_031377), we performed a time course study during testis development. The RT-PCR results indicated that, while a lower level the Pramel1 mRNA was expressed in the newborn testis, a higher level of constant expression was detected in 1-w- through 8-w-old testes
(Figure 3-1).
87 To determine whether the PRAMEL1 protein is expressed in testis, a western blot analysis was performed on whole testis lysate from mature mice using a PRAMEL1- specific antibody. As shown in Figure 3-2A, the PRAMEL1 antibody identified a specific band with a molecular weight of ~ 57 kDa, which is the predicted molecular weight for the mouse PRAMEL1 protein (acc. no. NP_113554). In addition, a very minor band (~
42 kDa) was observed (Figure 3-2 A). To test the specificity of the antibody, we performed a pre-absorption control with the peptide used to generate the PRAMEL1 antibody, in which no band was detected (Figure 3-2 A), suggesting that the antibody was
PRAMEL1-specific and the minor band required further investigation. A time course analysis of the PRAMEL1 protein further indicated that its expression in testes was age- dependent (Figure 3-2 B). The protein expression level was very low at the newborn stage (Figure 3-2 B), consistent with the low level of transcription detected by RT-PCR
(Figure 3-1). The expression was increased gradually from 1- to 3-w-old testes, and then remained constant after three weeks of age (Figure 3-2 B). As a positive control, a monoclonal β-actin (ACTB) antibody was used to detect ACTB, and an expected protein of 42 kDa was identified (Figure 3-2 A, B).
88
Figure 3-1. Temporal expression analysis of Pramel1 by RT-PCR during the mouse testis development. Low expression was observed in the newborn (Nb) testis, while constant high expression was observed in testes from 1- to 8-w-old mice. The mouse Actb gene was used as a positive control. M: marker.
89
Figure 3-2. Expression analysis of the mouse PRAMEL1 protein in testes. A. Western blot analysis of PRAMEL1. Protein extracts from adult mouse testis were subjected to western blot analysis using a mouse PRAMEL1 antibody. An immune- reactive protein with an expected molecular weight of ~ 57 kDa was detected in the testis, and a very minor band of ~ 42 kDa was also observed. As a positive and loading control a monoclonal ACTB antibody was used, and an expected immune-reactive protein of 42 kDa was observed. As a negative control, we performed a pre-absorption control by the peptide used to generate the PRAMEL1 antibody, and no band was detected. B. Time course analysis of the PRAMEL1 protein expression during testis development. Protein extracts from newborn (Nb), 1-w, 2-w, 3-w, 4-w and 8-w-old testes were subjected to western blotting with the mouse PRAMEL1 antibody. The expression level of the PRAMEL1 was gradually increased from Nb to 3-w and then remained constant after 3-w of age. Numbers on the left indicate the molecular weights of standard proteins.
90 Localization of the mouse PRAMEL1 protein during spermatogenesis
We investigated the cellular, spatial and temporal expression of the PRAMEL1 protein during spermatogenesis by indirect immunofluorescence microscopy. Initially, we examined the PRAMEL1 protein localization on cross-sections of testes in young mice, from newborn to 3-w of age. As shown in Figure 3, there was no specific staining pattern observed for the PRAMEL1 antibody across the section in 1-w-old testis (Figure 3-3 a, c), though weakly unspecific background staining was observed for both the PRAMEL1 antibody (Figure 3-3 a, c) and pre-absorbed peptide control (Figure 3-3 d). This might be a consequence of relatively low expression of the transcript and protein at this stage
(Figure 3-1 and Figure 3-2 B). Compared to the pre-absorbed peptide control where some non-specific staining was observed (Figure 3-3 i), specific staining was seen for the
PRAMEL1 antibody mainly in the cytoplasm of spermatocytes in 2-w-old testis (Figure
3-3 f, h). When the mice reached 3 w of age, a very unique and strong staining was detected by the PRAMEL1 antibody in the perinuclear region of round spermatids
(Figure 3-3 k, m), where a cap-like structure on the nuclear surface was observed (Figure
3-3 m).
91
Figure 3-3. Immunofluorescent localization of the mouse PRAMEL1 during early testis development. Cross-sections from 1-w (a-e), 2-w (f-j) and 3-w (k-o)-old mouse testes were stained with the mouse PRAMEL1 antibody. Although there was no specific signal observed across the seminiferous tubules in 1-w-old testis, specific staining (green) was observed in cytoplasm of spermatocytes (f, h) in 2-w-old testis and the perinuclear region of round spermatids (k, m) in 3-w-old testis. Panels a, f, k were the PRAMEL1 antibody staining. Panels b, g and l were counterstained with DAPI to visualize nuclei. Panels c, h and m were merged images for the PRAMEL1 and DAPI staining. The arrowheads point to an area with described signals. Panels d, i and n were peptide-preabsorbed antibody staining as negative control. Panels e, j and o were H&E staining to show the structure and cell types of the seminiferous tubules. Magnification is ×400. Scale bar = 20 µm.
92 4. Discussion
It has been reported in previous studies that different CT antigens are involved in different stages of spermatogenesis. Some CT antigens are expressed exclusively in one stage, such as SYCP1 (synaptonemal complex protein 1, also known as HOM-TES-14), which is restricted to meiotic prophase of spermatocytes, and SP17 (sperm protein 17), which is expressed in the mature spermatozoa only (Kong et al., 1995; Meuwissen et al.,
1992). Other CT antigens such as CTAG1 (cancer/testis antigen 1, also known as NY-
ESO-1) and TRO (trophinin, also known as magphinin or MAGE-D3), are expressed in several stages of spermatogenesis. CTAG1 is expressed in spermatogonia through pachytene spermatocytes (Jungbluth et al., 2001; Satie et al., 2002), while TRO is expressed in almost all types of germ cells from spermatogonia to mature spermatozoa
(Saburi et al., 2001). In the present study, we demonstrated that, like the Tro gene, the mouse Pramel1 is expressed broadly in different types of germ cells during spermatogenesis. The PRAMEL1 protein was first seen, though at a relatively low level, in the newborn testes by western blotting, suggesting the expression of PRAMEL1 in spermatogonia. A gradually increased level of PRAMEL1 was detected in testes at 1-, 2-,
3-w of age and maintained at the higher level in mature testes. This increase in protein expression coincides with the increase in RNA expression from newborn to 1-w-old testes, and could be explained by an up-regulation of the gene expression, or alternatively, by an increase in the number of germ cells as there is dramatic germ cell proliferation before the first wave of spermatogenesis.
93 Unlike the cancer cells in which the PRAME protein was localized in the nucleus, the mouse PRAMEL1 was first seen in the cytoplasm of spermatocytes in 2-w-old testes in this study. A dominant expression of the PRAMEL1 protein was then observed in the anterior region of (round, elongating and elongated) spermatids, coordinating with the morphological alterations of the acrosome, which suggests that the Pramel1 plays a role in acrosome development. Earlier studies in cancer cells demonstrated that PRAME functions either as a transcription suppressor of RA signaling (Epping et al., 2005), or as a transcription activator on promoter regions bound by NFY (Costessi et al., 2011, 2012).
However, our data suggest that Pramel1 may not participate in transcription regulation through RA- and NFY-mediated mechanisms because PRAMEL1 is localized to the cytoplasm of germ cells during spermatogenesis. In combination with our recent published data on the expression of the mouse Pramel1 in mature testis and spermatozoa, we conclude that PRAMEL1 may play a role in acrosome biogenesis. Our data provide a base for further studying on the functional role of Pramel1 during spermatogenesis.
Summary
This study focuses on the expression and function of a CT antigen gene family,
PRAME/PRAMEY, during spermatogenesis. Bovine PRAMEY was identified as a testis- and spermatozoa- specific protein. PRAMEY was mainly associated with acrosmal granule of the round spermatids during spermiognesis, and was finally localized in the acrosome and flagellum of the mature spermatozoa. Incubating the sperm with anti-
PRAMEY antibody resulted in a significant increase of the polyspermic fertilization, suggesting an important role of PRAMEY in the block to polyspermy. Co-IP experiment showed that PRAMEY interacted with the major regulator of sperm motility, phosphatase
PP1γ2, which indicated PRAMEY was involved in the PP1γ2 regulated sperm motility acquisition.
Bovine PRAMEY and mouse PRAMEL1 were chosen as representatives to compare the PRAME family members between different species. Bovine PRAMEY was expressed in the later stage of the first wave of spermatogenesis, while mouse
PRAMEL1was expressed from new born until mature testis. Mouse PRAMEL1 was expressed in the cytoplasm of pachytene spermatocytes in 2-week-old testis, and translocated to the acrosomal region of round spermatids in 3-week-old testis.
Furthermore, no expression of PRAMEL1 was detected in the acrosomal granule region of round spermatids, which was different from bovine PRAMEY. It is very interesting that PRAME family members in different species are both involved in spermatogenesis and acrosome formation, however, the specific pathway of biogenesis may be different.
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