DNA Stability of Stallion Sperm: Factors Affecting Chromatin Integrity in Individual Stallions

University of Veterinary Medicine Hannover

DNA stability of stallion sperm: Factors affecting chromatin integrity in individual stallions

Thesis Submitted in partial fulfillment of the requirements for the degree -Doctor of Veterinary Medicine- Doctor medicinae veterinariae (Dr. med. vet.)

by Saskia Schütze Haldensleben

Hannover 2015

Academic supervision: Prof. Dr. Harald Sieme Clinic for Horses Unit for Reproductive Medicine

Dr. Ir. Harriëtte Oldenhof Clinic for Horses Unit for Reproductive Medicine

1. Referee: Prof. Dr. Harald Sieme 2. Referee: Prof. Dr. Ralph Brehm

Day of the oral examination: 2015/08/31

A contribution from the Virtual Center for Reproduction Medicine, Lower Saxony

Meinen Eltern und Großeltern

CONTENTS

1 Introduction: aims and outline 9

2 Literature study 11 2.1. Sperm DNA-structure 11 2.2. Spermatogenesis and transit through the epididymis 12 2.3. Correlating sperm DNA integrity and fertility 13 2.4. Factors affecting sperm DNA integrity: oxidative damage 14 2.5. Methods for detecting chromatin integrity and DNA damage 15 2.5.1. Sperm Chromatin Structure Assay (SCSA) 16 2.5.2. Further assays for detecting DNA damage 17 2.5.3. Spectral analysis 19

3 Material and methods 21 3.1. Semen collection and processing 21 3.2. Isolation of epididymal sperm 22 3.3. Light microscopic evaluation of sperm morphology 23 3.4. Computer assisted sperm analysis (CASA) of sperm motility 23 3.5. Sperm chromatin structure assay (SCSA) 24 3.6. Induction of DNA damage by varying the duration of acid denaturation and exposure to Fenton’s Reaction 25 3.7. Fourier transform infrared spectroscopy (FTIR) 26 3.8. Treatment of sperm with disulfide-reducing and -stabilizing agents 26 3.9. Transmission electron microscopy 27 3.10. Statistical analysis 28

4 Results 29 4.1. Analysis of sperm from stallions with different fertility rates and SCSA analysis after induced DNA damage 29 4.1.1 Sperm morphology, motility and chromatin-stability for poor and

good fertility stallions 29 4.1.2. Induction of DNA-damage by altering the duration of exposure to acid denaturation 32 4.1.3. Induction of DNA-damage by exposure to oxidative stress using Fenton’s reaction 35 4.1.4. Fourier transform infrared spectroscopy (FTIR) on Fenton-treated sperm 38 4.2. Analysis of sperm with differences in chromatin packaging 40 4.2.1. Chromatin stability of epididymal sperm 40 4.2.2. Effects of disulfide-reducing and -stabilizing treatment on chromatin stability 45 4.3. Light and electron microscopic evaluation of alterations in sperm nuclear morphology 49

5 Discussion and Conclusions 53 5.1 Analysis of sperm from stallions with different fertility rates, and SCSA analysis after induced DNA damage 53 5.2. Analysis of sperm with differences in chromatin packaging 56 5.3. Conclusions 59

6 Summary 60

7 Zusammenfassung 62

8 References 65

9 Appendix 78

10 Danksagung 83

ABBREVIATIONS

a.u. arbitrairy units

ATR attenuated total reflection

CASA computer assisted sperm analysis

COMP αt cells outside the main population of αt

DA diamide

DBD-FISH DNA-breakage detection fluorescence in situ hybridization

DFI DNA fragmentation index

DIC differential interference contrast

DNA deoxyribonucleic acid

DTT dithiothreitol e.g. exempli gratia et al. et alia

Fig. figure

FTIR Fourier transform infrared spectroscopy

Fnt sln Fenton`s solution mRNA messenger ribonucleic acid

P1 protamine 1

P2 protamine 2

PBS phosphate buffered saline r.u. relative units

ROS reactive oxygen species

SCD sperm chromatin dispersion test

SCSA sperm chromatin structure assay

SH sulfhydryl

Tab. table

Td denaturation time

TEM transmission electron microscopy

TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling

WinMDI Windows Multiple Interface for Flow Cytometry

INTRODUCTION: AIMS ANS OUTLINE

1. INTRODUCTION: AIMS AND OUTLINE

In addition to their pedigree, athletic performance or appearance, stallions used for breeding are selected based on their reproductive performance. In the equine breeding industry, reproductive properties of individuals may vary to a great extent and therefore are critically examined. Parameters commonly used for evaluation of stallion fertility include total number of sperm present in an ejaculate, as well as sperm motility, morphology and membrane integrity characteristics. Recently, there is an increased interest in additional assays that give insights in sperm function, including tests for evaluating the ability of sperm to undergo capacitation and acrosome reaction in vitro as well as analysis of chromatin integrity (NEILD et al. 2005). It is suggested that DNA intactness does not necessarily correlate with the above mentioned sperm characteristics, whereas it is essential for successful fertilization of an oocyte and embryonic development (EVENSON et al. 2002, SAKKAS and TOMLINSON 2000). The sperm chromatin structure assay (SCSA) is most commonly used to evaluate chromatin intactness. With this assay, the susceptibility for in vitro DNA denaturation after treatment with acid is evaluated, via flow cytometric analysis of acridine orange stained samples (EVENSON et al. 1980, EVENSON and JOST 2000). With sperm maturation, and transit through the epididymis, DNA will be tightly packed. This occurs through replacement of histone proteins by protamines and increased formation of inter- and intramolecular disulfide bonds (WARD 1993). Chromatin intactness and stability after ejaculation is dependent on individual genetic as well as environmental factors. Intrinsic factors include alterations in protamination during spermiogenesis which can lead to altered DNA packing and consequently damage (GONZÁLEZ-MARIN et al. 2012) as well as reduced fertility (SPANO et al. 2000). Extrinsic factors include harms sperm are exposed to during preservation for artificial insemination, like exposure to diluents and cooling. Damage due to oxidative stress has been described to be a major cause for DNA fragmentation (AITKEN and ROMAN 2008, BAUMBER et al. 2003).

INTRODUCTION: AIMS ANS OUTLINE

The aims of the study described in this thesis were: (1) determine if sperm from stallions classified to be of poor or good fertility exhibited differences in stability towards induced DNA damage, and (2) determine if DNA packing affected DNA stability towards exposure to denaturation conditions or oxidative stress. To assess if differences in sperm DNA stability exist between stallions with poor and good fertility, sperm was exposed to acid denaturation for different durations as well as to different levels of oxidative stress using Fenton’s reagent. Subsequently, DNA intactness was analyzed using SCSA. It was determined if sperm from different individuals showed differences in denaturation kinetics or threshold levels for inducing damage. To evaluate if chromatin packing affected DNA stability, sperm granularity, intercalation of fluorescent dye and induced DNA damage was evaluated for immature spermatozoa isolated from different regions of the epididymis (caput, corpus and cauda) as well as ejaculated sperm treated with chemical compounds for reducing and stabilizing disulfide bonds in chromatin (dithiothreitol and diamide, respectively).

LITERATURE STUDY

2. LITERATURE STUDY

2.1. Sperm DNA-structure

Fertilization involves fusion of nuclei of the male and female gamete. Successful fertilization of an oocyte requires functional spermatozoa with intact chromatin. Chromatin consists of DNA folded around specific proteins. DNA molecules carry the genetic information of a cell and consist of nucleotides which are composed of desoxyribose residues with phosphate groups and an adenine, cytosine, guanine or thymine base attached through a nitrogen-bond. Nucleotides are connected to each other via their phosphate and hydroxyl groups. In addition, hydrogen bonds exist between bases giving rise to a three dimensional double helix structure with all bases located inside the helix and sugar-phosphate backbone on the outside (ALLIS et al. 2008). Initially, germ cells have a chromatin structure as somatic cells, where DNA is packed around histone nucleoproteins. Highly basic, arginine-rich protamines are formed during spermiogenesis and transit through the epididymis. These replace histone proteins upon sperm maturation (BALHORN 1982, WARD and COFFEY 1991) resulting in highly condensed DNA through additional formation of doughnut- shaped loops (WARD 1993). It has been suggested that this almost crystalline state protects sperm DNA during transport through the female reproductive tract (BJÖRNDAL and KVIST 2014). Tight packing of DNA with protamines is the result of their amino acid composition and interactions they can form. Protamines contain positively charged arginine residues which can interact with the negatively charged phosphodiester residues in the major groove of the DNA-backbone (BALHORN 2007). In addition, protamines contain cysteine residues with free SH-groups, which can form intra- and intermolecular disulfide bonds (WARD 1993). These SH-groups as well as histidine imidazole groups also bind Zn2+, which accumulates during spermiogenesis, facilitating further stabilization. Moreover, Zn2+ prevents formation of excess disulfide bonds, allowing immediate DNA unfolding upon fertilizing the oocyte (BJÖRNDAL

LITERATURE STUDY

and KVIST 2010). Two major types of protamines have been detected in mammals, including stallion (BALHORN 1982, 2007, GOSÁLVEZ et al. 2011). These have been designated protamine 1 (P1) and 2 (P2), and their presence has been correlated with sperm DNA stability (CASTILLO et al. 2011, PARADOWSKA-DOGAN et al. 2014). Whereas P1 is rich in arginine and cysteine residues, low numbers of these residues are present in P2. Lysine and cysteine lack SH-groups and consequently the ability to form disulfide bonds (KUMAROO et al. 1975).

2.2. Spermatogenesis and transit through the epididymis

Spermatogenesis is the process by which spermatozoa are formed in the seminiferous epithelium of the testis, through divisions and differentiation of germ cells. Three phases can be distinguished: (i) spermacytogenesis, (ii) meiosis, and (iii) spermiogenesis (reviewed in JOHNSON et al. 1997). During spermacytogenesis, stem cell spermatogonia undergo mitotic divisions. These spermatogonia will form primary spermatocytes and later, via meiosis, haploid spermatids. During spermiogenesis, spermatids differentiate into spermatozoa, as evident from morphological changes. During so-called maturation in the epididymis, and transit through the caput, corpus and cauda of the epididymis, they become progressively motile and obtain their fertilizing potential. Furthermore, in this phase, chromatin is dramatically remodeled to form a highly condensed state through replacement of histones by sperm DNA specific protamines (BALHORN 1982, WARD 1993, GOLAN et al. 1996). Different forms of sperm DNA packing can be observed during maturation. Histones are maintained, directly replaced by sperm specific protamines, or initially replaced by so-called transition proteins that are substituted by protamines at a later time point (WOUTERS-TYROU et al. 1980). A testis-specific histone (Ht1) was found in immature spermatozoa of humans (STEGER et al. 1998) and stallions (CAVALCANTI et al. 2009). It is suggested to facilitate the replacement of histones by protamines resulting in increased DNA packing and stability, whereas in stallions a prolonged expression of Ht1 was detected during spermatogenesis with individual

LITERATURE STUDY

age-dependent variations, probably explaining peripubertal infertility (CAVALCANTI et al. 2009).

2.3. Correlating sperm DNA integrity and fertility

Several studies exist in which sperm quality has been correlated with fertility rates (SPANO et al. 2000, SAKKAS and TOMLINSON 2000, LOVE and KENNEY 1998, 1999). Parameters typically used for describing sperm quality include sperm morphology, motility and plasma and acrosomal membrane integrity, as well as ability to undergo capacitation and acrosome reaction in vitro (NEILD et al. 2005). In addition to the above mentioned parameters describing sperm function, EVENSON et al. (1980) stated that DNA intactness is an important marker foreseeing successful fertilization. Sperm chromatin fragmentation has been observed in immature as well as ejaculated sperm. DNA is especially susceptible for damage during spermatogenesis, when chromatin remodeling is not yet completed. DNA nicks as formed by accident during chromatin remodeling and sperm development can be either repaired by topoisomerase activity or lead to DNA strand breaks in mature spermatozoa. Further intrinsic causes for sperm DNA damage can be apoptosis or oxidative stress triggered by cell metabolism (GONZÁLEZ-MARIN et al. 2012). In addition to intrinsic causes, damage can occur due to environmental factors sperm are exposed to after ejaculation or during processing for use for artificial insemination. Extrinsic factors causing damage include storage temperature, composition of extenders used during processing, and infections or oxidative stress following ejaculation (GONZÁLEZ-MARIN et al. 2012). Morphological abnormalities are commonly found for spermatozoa with defects in chromatin structure or damaged DNA (SAKKAS and TOMLINSON 2000). Abnormal protamination, like persisted histone-bond DNA or a lack in protamines, has been described to result in abnormal sperm head shape and size as well as increased numbers of nuclear vacuoles, which in turn interfere with capacitation and fertilization (UTSUNO et al. 2014). Furthermore, presence of large amounts of DNA

LITERATURE STUDY

bound to histones and the relative amounts of protamine 1 and 2 in sperm have been correlated with the extent of DNA packing and fertilizing potential (WARD 2010, CAVALCANTI et al. 2009). In human spermatozoa the protamine 1 versus 2 protein ratio (P1/P2) was determined to be approximately 1/1 (CORZETT et al. 2002). For other species different ratios were determined or even a complete absence of P2 (BALHORN 2007). Studies performed by GOSÁLVEZ et al. (2011) revealed that spermatozoa from mammals containing both P1 and P2 showed higher susceptibility for DNA fragmentation as compared to those containing only P1. STEGER et al. (2001) revealed a decreased amount of P1 and P2 mRNA in spermatids from infertile men. For stallions P1/P2 mRNA ratios were determined and correlated with fertility rates by PARADOWSKA-DOGAN et al. (2014).

2.4. Factors affecting sperm DNA integrity: oxidative damage

After ejaculation and passage through the female reproductive tract or when subjected to preservation procedures, sperm are exposed to changing environmental conditions. Damage due to exposure to oxidative stress has been described to be a major factor causing membrane damage as well as DNA fragmentation (AITKEN and ROMAN 2008, WNUK et al. 2010). Reactive oxygen species (ROS) are a natural byproduct of oxygen metabolism and are normally controlled by the presence and action of antioxidants in the seminal plasma. Damaging reactions can take place in a situation of imbalance, when concentrations of ROS increase or a depletion of antioxidants occurs (AMARAL et al. 2013). Furthermore, ROS accumulation may be the result of pathologic processes including infections, increased physical exertion or testicular torsion (AITKEN and ROMAN 2008). Spermatozoa have membranes that contain high amounts of polyunsaturated fatty acids while they lack antioxidants. This makes them especially susceptible for oxidative attack (AGARWAL et al. 2003). The double bonds in fatty acid chains include reactive methylene groups which can react with hydrogen peroxide resulting in formation of reactive hydroxyl radical (OH∙) or - superoxide anion (∙O2 ) groups. These groups can react further in a chain reaction,

LITERATURE STUDY

which is only stopped when non-radical end products are formed (AGARWAL et al. 2003). In addition to lipid peroxidation, DNA can be subject for oxidative damage reactions. Types of DNA damage include single or double strand breaks, which can lead to chromosomal fragmentation, or crosslinks and mismatches (GONZÁLEZ- MARIN et al. 2012). A few mechanisms are described to be involved in repairing DNA damage in haploid cells, including base excision repair and DNA double strand break repair. Furthermore, repair mechanisms from the oocyte will be available upon fertilization and formation of a zygote (GONZÁLEZ-MARIN et al. 2012). Damaged sperm DNA is described to contain the bas adduct 8-hydroxy-2`-deoxygenase (8- OHdG), which can be repaired by 8-oxoguanine glycosylase (AITKEN et al. 2014; WNUK et al. 2010). Damage caused by ROS with sperm processing can be counteracted by addition of antioxidants, which function as scavengers or can react with ROS therewith minimizing oxidative attack on sperm biomolecules. Examples of antioxidants commonly used are α-tocopherol and catalase (BAUMBER et al. 2003). Also, sperm clean-up and selection procedures can be employed for removal of sources for ROS production. Procedures for removal of seminal plasma and damaged sperm include migration (swim up) and filtration (e.g. using glass wool or Sephadex) methods, as well as ordinary and density gradient centrifugation (SIEME et al. 2003).

2.5. Methods for detecting chromatin integrity and DNA damage

Different methods can be applied for evaluation of DNA integrity (see figure 2.5.2.). The most commonly used assays involve a denaturation step to remove nuclear proteins and induce damage prior to staining with a (non-)fluorescent dye which binds DNA. After this, fragmented DNA is visualized by use of (fluorescence) microscopy or flow cytometric analysis. Commonly used tests include: the halo-test or sperm chromatin dispersion test (SCD) (FERNÁNDEZ et al. 2005), the ‘comet assay’ (OSTLING and JOHANSON 1984, HUGHES et al. 1996), and TUNEL-assay

LITERATURE STUDY

(GORCZYCA et al. 1993). These are described in detail below. In addition to tests that include a sample preparation, non-invasive spectroscopic techniques can be employed (SÁNCHEZ et al. 2012).

2.5.1. Sperm Chromatin Structure Assay (SCSA)

The sperm chromatin structure assay (SCSA) as originally described by EVENSON et al (1980) is the gold standard for assessing chromatin integrity. A treatment with acid (pH 1.2, for 30 s) is performed after which samples are stained with acridine orange, and analyzed using flow cytometry. Acridine orange specifically stains nucleic acids. When it intercalates into double-stranded intact DNA as a monomer it fluoresces green, upon excitation. In contrast, when bound as an aggregate to single stranded damaged DNA it emits orange fluorescence (KOSOWER et al. 1992). Green and red fluorescence intensities for a sperm sample are detected using flow cytometry: typically using a 488 nm laser for excitation and specific filters for detecting fluorescence. When green versus orange/red fluoresence dot plots are created for sperm particles, different sperm populations can be recognized (see Figure 2.5.1.). Sperm with higher DNA fragmentation exhibit higher red fluorescence, and the relative proportion can be determined using specific software. With such software different parameters are derived, according to EVENSON and JOST (2000), as described in detail below. The extend of fragmented DNA is calculated by dividing red fluorescence by the total (red plus green) fluorescence, for each sperm (10000 in total), and is designated as αt. A histogram on this (number versus αt) can be created, in which different populations can be recognized. The percentage of cells outside the main population of αt is designated as COMP αt, and is indicative for cells with damaged DNA. COMP αt is also referred to as DNA fragmentation index (DFI) and is considered the most valuable parameter for fertility assessment (EVENSON et al. 2002). Parameters derived from SCSA analysis have been correlated with fertility rates for human sperm (EVENSON et al. 1980, SPANO et al. 2000). For stallions,

LITERATURE STUDY

high green

fluorescence

cells outside the main populatin (COMP αt)

green fluoresence green (channel)

red fluoresence (channel)

Figure 2.5.1. SCSA scatter plot (drawn with WinMDI) showing green vs. red fluorescence intensities (channel) of 10000 cells measured of a native sperm sample. The red area is demonstrating the gated region of cells outside the main population, which is calculated as DFI (%).

LOVE (2005) introduced a classification for their fertility based on studies done in which fertility rates were correlated with SCSA data. In this classification, fertile stallions showed an average DFI value of 12%, while subfertile and infertile stallions had DFI values of approximately 17% and 25%, respectively.

2.5.2. Further assays for detecting DNA damage

With the so-called halo-test sperm DNA fragmentation is visualized microscopically for single cells. Therefore, spermatozoa are treated with acid lysing solution, after which they are immersed in agarose and stained with a DNA intercalating fluoresescent dye like propidium iodide. The result is that nuclei are seen as fluorescent extended halos because of DNA dispersion into the agarose and intercalating dye in intact DNA. In case of fragmented DNA, these characteristic halos are missing (FERNÁNDEZ et al. 2005). The sperm chromatin dispersion test (SCD) is a variation on the halo-test. It includes an additional step for lysis/denaturation of proteins prior to staining and microscopic inspection

LITERATURE STUDY

(FERNÁNDEZ et al. 2005). This assay is commercially available as Halomax (from Halotech DNA SL, Madrid, Spain). With this assay, spermatozoa with fragmented DNA are claimed to show a large and spotty halo of chromatin dispersion, whereas spermatozoa with non-fragmented DNA show a small and compact halo. Halo-test approaches are simple and fast to perform, but only allow a small number of cells for microscopic evaluation. Furthermore, although halo-size can be measured for quantification, which is rather subjective. The ‘comet assay’ developed by OSTLING and JOHANSSON (1984) employs single cell gel electrophoresis on an agarose coated microscope slide. This is done for detecting fragmented DNA as a ‘comet tail’ separated from the intact DNA; after denaturation under alkaline conditions, staining with DNA intercalating fluorescent dye, and separation through subjection to a directed electric field (HUGHES et al. 1996). As with the halo-test, this assay can be used for rapid analysis of DNA damage in single cells. However, sperm DNA fragmentation in response to exposure to alkali conditions has not been correlated with fertility rates (SINGH et al. 1989). In addition to fluorescent DNA intercalating dyes, non-fluorescent stains like toluidine blue can be used to detect DNA strand breaks and fragmentation. As described above, prior to staining, sperm are treated with a protein reducing/denaturing agent like dithiothreitol (DTT). With light microscopic evaluation of samples, orthochromatic and metachromatic blue stained sperm can be distinguished representing sperm with nuclei containing intact and fragmented DNA, respectively (KRZANOWSKA 1982, BARRERA et al. 1993). DNA breaks can be specifically detected using the DNA-breakage detection fluorescence in situ hybridization assay (DBD-FISH). With this assay, samples are embedded in agarose and incubated in alkaline buffer, after which they are allowed to hybridize with fluorescently labeled DNA fragments representing the whole genome (SCHLEGEL and PADUCH 2005). Such fluorescently labeled DNA probes bind to complementary single stranded DNA fragments as present in the sample. CORTÉS-GUTIÉRREZ et al. (2014) used this method to detect alkali-labile sites in equine spermatozoa. Also, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay can be used for detecting DNA strand breaks. Here,

LITERATURE STUDY

samples are incubated with fluorescently labeled deoxyuridine triphosphate nucleotides and the enzyme terminal deoxynucleotidyl transferase for incorporation of such nucleotides in case of presence of DNA nicks. Presence of incorporated fluorescent nucleotides can be assessed by flow cytometry or fluorescence microscopy (GORCZYCA et al. 1993). Differences in chromatin packing due to different protamination have been previously evaluated by means of level of intercalation and staining with dyes like Chromomycin A3. For this dye it has been suggested that it cannot bind very tightly packed chromatin, condensed by protamines, resulting in decreased fluoresence (MANICARDI et al. 1995). UTSUNO et al. (2014) used this method for evaluation of the protamine status in subfertile men.

2.5.3. Spectral analysis

All assays described above require treatment of sperm prior to analysis. In contrast, spectroscopic techniques, like Raman and Fourier infrared transform (FTIR) microspectroscopy, can be used without a sample preparation. These techniques rely on interactions between light and molecular groups present within the sample. Spectra as collected with such studies provide information on occurrence and conformation of endogenous biomolecules in cells and/or tissues (SÁNCHEZ et al. 2013, WOLKERS and OLDENHOF 2010). Raman microspectroscopy has been employed for detection of specific spectral changes in bands resulting from phosphate groups in the DNA-backbone of sperm, in response to UV-irradiation (MALLIDIS et al. 2011). Also, specific changes in DNA-bands in Raman as well as FTIR spectra have been reported and correlated with different levels of induced oxidative DNA damage (SÁNCHEZ et al. 2012).

LITERATURE STUDY

Figure 2.5.2. Overview of different assays for detecting DNA damage including sample preparation and evaluation. Single assays are described in detail with references in section 2.5.

MATERIALS AND METHODS

3 MATERIALS AND METHODS

3.1 Semen collection and processing

Semen was collected from Hanovarian warmblood stallions that were held at the National Stud of Lower Saxony in Celle, Germany. Semen samples used for the studies described here were aliquots from routine semen collections performed for the commercial artificial insemination program of the stud. Stallions were kept in box stalls bedded with straw, were fed three times a day with grain and hay, and had access to water ad libitum. Stallions were held according to national regulations and institutional animal care and use protocols. Semen collections took place every other day. Prior to use for experiments, collections were performed for two weeks to stabilize the extra gonadal sperm reserves. Semen was collected using an artificial vagina and a breeding phantom (both model ‘Hannover’; Minitüb, Tiefenbach, Germany). Each ejaculate was filtered to remove the gel fraction. Semen was evaluated for its density directly after collection using a NucleoCounter SP-100 (ChemoMetec A/S, Allerød, Denmark), and diluted with pre-warmed (37 °C) skim milked extender (INRA-82; VIDAMENT et al. 2000) to a concentration of 100 × 106 sperm mL-1. Diluted semen was centrifuged for 10 min at 600 × g in conical tubes, the supernatant was removed and the sperm pellet was resuspended with fresh INRA-82. Centrifuged samples were used for analysis of sperm motility and morphology as described below. For assessment of DNA integrity/stability and treatments affecting these, samples were centrifuged once more and sperm was resuspended in phosphate buffered saline solution (PBS; 137 mM NaCl, 27 mM KCl,

10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). For experiments in which sperm characteristics were correlated with stallion fertility rates, three ejaculates were collected from each of ten stallions (5-19 years). Collections took place from October until December 2013, during the non-breeding season. Stallions were divided into two groups, consisting of five stallions each, based on fertility data available via the records of the stud. Stallions were considered to be of good fertility when foaling rates were higher than 50%. In the current studies,

MATERIALS AND METHODS

stallions classified as good fertility stallions (15±6 years) had a non-return rate of 59±7% and foaling rate of 54±3%, whereas stallions classified as poor fertility stallions (10±6 years) had a non-return rate of 45±6% and foaling rate of 43±8%. For treatment of sperm with disulfide-reducing and -stabilizing agents three pooled sperm samples were used. Each pooled sample was prepared using semen from four different stallions, by mixing equal volumes of diluted semen (100 × 106 sperm mL-1 in INRA-82). Collections for this took place March-July 2014.

3.2 Isolation of epididymal sperm

Immature sperm was collected from the epididymis obtained from six different stallions (2-20 years). Stallions were castrated under general anesthesia, and epididymal sperm was isolated according to BRUEMMER (2006). In brief, after the epididymis was separated from the testis and dissected free from the fascia, the epididymis was divided into three sections; via ligating the epididymal caput, corpus and cauda regions. The proximal ductus deferens was ligated as high as possible to prevent loss of sperm. Sperm was collected via retrograde flushing of the three sections. This was done by inserting a 16-gauge needle into the lumen of the previously ligated part. The needle was attached to a syringe containing PBS, for use as flush-medium. An incision was made at the opposite end of the ligated region, using a scalpel, where PBS containing epididymal sperm was collected in a tube during flushing of the ligated section. A gentle amount of pressure was used in case of retrieving sperm from the epididymal caput and corpus regions. In addition, these regions were incised with a scalpel and smears on glass slides were collected. Diluted samples were centrifuged (10 min at 600 × g) and the sperm pellet was resuspended in a small volume of fresh PBS. Sperm concentrations were ~10-50 × 106 sperm mL-1 in case of samples from the caput and corpus regions, and ~500 × 106 sperm mL-1 for the cauda region. For two out of six stallions, an ejaculate could be collected one day before the castration took place. The ejaculate was split for use for analysis of ejaculated sperm, and recovery of seminal plasma. For isolation of seminal plasma, semen was

MATERIALS AND METHODS

centrifuged three-times 10 min at 3400 × g, after which the supernatant was recovered. The supernatant was passed through a 0.2 µm filter, and stored at 4 °C until use. Seminal plasma was added to epididymal sperm from the caudal region (~500 × 106 sperm mL-1 in PBS), at a 10:1 volume ratio, and incubated for 30 min at room temperature before plunging in liquid nitrogen.

3.3 Light microsopic evaluation of sperm morphology

Percentages of morphologically abnormal sperm were determined according to BRITO (2007). A 100 µL sperm sample was stained by adding 300 µL nigrosin- eosin staining solution (0.7% eosin, 10% nigrosin, 3.75 mM Na2HPO4, 1.88 mM

KH2PO4, 5.78 mM potassium sodium tartrate, 3.75 mM glucose) according to DOTT and FOSTER (1972) with minor modifications. After 30 s incubation in staining solution, smears were prepared and dried. Two hundred sperm per sample were evaluated using phase contrast microscopy and a 1000× magnification with oil immersion. In addition, for evaluation of epididymal sperm and sperm treated with various agents as described below, samples were fixed by adding an equal volume of buffered formal citrate solution (1.48% formaldehyde, 99 mM sodium citrate) according to DOTT and FOSTER (1975). Fixed samples were stored at 4 °C until light microscopic evaluation; using phase contrast as well as differential interference contrast (DIC) optics (Axioskop 50, Zeiss, Jena, Germany).

3.4 Computer assisted sperm analysis (CASA) of sperm motility

Computer assisted sperm analysis (CASA; Spermvision; Minitüb, Tiefenbach, Germany) was used for assessment of motility characteristics of sperm. The setup used included a microscope with a temperature-controlled stage that was maintained at 37 °C, and a camera for recording motility patterns with 60 frames per s. Software settings were according to the instructions provided by the manufacturer. A 500 µL sperm sample (100 × 106 sperm mL-1 in INRA-82) was incubated for 5 min at 37 °C,

MATERIALS AND METHODS

after which 3 µL of the sample was added drop-wise into a chamber of a Leja 20 micron four chamber slide (Leja Products BV, Nieuw Vennep, The Netherlands), and motility measurements were done while maintaining the sample at 37 °C. Percentages of motile sperm were calculated as mean values from counts of eight microscopic fields. Sperm were considered progressively motile when the average path velocity was greater than 40 µm s-1 and the straightness was greater than 0.5 (relative units).

3.5 Sperm chromatin structure assay (SCSA)

The sperm chromatin structure assay (SCSA) was used to evaluate nuclear chromatin integrity. In this assay, sperm is treated with acid after which the level of induced DNA denaturation is determined (EVENSON and JOST 2000). In all cases, SCSA analysis was done using sperm samples which were centrifuged and resuspended in PBS at ~100 × 106 sperm mL-1 prior to plunging and storage in liquid nitrogen. After thawing in a 37 °C water bath, samples were diluted in TNE buffer (0.15 M NaCl, 0.01 M TRIS-HCl, 1 mM disodium EDTA, pH 7.4) at approximately 2 × 106 sperm mL-1. From this an aliquot of 200 μL was taken and 400 μL acid solution (0.08 M HCl, 0.15 M NaCl, 0.1% Triton X-100, pH 1.2) was added. For standard measurements, this was mixed for exactly 30 s using a vortex, after which 1.2 mL acridine orange (Polysciences, Warrington, PA, USA) staining solution (0.15 M NaCl,

0.037 M citric acid, 0.126 M Na2HPO4, 0.0011 M disodium EDTA, pH 6.0; containing 6 μg mL-1 acridine orange) was added to stop the denaturation reaction. Samples were placed on ice for an additional 3 min, after which 10000 cells were analyzed with an average flow rate of 200-300 particles per second using a FACScan flow cytometer (Becton-Dickinson, Heidelberg, Germany). This flow cytometer contains a 488 nm argon ion laser of 15 mW for excitation and a filter setup for detecting green (band pass 530/30 nm filter), orange (band pass 582/42 nm filter) and red (long pass 650 nm) fluorescence. The DNA fragmentation index (DFI) was determined as described by EVENSON et al. (2002), from numbers of sperm with single and double stranded DNA, using DAS software (BEISKER 1994). In addition, WinMDI software

MATERIALS AND METHODS

(Windows Multiple Interface for Flow Cytometry, version 2.9, developed by J. Trotter, at The Scripps Research Institute, La Jolla, CA, USA) was used for display of flow cytometry data and additional analyses.

3.6 Induction of DNA damage by varying the duration of acid denaturation and exposure to Fenton’s reaction

In addition to performing the SCSA assay as described above, according to EVENSON and JOST (2000) with a 30 s incubation with acid solution, we varied the incubation period with acid solution (Td: denaturation time). Samples were mixed with acid solution, and then incubated on ice for up to 10 min after which neutralizing staining solution was added. To induce sperm DNA damage via exposure to extreme oxidative stress, Fenton’s reaction was used. This chemical treatment is based on the following reactions: 2+ 3+ - 3+ 2+ + (1) Fe + H2O2 → Fe + ∙OH + OH ; (2) Fe + H2O2→ Fe + ∙OOH + H Different levels of oxidative damage were obtained through adding different volume percentages of Fenton’s solution (3% H2O2, 50 mM iron-(II)-sulfate) to sperm samples. Hundred µL sperm sample (100 × 106 sperm mL-1 in PBS) was added in an

Eppendorf tube, and different amounts of 3% H2O2 and 50 mM iron-(II)-sulfate (at a 1:1 ratio) were added. PBS was added to end up with a final volume of 300 µL, while iron sulfate solution was added the latest to start the reaction. This resulted in final concentrations of 33 × 106 sperm mL-1 and 0-67% Fenton’s solution. Samples were incubated for 30 min on ice, after which 1000 µL PBS of 4 °C was added. Samples were centrifuged in a microfuge (10 min at 600 × g), the supernatant was removed, and the pellet was resuspended in 200 µL PBS. These samples were shock-frozen and stored in liquid nitrogen for later analysis.

MATERIALS AND METHODS

3.7 Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) was used to evaluate possible changes in the DNA region of the infrared spectrum; in response to induced oxidative damage using Fenton’s reaction, as previously described (SÁNCHEZ et al. 2012). Reactions were done as described above, using a larger final volume such that a sample could be stored as aliquots in liquid nitrogen. One aliquot was used for SCSA analysis, for determining DFI values, while the other aliquot was used for FTIR. Prior to FTIR measurements, samples were thawed at room temperature (~20 min), and sperm were collected via centrifugation in a microfuge (2 min, 1000 × g). The supernatant was removed and the pellet was resuspended in 2 µL PBS. Infrared spectra were recorded using a Perkin-Elmer 100 FTIR spectrometer (PerkinElmer, Norwalk, CT, USA), equipped with a triglycine sulfate (TGS) detector and an attenuated total reflection (ATR) accessory with diamond/ZnSe crystal. Acquisition parameters were: 4 cm-1 resolution, eight co-added interferograms, and a -1 4000-900 cm wavenumber range. An automatic CO2/H2O vapor correction algorithm was used during recording of the spectra. Two µL sperm sample was transferred onto the ATR sample holder where it was air-dried for 10 min before spectra acquisition. Spectra analysis and display were carried out using Perkin-Elmer software (PerkinElmer, Norwalk, CT, USA), and Omnic software (Thermo-Nicolet,

Madison, WI, USA). The bands emerging from the DNA-phosphate (PO4) backbone can be found in the spectral region between 900 and 1200 cm-1.

3.8 Treatment of sperm with disulfide-reducing and -stabilizing agents

Dithiothreitol (DTT) was used as an agent to reduce disulfide bonds in the DNA backbone of sperm chromatin, while diamide was used as a stabilizing agent. Both DTT and diamide (Sigma-Aldrich, St. Louis, MO, USA) were prepared as 1 M stock solutions in PBS, and stored at -20 °C until use. 250 µL sperm sample (100 × 106 sperm mL-1 in PBS) was added in an Eppendorf tube, and different amounts of DTT or diamide stock solution were added. PBS was added to end up with a final

MATERIALS AND METHODS

volume of 500 µL, and final concentrations of 50 × 106 sperm mL-1 and 0-15 mM DTT or 0-10 mM diamide. Samples were incubated for 30 min at room temperature, after which 500 µL PBS was added. Samples were centrifuged (10 min, 600 × g), the supernatant was removed and the pellet was resuspended in 250 µL PBS. Samples were shock-frozen and stored in liquid nitrogen for later analysis.

3.9 Transmission electron microscopy

Transmission electron microscopy (TEM) was used to evaluate sperm ultrastructure, with special emphasis on the nucleus. Fixation and embedding of samples was performed directly after treatment with DTT (10 mM), diamide (10 mM) and Fenton solution (67%) as described above. Sperm were collected by centrifugation after which the supernatant was removed and the pellet was resuspended in fixation solution (5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2). The fixation solution was removed by three washes with 0.1 M cacodylate buffer at 4 °C. Subsequently, samples were incubated in a 1% OsO4-solution (Science Services, München, Germany), for 2 h at 4 °C, and washed with 0.1 M cacodylate buffer. Dehydration of fixed material was through a graded ethanol series (30, 50, 70, 90, and 100% twice) for 20 min in each grade. After this, samples were infiltrated with epon. Therefore, samples were incubated in propylene oxide for 20 min, followed by 20 min incubation in a mixture of propylene oxide and epon, supplemented with 2,4,6-Tris(dimethyl-aminomethyl)phenol, and overnight incubation in epon. Embedded material was stored at room temperature. Sections of 70-90 nm were cut using a microtome (Ultracut E, Leica Microsystems, Bensheim, Germany) equipped with a diamond knife, and transferred onto copper grids (Plano, Wetzlar, Germany). Prior to electron microscopic evaluation, sections on grids were treated with uranyl acetate and lead citrate. A transmission electron microscope (Zeiss EM 10 C, Oberkochen, Germany) at 80 kV was used to evaluate sperm with a 16000x magnification.

MATERIALS AND METHODS

3.10 Statistical analysis

Statistical analysis was done using ‘SAS’ software (SAS Institute Inc., Cary, NC, USA), according to advise given by a statistician at the Institute for Biometry and Epidemiology of the University of Veterinary Medicine Hannover. Data were tested for normal distribution using the Kolmogorov-Smirnov test, after which differences between fertility groups and correlations between sperm parameters, differences for induced DNA damage as well as differences in DNA stability for epididymal sperm and sperm treated with chromatin modifying compounds were analyzed. For descriptive statistics, the mean and standard deviation were calculated. Differences between treatments were tested by ANOVA with repeated measurements. Correlations were analyzed using Pearson correlation coefficient analysis. Differences were taken to be statistically significant when p<0.05.

RESULTS

4 RESULTS

4.1 Analysis of sperm from stallions with different fertility rates, and SCSA analysis after induced DNA damage

4.1.1 Sperm morphology, motility and chromatin-stability for poor and good fertility stallions

In figure 4.1.1., sperm characteristics are shown for ten stallions (three ejaculates each) which were divided into two groups based on their fertility. Stallions were classified to be fertile when NNR was > 50% and sub-fertile when NNR was < 50%. Percentages of morphologically abnormal sperm ranged from 41-68% and 28- 48% for stallions classified as poor and good fertility stallions, respectively, while percentages of progressively motile sperm ranged from 8-39% and 39-60%. DFI- values varied from 16-21% for stallions classified as poor fertility stallions, while DFI- values varied from 7-21% for good fertility stallions. Differences between fertility groups were significant for sperm morphology, motility as well as DNA integrity

(p<0.05). DFI-values as well as percentages of morphologically abnormal sperm were found to increase with increasing stallion age and with decreasing fertility (figure 4.1.2 A, B). Furthermore, DFI-values positively correlated with percentages of morphologically abnormal sperm and progressive sperm motility negatively correlated with abnormal sperm morphology and fertility (figure. 4.1.2. C, D).

RESULTS

Figure 4.1.1. Sperm characteristics including chromatin integrity (A) and percentages of morphologically abnormal (B) and progressively motile sperm (C), as determined for ten different stallions (three ejaculates per stallion). Data are presented as means ± standard deviations, for individual stallions (A-C’) as well as for stallions divided into two groups according to their fertility (red bars: poor fertility, green bars: good fertility, blue bars: all stallions). The DNA fragmentation index (DFI) was determined as a measure for chromatin integrity, using the sperm chromatin structure assay, sperm morphology was evaluated via microscopic evaluation of nigrosin-eosin stained dried specimens, while computer assisted sperm analysis was used for analysis of sperm motility characteristics. Differences between fertility groups were significant for sperm morphology, motility as well as DNA integrity (p<0.05).

RESULTS

n: 30 n: 30 r: 0,5870 r: 0,5958 p<0,05 p<0,05

n: 30 n: 30 r: 0,7860 r: -0,6946 p<0,05 p<0,05

Figure 4.1.2. Chromatin integrity (DFI-values in %) and percentages of morphologically abnormal sperm were determined for individual stallions and were plotted versus their age (A and B, respectively). In addition, DFI-values and percentages of progressively motile sperm were plotted versus percentages of morphologically abnormal sperm as determined for the same sample (C and D, respectively). Data from three ejaculates for ten stallions are presented; for stallions of poor (red squares) and good (green squares) fertility (five stallions each).

RESULTS

4.1.2 Induction of DNA-damage by altering the duration of exposure to acid denaturation

Whereas with the standard SCSA protocol (EVENSON and JOST 2000) an acid denaturation time (Td) of 30 s is used, we tested incubation times up to ten minutes. This was done to obtain insights in maximum levels of DNA-damage that could be induced in sperm from different stallions. From a representative sample green versus red fluorescence intensity scatter plots were drawn for each denaturation time using WinMDI to illustrate changes in fluorescence intensities (figure 4.1.3.). It was found that green fluorescence only exhibited a small increase with longer denaturation times (figure 4.1.4A). The green fluorescence intensity was 1.07-fold higher for incubations with a Td of 10 min as compared to 30 s, both for poor and good fertility stallions. In contrast, the red fluorescence intensity, indicative for sperm with damaged DNA, drastically increased with increasing incubation times with acid solution (p<0.05), while exhibiting differences between poor and good fertility stallions (figure 4.1.4B). For poor fertility stallions, the red fluorescence intensity was 1.30-fold increased for incubations with a Td of 10 min as compared to the intensity after 30 s, whereas this was only 1.22-fold in case of good fertility stallions. This is also illustrated when plotting alpha-T values versus the denaturation period (figure 4.1.4C). DFI-values were also derived and found to increase with increasing incubation times in acid solution, reaching a plateau after about 1 min, for sperm from both fertility groups (figure 4.1.4D). DFI-values for poor fertility stallions, however, were about 2-fold higher as those for good fertility stallions for all denaturation incubation periods tested. DFI-values of 16% and 32% were determined for a denaturation time of 10 min, for good and poor fertility stallions, respectively. The difference in DFI- values amongst good and poor fertility stallions was highly significant at a Td of 3 min, 5min and 10 min (p<0.001).

RESULTS

Figure 4.1.3. Representative green (Fl-1) versus red (Fl-3) fluorescence intensity scatter plots, obtained by flow cytometric analysis with SCSA while varying the incubation period in acid solution (denaturation time or Td; A: 0 s, B: 30 s, C: 1 min, D: 3 min, E: 5 min, F: 10 min). Fluorescence intensities are plotted as mean channel number, WinMDI software was used for display of scatter plots.

RESULTS

Figure 4.1.4. Flow cytometric analysis with SCSA, for sperm incubated for different periods in acid solution (Td: denaturation time). Green (A) and red (B) fluorescence intensities of sperm after such treatments are plotted versus the denaturation time. In addition, alpha-t (C) and DFI (D) values were derived and plotted versus the denaturation time. Data points represent mean values ± standard deviations; as determined for stallions of poor (red squares) and good (green squares) fertility (five stallions each, three ejaculates per stallion). DFI percentages differed significantly between fertility groups (p<0.05).

RESULTS

4.1.3 Induction of DNA-damage by exposure to oxidative stress using Fenton’s reaction

Sperm samples were treated with increasing concentrations of Fenton’s solution, after which SCSA was performed. Representative green versus red fluorescence intensity scatter plots as obtained after such analyses are shown in figure 4.1.5. No changes in the green fluorescence intensity were seen for samples exposed to increasing volumes of Fenton’s solution (figure 4.1.6A), whereas red fluorescence increased up to 3-fold (figure 4.1.6B). Red fluorescence was higher for poor fertility stallions as compared to good fertility stallions when exposed Fenton’s solution volumes ranging from 17-50%, but similar intensities were attained with exposure to 67% Fenton’s solution. This is also illustrated when plotting alpha-T values versus the volume of Fenton’s solution sperm were exposed to (figure 4.1.6C). DFI-values were determined and found to increase up to 99% in case of exposure to 67% Fenton’s solution, both for good and poor fertility stallions (figure 4.1.6D). A dose dependent increase in DFI values was seen. When exposed to lower concentrations of Fenton’s solution, however, DFI values were higher for poor fertility stallions as compared to good fertility stallions. Differences between fertility groups were most manifestly after exposure to 17% and 33% Fenton`s solution (p<0.05).

RESULTS

A B C

Fnt-sln: 0% Fnt-sln:: 17% Fnt-sln: 33%

D E F

Fnt-sln: 50% Fnt-sln: 60% Fnt-sln: 67% green fluoresence (channel) fluoresence green

red fluoresence (channel)

Figure 4.1.5. Representative green (Fl-1) versus red (Fl-3) fluorescence intensity scatter plots, obtained by flow cytometric analysis with SCSA, after exposure to different volume percentages of Fenton’s solution (Fnt-sln; A: 0%, B: 17%, C: 33%, D: 50%, E: 60%, F: 67 %). Fluorescence intensities are plotted as mean channel number, WinMDI software was used for display of scatter plots.

RESULTS

A B

C D

Figure 4.1.6. Flow cytometric analysis with SCSA, after exposure of sperm to oxidative stress by incubation with different volume percentages of Fenton’s solution (Fnt-sln). Green (A) and red (B) fluorescence intensities are plotted versus the volume of Fenton’s solution sperm were incubated with. In addition, alpha-t (C) and DFI (D) values were derived and plotted versus the Fenton’s solution- volume. DFI values were found to increase with increasing concentrations of Fenton`s solution. Data points represent mean values ± standard deviations; as determined for stallions of poor (red squares) and good (green squares) fertility (five stallions each, three ejaculates per stallion).

RESULTS

4.1.4. Fourier transform infrared spectroscopy (FTIR) on Fenton-treated sperm

In addition to SCSA analysis we collected infrared spectra of sperm samples treated with increasing concentrations of Fenton’s solution, to see if different levels of induced oxidative stress showed differences in the characteristic bands arising from the DNA-backbone, similar as previously found by SÁNCHEZ et al. (2012) for human sperm. In figure 4.1.7.A a full spectrum (400-900 cm-1 spectral range) is shown for a hydrated and dried sperm sample in which characteristic bands arising from biomolecules present in the sample are shown. The CH2 stretching bands mainly arising from membrane lipids, while the amide-I and –II bands arise from the C=O and N-H groups from proteins. Furthermore, the OH-stretching, the H2O-libration and bending combination band as well as the H2O scissoring band arise from water. Figure 4.1.7B shows an enlargement of the 1150-900 cm-1 spectral region for non- treated sperm (with a DFI-value of 18%) as well as sperm exposed to different volumes of Fenton’s solution (with DFI-values increasing up to 100%). This region contains characteristic bands from PO-vibrations predominantly arising from of nucleic acids and lipids. Drastic changes can be seen in this region with exposure to increasing volumes of Fenton’s solution and concomitant induced DNA-damage.

RESULTS

0,6 0,6 A BB

F6 0,5 100% DFI 69% DFI 26% DFI 18% DFI 0,5

0,4 0,4

0,3 0,3

0,2 0,2

absorbance(a.u.) absorbance(a.u.)

0,1 0,1

0,0 0,0 3900 3400 2900 2400 1900 1400 900 1150 1100 1050 1000 950 900 wavenumber(a.u.) wavenumber(a.u.)

Figure 4.1.7 In panel A, in situ infrared absorption spectra of sperm in the 4000-900 cm-1 region are shown. Panel B shows an enlargement of the 1150-900 cm-1 spectral region is shown for non-treated (orange line) sperm as well as sperm exposed to different volumes of Fenton’s solution (blue line: 17%, green line: 50%, red line: 67%). This region contains characteristic bands from PO-vibrations arising from of nucleic acids and lipids.

RESULTS

4.2. Analysis of sperm with differences in chromatin packaging

4.2.1. Chromatin stability of epididymal sperm

To evaluate changes in chromatin stability during sperm maturation, SCSA was done for sperm samples isolated from the epididymal caput, corpus and cauda. Representative side versus forward scatter and green versus red fluorescence intensity plots as obtained after such analyses are shown in figure 4.2.1. Side and forward scatter properties of sperm, indicating granularity and size respectively, showed clear differences during sperm maturation. Forward and side scatter properties of sperm were found to be higher for sperm from the epididymal caudal region as compared to the corpus and caput regions, indicating an increase in size and granularity during development (figure 4.2.2B). Green and red fluorescence were highest for sperm from the epididymal caput and lowest for sperm from the cauda (figure 4.2.2C). This likely indicates increased accessibility of sperm DNA for acridine orange and fluorescence in case of samples with a lower granularity. For epididymal sperm, induced DNA-damage with SCSA was evaluated by: (1) determining DFI- values according to EVENSON et al. (2002), without elimination of sperm in the so- called high green region, as well as (2) determining percentages of damaged sperm directly in the green versus red fluorescence intensity scatter plot as illustrated in figure 4.2.3A. For sperm from the epididymal caput, percentages of sperm with induced DNA-damage were about three times higher as those for epididymal sperm from the corpus and cauda regions (9.5% versus 3.1-3.6%). DFI-values revealed slightly higher values, but illustrated a similar trend (figure 4.2.3B). In figure 4.2.4A, side scatter properties are shown for native/ejaculated sperm and epididymal sperm isolated from the cauda region. The latter was directly used or incubated with seminal plasma prior to analysis. Interestingly, the granularity of sperm from the epididymal cauda decreased upon incubation with seminal plasma, resulting in similar values as determined for native/ejaculated sperm. In figure 4.2.4B, the effect of increasing the incubation time in acid solution with SCSA is shown. Percentages of sperm with damaged DNA increased up to 12% at a Td of 5 min for

RESULTS

ejaculated sperm, whereas this was only 2.7% in case of caudal epididymal sperm. When incubated with seminal plasma, percentages of DNA-damaged caudal epididymal sperm increased up to 5.8% at a Td of 5 min.

RESULTS

A B C D

ep. sperm - caput ep. sperm - corpus ep. sperm - cauda ej. sperm - native

side scatter (channel) sidescatter

forward scatter (channel)

E F G H

ep. sperm - caput ep. sperm - corpus ep. sperm - cauda ej. sperm - native

green fluoresence (channel) fluoresence green

red fluoresence (channel)

Figure 4.2.1. Flow cytometric analysis with SCSA, of immature sperm isolated from different regions of the epididymis (A, E: caput, B, F: corpus, C, G: cauda) as well as ejaculated/native sperm (D, H). Representative forward (FSC) versus side scatter (SSC) plots (A-D) and green (Fl-1) versus red (Fl-3) fluorescence (E-H) plots are shown. Scatter properties and fluorescence intensities are plotted as channel numbers, WinMDI software was used for display of scatter plots.

RESULTS

b b

a a a

b b a

b b

Figure 4.2.2. Flow cytometric analysis with SCSA, of immature sperm isolated from the caput (purple bars), corpus (green bars) and cauda (red bars) regions of the epididymis. Forward (A) and side (B) scatter properties, as well as green (C) and red fluorescence intensities (D) were determined for epididymal sperm. Mean values ± standard deviations are presented, determined for sperm from six different stallions. Values with different subscript letters differ significantly (p<0.05).

RESULTS

Figure 4.2.3. Induced DNA-damage in epididymal sperm, analyzed using SCSA. In panel A, a representative green versus red fluorescence scatter plot is shown, with indicated regions for all sperm (R1, in blue) as well as sperm with induced DNA-damage (R2, in red). Percentages of sperm with induced DNA-damage were determined as the percentage of sperm in R2 (B, left panel) as well as DFI-values according to EVENSON et al (2002); for epididymal sperm from the caput (purple bars), as compared to corpus (green bars) and cauda (red bars) regions. Mean values ± standard deviations are presented, determined for sperm from six different stallions.

Figure 4.2.4. Sperm from the epididymal cauda without further treatment (red bars) and after incubation with seminal plasma (light blue bars), as well as ejaculated/native sperm (blue bars), were analyzed using SCSA with different incubation periods with acid solution. In panel A, percentages of sperm with induced DNA-damage are plotted versus the denaturation period sperm were exposed to, while side scatter properties of these samples (for Td: 30 s) are shown in panel B. Mean values ± standard deviations are presented for sperm from two different stallions.

RESULTS

4.2.2. Effects of disulfide-reducing and -stabilizing treatment on chromatin stability

Dithiothreitol (DTT) and diamide (DA) were used to reduce and stabilize disulfide-bonds within the DNA backbone, respectively, after which chromatin stability was tested using SCSA. Representative side versus forward scatter and green versus red fluorescence intensity plots as obtained after such analyses are shown in figure 4.2.5 and 4.2.6. It should be noted that DTT-treatment resulted in aggregation of sperm, especially with high concentrations (see figure 4.2.5 B). When sperm was treated with increasing concentrations of DTT, green fluorescence increased up to 2.79-fold. In contrast, diamide treatment did not affect green fluorescence intensity (figure 4.2.7A). Also, red fluorescence increased with exposure to increasing DTT- concentrations (up to 4.51-fold), whereas no differences were seen for sperm treated with diamide (figure 4.2.7B). Side scatter properties of sperm were found to be decreased after DTT-treatment, whereas diamide-treatment did not affect sperm granularity (figure 4.2.7.C). Percentages of sperm with induced DNA-damage increased with exposure to higher DTT-concentrations (figure 4.2.7.D). Side scatter properties as well as green fluorescence intensities changed significantly with increasing concentrations of DTT (p<0.05), whereas induced DNA damage and red fluorescence intensities increased significantly after treatment with at least 2.5 mM DTT (p<0.05).

RESULTS

A B

0 mM DTT 10 mM DTT

side scatter (channel) sidescatter

forward scatter (channel)

C D

0 mM DTT 10 mM DTT

green fluoresence (channel) fluoresence green

red fluoresence (channel)

Figure 4.2.5. Flow cytometric analysis with SCSA, of non-treated sperm (A, C) and sperm treated with 10 mM of the disulfide-reducing agent dithiothreitol (DTT). Representative forward versus side scatter plots are shown (A, B), as well as versus red fluorescence intensity plots (C, D). Scatter properties and fluoresence intensities are plotted as channel numbers, WinMDI software was used for display of scatter plots.

RESULTS

A B

0 mM diamide 10 mM diamide

side scatter (channel) sidescatter

forward scatter (channel)

C D

0 mM diamide 10 mM diamide

green fluoresence (channel) fluoresence green

red fluoresence (channel)

Figure 4.2.6. Flow cytometric analysis with SCSA, of non-treated sperm (A, C) and sperm treated with 10 mM of the disulfide-stabilizing agent diamide (B, D). Representative forward versus side scatter plots are shown (A, B), as well as green versus red fluorescence intensity plots (C, D). Scatter properties and fluoresence intensities are plotted as channel numbers, WinMDI software was used for display of scatter plots.

RESULTS

Figure 4.2.7. SCSA was used to determine induced DNA-damage (A), side scatter properties (B), as well as green (C) and red (D) fluorescence intensities; for sperm exposed to increasing concentrations of DTT (green squares) or diamide (red squares). Mean values ± standard deviations are presented, determined for three pooled sperm samples (each consisting of sperm from four different stallions).

RESULTS

4.3. Light and electron microscopic evaluation of alterations in sperm nuclear morphology

Light microscopy was used to study morphology of epididymal sperm (figure 4.3.1.) and alterations in sperm (head) morphology after treatment with DTT and Fenton`s solution in comparison to non-treated sperm (figure 4.3.2.). Epididymal sperm showed cytoplasmic droplets, whereof proximal droplets were primarily found in sperm collected from the epididymal caput and distal droplets in sperm from the epididymal corpus and cauda region. Overall epididymal samples showed a morphologically homogeneous sperm population with similar size and head morphology. Sperm treated with DTT and Fenton`s solution exhibited alterations in sperm morphology including fractured midpieces and tails, swollen heads, membrane defects and vacuoles located at the head surface (figure 4.3.2. B, C). Transmission electron microscopy was used study the nuclear ultrastructure of native (non-treated) sperm as well as sperm treated with DA, DTT or Fenton’s solution (figure 4.3.3.) to modify chromatin interactions or induce DNA damage. Small nuclear vacuoles as well as alterations in membrane intactness were seen in all samples. Fenton-treated sperm, however, shows more damaged membrane structures and loosened nuclear material at the edges of the nucleus. DTT-treated sperm showed less dense nuclear structure with an increased number of vacuoles, as compared to non-treated and DA-treated samples.

RESULTS

A B C

caput corpus cauda

DIC

D E F

caput corpus cauda

phase contrast phase

Figure 4.3.1. Microscopic images of sperm isolated from the epididymal caput (A, D), corpus (B, E) and cauda (C, F) region. Panels A-C and D-F show images as obtained using differential interference contrast (DIC) and phase contrast optics, respectively.

RESULTS

A B C

native sperm DTT-treated Fnt-treated

DIC

D E F

native sperm DTT-treated Fnt-treated

phase contrast phase

Figure 4.3.2. Microscopic images of native sperm (A, D) and sperm treated with DTT (B, E) or Fenton’s solution (C, F). Panels A-C and D- F show images as obtained using differential interference contrast (DIC) and phase contrast optics, respectively.

RESULTS

A B C non-treated non-treated non-treated

D E F diamide-treated diamide-treated diamide-treated

G H I DTT-treated DTT-treated DTT-treated

J K L Fnt-treated Fnt-treated Fnt-treated

Figure 4.3.3. Transmission electron microscopic images of native sperm (A-C) as well as sperm treated with DA (D-F), DTT (G-I) or Fenton’s solution (J-L). Scale bars indicate 250 nm.

DISCUSSION AND CONCLUSIONS

5. DISCUSSION AND CONCLUSIONS

5.1. Analysis of sperm from stallions with different fertility rates and SCSA analysis after induced DNA damage

Stallions that have normal sperm motility and morphology characteristics are not necessarily fertile. It has been suggested that sperm DNA intactness is a more valuable parameter for foreseeing successful fertilization (EVENSON et al. 2002, SPANO et al. 2000, COLENBRANDER et al. 2003). The aim of the first part of the studies described in this thesis was to determine if sperm from different stallions, classified according to their fertility rates (sub-fertile when NRR < 50%, fertile when > 50%), exhibited differences in sperm characteristics as well as stability towards induced DNA damage. It was found that poor fertility stallions exhibited higher percentages of morphologically abnormal sperm and lower percentages of motile sperm, as compared to good fertility stallions. Furthermore, these two parameters were positively correlated. For stallions, sperm characteristics like increased abnormal sperm morphology and motility are commonly correlated with decreased non-return rates or foaling rates (SULLIVAN et al. 1997, VAN BUITEN et al. 1998, MORRIS and ALLEN 2002). It can be expected that motility characteristics of morphologically abnormal sperm are affected, e.g. due to abnormal midpiece and flagellar bending. Abnormal motility as well as malformed acrosomal structures could diminish successful penetration of the female reproductive tract and fertilization of an oocyte (NEILD et al. 2005, MCPARTLIN et al. 2009). In our studies we also found that DFI values were higher for stallions classified to be of poor fertility, as compared to good fertility stallions. This is in agreement with findings of others, determined for sperm from multiple species including human and stallion (SPANO et al. 2000, SAKKAS and TOMLINSON 2000, LOVE and KENNEY 1998, EVENSON et al 2002). Furthermore, in addition to our findings, there are several reports on correlations between DNA fragmentation levels and motility parameters (GIWERCMAN et al. 2003) as well as sperm morphology properties (MORRELL et al. 2008).

DISCUSSION AND CONCLUSIONS

Abnormal sperm morphology has been described to coincide with altered DNA packing. Studies by SAKKAS and TOMLINSON (2000) and UTSUNO et al. (2014) described a correlation between DNA fragmentation and head shape abnormalities as well as increased numbers of nuclear vacuoles. Non-complete substitution of histones by protamines, presence of large amounts of DNA bound to histones in sperm, and relative amounts of protamine 1 and 2 in sperm, have been correlated with the extent of DNA packing and fertilizing potential (WARD 2010). Studies performed by GOSÁLVEZ et al. (2011) revealed higher percentages of sperm with DNA damage in spermatozoa from mammals containing both P1 and P2 as compared to those containing only P1. Abnormal P1/P2 mRNA ratios in sperm have been suggested to be a marker for decreased fertility in humans (CASTILLO et al. 2011, UTSUNO et al. 2014). For stallions, P1/P2 mRNA ratios were determined and correlated with fertility rates by PARADOWSKA-DOGAN et al. (2014). Since fertility typically decreases with age, sperm abnormal morphology and DNA intactness also correlate with stallion age (DOWSETT and KNOTT 1996).

DNA nicks formed by accident during chromatin remodeling with sperm development can lead to DNA strand breaks in mature spermatozoa. Further causes for decreased DNA intactness in mature sperm include damage due to oxidative stress or exposure to environmental changes (e.g. temperature and osmotic changes) after ejaculation and preservation for artificial insemination (GONZÁLEZ- MARIN et al. 2012). In this study, chromatin intactness was evaluated using SCSA according to EVENSON and JOST (2000); which includes incubation in acid solution to provoke DNA denaturation. In our studies, we determined if sperm from individual stallions exhibited differences in stability towards induced DNA damage and if this correlated with fertility. DNA fragmentation was induced: (1) via incubation for different durations in acid solution with SCSA, or (2) exposure to different amounts of Fenton’s reagent to induce reactive oxidative species. We found significant differences in susceptibility for acid induced DNA damage of sperm from individual stallions. When using a standard incubation time of 30 s, mean DFI values varied between 12-23%. These values increased with higher

DISCUSSION AND CONCLUSIONS

differences between individuals when using an incubation time of 5 min: values varied between 15-32%. Plots in which DNA fragmentation was plotted versus the duration of the acid incubation revealed similar kinetics for all stallions tested, with maximum DFI levels after 1-5 minutes. According to EVENSON et al. (1980), who developed the SCSA protocol for human sperm, an acid incubation time of 30 seconds should be employed. According to our results, when using stallion sperm, an incubation time of 5 minutes would allow determination of the maximum amount of induced DNA damage, with largest differences amongst individuals. In addition to DFI-values, we followed changes in green and red fluorescence. Green fluorescence was found to increase with increasing denaturation time. This is likely the result of increased accessibility for acridine orange. However, no differences in green fluorescence were seen amongst individuals. Red fluorescence of acridine orange exhibited similar trends as seen for DFI values, suggesting that it can be used as a measure for induced DNA damage. The additive value of expressing changes in red fluorescence as alpha-T values is, that a normalization is performed towards the sum of red and green fluorescence (EVENSON and JOST 2000) When exposing to different amounts of Fenton’s solution, a dose-dependent increase in DNA damage was found with increasing concentrations of Fenton’s solution. Different individuals exhibited differences in the threshold for inducing damage. Differences among stallions were most significant after treatment with intermediate concentrations of Fenton’s solution (33-50%), while in all cases DFI values up to 100% could be obtained with exposure to maximum oxidative stress (with 67% Fenton’s solution). Differences between individuals for iatrogenic DNA damage were also found by LÓPEZ-FERNÁNDEZ et al. (2007) and GOSÁLVEZ et al. (2011), who incubated sperm samples for prolonged periods at 37°C during which they evaluated DNA fragmentation. Interestingly, they found DNA fragmentation to increase up to 100% when samples were incubated for 48 h at 37°C. It should be noted, however, that they used the sperm chromatin dispersion test (LÓPEZ- FERNÁNDEZ et al. 2007) as well as the Halo test (GOSÁLVEZ et al. 2011) to evaluate the extent of DNA fragmentation. LOVE et al (2002) performed SCSA analysis for stallion sperm samples stored for up to 2 d at 37°C. Moreover, for

DISCUSSION AND CONCLUSIONS

storage at 5°C, they found that chromatin quality declined more rapidly (DFI values increased) for sperm from poor fertility stallions as compared to good fertility stallions. BAUMBER et al. (2003) reported increasing DNA fragmentation after exposure to oxidative stress using the xanthine/xanthine oxidase system to induce ROS formation and comet assay for evaluating DNA fragmentation. Several studies exist in which DNA intactness evaluated with SCSA are corroborated with alternative methods to evaluate DNA fragmentation (SAKKAS and TOMLINSON 2000, FERNÁNDEZ et al. 2005, SIMON et al. 2014). However, it maintains to be determined how absolute numbers obtained via different methods in different labs correlate and reference samples or treatments can be employed to standardize. With FTIR microspectroscopy of Fenton-treated sperm samples we found drastic changes in bands arising from endogenous biomolecules including nucleic acids, indicating massive damage. Similar results were obtained by SÁNCHEZ et al (2012) and HUANG et al. (2014) for human sperm. The drastic increase in the band around 950- 1000 cm-1 with increased exposure to reactive oxygen species, seen here for stallion sperm, likely results from an increase in the amount of free phosphate groups released from the DNA backbone (MELLO and VIDAL 2012). In addition to FTIR, Raman microspectroscopy is receiving increasing interest as a method to evaluate sperm DNA biomolecular structure and damage for foreseeing fertilizing potential (HUSER et al. 2009, MALLIDIS et al. 2011, SANCHEZ et al. 2012).

5.2. Analysis of sperm with differences in chromatin packaging

With sperm maturation, and transit through the epididymis, DNA will be tightly packed. This occurs through replacement of histone proteins by protamines and increased formation of inter- and intramolecular disulfide bonds (WARD 1993). The aim of the second part of the studies described in this thesis was to determine if DNA packing affected DNA stability towards exposure to denaturation conditions or oxidative stress. Therefore, sperm granularity, intercalation of fluorescent dye and induced DNA damage were evaluated for (1) immature spermatozoa isolated from three different regions of the epididymis (caput, corpus and cauda), as well as (2)

DISCUSSION AND CONCLUSIONS

sperm treated with chemical compounds for reducing and oxidizing disulfide bonds in chromatin (dithiothreitol and diamide, respectively). We determined that with maturation, sperm granularity as well as size increased, which coincided with decreased accessibility for the DNA binding dye acridine orange and decreased susceptibility for inducing DNA damage via acid denaturation (decreased green and red fluorescence, respectively). Reduced intercalation of acridine orange, likely results from more tight packing of the DNA with protamines during transit through the epididymis. This has already been described for human sperm by KOSOWER et al. (1992) and GOLAN et al. (1996). Furthermore, we determined that decreased accessibility coincides with increased stability of sperm DNA towards induced damage. In addition to intrinsic properties (sperm developmental stage) affecting DNA stability towards induced damage, we found that addition of seminal plasma had an effect on DNA intactness after acid denaturation. When sperm from the epididymal cauda were incubated with seminal plasma this resulted in an increased percentage of sperm with damaged DNA and decreased granularity, comparable to that of ejaculated sperm. Seminal plasma might be a source for ROS and damaging reactions which might result in unpacking of DNA and susceptibility for damaging reactions (WNUK et al. 2010, GONZÁLEZ-MARIN et al. 2012). LOVE et al. (2005) determined that DNA intactness is negatively affected by when incubated with increasing amounts of seminal plasma. Sperm DNA packing can also be affected by employment of chemical compounds for reducing and oxidizing SH- and disulfide groups (dithiothreitol and diamide, respectively) in chromatin (KOSOWER et al. 1992, BARRERA et al. 1993, ANDREETTA et al. 1995). After treatment with DTT we determined that sperm exhibited a reduced granularity coinciding with higher accessibility for acridine orange and increased numbers of sperm with acid denatured DNA. These results are in agreement with the findings of KOSOWER et al. (1992), and confirm that DTT treatment results in less packed/more loose chromatin and consequently DNA becomes more susceptible for inducing damage. In contrast, stabilization of disulfide bonds would ensure DNA stability; as occurs in nature through crosslinking by

DISCUSSION AND CONCLUSIONS

protamines. Oxidation of free thiol groups at the cysteine residues of protamines by diamides would result in stabilizing disulfide bonds. When treating ejaculated sperm with diamide, we did not find a beneficial effect: sperm granularity and resistance towards induced DNA damage were similar as for non-treated sperm. This can be the result of the sperm samples used; for which the number of free SH-groups available for further stabilization was limited. Diamide-treatment for stabilization of sperm DNA has been used for epididymal sperm (KANEKO et al. 2003). Furthermore, it remains to be determined if diamide treatment has beneficial effects for stabilizing sperm DNA of stallions exhibiting poor fertility rates (KOSOWER 1992, KANEKO et al. 2003). In addition to flow cytometric analysis of acridine orange stained sperm after acid denaturation, we used transmission electron microscopy; to obtain more insights in sperm nuclear morphology and ultrastructure after induced damage (Fenton- treatment, DTT-treatment) or stabilization (diamide-treatment). It should be noted that the fixation and embedding procedure needed for TEM analyses resulted in additional artefacts. Interestingly, we found that sperm exposed to Fenton’s reagent exhibiting DFI-values of 100% did not show drastic changes in density or ultrastructure of the inner part of the nucleus itself, as compared to non-treated samples. However, more severe abnormalities in the sperm membranes were seen; likely caused by lipid peroxidation. We suggest that such decreased membrane intactness allows for access to sperm DNA, especially at the edges of the nucleus. Samples treated with DTT displayed more intact nuclear membranes and a less dense/brighter nuclear structure with increased numbers of vacuoles. Such ultrastructural changes in sperm chromatin electron density with TEM have been observed before (IRANPOUR 2014), and resembles a less dense packing as also observed for immature sperm (DIAS et al. 2006) likely caused by reduction of disulfide bonds in the chromatin structure. Diamide-treated sperm looked similar as non-treated sperm samples, which corroborates our findings on granularity properties as determined using flow cytometry.

DISCUSSION AND CONCLUSIONS

5.3. Conclusions

We determined that for stallions with poor fertility rates, percentages of morphologically abnormal sperm and DFI values were increased, while sperm progressive motility was decreased. Moreover, for poor fertility stallions we found the highest levels of induced DNA damage after acid treatment and lowest threshold levels of Fenton’s reagent for inducing damage via exposure to oxidative attack. We found that DFI values after 5 min acid treatment with SCSA, instead of the commonly used 30 s, resulted in maximum levels of induced DNA damage with largest differences amongst individuals. Exposure to Fenton’s reaction finally resulted in 100% DNA fragmentation for all stallions. Red fluorescence of acridine orange showed similar trends as DFI and alpha-t values and thus might be useful for indicating DNA intactness. In our studies we correlated accessibility for the dye acridine orange and acid induced DNA damage with the degree of chromatin packing. With transit through the epididymis and maturation sperm granularity increased which coincided with a decreased green and red fluorescence of acridine orange, indicative for accessibility and induced DNA damage, respectively. Treatment with DTT for reducing disulfide bonds in sperm chromatin caused a reduced sperm granularity and increased fluorescence and access of acridine orange with higher numbers of sperm with damaged DNA after acid denaturation. Diamide-treatment of ejaculated sperm did not show improved DNA stability in our experiments. Taken together, we conclude that DNA packing affects accessibility of chromatin for inducing DNA damage, which in turn might explain differences in fertility rates amongst stallions.

SUMMARY

6 SUMMARY

Saskia Schütze (2015):

DNA-stability of stallion sperm: Factors affecting chromatin integrity in individual stallions

Stallions exhibit great inter-individual variation in fertility rates. DNA intactness is essential for successful fertilization of an oocyte and embryonic development, and therefore analysis of sperm DNA structure might be used to foresee fertilizing potential or explain subfertility. Moreover, insights in factors affecting chromatin intactness and susceptibility for DNA damage might give rise to approaches for counteracting decreased chromatin intactness and fertility rates. Chromatin packing, through interactions between DNA and sperm specific protamines via disulfide bonds has been suggested to be one of the factors affecting chromatin stability. The aim of the first part of this study was to determine if sperm from stallions classified according to their fertility exhibit differences in chromatin intactness and stability towards induced DNA damage. Sperm DNA damage was induced via exposure to acid denaturation for different durations as well as exposure to different concentrations of Fenton’s reagent to provoke oxidative stress/damage. DNA intactness was analyzed using the sperm chromatin structure assay (SCSA), which involves acid denaturation and flow cytometric analysis of acridine orange stained sperm for discriminating between intact and damaged DNA. It was found that poor fertility stallions exhibited higher percentages of morphologically abnormal sperm, lower percentages of motile sperm, and a decreased DNA intactness with SCSA, as compared to good fertility stallions. Furthermore, the susceptibility for acid denaturation and oxidative stress induced sperm DNA damage was increased with decreasing fertility rates. DNA fragmentation reached a plateau with acid denaturation for 5 minutes, with largest differences amongst individuals. A dose- dependent increase in DNA damage was found with exposure to Fenton’s reaction and oxidative stress, with individual differences in the threshold for inducing damage,

SUMMARY

while ultimately resulting in DNA fragmentation indices of 100%. In addition to SCSA analysis, we collected Fourier transform infrared (FTIR) spectra of sperm samples treated with increasing concentrations of Fenton’s solution and determined that induced oxidative stress resulted in alterations in characteristic vibration bands arising from phosphate groups as present in the DNA-backbone. The aim of the second part of this study was to determine if sperm DNA stability towards induced damage depends on the extent of chromatin packing (DNA- protein interactions). Therefore, immature spermatozoa isolated from the epididymis (caput, corpus and cauda regions) were analyzed, as well as sperm treated with chemical compounds for reducing and oxidizing disulfide bonds in chromatin (dithiothreitol and diamide, respectively). It was found that sperm granularity increased with maturation, which coincided with decreased accessibility for the DNA binding dye acridine orange and decreased susceptibility for induced DNA damage via acid denaturation. Treatment with dithiothreitol (DTT) resulted in sperm with reduced granularity coinciding with higher accessibility for acridine orange and increased numbers of sperm with acid denatured DNA, as compared to non-treated sperm or sperm treated with diamide. Inspection of the sperm ultrastructure using transmission electron microscopy, confirmed that the sperm nucleus displayed a less dense ultrastructure after reduction of disulfide bonds, whereas treatment with Fenton’s reagent resulted in severe damage of the nuclear membrane. Taken together, we conclude that DNA packing affects susceptibility of chromatin for inducing DNA damage, which in turn might explain differences in fertility rates amongst stallions.

ZUSAMMENFASSUNG

7 ZUSAMMENFASSUNG

Saskia Schütze (2015):

DNA-Stabilität von Hengstspermien: Einflussfaktoren auf die Chromatinintegrität individueller Hengste

Bei Hengsten zeigen sich große individuelle Unterschiede in ihren Befruchtungsraten. Da die Integrität der DNA wesentlich für eine erfolgreiche Befruchtung und Entwicklung des Embryos ist, kann die DNA-Struktur als Parameter für das Befruchtungspotenzial genutzt werden oder mögliche Subfertilität erklären. Zudem bieten Informationen über die die Chromatinintegrität beeinflussenden Faktoren sowie die unterschiedliche Anfälligkeit der Spermien für DNA-Schädigung neue Ansätze zur Aufklärung verminderter Befruchtungsraten. Die Chromatinpackung, die durch Interaktionen zwischen der DNA und spermienspezifischen Protaminen sowie durch Disulfidbrücken zwischen den Protaminen vermittelt wird, ist hierbei vermutlich ein wesentlicher Faktor. Im ersten Teil der Studie war das Ziel herauszufinden, ob Spermien von Hengsten, die hinsichtlich ihrer Befruchtungsfähigkeit eingestuft wurden, Unterschiede in der Chromatinintegrität bzw. –stabilität gegenüber induzierter Schädigung aufweisen. Die Spermien-DNA wurde einerseits durch Säuredenaturierung mit verschiedenen Einwirkzeiten und andererseits durch Inkubation mit Fenton´s Reagent, zur Provokation von oxidativem Stress, geschädigt. Anschließend wurde die DNA- Integrität mit Hilfe des Spermien-Chromatin-Struktur-Assay (SCSA) ermittelt. Dieser beruht auf einer Proteindenaturierung mittels Säure sowie einer anschließenden durchflusszytometrischen Analyse nach Anfärben mit Acridine Orange und ermöglicht eine Unterscheidung zwischen intakter und geschädigter DNA. Es wurde nachgewiesen, dass Hengste mit schlechten Befruchtungsraten einen höheren Anteil an morphologisch abweichenden Spermien, weniger vorwärts bewegliche Spermien und einen erhöhten Anteil an Spermien mit geschädigter DNA im Vergleich zu

ZUSAMMENFASSUNG

Hengsten mit guten Befruchtungsraten aufweisen. Weiterhin wurde festgestellt, dass sich die Empfindlichkeit für säureinduzierte sowie durch oxidativen Stress verursachte DNA-Schädigung mit abnehmenden Befruchtungsraten erhöht. Nach einer Säureeinwirkzeit von 5 min erreichte das Ausmaß der DNA-Fragmentation ein Plateau, bei welchem die größten Unterschiede zwischen den Individuen nachweisbar waren. Eine Dosis-abhängige Zunahme der DNA-Schädigung wurde mit zunehmenden Konzentrationen an Fenton`s Reagent deutlich, wobei individuell verschiedene Schwellenwerte für das Induzieren von Schädigung bestanden. Letztendlich konnte jedoch bei allen Hengsten 100% DNA-Schädigung durch oxidativen Stress induziert werden. Zusätzlich zu den SCSA Messungen wurden Fourier-Transform-Infrarot Spektra (FTIR) von Fenton-behandelten Spermaproben angefertigt, bei denen nach oxidativer Schädigung deutliche Unterschiede in den Molekülbewegungen der DNA-charakteristischen Phosphatgruppen erkennbar waren. Ziel des zweiten Teils der Studie war herauszufinden, ob die DNA-Stabilität gegenüber induzierter Schädigung abhängig vom Grad der Chromatinpackung ist. Dafür wurden unreife Spermien aus dem Nebenhoden (Nebenhodenkopf, -körper, - schwanz) isoliert und analysiert sowie Spermien untersucht, die mit chemischen Verbindungen behandelt wurden, welche Disulfidbrücken im Chromatin reduzieren bzw. oxidieren (Dithiothreitol bzw. Diamide). Es wurde ermittelt, dass sich die Granularität der Spermien mit zunehmender Reife erhöht, welche die verminderte Zugänglichkeit für den DNA-bindenden Farbstoff Acridine Orange und die verminderte Empfindlichkeit für induzierbare Schädigung durch Säuredenaturierung erklärt. Eine Behandlung mit Dithiothreitol (DTT) resultierte in verminderter Granularität der Spermien, was eine erhöhte Zugänglichkeit für Acridine Orange und die zunehmende Anzahl an Spermien mit geschädigter DNA, im Gegensatz zu unbehandelten Spermien oder zu solchen, die mit Diamiden behandelt worden sind, bestätigt. Außerdem konnten die Ergebnisse mit elektronenmikroskopischen Bildern belegt werden, auf denen eine aufgelockerte Chromatinstruktur nach Reduktion der Disulfidbrücken sichtbar war. Alles in allem lässt sich schlussfolgern, dass die Packung der DNA die Empfindlichkeit des Chromatins gegenüber induzierter

ZUSAMMENFASSUNG

Schädigung beeinflusst, wodurch wiederum Unterschiede in den Befruchtungsraten von Hengsten erklärt werden können.

REFERENCES

8 REFERENCES

AGARWAL, A., R. A. SALEH and M. A. BEDAIWY (2003): Role of reactive oxygen species in the pathophysiology of human reproduction Fertil. Steril. 79 (4), 829-843

AITKEN, J. D. and S. D. ROMAN (2008): Antioxidant systems and oxidative stress in the testes Oxid. Med. Cell. Longev. 1, 15-24

AITKEN, R. J., T.B. SMITH, M. S. JOBLING, M. A. BAKER and G. N. DE IULIIS (2014): Oxidative stress and male reproductive health Asian J. Androl.16, (31–38)

ALLIS, D., A. BIRD, S. GASSER, E. GREEN, D. KOSHLAND, U. LAEMMLI, M. LYNCH, H. MADHANI, E. MARGOLIES, G. NARLIKAR and M. OLSON (2008): DNA, chromosomes and genomes in: ALBERTS B., A. JOHNSON, J. LEWIS, M. RAFF, K. ROBERTS, P. WALTER: Molecular biology of the cell Garland Science, New York, 5th Edition, pages 195-263

AMARAL, S., K. REDMANN, V. SANCHÉZ, C. MALLIDIS, J. RAMALHO-SANTOS and S. SCHLATT (2013): UVB irradiation as a tool to assess ROS-induced damage in human spermatozoa Andrology 1, 707–714

ANDREETTA, A. M., J. C. STOCKERT and C. BARRERA (1995): A simple method to detect sperm chromatin abnormalities: cytochemical mechanism and possible value in predicting semen quality in assisted reproductive procedures Int. J. Androl. 18, 23-8

REFERENCES

BALHORN, R. (1982): A model for the structure of chromatin in mammalian sperm J. Cell. Biol. 93, 298-305

BALHORN, R. (2007): The protamine family of sperm nuclear proteins Genome Biol. 8 (9): 227

BARRERA, C., A. B. MAZZOLLI, C. PELLING and J. C. STOCKERT (1993): Metachromatic staining of human sperm nuclei after reduction of disulfide bonds Acta Histochem. 94 (2), 141-9

BAUMBER, J., B. A. BALL, J. J. LINFOR and S. A. MEYERS (2003): Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa J. Androl. 24 (4), 621-628

BEISKER, W. (1994): A new combined integral-light and slit-scan data analysis system (DAS) for flow cytometry Comput. Methods Programs Biomed. 42, 15-26

BJÖRNDAL, L. and U. KVIST (2010): Human sperm chromatin stabilization: a proposed model including zinc bridges Mol. Hum. Reprod. 16 (1), 23-29

BJÖRNDAL, L. and U. KVIST (2014): Structure of chromatin in spermatozoa in: BALDI, E. and M. MURATORI: Genetic damage in human spermatozoa Springer, New York, pages 1-11

REFERENCES

BRITO, L. F. C. (2007): Evaluation of stallion sperm morphology Clinical Techniques in Equine Practice 6, 249-264

BRUEMMER, J. E. (2006): Collection and freezing of epididymal stallion sperm Vet. Clin. Equine 22, 677-682

CASTILLO, J., L. SIMON, S. DE MATEO, S. LEWIS, R. OLIVA (2011): Protamine/DNA ratios and DNA damage in native and density gradient centrifuged sperm from infertile patients J. Androl. 32 (3), 324-332

CAVALCANTI, M. C. O., M. RIZGALLA, J. GEYER, K. FAILING, L. F. LITZKE and M. BERGMANN (2009): Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis Anim. Reprod. Sci. 111, 220–234

COLENBRANDER, B., B. M. GADELLA and T. A. E. STOUT (2003): The predictive value of semen analysis in the evaluation of stallion fertility Reprod. Dom. Anim. 38, 305–311

CORTÉS-GUTIÉRREZ, E. I., M. I. DÁLIVA-RODRÍGUEZ, C. LÓPEZ-FERNÁNDEZ, J. L. FERNÁNDEZ, F. CRESPO and J. GOSÁLVEZ (2014): Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa Theriogenology 81 (2), 321–325

REFERENCES

CORZETT, M., J. MAZRIMAS, R. BALHORN (2002): Protamine 1: Protamine 2 stoichiometry in the sperm of eutherian mammals Mol. Reprod. Dev. 61, 519-527

DIAS, G. M., C. A. RETAMAL, L. TOBELLA, A. C. V. ARNHOLDT and M. L. LÓPEZ (2005): Nuclear status of immature and mature stallion spermatozoa Theriogenology 66, 354-365

DOTT, H. M. and G. C. FOSTER (1972): A technique for studying the morphology of mammalian spermatozoa which are eosinophilic in a differential 'life-dead' stain J. Reprod. Fertil. 29, 443–445

DOTT, H. M. and G. C. FOSTER (1975): Preservation of differential staining of spermatozoa by formol citrate J. Reprod. Fertil. 45, 57–60

DOWSETT, K. F. and L. M. KNOTT (1996): The influence of age and breed on stallion semen Theriogenology 46 (3), 397-412

EVENSON, D. P., Z. DARZYNKIEWICZ and M. R. MELAMED (1980): Relation of mammalian sperm chromatin heterogeneity to fertility Science 210 (4474), 1131-1133

EVENSON, D. P. and L. K. JOST (2000): Sperm chromatin structure assay is useful for fertility assessment Methods Cell Sci. 22, 169-189

REFERENCES

EVENSON, D. P., K. L. LARSON and L. K. JOST (2002): Sperm Chromatin Structure Assay: Its clinical use for detecting DNA fragmentation in male infertility and comparisons with other techniques J. Androl. 23 (1), 25-43

FERNÁNDEZ, J. L., L. MURIEL, V. GOYANES, E. SEGRELLES, J. GOSÁLVEZ, M. ENCISO, M. LAFROMBOISE and C. DE JONGE (2005): Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test Fertil. Steril. 84 (4), 833-842

GIWERCMAN, A., J. RICHTHOFF, H. HJOLLUND, J. P. BONDE, K. JEPSON, B. FROHM and M. SPANO (2003): Correlation between sperm motility and sperm chromatin structure assay parameters Fertil. Steril. 80 (6), 1404-1412

GOLAN, R, T. G. COOPER, Y. OSCHRY, F. OBERPENNIG, H. SCHULZE, L. SHOCHAT and L. M. LEWIN (1996): Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry Hum. Reprod. 11 (7), 1457-1462

GONZÁLEZ-MARÍN, C., J. GOSÁLVEZ and R. ROY (2012): Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells Int. J. Mol. Sci. 13, 14026-14052

GORCZYCA, W., J. P. GONG, Z. DARZYNKIEWICZ (1993): Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays Cancer Res. 53 (8), 1945-1951

REFERENCES

GOSÁLVEZ, J., C. LÓPEZ-FERNÁNDEZ, J. L. FERNÁNDEZ, A. GOURAUD and W. V. HOLT (2011):

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species Mol. Reprod. Dev. 78, 951-961

HUANG, Z., H. XIAO, T. QI, Z. HU, H. LI, D. CHEN, Y. XU, J. CHEN (2014):

Antioxidative protective effect of Icariin on the FeSO4/H2O2-damaged human sperm based on confocal Raman micro-spectroscopy J. Huazhong Univ. Sci. Technol. Med. Sci. 34, 755-780

HUGHES, C. M., S. E. LEWIS, V. J. MCKELVEY-MARTIN and W. THOMPSON (1996): A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay Mol. Hum. Reprod. 2 (8), 613-9

HUSER, T., C. A. ORME, C. W. HOLLARS, M. H. CORZETT and R. BALHORN (2009): Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells J. Biophotonics 2, 522-332

IRANPOUR, F. G. (2014): The effects of protamine deficiency on ultrastructure of human sperm nucleus Adv. Biomed. Res. 3 (1), 1-6

JOHNSON, L., T. L. BLANCHARD, D. D. VARNER and W. L. SCRUTCHFIELD (1997): Factors affecting spermatogenesis in the stallion Theriogenology 48, 1199-1216

REFERENCES

KANEKO, T., D. G. WITTINGHAM, J. W. OVERSTREET and R. YANAGIMACHI (2003): Tolorance of the mouse sperm nuclei to freeze-drying depends on their disulfide status Biol. Reprod. 69, 1859-1862

KOSOWER, N. S., H. KATAYOSE and R. YANAGIMACHI (1992): Thilol-dislufide status and Acridine Orange fluorescence of mammalian sperm nuclei J. Androl. 13 (4), 342-348

KRZANOWSKA, H. (1982):

Toluidine blue staining reveals changes in chromatin stabilization of mouse spermatozoa during epididymal maturation and penetration of ova

J. Reprod. Fertil. 64 (1), 97-101

KUMAROO, K. K., G. JAHNKE and J. L. IRVIN (1975): Changes in basic chromosomal proteins during spermatogenesis in the mature rat Arch. Biochem. Biophys. 168 (2), 413–424

LÓPEZ-FERNÁNDEZ, C., F. CRESPO, F. ARROYO, J. L. FERNÁNDEZ, P. ARANA, S. D. JOHNSTON and J. GOSÁLVEZ (2007): Dynamics of sperm DNA fragmentation in domestic animals II. The stallion Theriogenology 68, 1240-1250

LOVE, C. C. and R. M. KENNEY (1998): The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion Theriogenology 50 (6), 955–972

REFERENCES

LOVE, C. C. and R. M. KENNEY (1999): Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion

Biol. Reprod. 60, 615-620

LOVE, C.C., J. A. THOMPSON, V. K. LOWRY and D. D. VARNER (2002): Effect of storage time and temperature on stallion sperm DNA and fertility Theriogenology 57, 1135-1142

LOVE, C. C., S. P. BRINSKO, S. L. RIGBY, J. A. THOMPSON, T. L. BLANCHARD and D. D. VARNER (2005): Relationship of seminal plasma level and extender type to sperm motility and DNA integrity Theriogenology 63, 1584–1591

LOVE, C. C. (2005):

The sperm chromatin structure assay: A review of clinical applications

Anim. Reprod. Sci. 89, 39-45

MALLIDIS, C., J. WISTUBA, B. BLEISTEINER, O. S. DAMM, P. GROß, F. WÜBBELING, C. FALLNICH, M. BURGER and S. SCHLATT (2011): In situ visualization of damaged DNA in human sperm by Raman microspectroscopy Hum. Reprod. 26 (7), 1641–1649

MANICARDI, G. C., P. G. BIANCHI, S. PANTANO, P. AZZONI, D. BIZZARO, U. BIANCHI and D. SAKKAS (1995): Presence of endogenous nicks in DNA of ejaculated human spermatozoa and its relationship to Chromomycin A3 accessibility Biol. Reprod. 52, 864-867

REFERENCES

MCPARTLIN, L., A., S. S. SUAREZ, C. A. CZAYA, K. HINRICHS and S. J. BEDFORD-GUAUS (2009): Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes Biol. Reprod. 81, 199-206

MELLO, M. L. S. and B. C. VIDAL (2012): Changes in the infrared microspectroscopic characteristics of DNA caused by cationic elements, different base richness and single-stranded form PLOS ONE 7, e43169

MORRELL, J.M., A. JOHANNISSON, A. M. DALIN, L HAMMAR, T. SANDEBERT and H. RODRIGUEZ-MARTINEZ (2008): Sperm morphology and chromatin integrity in Swedish Warmblood stallions and their relationship to pregnancy rates Acta Vet. Scand. 50, 1-7

MORRIS, L. H. and W. R. ALLEN (2002): Reproductive efficiency of intensively managed Thoroughbred mares in Newmarket Equine Vet J. 34 (1), 51-60

NEILD, D. N., B. M. GADELLA, A. AGÜERO, T. A. E. STOUT and B.

COLENBRANDER (2005): Capacitation, acrosome function and chromatin structure in stallion sperm Anim. Reprod. Sci. 89, 47–56

OSTLING, O. and K. J. JOHANSON (1984): Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells Biochem. Biophys. Res. Commun. 123 (1), 291-8

REFERENCES

PARADOWSKA-DOGAN, A., A. FERNANDEZ, M. BERGMANN, K. KRETZER, C. MALLIDIS, M. VIEWEG, P. WALISZEWSKI, M. ZITZMANN, W. WEIDNER, K. STEGER and S. KLIESCH (2014): Protamine mRNA ratio in stallion spermatozoa correlates with mare fecundity

Andrology 2, 521-530

SAKKAS, D. and M. TOMLINSON (2000):

Assessment of sperm competence Semin. Reprod. Med. 18 (2), 133-140

SÁNCHEZ, V., K. REDMAN, J. WISTUBA, F. WUEBBELING, M. BURGER, H. OLDENHOF, W. F. WOLKERS, S. KLIESCH, S. SCHLATT and C. MALLIDIS (2012): Oxidative DNA damage in human sperm can be detected by Raman microspectroscopy Fertil. Steril. 98, 1124-1129

SÁNCHEZ, V., J. WISTUBA and C. MALLIDIS (2013): Semen analysis: update on clinical value, current needs and future perspectives Reproduction 146, 249-258

SCHLEGEL, P. N. and D. A. PADUCH (2005):

Yet another test of sperm chromatin structure

Fertil. Steril. 84 (4), 854-859

SIEME, H., G. MARTINSSON, H. RAUTERBERG, K. WALTER, C. AURICH, R.

PETZOLDT and E. KLUG (2003): Application of techniques for sperm selection in fresh and frozen-thawed stallion semen Reprod. Dom. Anim. 38, 134–140

REFERENCES

SIMON, L., L. LIU, K. MURPHY, S. GE, J. HOTALING, K. I. ASTON, B. EMERY and D. T. CARRELL (2014): Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment Hum. Reprod. 29 (5), 904-917

SINGH, N. P., D. B. DANNER, R. R. TICE, M. T. MCCOY, G. D. COLLINS and E. L. SCHNEIDER (1989): Abundant alkali-sensitive sites in DNA of human and mouse sperm Exp. Cell Res. 184 (2), 461-70

SPANO, M., J. P. BONDE, H. I. HJOLLUND, H. A. KOLSTAD, E. CORDELLI and G. LETER (2000): Sperm chromatin damage impairs human fertility Fertil. Steril. 73 (1), 43-50

STEGER, K., T. KLONISCH, K. GRAVENIS, B. DRABENT, D. DOENECKE and M. BERGMANN (1998): Expression of mRNA and protein of nucleoproteins during human spermiogenesis Mol. Hum. Reprod. 4 (10), 939-945

STEGER, K., K. FAILING, T. KOLINSCH, H.M. BEHRE, M. MANNING, W. WEIDNER, L. HERTLE, M. BERGMANN and S. KLIESCH (2001): Round spermatids from infertile men exhibit decreased protamine-1 and -2 mRNA Hum. Reprod. 16 (4), 709-716

SULLIVAN, J. J., P. C. TURNER, L. C. SELF, H. B. GUTTERIDGE and D. E. BARTLET (1997): Survey of reproductive efficiency in the Quarter-horse and Thoroughbred J. Reprod. Fertil. 23, 315-8

REFERENCES

UTSUNO, H., T. MIYAMOTO, K. OKA and T. SHIOZAWA (2014):

Morphological alterations in protamine-deficient spermatozoa

Hum. Reprod. 29 (11), 2374-2381

VAN BUITEN, A., J. L. REMMEN and B. COLENBRANDER (1998): Fertility of Shetland pony stallions used in different breeding systems: a retrospective study Vet Q. 20 (3), 100-3

VIDAMENT, M., P. ECOT, P. NOUE, C. BURGEOIS, M. MAGISTRINI and E. PALMER (2000): Centrifugation and addition of glycerol at 22°C instead of 4°C improve post-thaw motility and fertility of stallion spermatozoa Theriogenology 54 (6): 907–919

WARD, W. S. and D.S. COFFEY (1991):

DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells

Biol. Reprod. 44, 569-574

WARD, W. S. (1993): Desoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa Biol. Reprod. 48, 1193-1201

WARD, W. S. (2010): Function of sperm chromatin structural elements in fertilization and development Mol. Hum. Reprod. 16, 30-36

REFERENCES

WNUK, M., A. LEWINSKA, B. OKLEJEWICZ, G. BARTOSZ, M. TISCHNER and M. BUGNO-PONIEWIERSKA (2010): Redox status of equine seminal plasma reflects the pattern and magnitude of DNA damage in sperm cells Theriogenology 74, 1677-1684

WOLKERS, W. F. and H. OLDENHOF (2010): In situ FTIR studies on mammalian cells Spectroscopy 24, 525–534

WOUTERS-TYROU, D., A. MARTINAGE, P. CHEVAILLIER and P. SAUTIÈRE (1980): Nuclear basic proteins in spermiogenesis Biochimie 80, 117-128

APPENDIX

9 APPENDIX

9.2. Supplemental tables and figures

Table 9.1. Stallion age and non-return-rates (NRR), and the sperm characteristics: DNA-fragmentation-index (via SCSA analysis with denaturation time of 30 s and 5 min), and percentages of morphologically abnormal and progressively motile sperm. Averages ± standard deviations were determined from three ejaculates for each of ten stallions (H1-10). Group 1 and 2 (G1: H1-5; G2: H6-10) represent groups of poor and good fertility stallions, respectively. Values with different subscript letters differ significantly (p<0.05). stallion age (a) NNR (%) DFI (%) DFI (%) MAS (%) PMS (%) Td 30 s Td 5 min H1 16 45.40 22.52 25.38 68.33 38.99 H2 5 50.00 16.09 35.75 40.50 34.19 H3 16 34.55 20.63 27.86 40.83 29.00 H4 17 44.78 36.31 49.61 64.67 8.33 H5 19 49.15 19.58 21.64 49.50 30.69 G1 14.60± 44.78± 23.03± 32.05± 52.77± 28.24± 5.50 6.15 7.78 a 11.09 a 13.11 a 11.77 a H6 7 59.71 6.99 8.76 32.83 59.99 H7 7 54.92 10.16 16.35 30.00 54.18 H8 15 57.53 20.69 23.11 47.67 38.99 H9 14 70.00 10.39 13.44 28.50 54.70 H10 5 53.42 10.88 14.08 28.00 58.16 G2 9.60± 59.12± 11.82± 15.15± 33.40± 53.21± 4.56 6.55 5.19 b 5.24 b 8.9 b 8.31 b

APPENDIX

100

DFI (%) DFI 40

0 0 1 2 3 4 5 6 7 8 9 10 Td (min)

100

90 B

DFI (%) DFI 40

0 0 10 20 30 40 50 60 Fnt sln (v-%)

Figure 9.1. SCSA analysis of sperm incubated for different periods in acid solution (A) and after exposure to different volumes of Fenton’s reagent (B). DFI values were derived and plotted versus the denaturation time or volume of Fenton´s solution. Plots are shown for ten different stallions of poor (red squares) and good (green squares) fertility (five stallions each, three ejaculates per stallion).

APPENDIX

Table 9.2. Data obtained with SCSA analysis of immature sperm, isolated from the caput corpus and cauda regions of the epididymis. Sperm side and forward scatter properties, as well as green and red fluorescence intensities and percentages of damaged sperm were determined. Mean values ± standard deviations are presented, determined for sperm from six different stallions. Values with different subscript letters differ significantly (p<0.05). localization side forward green red damaged scatter scatter fluorescence fluorescence sperm (channel) (channel) (channel) (channel) (%) epididymal 13.48±3.2a 19.28±2.2 a 710.46±131.6 a 201.81±71.5a 12.25±11.4a caput epididymal 18.87±5.5 a 21.24±2.9 a 523.08±81.1b 129.17±27.7 b 3.63±2.6a corpus epididymal 27.90±2.2 b 28.16±1.4 b 479.90±37.4 b 117.89±14.1b 3.07±2.4a cauda

Table 9.3. Data obtained with SCSA analysis of sperm that were treated with either non-treated, or treated with 10 mM diamide (DA) or DTT. Sperm side scatter properties, green and red fluorescence intensities and percentages of damaged sperm were determined. Mean values ± standard deviations are presented, determined for three pooled sperm samples (each consisting of sperm from four different stallions). Values with different subscript letters differ significantly (p<0.05). treatment side scatter forward green red damaged (channel) scatter fluorescence fluorescence sperm (channel) (channel) (channel) (%) non- 25.64±1.3a 25.30±0.1 a 456.52±20.6 a 115.74±9.9 a 7.57±4.3 a treated

DA 24.26±3.8 a 25.56±1.8 a 422.65±95.5 a 106.64±27.0 a 6.92±1.2a (10 mM)

DTT 17.45±1.0 b 25.88±3.5 a 872.44±120.8 b 284.04±95.4 b 18.18±8.8 b (10 mM)

APPENDIX

9.2. List of figures

Fig. 2.5.1. SCSA scatter plot demonstrating the gated region of cells outside the main population, calculated as DFI (%) Fig. 2.5.2. Different assays for detecting DNA damage Fig. 4.1.1. Sperm characteristics of stallions with different fertility rates Fig. 4.1.2. Correlation plots of sperm characteristics Fig. 4.1.3. Representative green versus red fluorescence intensity scatter plots after varying the incubation period in acid solution Fig. 4.1.4. SCSA analysis data after different incubation durations of sperm in acid solution Fig. 4.1.5. Representative green versus red fluorescence intensity scatter plots after exposure to different volume percentages of Fenton’s solution Fig. 4.1.6. SCSA analysis data after exposure of sperm to oxidative stress by incubation with different volume percentages of Fenton’s solution Fig. 4.1.7 In situ infrared absorption spectra of sperm Fig. 4.2.1. Representative scatter plots from SCSA analysis of immature sperm isolated from different regions of the epididymis Fig. 4.2.2. SCSA analysis data of epididymal sperm Fig. 4.2.3. Induced DNA damage in epididymal sperm Fig. 4.2.4. Induced DNA damage in sperm from the epididymal cauda without further treatment and after incubation with seminal plasma, as compared to native sperm Fig. 4.2.5. Representative scatter plots from SCSA analysis of non-treated sperm and sperm treated with DTT Fig. 4.2.6. Representative scatter plots from SCSA analysis of non-treated sperm and sperm treated with DA Fig. 4.2.7. SCSA analysis data of sperm exposed to increasing concentrations of DTT or DA

APPENDIX

Fig. 4.3.1. Microscopic images of epididymal sperm using differential interference contrast (DIC) and phase contrast optics Fig. 4.3.2. Microscopic images of native sperm and sperm treated with DTT or Fenton’s solution using interference contrast (DIC) and phase contrast optics Fig. 4.3.3. Transmission electron microscopic images of native sperm as well as sperm treated with DA, DTT or Fenton’s solution Fig. 9.1. Threshold levels for inducing DNA damage via incubation for different periods in acid solution and after exposure to oxidative stress, as determined for ten stallions

9.3. List of tables

Tab. 9.1 Sperm characteristics determined from three ejaculates for each of ten stallions divided in two fertility groups (H1-10). Tab. 9.2 SCSA analysis data of immature sperm isolated from the caput corpus and cauda regions of the epididymis, as determined for sperm from six different stallions. Tab. 9.3 SCSA analysis data of sperm after exposure to 10 mmol DTT or DA, as determined for three pooled sperm samples with four stallions each.

DANKSAGUNG

10 DANKSAGUNG

Mein erster Dank gilt meinem Doktorvater Herrn Prof. Dr. Harald Sieme für die Überlassung dieses interessanten Themas und die allzeit gewährte Unterstützung bei den Messungen und der Schreiberei.

Dr. Harriëtte Oldenhof danke ich von ganzem Herzen für die gewissenhafte und umsichtige Betreuung der Doktorarbeit sowie den stets optimistischen Beistand durch alle Hochs und Tiefs, die mit ordentlichem Kaffee und Nervennahrung überwindbar wurden.

Ebenfalls möchte ich Dr. Axel Brockmann und Dr. Gunilla Martinsson sowie allen Mitarbeitern des Landgestüts in Celle für die gute Zusammenarbeit danken. Ein spezieller Dank gilt hierbei meinen Mitdoktoranden und den Kollegen der Stationen Celle und Adelheidsdorf für eine tolle und unvergessliche Zeit mit vielen gewonnenen Freundschaften.

Weiterhin danke ich Herrn Prof. Dr. Willem F. Wolkers für die Hilfe bei den spektroskopischen Messungen. Frau Kerstin Rohn danke ich für die Anfertigung etlicher elektronenmikroskopischer Bilder. Ein Dank geht auch an Herrn Dr. Karl Rohn für die Beratung bei statistischen Problemen. Außerdem danke ich Hanna Thode für die unermüdliche Hilfe bei unzähligen SCSA Messungen.

Ganz besonders möchte ich meinen Freundinnen für das Miteinander, das Antreiben, Zurückholen und Aufmuntern danken.

Mein letzter und größter Dank gilt meinen Eltern und Großeltern, meinem Bruder und ganz besonders meinem tollen Freund für die unermüdliche Unterstützung in jeglicher Hinsicht über die Jahre des Studiums samt Doktorarbeit.