The sperm chromatin structure assay (SCSA), as developed by Evenson et al.(1980), utilizes flow cytometry to quantify the susceptibility of sperm chromatin to in situ acid denaturation via the metachromatic properties of acridine orange. SCSA is repeatable and has been used to distinguish between fertile and subfertile males in different species; however, it does not permit morphological evaluation of cells. In the present study, the SCSA was modified for the fluorescence/differential interference contrast (DIC) microscope to examine morphology and chromatin stability on the same cell. Semen from six Holstein bulls was collected twice weekly for six weeks. Semen was cryopreserved after collection. A 48-hr scrotal insulation was applied after the first three collections to exert a mild thermal insult to the testes; this induces specific spermatozoal morphological abnormalities to appear in a predictable chronological order, as determined by Vogler et al. (1993). Using DIC optics, sperm head morphology was classified as normal, slightly misshapen, pyriform, severely misshapen, or tailless. Vacuolization in the head region was scored separately as apical, diadem, or random. SCSA and modified-SCSA for fluorescence microscopy were used to assess chromatin instability in the samples. The SCSA parameter of 'cells outside the main population of alpha t' (%COMP alpha t) and the modified-SCSA parameter of '% cells shifted from green' were positively correlated (r=0.84; P<0.01). Both variables were positively correlated with the appearance of tailless, pyriform, severely misshapen, and randomly vacuolated cells (P< 0.01), but not with the appearance of diadems or apical vacuoles. Also, the fluorescence microscope detected a significant shift from green in normally shaped cells appearing in morphologically abnormal ejaculates (P<0.01). These results demonstrate that scrotal insulation-induced morphological abnormalities in spermatozoa signify a perturbation in chromatin structure, and that the chromatin perturbation extends into normally shaped cells in the same ejaculate. / Master of Science

<p>The chromatin integrity in sperm cells is vital for successful pregnancy. In this</p><p>study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization</p><p>(IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were</p><p>purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test</p><p>Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation</p><p>Index (DFI) of 30 % and the results correlated with fertilization, cleavage and</p><p>pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower</p><p>cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro</p><p>may be independent of the degree of DNA-damage, the embryonic development could be</p><p>seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a</p><p>DFI threshold of 30 %.</p>

During spermiogenesis, somatic chromatin is remodeled and a vast majority (> 90%) of DNA histones are replaced by short arginine-rich peptides called protamines. This compaction is immense, with protamine-DNA self-assembly in sperm chromatin resulting in a final volume roughly 1/6th of a somatic nucleus. This near crystalline organization of the DNA in sperm is thought crucial both for the transport of the paternal genes as well as for the protection of genetic information as sperm chromatin is transcriptionally inactive and all DNA repair mechanisms are shut down. Chapter 1 will include an overview of the topics discussed in this document, including: sperm chromatin, Sperm chromatin remodeling, DNA damage, and the effect of DNA damage to sperm DNA. Chapter 2 will contain a brief overview of the techniques used within this study. This includes: Small-angle X-ray Scattering, gel electrophoresis, DNA precipitation assays, and ethidium bromide dissociation assays. In chapter 3, we will discuss the effect of DNA packaging on the accessibility of free radicals to damage condensed DNA. A variety of polycations were used to condense plasmid DNA in reconstituted samples. After condensation, the DNA-polycation condensates were exposed to 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) for 1 hour, decondensed, and the plasmid DNA examined by gel electrophoresis. By comparing the intensities of the supercoiled, open coiled and linear bands, we were able to identify the presence of single-strand nicks and double-strand breaks in DNA. DNA packaging densities for all polycation-DNA systems were determined by small-angle X-Ray scattering (SAXS). Our results show that for similar length polycations, the amount of oxidative damage scales directly with the DNA packaging with more tightly condensed DNA being damaged less. However, our results also show that DNA damage is also dependent on polycation length, with DNA condensed by shorter polycations being damaged more than DNA condensed with longer polycations even at similar packaging densities. Protamine has long been thought to play a role in protecting spermatic DNA from damaging agents in vivo. However, the relationship between the hypercondensation of sperm chromatin, the DNA integrity, and the transfer of epigenetic information from sperm to oocyte and potential to alter gene expression in the early embryo are poorly understood. In Chapter 4, we examine how underprotamination affects free radical accessibility and DNA stability in reconstituted sperm chromatin. Specifically, reconstituted salmon protamine- plasmid DNA condensates (polyplexes) were formed at precise protamine/DNA ratios and subsequently subjected to exposure to AAPH free radicals. Agarose gel electrophoresis was then used to assess DNA damage by observing topology alternations in the decondensed polyplexes. FPG-DNA glycosylase has also been used to more accurately determine oxidative damage beyond just nicks and double-strand breaks in the various condensed states. We show that higher levels of protamination correlate to greater levels of protection to the DNA from oxidative damage up until full charge compensation. Furthermore, we also demonstrate that poorly compacted chromatin could be recovered by the introduction of small cationic peptides in underprotaminated condensates as well as actual sperm nuclei. SAXS studies were performed to show that the introduction of cationic peptides resulted in tighter DNA packaging densities in the underprotaminated sperm chromatin. In Chapter 5, we examine the role of disulfide bonds on DNA packaging in mammalian sperm chromatin. Mammalian protamine, unlike fish, are known to have cysteine residues capable of forming inter- and intra-protamine disulfide bonds. In bull, prior work had shown evidence for the formation of a unique hairpin secondary structure due to the folding of the ends of the protamine molecule by intramolecular disulfide linkages. Between folds is an arginine-rich region known as the DNA binding region. The DNA binding region has a local arginine fraction (~60-75%) that is much higher than the arginine fraction within the full bull protamine sequence (~50%). Previous work by the DeRouchey lab has shown that the percent arginine was crucial for DNA condensation in small arginine-rich peptides. We hypothesize that the fraction of arginine is also critical to DNA remodeling in sperm chromatin. SAXS studies showed that disulfide bond reduction resulted in complete decondensation of bull sperm nuclei. Here, we have used cysteine alkylation chemistry to add neutral or charged functional groups to the protamine cysteine, thereby inhibiting the formation of these disulfide bonds. This chemistry both prevents the formation of the hairpin as well as modifies the overall charge of the protamine. Through ethidium bromide exclusion assays, we measured binding of these altered protamines to calf thymus DNA and determined that a percent cationic charge of above 50% is necessary for the protamine to effectively condense DNA. In addition, we show that DNA condensation of bull protamine with the hairpin is nearly identical to piscine protamines which have no disulfide linkages but a net arginine fraction of 60-75%. Upon disruption of the hairpin, however, complete condensation does not occur despite a net charge on the protamine of +26.

Artificial insemination (AI) with sex-sorted bottlenose dolphin spermatozoa provides female calves for obtaining more cohesive social groups and optimum genetic management of captive populations. However, distance of animals to the sorting facility represents a limit to the procedure. Although one bottlenose dolphin calf has been born using spermatozoa from frozen-thawed, sorted and recryopreserved spermatozoa, critical evaluation of the steps involved in this process is required to maximize its efficiency for future AIs and expansion of the technology to other species. Two experiments were designed to determine the efficiency of the sorting process and the quality of frozen-thawed bottlenose dolphin spermatozoa during sorting and recryopreservation. In experiment 1, the effect of two washing media (with and without 4 percent egg yolk, v/v) following density gradient centrifugation (DGC) on sperm recovery rate and in vitro characteristics of cryopreserved spermatozoa was examined. In experiment 2, cryopreserved semen was used to compare the effects of two recryopreservation methods (conventional straw freezing and directional freezing) on in vitro sperm characteristics of control (non-sorted) and sorted spermatozoa. Egg yolk supplementation of the washing medium in experiment 1 did not influence (P > 0.05) the sperm recovery rate, however, sperm motility parameters and viability were improved (P < 0.05). For Experiment 2, motility parameters and viability were influenced by stage of sex-sorting process, sperm type (non-sorted and sorted) and freezing method (P < 0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P < 0.05) motility parameters over the 24 h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of nonsorted spermatozoa, as assessed by the SCSA, remained unchanged throughout the process. However, a possible interaction between Hoechst 33342 and acridine orange was observed in sorted samples. After recryopreservation, viability of sorted spermatozoa was higher (P < 0.05) than that of non-sorted spermatozoa across all time points. The percentages of viable spermatozoa determined by light (eosin-nigrosin) and fluorescence microscopy (propidium iodide) techniques were correlated (R^2=0.79, P < 0.001). Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

The chromatin integrity in sperm cells is vital for successful pregnancy. In this study DNA-damage was evaluated in sperm cells from 50 men attending In Vitro Fertilization (IVF) or Intra Cytoplasmic Sperm Injection (ICSI) treatment. Male semen samples were purified with a two-shift gradient before the sperm cells were treated with the Halosperm® Test Kit and evaluated for DNA-damage. The samples were divided in two groups according to DNAFragmentation Index (DFI) of 30 % and the results correlated with fertilization, cleavage and pregnancy rate. Men with DFI ≥ 30 % had a higher fertilization and pregnancy rate and a lower cleavage rate compared to men with DFI ≤ 30 %. The conclusions were that fertilization in vitro may be independent of the degree of DNA-damage, the embryonic development could be seriously disrupted by damaged sperm cells, and the pregnancy rate showed no correlation to a DFI threshold of 30 %.