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Male fertility is an important factor in reproductive success which is crucial for the survival of a sexually reproducing lineage or species. In mammals, male reproduction is complex and needs many physiological processes to be successful for reaching sexual maturity, spermatogenesis, sperm maturation, finding and mating a female in heat, and ejaculation of the sperm and the seminal plasma. In the female reproductive tract, the viable and fertile sperm need to be able to reach the place of fertilization. Changes in sperm physiology like hyperactivation, capacitation, and acrosome reaction are required to occur at the correct place and in coordination with the female mate. All those processes are influenced and controlled to a certain extent by genetic variants. Reproduction is as well crucial for livestock farming. Because of the wide-spread use of artificial insemination, records of sperm quality and insemination success are available for many bulls. Linking this phenotypic information with SNP array and whole-genome genotypes enables to quantify heritability and map trait associated loci and candidate causal variants. This thesis investigated genetic variation affecting male fertility traits in Brown Swiss (BSW) dairy cattle using comprehensive semen quality and insemination success records. Chapter 2 reports on a recessive quantitative trait locus (QTL) for sperm motility, sperm head and tail morphology, and insemination success that is located within a 2.38 Mb segment of extended homozygosity on bovine chromosome 6. Haplotype-based genome-wide association testing between high density level SNP array data (607,511 SNPs) and various semen quality phenotypes from 794 BSW bulls revealed strong association at the QTL region. Homozygosity for the top QTL haplotype reduces insemination success by 0.99 phenotypic standard deviations. Whole-genome sequencing data of 42 BSW bulls showed that a synonymous variant (BTA6:58373887C>T, rs474302732) in WDR19 was compatible with the inheritance of the top haplotype. Although annotated as a synonymous variant, the investigation of transcriptome data revealed that it activates a cryptic splice site leading to the truncation of three evolutionarily conserved amino acids from the protein. WD repeat-containing protein 19 is part of the intraflagellar transport complex that is essential for the formation and physiological function of motile cilia and flagella. Impaired function of this protein is likely causal for the impaired motility and lower fertility in the homozygous state. Surprisingly, the variant has a high frequency of 24% in the BSW population, leading to an expected proportion of 5.8% of all bulls carrying the variant in homozygous state assuming random mating at this locus. Even subtle reductions in sperm quality due to genetic aetiology can reduce insemination success. Chapter 3 revealed four additional recessive QTL for insemination success in Brown Swiss cattle located on BTA1, 18, 25, and 26. By conditioning the haplotype-based genome wide association study on the known QTL on BTA6, the additional signals were stronger and only then met the significance threshold in case of the QTL on BTA18 and 25. The top associated haplotypes segregate at rather high frequency (0.18, 0.34, 0.09, and 0.26) in the BSW population. They reduce the insemination success by 0.80, 0.28, 1.09, and 0.52 phenotypic standard deviations, for BTA1, BTA18, BTA25, and BTA26, respectively. Possible reasons for the reduced insemination success were investigated using comprehensive sperm quality data. However, only the QTL on BTA1 is associated with an increased proportion of sperm with an abnormal head shape, possibly explaining the mechanism of reduced insemination success in homozygous bulls. We screened for candidate variants using whole-genome sequence variants of 125 bulls with insemination success phenotypes and imputed sequence variants for all bulls with phenotypes. A missense variant in SPATA16 (spermatogenesis associated protein 16) was strongly associated with this QTL. But non-coding variants were stronger associated, suggesting that regulatory variants are more likely to be causal for the QTL variation. None of the other QTL showed an association with any of the analysed sperm quality traits. Coding candidate variants compatible with the top haplotypes were located in ENSBTAG00000006717 (encoding ATP-binding cassette sub-family A member 3-like protein) and VWA3A (encoding von Willebrand factor A domain-containing protein 3A) on BTA25 and in ENSBTAG00000019919 (bovine ortholog of CFAP46 encoding cilia and flagella associated protein 46) on BTA26. Likewise, non-coding variants were stronger associated, e.g., a variant downstream SYCE1 (synaptonemal complex central element protein 1) on BTA26. In summary, this chapter showed that recessive QTL explain a considerable fraction of quantitative variation of male fertility in BSW bulls. Chapter 4 reports on a recessive loss-of-function variant in QRICH2 leading to male infertility in vivo because of immotile sperm with abnormal flagellum and head morphology. In sperm quality data of 70,990 ejaculates from 1343 BS collected at the Swiss artificial insemination centre Swissgenetics, we recognized 7 bulls that never produced ejaculates suitable for artificial inseminations and that had severely impaired semen quality. Among those, one bull presented severe oligoasthenoteratozoospermia. Whole-genome sequence screening and comparison with 397 fertile bulls revealed a 1-bp deletion in QRICH2 (glutamine rich 2) causing a frameshift and a premature stop codon (BTA19:55436705TC>T, ENSBTAT00000018337.1:c.4929del) that was homozygous only in this infertile bull. Through mapping the 1-bp deletion to a SNP array genotype derived haplotype segregating at a frequency of 5% in the BSW population, we identified a second bull that was homozygous for the 1-bp deletion. Confirming the disorder of the first bull, this bull produced immotile sperm with multiple morphological abnormalities of the flagella and head at low sperm concentration. The most frequent abnormality observed in the ejaculates of this bull were shortened, abnormally shaped flagella. Transmission electron microscopy imaging revealed that the ultrastructure of the sperm flagella was affected, often resulting in incomplete microtubule structures and a generally disorganized assembly of the sperm flagella. Bulls homozygous for the 1-bp deletion can now be identified early on through customized genotyping. This thesis leveraged comprehensive genotype and phenotype data to study the effects of genetic variation on male fertility traits in cattle. Different approaches were applied to detect 5 QTL affecting insemination success and one loss-of-function variant leading to severely disturbed sperm quality. All six QTL/variants are recessively inherited which implies that the fertility is only impaired in homozygous bulls. As heterozygous carrier bulls and females are not affected, such recessive alleles can segregate undetected for a long time in the population. Monitoring known alleles and putting constraints on future inbreeding are possible approaches to counteract the negative effects on male fertility. Reduced sperm quality and a higher proportion of ejaculates per bull not meeting the requirements for artificial insemination can be indicative of a genetic defect and the detected reduction of sperm quality might just be the tip of the iceberg of the overall effect. The effect of QTL reducing sperm quality and insemination success is likely underestimated using records from artificial insemination bulls as ejaculates and bulls not meeting the minimum quality requirements are excluded from breeding. Minimum quality requirements and the exclusion of bulls with low sperm quality are thus as well useful when dealing with genetic aetiology of impaired sperm quality.

Gametes of gray treefrogs, Dryophytes chrysoscelis, are deposited into freshwater ponds. Sperm undergo spermatogenesis and maturation beginning in the seminiferous tubules and migrating to the lumen. In mammals and fishes, these cells are immotile within the isosmotic fluid of the testes and have motility activated by exposure to a hyper- or hypoosmotic medium. Water flows into or out of the sperm cell, altering intracellular ionic concentrations, and ultimately stimulates flagellar movement. We tested the hypothesis that exposure to a hypotonic environment activates motility of gray treefrog sperm. We also hypothesized that osmotic water uptake is facilitated by expression of water channel proteins from the aquaporin family. To test these hypotheses, we collected sperm from captive treefrogs maintained with food and water at 22¿C and assessed motility of sperm immersed in hypoosmotic solutions of 200mOsm/L, 100mOsm/L, 75mOsm/L, 50mOsm/L, 25mOsm/L, and 10mOsm/L. A significant peak in reactivation percentages was seen at 50mOsmL (two-way ANOVA: P

Introduction: In animals, the process of fertilization requires that a motile sperm interact with an egg. In most sperm, including those of insects, the motility apparatus is a eukaryotic flagellum and its regulation results from a series of tightly regulated molecular events. The flagellum is a biological nano-machine that is widely conserved through evolution. In recent studies using the mosquito Culex quinquefasciatus, Thaler et. al. (2013) observed a series of three flagellar waveforms that progressed from activation to full progressive motility. This same activation pattern occurred in sperm from a related species, Culex pipiens. The three distinct waveforms observed in vitro were: a low amplitude, low velocity, and high frequency waveform (A), a high amplitude, high velocity, and low frequency waveform (C), and a low velocity intermediate waveform that had superimposed features from both waveforms A and C (B). Based on these findings, we are interested in identifying the molecular switch responsible for the waveform transitions during sperm motility in C. pipiens. Here, we report our studies on C. pipiens with the aim of modeling the mosquito sperm flagellum as a nano-machine.Materials and Methods: Mosquitoes were euthanized by placing them in a chamber containing a piece of cotton soaked in chloroform. Seminal vesicles and accessory glands were removed while in PBS solution. They were then placed on a glass slide with a drop of insect Ringer’s solution and a coverslip. In some cases, only the seminal vesicles were used. Pressure was applied to the coverslip to break open accessory glands thereby activating sperm. Data was acquired with a Nikon Labphot Microscope at 10x magnification using phase contrast optics and a DAGE-MTI CCD 100 camera. Images were captured using Scion Image and processed using ImageJ to quantify wave parameters.Results and Discussion: Using phase contrast microscopy and image processing methodologies we obtained parameters for flagellar wavelength and amplitude as well as progressive velocity for sperm displaying waveforms A and C. In addition, we were able to determine the beat frequency for waveform C as well as the dimensions of the sperm head and tail. Once all the desired parameters have been measured with a large sample size, average parameters for each waveform, A and C, will be used to test current physical models. This will provide insight into the development of a mathematical model for this system that will describe the regulation of flagellar motion in both two- and three-dimensions.Conclusion: The results for the parameters describing waveforms A and C provide confidence in obtaining values that are accurate in order to develop a mathematical model for sperm motility. The model can then be combined with molecular events that occur during motility in order to provide a deeper understanding of flagellar motion. The eukaryotic flagellum serves as an example of a naturally occurring cellular motor and with a better understanding of the mechanism, can aid in the design of nano-scale biomimetic devices.

In mammals, sperm attain motility and the ability to fertilize eggs during their passage through the epididymis. Changes in motility characteristics, called hyperactivation, occur in the female reproductive tract. Serine/threonine protein phosphorylation controlled by protein kinases and protein phosphatases has been identified as an important mechanism involved in sperm maturation and function including motility. This study focuses on the serine/threonine phosphatases present in mammalian spermatozoa, PP2A and PP1γ2. The first part of this study confirms the presence of PP2A in sperm and shows that it undergoes marked changes in methylation (Leu 309), tyrosine phosphorylation (Tyr 307) and catalytic activity during epididymal sperm maturation. Catalytic activity of PP2A declined as PP2A was demethylated during epididymal sperm maturation. Further, inhibition/demethylation of PP2A in caudal sperm resulted in increased phosphorylation of glycogen synthase kinase-3 and sperm motility parameters resembling hyperactivation. The results show for the first time that changes in PP2A activity due to methylation and tyrosine phosphorylation occur in sperm and that these changes may play an important role in the regulation of sperm function.The second part of the study focuses on unraveling the significance of the testis specific PP1γ2 in spermatogenesis and sperm function. PP1γ1 and PP1γ2 are alternately spliced transcripts of the same gene Ppp1cc and are identical except at their extreme C-termini. While PP1γ1 is ubiquitous in somatic cells, PP1γ2 is expressed exclusively in male germ cells. Ppp1cc knockout male mice (-/-) are sterile due to severely impaired sperm morphogenesis. Fertility can be restored in Ppp1cc -/- mice by high levels of transgenic PP1γ2 expression in germ cells of testis. Similarly, we wanted to determine if PP1γ1 is capable of restoring sperm morphogenesis and sperm function in Ppp1cc -/- mice. We generated four different transgenic Rescue mice lines. The first three transgenic lines were generated using cDNA of PP1γ1 mRNA including the initial sequence or entire intron 7. Lack of fertility in these three lines was attributed to low levels of transgenic PP1γ1 in testis probably due to instability of the PP1γ1 mRNA containing intron 7 in testis. Testis expression of PP1γ1 occurred at significant levels only when the transgene lacked the intron7. Levels of the PP1γ1 transgene in this line (Rescue IV) were comparable to PP1γ2 levels in Ppp1cc +/- mice testes. Sperm morphogenesis was restored with the PP1γ1 transgene in Ppp1cc -/- mice but small percentage of these males remained infertile due to altered sperm motility. The results of this study suggest that despite the ability of PP1γ1 to support sperm morphogenesis, it is detrimental for male fertility and hence excluded in differentiating spermatogenic cells by alternate splicing and miRNA mediated instability of its transcript.

In the mammalian sperm, regulation of intracellular pH (pHi) is essential for normal motility. Sodium hydrogen exchangers (NHEs) are integral membrane proteins that catalyze the electroneutral exchange of one extracellular Na+ for an intracellular H+ and are therefore important regulators of pHi. Of the several recognized NHE isoforms, NHE1, NHE5, NHE10 and the more recently identified NHEDC1 are known to be localized to the mature sperm flagellum. The focus of the first part of this dissertation is on NHEDC1, which was first partially characterized in humans. Subsequent studies in the mouse suggest that NHEDC1 is important for normal sperm motility and fertility. However, the characterization of NHEDC1 as a functional NHE has not been reported therefore the second chapter of this dissertation focuses on the gene cloning and functional characterization of the human orthologue of NHEDC1. Moreover, we demonstrate the existence of the rat orthologue of NHEDC1 and determine its and tissue distribution. Furthermore, since no studies have been done to determine the gene regulatory mechanisms of NHEDC1, the third chapter of this dissertation focuses on elucidating whether the epigenetic mechanism of DNA methylation plays a role in the regulation of NHEDC1 expression. The second part of this dissertation focuses on determining the role of ENaC a in sperm capacitation. Using the relatively non-specific inhibitors 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and amiloride to inhibit ENaC, previous studies have been interpreted to show that a decrease in Na+ permeability as a result of closure of ENaC is responsible for capacitation-associated hyperpolarization. However, these compounds can also inhibit other Na+ transporters in sperm. Therefore, using a mouse model with ENaC a alleles deleted only in the sperm we sought to categorically determine the role of ENaC a in sperm capacitation. Studying the role of Na+ ion transporters in sperm physiology is important as it may be possible to exploit them as targets for design of novel male factor contraceptive agents or for treatment of male infertility.

The studies that comprise this thesis were performed in an attempt to identify and eliminate stressful conditions that may lead to infertility in the male bovine. The first project was performed to determine if ergot alkaloids directly affect bovine sperm motility. Percentage of motile spermatozoa was affected (P = 0.015) by a three-way interactions between time, concentration, and alkaloid. Ergotamine and dihydroergotamine decreased (P = 0.01) sperm motility in a concentration and time dependant manner and ergonovine had a minimal effect on overall sperm motility. The number of static spermatozoa also was affected (P < 0.01) by a three way interaction and increased as ET and DHET concentrations increased. Percentages of progressively motile and rapidly motile spermatozoa decreased (P < 0.01) in a two way interaction between alkaloid and concentration. Overall, sperm motility was decreased by ET and DHET. Furthermore, the qualities of motility as represented by progressive, rapid, path velocity (VAP), progressive velocity (VSL), track speed (VCL), beat frequency (BCF), lateral amplitude (ALH), straightness (STR), elongated, and area were decreased by those alkaloids. These data verify that ergot alkaloids commonly found in toxic tall fescue are detrimental to bovine spermatozoa. In the second study two antioxidants (alpha-tocopherol and ascorbic acid) were added to bovine sperm culture media and cryopreservation extender. The antioxidant capabilities of these two vitamins could help reduce free radical production and help preserve sperm viability. There was an interactive effect between ascorbic acid concentration and storage method on the bovine sperm motility characteristics: motile, progressive, rapid, track speed (VCL), and straightness (P < 0.05). A bull by ascorbic acid interaction (P < 0.05) was observed for motile, progressive, rapid, path velocity (VAP), progressive velocity (VSL), and VCL characteristics. Alpha tocopherol had no affects on sperm motility characteristics. Lipid peroxidation was affected (P < 0.01) by bull and ascorbic acid. Malondialdehyde concentrations for ascorbic acid treatments (0, 5, 10, 20 mM) were 3.25, 4.2, 2.96, and 2.15 ìM respectively. Results from the second study indicate that the addition of ascorbic acid may reduce sperm motility, but help prevent lipid peroxidation.

A protein’s biological activity can be modified by non-protein co-factors. Co-factors bind to or are covalently linked to an enzyme to assist in biochemical transformations. An enzyme and a co-factor together form an enzymatically active conjugated protein called the holozyme. Co-factors can be organic (coenzymes) or inorganic (metals). They can also be classified according to their ability to bind to enzymes. Loosely-bound cofactors are termed coenzymes and tightly-bound cofactors are termed prosthetic groups. The primary difference between a prosthetic group and coenzyme is; the prosthetic group remains attached to the apoenzyme while undergoing oxidation and reduction while coenzymes may undergo reduction while attached to one apoenzyme, and then migrate to another apoenzyme where it can be oxidized. NAD, NADP and CoA are examples of coenzymes whereas hemes, flavins and biotin are prosthetic groups.Biotin is a cofactor responsible for carbon dioxide transfer in several carboxylase enzymes. It is covalently attached to the active sites of the metabolic carboxylases. Using biotin cofactor as a mobile carboxyl carrier these metabolic enzymes generally capture CO2 from bicarbonate ion and catalyze transfer of this carboxylate to organic acids to form various cellular metabolites[10] . Biotin protein ligase (BPL), also known as holocarboxylase synthetase (EC 6.3.4.15), is the enzyme that enables covalent attachment of biotin to the carboxylases. Post-translationally, biotin forms an amide linkage with specific lysine residue of newly synthesized carboxylases with the help of BPL[10]. The image below shows steps of the biotin protein ligase reaction.Role of biotin in carboxyl group transfer.Biotin is the only prosthetic group that facilitates the transfer of a carboxyl group. The role of biotin is to act as a mobile carboxyl group carrier, transporting the carboxyl group from the site of the carboxyl donor to the carboxyl group acceptor enzyme. Common carboxyl group donors are HCO3-, oxaloacetate, or methylmalonyl CoA, and carboxyl group accepter enzymes are pyruvate, acetyl CoA, propionyl CoA.Biotin-dependent carboxylases. The four biotin-dependent carboxylases in mammals are acetyl-CoA carboxylase (E.C. 6.4.1.2), pyruvate carboxylase (E.C. 6.4.1.1), propionyl-CoA carboxylase, (E.C. 6.4.1.3), and b-methylcrotonyl-CoA carboxylase (E.C. 6.4.1.4). Acetyl-CoA carboxylase (ACC) is found mainly in the cytosol while pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC) and methylcrotonyl-CoA carboxylase (MCC) are present in the mitochondria [6] (Figure 1).The biotin-dependent carboxylases play a crucial role in cell metabolism. ACC controls fatty acid synthesis in the cell cytosol by providing the substrate malonyl-CoA[1] (Figure 1). ACC may also play an important role in biotin storage [1]. PC is a key enzyme in gluconeogenesis [3] and provides a tricarboxylic acid cycle intermediate [1]. PCC catalyzes an essential step in the metabolism of amino acids such as isoleucine and methionine, odd-chain fatty acids, and breakdown products of dietary carbohydrates [1]. MCC carboxylase catalyzes an essential step in leucine metabolism.

Sperm from a semi-aquatic insect, the water strider Aquarius remigis, are unusually long and possess a complex acrosome and flagellum. Like other animal systems, water strider sperm that emerge from the testis are incapable of fertilizing an egg and must undergo several highly regulated developmental steps in both the male and female reproductive tracts. However, in contrast to well-studied model animals such as mammals and echinoderms, little is known about these events in insects. In this dissertation, I describe the post-meiotic events in Aquarius remigis using biochemical and biophysical methodologies to follow the assembly, structure, and fate of the acrosome and the flagellum from spermatogenesis through fertilization and into early embryonic development. In contrast to other long insect sperm, half of the length of the A. remigis sperm consists of an acrosomal matrix that emerges as a 300 µm helical structure followed by a 2200 µm linear region. This unusually long acrosome contains an intrinsically fluorescent molecule with properties consistent with those of Flavin Adenine Dinucleotide (FAD). Biophysical analyses showed that FAD is immobilized and oriented during acrosome formation and may be involved in the formation of disulfide bond formation during its assembly. Further, using the intrinsic fluorescence as a marker, I followed the fate of the acrosomal matrix through fertilization and observed it inside the fertilized egg where it remained structurally intact through gastrulation. The acrosomal matrix may play roles in sperm transport and fertilization. Similar to the proximal acrosomal process, the axoneme and its associated structures, appears helical. I describe a unique motility pattern in which the flagellum loops back upon itself and forms a coil. This structure can then twist and undergo forward progressive motility with the loop acting as the anterior end. A. remigis sperm are quiescent in the seminal vesicles, but sperm motility was initiated by specific proteases or phosphatase inhibitors. Further, a broad spectrum kinase inhibitor blocked sperm motility initiation by trypsin. These results suggest that quiescence is maintained by high levels of endogenous phosphatase activity and that activation of motility is regulated by protein phosphorylation through the action of one or more kinases.

The Na+/H+ Exchanger (NHE) is a trans-membrane antiporter that regulates the pH of the cell by allowing for the exchange of a H+ out of the cell as a Na+ falls back into the cell. NHE-regulated intracellular pH levels have been theorized to affect the motility of vertebrate sperm. NHE1 and NHE5 isoforms have been previously localized to sperm, although a novel NHE isoform (mNHE10) has recently been discovered and localized to mice sperm. While the sequenced homology of the protein categorizes it as an NHE, it has yet to be characterized as a functional antiporter. To characterize the mouse NHE10, its gene must first be cloned and expressed in a eukaryotic cell line. Then its proton-extruding function can be characterized through the method of “proton suicide”. Future studies may lead to the possibility of targeting the mNHE10 protein in future male infertility or contraceptive treatments.