A multi-megabase copy number gain causes maternal transmission ratio distortion on mouse chromosome 2.

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 - 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system.

Sperm function, protein phosphorylation, and metabolism differ in mice lacking successive sperm-specific glycolytic enzymes.

Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDHS) and phosphoglycerate kinase 2 (PGK2), two isozymes restricted to the male germline, catalyze successive steps in the glycolytic pathway in mammalian sperm. Although gene targeting of each isozyme demonstrated that glycolysis is required for normal sperm motility and male fertility, the phenotype of mice lacking GAPDHS is more severe than that of mice lacking PGK2. This study examined sperm function, signaling pathways, and metabolism to investigate factors that contribute to the phenotypic differences between these knockout models. Sperm from the two knockouts exhibited comparable deficits in zona binding, in vitro fertilization with or without zona drilling, and capacitation-dependent tyrosine phosphorylation. In contrast, signaling and metabolic differences were apparent prior to capacitation. Phosphorylation of sperm protein phosphatase 1, which has been associated with the acquisition of motile capacity during epididymal maturation, was deficient only in GAPDHS-null sperm. Carnitine, choline, phosphocholine, and taurine were elevated in sperm from both knockouts immediately after collection from the epididymis. However, only carnitine levels in PGK2-null sperm were significantly different from wild-type sperm, while all four metabolites were significantly higher in GAPDHS-null sperm. We confirmed that glycolysis is required for robust hyperactivation, but found that the motility of PGK2-null sperm improved to levels comparable to wild-type sperm with pyruvate as the sole metabolic substrate. This nonglycolysable substrate did not improve progressive motility in GAPDHS-null sperm. These results identify multiple signaling and metabolic defects that are likely contributors to male infertility and the absence of progressive sperm motility seen in mice lacking GAPDHS.

CASAnova: a multiclass support vector machine model for the classification of human sperm motility patterns.

The ability to accurately monitor alterations in sperm motility is paramount to understanding multiple genetic and biochemical perturbations impacting normal fertilization. Computer-aided sperm analysis (CASA) of human sperm typically reports motile percentage and kinematic parameters at the population level, and uses kinematic gating methods to identify subpopulations such as progressive or hyperactivated sperm. The goal of this study was to develop an automated method that classifies all patterns of human sperm motility during in vitro capacitation following the removal of seminal plasma. We visually classified CASA tracks of 2817 sperm from 18 individuals and used a support vector machine-based decision tree to compute four hyperplanes that separate five classes based on their kinematic parameters. We then developed a web-based program, CASAnova, which applies these equations sequentially to assign a single classification to each motile sperm. Vigorous sperm are classified as progressive, intermediate, or hyperactivated, and nonvigorous sperm as slow or weakly motile. This program correctly classifies sperm motility into one of five classes with an overall accuracy of 89.9%. Application of CASAnova to capacitating sperm populations showed a shift from predominantly linear patterns of motility at initial time points to more vigorous patterns, including hyperactivated motility, as capacitation proceeds. Both intermediate and hyperactivated motility patterns were largely eliminated when sperm were incubated in noncapacitating medium, demonstrating the sensitivity of this method. The five CASAnova classifications are distinctive and reflect kinetic parameters of washed human sperm, providing an accurate, quantitative, and high-throughput method for monitoring alterations in motility.

Structural analyses to identify selective inhibitors of glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme.

STUDY HYPOTHESIS: Detailed structural comparisons of sperm-specific glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) and the somatic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozyme should facilitate the identification of selective GAPDHS inhibitors for contraceptive development. STUDY FINDING: This study identified a small-molecule GAPDHS inhibitor with micromolar potency and >10-fold selectivity that exerts the expected inhibitory effects on sperm glycolysis and motility. WHAT IS KNOWN ALREADY: Glycolytic ATP production is required for sperm motility and male fertility in many mammalian species. Selective inhibition of GAPDHS, one of the glycolytic isozymes with restricted expression during spermatogenesis, is a potential strategy for the development of a non-hormonal contraceptive that directly blocks sperm function. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Homology modeling and x-ray crystallography were used to identify structural features that are conserved in GAPDHS orthologs in mouse and human sperm, but distinct from the GAPDH orthologs present in somatic tissues. We identified three binding pockets surrounding the substrate and cofactor in these isozymes and conducted a virtual screen to identify small-molecule compounds predicted to bind more tightly to GAPDHS than to GAPDH. Following the production of recombinant human and mouse GAPDHS, candidate compounds were tested in dose-response enzyme assays to identify inhibitors that blocked the activity of GAPDHS more effectively than GAPDH. The effects of a selective inhibitor on the motility of mouse and human sperm were monitored by computer-assisted sperm analysis, and sperm lactate production was measured to assess inhibition of glycolysis in the target cell. MAIN RESULTS AND THE ROLE OF CHANCE: Our studies produced the first apoenzyme crystal structures for human and mouse GAPDHS and a 1.73 Å crystal structure for NAD(+)-bound human GAPDHS, facilitating the identification of unique structural features of this sperm isozyme. In dose-response assays T0501_7749 inhibited human GAPDHS with an IC50 of 1.2 µM compared with an IC50 of 38.5 µM for the somatic isozyme. This compound caused significant reductions in mouse sperm lactate production (P= 0.017 for 100 µM T0501_7749 versus control) and in the percentage of motile mouse and human sperm (P values from <0.05 to <0.0001, depending on incubation conditions). LIMITATIONS, REASONS FOR CAUTION: The chemical properties of T0501_7749, including limited solubility and nonspecific protein binding, are not optimal for drug development. WIDER IMPLICATIONS OF THE FINDINGS: This study provides proof-of-principle evidence that GAPDHS can be selectively inhibited, causing significant reductions in sperm glycolysis and motility. These results highlight the utility of structure-based drug design and support further exploration of GAPDHS, and perhaps other sperm-specific isozymes in the glycolytic pathway, as contraceptive targets. LARGE SCALE DATA: None. Coordinates and data files for three GAPDHS crystal structures were deposited in the RCSB Protein Data Bank (http://www.rcsb.org). STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the National Institutes of Health (NIH), USA, including U01 HD060481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research from the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and TW/HD00627 from the NIH Fogarty International Center. Additional support was provided by subproject CIG-05-109 from CICCR, a program of CONRAD, Eastern Virginia Medical School, USA. There are no conflicts of interest.

Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation.

Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.

Classification of mouse sperm motility patterns using an automated multiclass support vector machines model.

Vigorous sperm motility, including the transition from progressive to hyperactivated motility that occurs in the female reproductive tract, is required for normal fertilization in mammals. We developed an automated, quantitative method that objectively classifies five distinct motility patterns of mouse sperm using Support Vector Machines (SVM), a common method in supervised machine learning. This multiclass SVM model is based on more than 2000 sperm tracks that were captured by computer-assisted sperm analysis (CASA) during in vitro capacitation and visually classified as progressive, intermediate, hyperactivated, slow, or weakly motile. Parameters associated with the classified tracks were incorporated into established SVM algorithms to generate a series of equations. These equations were integrated into a binary decision tree that sequentially sorts uncharacterized tracks into distinct categories. The first equation sorts CASA tracks into vigorous and nonvigorous categories. Additional equations classify vigorous tracks as progressive, intermediate, or hyperactivated and nonvigorous tracks as slow or weakly motile. Our CASAnova software uses these SVM equations to classify individual sperm motility patterns automatically. Comparisons of motility profiles from sperm incubated with and without bicarbonate confirmed the ability of the model to distinguish hyperactivated patterns of motility that develop during in vitro capacitation. The model accurately classifies motility profiles of sperm from a mutant mouse model with severe motility defects. Application of the model to sperm from multiple inbred strains reveals strain-dependent differences in sperm motility profiles. CASAnova provides a rapid and reproducible platform for quantitative comparisons of motility in large, heterogeneous populations of mouse sperm.

Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) is expressed at high yield as an active homotetramer in baculovirus-infected insect cells.

The sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) isoform is a promising contraceptive target because it is specific to male germ cells, essential for sperm motility and male fertility, and well suited to pharmacological inhibition. However, GAPDHS is difficult to isolate from native sources and recombinant expression frequently results in high production of insoluble enzyme. We chose to use the Bac-to-Bac baculovirus-insect cell system to express a His-tagged form of human GAPDHS (Hu his-GAPDHS) lacking the proline-rich N-terminal sequence. This recombinant Hu his-GAPDHS was successfully produced in Spodoptera frugiperda 9 (Sf9) cells by infection with recombinant virus as a soluble, enzymatically active form in high yield, >35 mg/L culture. Biochemical characterization of the purified enzyme by mass spectrometry and size exclusion chromatography confirmed the presence of the tetrameric form. Further characterization by peptide ion matching mass spectrometry and Edman sequencing showed that unlike the mixed tetramer forms produced in bacterial expression systems, human his-GAPDHS expressed in baculovirus-infected insect cells is homotetrameric. The ability to express and purify active human GAPDHS as homotetramers in high amounts will greatly aid in drug discovery efforts targeting this enzyme for discovery of novel contraceptives and three compounds were identified as inhibitors of Hu his-GAPDHS from a pilot screen of 1120 FDA-approved compounds.

Frequent and recent retrotransposition of orthologous genes plays a role in the evolution of sperm glycolytic enzymes.

BACKGROUND: The central metabolic pathway of glycolysis converts glucose to pyruvate, with the net production of 2 ATP and 2 NADH per glucose molecule. Each of the ten reactions in this pathway is typically catalyzed by multiple isozymes encoded by a multigene family. Several isozymes in this pathway are expressed only during spermatogenesis, and gene targeting studies indicate that they are essential for sperm function and male fertility in mouse. At least three of the novel glycolytic isozymes are encoded by retrogenes (Pgk2, Aldoart1, and Aldoart2). Their restricted expression profile suggests that retrotransposition may play a significant role in the evolution of sperm glycolytic enzymes. RESULTS: We conducted a comprehensive genomic analysis of glycolytic enzymes in the human and mouse genomes and identified several intronless copies for all enzymes in the pathway, except Pfk. Within each gene family, a single orthologous gene was typically retrotransposed frequently and independently in both species. Several retroposed sequences maintained open reading frames (ORFs) and/or provided evidence of alternatively spliced exons. We analyzed expression of sequences with ORFs and <99% sequence identity in the coding region and obtained evidence for the expression of an alternative Gpi1 transcript in mouse spermatogenic cells. CONCLUSIONS: Our analysis detected frequent, recent, and lineage-specific retrotransposition of orthologous glycolytic enzymes in the human and mouse genomes. Retrotransposition events are associated with LINE/LTR and genomic integration is random. We found evidence for the alternative splicing of parent genes. Many retroposed sequences have maintained ORFs, suggesting a functional role for these genes.

Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice.

Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.

A-kinase anchoring protein 4 has a conserved role in mammalian spermatogenesis.

A-kinase anchor protein 4 (AKAP4) is an X-linked member of the AKAP family of scaffold proteins that anchor cAMP-dependent protein kinases and play an essential role in fibrous sheath assembly during spermatogenesis and flagellar function in spermatozoa. Marsupial spermatozoa differ in structural organization from those of eutherian mammals but data on the molecular control of their structure and function are limited. We therefore cloned and characterized the AKAP4 gene in a marsupial, the tammar wallaby (Macropus eugenii). The gene structure, sequence, and predicted protein of AKAP4 were highly conserved with that of eutherian orthologues and it mapped to the marsupial X-chromosome. There was no AKAP4 expression detected in the developing young. In the adult, AKAP4 expression was limited to the testis with a major transcript of 2.9 kb. AKAP4 mRNA was expressed in the cytoplasm of round and elongated spermatids while its protein was found on the principal piece of the flagellum in the sperm tail. This is consistent with its expression in other mammals. Thus, AKAP4 appears to have a conserved role in spermatogenesis for at least the last 166 million years of mammalian evolution.

Germ cell survival through carbohydrate-mediated interaction with Sertoli cells.

Spermatogenesis is a precisely regulated process in which the germ cells closely interact with Sertoli cells. The molecular basis of this cell-cell adhesion is unknown. Here, we demonstrate that targeted disruption of Man2a2, a gene encoding alpha-mannosidase IIx (MX), an enzyme that forms intermediate asparagine-linked carbohydrates (N-glycans), results in Man2a2 null males that are largely infertile. The Man2a2 null spermatogenic cells fail to adhere to Sertoli cells and are prematurely released from the testis to epididymis. We identified an N-glycan structure that plays a key role in germ cell-Sertoli cell adhesion and showed that a specific carbohydrate was required for spermatogenesis.

CIB1 is essential for mouse spermatogenesis.

CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.

X-ray analysis of phosphoglycerate kinase 2, a sperm-specific isoform from Mus musculus.

Phosphoglycerate kinase 2 (PGK2) is an isozyme of the glycolytic pathway that provides ATP required for sperm motility. It is encoded by an autosomal retrogene that is expressed only during spermatogenesis, concomitant with the inactivation of the X-linked Pgk1 gene. PGK2 from the mouse, Mus musculus, has been overexpressed from a plasmid in bacteria and purified. It was crystallized in three forms: as the apoenzyme, as a complex with 3-phosphoglycerate (3PG), and as a complex with 3PG and ATP. The crystal structures were solved to 2.7, 2.0, and 2.7 A resolutions, respectively. The overall fold is nearly identical with previously solved mammalian PGK1 molecules. The apoenzyme is in the "open" form; that is the N-terminal domain that can bind 3PG and the C-terminal domain that binds ATP are too far apart for the substrates to interact. Binding 3PG causes a 13 degree rotation that partially closes the structure and causes helix 13, which is disordered in the unliganded structure, to stabilize. Binding ATP leaves the protein in the open configuration but also causes helix 13 to be ordered. Sequence alignment suggests that the active site of PGK2 is essentially identical to that of the cytoplasmic PGK1, but significant differences accumulate on a side of the C-terminal domain away from the active site. These changes may mediate the binding of this isoform to other proteins within the sperm flagellum, while still allowing the hinging action between the domains that is essential to catalytic activity.

Spermatogenic cell-specific type 1 hexokinase is the predominant hexokinase in sperm.

Hexokinase is the first enzyme in the glycolytic pathway and utilizes ATP to convert glucose to glucose-6-phosphate (G6P). We previously identified three variant transcripts of Hk1 that are expressed specifically in spermatogenic cells, have different 5' untranslated regions, and encode a protein (HK1S, spermatogenic cell-specific type 1 hexokinase) in which the porin-binding domain (PBD) of HK1 is replaced by a novel N-terminal spermatogenic cell-specific region (SSR). However, the level of expression of the individual variant transcripts or of the other members of the hexokinase gene family (Hk2, Hk3, and Gck) in spermatogenic cells remains uncertain. We show that Hk1, Hk2, and Hk3 transcripts levels are quite low in spermatocytes and spermatids and Gck transcripts are relatively abundant in spermatids, but that glucokinase (GCK) is not detected in spermatozoa. Using real time RT-PCR (qPCR) with primers specific for each of the three variant forms and RNA from whole testis and isolated germ cells, we found that transcripts for Hk1_v2 and Hk1_v3, but not for Hk1_v1, are relatively high in spermatids. Similar results were seen using spermatogenic cells isolated by laser-capture microdissection (LCM). Immunoblotting studies found that HK1S is abundant in sperm, and immunostaining confirmed that HK1S is located mainly in the principal piece of the sperm flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found. These results strongly suggest that HK1, HK2, HK3, and GCK are unlikely to have a role in glycolysis in sperm and that HK1S encoded by Hk1_v2 and Hk1_v3 serves this role.

Bicarbonate-induced phosphorylation of p270 protein in mouse sperm by cAMP-dependent protein kinase.

Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility.

Male Infertility Is Responsible for Nearly Half of the Extinction Observed in the Mouse Collaborative Cross.

The goal of the Collaborative Cross (CC) project was to generate and distribute over 1000 independent mouse recombinant inbred strains derived from eight inbred founders. With inbreeding nearly complete, we estimated the extinction rate among CC lines at a remarkable 95%, which is substantially higher than in the derivation of other mouse recombinant inbred populations. Here, we report genome-wide allele frequencies in 347 extinct CC lines. Contrary to expectations, autosomes had equal allelic contributions from the eight founders, but chromosome X had significantly lower allelic contributions from the two inbred founders with underrepresented subspecific origins (PWK/PhJ and CAST/EiJ). By comparing extinct CC lines to living CC strains, we conclude that a complex genetic architecture is driving extinction, and selection pressures are different on the autosomes and chromosome X Male infertility played a large role in extinction as 47% of extinct lines had males that were infertile. Males from extinct lines had high variability in reproductive organ size, low sperm counts, low sperm motility, and a high rate of vacuolization of seminiferous tubules. We performed QTL mapping and identified nine genomic regions associated with male fertility and reproductive phenotypes. Many of the allelic effects in the QTL were driven by the two founders with underrepresented subspecific origins, including a QTL on chromosome X for infertility that was driven by the PWK/PhJ haplotype. We also performed the first example of cross validation using complementary CC resources to verify the effect of sperm curvilinear velocity from the PWK/PhJ haplotype on chromosome 2 in an independent population across multiple generations. While selection typically constrains the examination of reproductive traits toward the more fertile alleles, the CC extinct lines provided a unique opportunity to study the genetic architecture of fertility in a widely genetically variable population. We hypothesize that incompatibilities between alleles with different subspecific origins is a key driver of infertility. These results help clarify the factors that drove strain extinction in the CC, reveal the genetic regions associated with poor fertility in the CC, and serve as a resource to further study mammalian infertility.

The Founder Strains of the Collaborative Cross Express a Complex Combination of Advantageous and Deleterious Traits for Male Reproduction.

Surveys of inbred strains of mice are standard approaches to determine the heritability and range of phenotypic variation for biomedical traits. In addition, they may lead to the identification of novel phenotypes and models of human disease. Surprisingly, male reproductive phenotypes are among the least-represented traits in the Mouse Phenome Database. Here we report the results of a broad survey of the eight founder inbred strains of both the Collaborative Cross (CC) and the Diversity Outbred populations, two new mouse resources that are being used as platforms for systems genetics and sources of mouse models of human diseases. Our survey includes representatives of the three main subspecies of the house mice and a mix of classical and wild-derived inbred strains. In addition to standard staples of male reproductive phenotyping such as reproductive organ weights, sperm counts, and sperm morphology, our survey includes sperm motility and the first detailed survey of testis histology. As expected for such a broad survey, heritability varies widely among traits. We conclude that although all eight inbred strains are fertile, most display a mix of advantageous and deleterious male reproductive traits. The CAST/EiJ strain is an outlier, with an unusual combination of deleterious male reproductive traits including low sperm counts, high levels of morphologically abnormal sperm, and poor motility. In contrast, sperm from the PWK/PhJ and WSB/EiJ strains had the greatest percentages of normal morphology and vigorous motility. Finally, we report an abnormal testis phenotype that is highly heritable and restricted to the WSB/EiJ strain. This phenotype is characterized by the presence of a large, but variable, number of vacuoles in at least 10% of the seminiferous tubules. The onset of the phenotype between 2 and 3 wk of age is temporally correlated with the formation of the blood-testis barrier. We speculate that this phenotype may play a role in high rates of extinction in the CC project and in the phenotypes associated with speciation in genetic crosses that use the WSB/EiJ strain as representative of the Mus muculus domesticus subspecies.

Fibrous sheath of mammalian spermatozoa.

The fibrous sheath is a unique cytoskeletal structure surrounding the axoneme and outer dense fibers and defines the extent of the principal piece region of the sperm flagellum. It consists of two longitudinal columns connected by closely arrayed semicircular ribs that assemble from distal to proximal throughout spermiogenesis. The fibrous sheath is believed to influence the degree of flexibility, plane of flagellar motion, and the shape of the flagellar beat. Nearly half of the protein in fibrous sheaths isolated from mouse sperm is AKAP4. This protein and two others, AKAP3 and TAKAP-80, have anchoring sites for cAMP-dependent protein kinase. AKAP3 also anchors ropporin, a spermatogenic cell-specific protein that is linked through rhophilin to the small GTPase Rho. Other proteins associated with the fibrous sheath include two enzymes in the glycolytic pathway. Glyceraldehyde 3-phosphate dehydrogenase-s (GAPDS) is the product of a gene expressed only in spermatogenic cells, while hexokinase type 1-s (HK1-S) is derived from alternative transcripts present only in spermatogenic cells. Most of the other glycolytic enzymes in sperm have unique structural or functional properties. The fibrous sheath also contains a spermatogenic cell-specific member of the mu-class glutathione S-transferase family (GSTM5) and an intermediate filament-like protein (FS39). These and other observations indicate that the fibrous sheath functions as a scaffold for proteins in signaling pathways that might be involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction and for enzymes in the glycolytic pathway that provide energy for the hyperactivated motility of sperm that allows them to penetrate the zona pellucida.

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS).

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z' values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents.

Mice lacking cyclin-dependent kinase inhibitor p19Ink4d show strain-specific effects on male reproduction.

p19(Ink4d) is a member of the INK4 family of cyclin-dependent kinase inhibitors, which are important negative regulators of the G1-phase cyclin-dependent kinases CDK4 and CDK6. On a mixed C57BL/6 x 129P2/OlaHsd background, mice deficient for p19(Ink4d) exhibited defects in male reproductive function including testicular atrophy, alteration in serum follicle stimulating hormone, qualitative increase in germ cell apoptosis, and delayed kinetics of meiotic prophase markers (Zindy et al., 2001. Mol Cell Biol 21:3244-3255; Zindy et al., 2000. Mol Cell Biol 20:372-378). In this study, a quantitative assessment of these aspects of reproductive capacity demonstrated relatively mild deficits in p19(Ink4d-/-) males compared to controls. These effects did not dramatically worsen in older males although some seminiferous tubule defects were observed. Following marker-assisted backcrossing into the C57BL/6 background, p19(Ink4d-/-) males did not display defects in testis weights, sperm numbers, serum FSH, germ cell apoptosis, or kinetics of selected meiotic prophase markers. These studies indicate that a reduction in Ink4 family function by the loss of p19(Ink4d) is sufficient to induce mild reproductive defects in male mice with a mixed genetic background, but not in the C57BL/6 genetic background.