Genes in the first and fourth inversions of the mouse t complex synergistically mediate sperm capacitation and interactions with the oocyte.

The t haplotypes (t) are recent evolutionary derivatives of an alternate form of the mouse t complex region located at the proximal end of chromosome 17. This variant form of approximately 1% of the mouse genome is a source of mutations altering numerous sperm functions crucial for fertilization. Males that carry two t haplotypes (t/t) are invariably sterile. t haplotypes contain four inversions relative to the wild-type t complex (+), so that in matings involving a +/t heterozygote, t is usually transmitted as a single unit. However, rare recombinants have been recovered, which carry only part of the t genotype and express only some of the t-dependent phenotypes. Use of these partial t haplotypes in genetic crosses has resulted in the general location of the two major t male sterility factors, S1 and S2, within inversions 1 and 4, respectively. Since sterility can result from a plethora of sperm defects, we have made a detailed study of various functional parameters of sperm from mice carrying S1 or S2 heterozygously or homozygously or in combination. Both S1 and S2 contain mutations altering sperm functions, including motility, capacitation, binding to the zona pellucida, binding to the oocyte membrane, and penetration of the zona pellucida-free oocyte. Therefore it seems clear that each of these factors contains multiple genes contributing to sterility. Furthermore, our results indicate that genes within S1 interact with genes in S2 for all sperm functions examined. However, S1 and S2 genes affecting motility interact in a purely additive fashion, while S1 and S2 genes affecting most other sperm characteristics interact in a synergistic manner. Additionally, the patterns of synergism between S1 and S2 for abnormalities in capacitation, sperm-oolemma binding, and zona-free oocyte penetration are nearly identical. This suggests that these three defects are caused by mutation of the same gene within each sterility factor. These findings will not only be instrumental in matching the various t haplotype sperm defects to candidate genes for S1 and S2, but will facilitate a more comprehensive understanding of the cellular and genetic mechanisms underlying t haplotype male sterility.

Evidence for the involvement of calmodulin in mouse sperm capacitation.

Although Ca(2+) is of fundamental importance in mammalian sperm capacitation, its downstream targets have not been definitively demonstrated. The purpose of this study was to use the calmodulin (CaM) antagonists W7 and calmidazolium (CZ) to investigate the possible role of CaM, a Ca(2+)-specific binding protein, in capacitation. Sperm membrane changes associated with capacitation were assessed by the B pattern after chlortetracycline staining and by the ability to undergo the acrosome reaction (AR) in response to lysophosphatidylcholine (LPC). The percentage of B pattern sperm was significantly inhibited by W7 or CZ in a concentration-dependent manner. At 100 microM W7 or 10 microM CZ, these inhibitors also significantly reduced the sperm's ability to undergo the LPC-induced AR. Inhibition of the B pattern and the LPC-induced AR was overcome by exogenous cAMP analogues. Treatment of the sperm with 100 microM W7 also resulted in a significant decrease in their ability to fertilize eggs in vitro. At 100 microM, W5, a less potent dechlorinated W7 analogue, had no effect on the B pattern, LPC-induced AR, or fertilization competence. Sperm viability and protein tyrosine phosphorylation were not substantially affected by 100 microM W7 (relative to 100 microM W5) or 10 microM CZ; however, the percentages of motile and hyperactivated sperm were significantly reduced. The antagonist-inhibited sperm motility was restored by dilution in control medium, but not by cAMP analogues. These results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers.

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as "curlicue" is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized "curlicue" and a flagellar developmental defect, "whipless", to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both "curlicue" and "whipless" to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both "whipless" and "curlicue" at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or "whipless" animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility.

An improved mouse sperm-oocyte plasmalemma binding assay: studies on characteristics of sperm binding in medium with or without glucose.

Sperm binding to the oocyte plasmalemma is crucial to subsequent steps in fertilization. However, the usual in vitro assay for sperm-oocyte binding does not distinguish between nonspecific attachment and specific binding leading to fusion and penetration. Since zona pellucida-free pronuclear zygotes should not bind sperm (because of the block to polyspermy at the level of the oocyte membrane), a procedure has been developed to remove virtually all sperm from zona-free pronuclear zygotes (2PNZ). After six washes with a 90-microm-bore pipette, there were 0.5 +/- 0.2 sperm/2PNZ (n = 83). Therefore, these washing conditions were used to define sperm-oocyte binding. The relationship of binding to oocyte penetration was determined for outbred mouse oocytes coincubated in complete medium with (B6 x 129)F1 hybrid sperm (10(7)/ml). Binding was maximal (29 +/- 5 sperm per oocyte) during the first 30 minutes but decreased significantly to 4.6 +/- 1.4 by 60 minutes of coincubation (over 10 trials). Oocyte penetration was 99 +/- 1% by 30 minutes, while the number of decondensed sperm nuclei per oocyte increased significantly to 7.5 +/- 0.6 at 60 minutes. These data suggest that the block to polyspermy involves detachment of bound sperm. Similar coincubations were carried out in medium without glucose (NoG), as this medium has been reported to inhibit fusion without affecting binding. However, binding was only 11 +/- 2 at 30 minutes but increased to 25 +/- 4 at 60 minutes, suggesting that binding was retarded in NoG. When gametes were coincubated in NoG for 5 hours, about half of the oocytes were penetrated, suggesting that the lack of glucose did not inhibit fusion but instead delayed it. These data suggest that if sperm binding is to be determined in complete medium, the time of the block to polyspermy should be determined prior to selecting the appropriate time to assay binding. Furthermore, using the same coincubation period for the binding assay in control and treated sperm may not be appropriate if the treatment alters the time of maximal binding.

Mice carrying two t haplotypes: sperm populations with reduced Zona pellucida binding are deficient in capacitation.

Capacitation is the unique process by which mammalian sperm become capable of undergoing the acrosome reaction (AR). An approach to studying sperm capacitation is to identify mutations altering this process. Male mice carrying two t haplotypes are sterile, with poor sperm motility, reduced zona pellucida binding, and an inability to penetrate zona-free oocytes. The objective of this study was to examine sperm capacitation and its potential relationship to zona pellucida binding in mice of the same genetic strain carrying none, one, or two t haplotypes. Sperm capacitation was assessed by the B pattern of staining by chlortetracycline (CTC) and by the ability of sperm to undergo the lysophosphatidylcholine (LPC)-induced AR. The CTC assay demonstrated that sperm capacitation from t/+ mice was similar to that from +/+ mice, but sperm from t/t mice were deficient. LPC induced the AR of capacitated sperm, but not noncapacitated sperm, in a concentration-dependent manner. Sperm from t/t mice were also deficient in the LPC-induced AR. Thus, by two independent assays, sperm from t/t mice were shown to be deficient in capacitation. To determine whether a deficiency in capacitation could influence zona binding, the ability of capacitated versus noncapacitated sperm to bind to the zona pellucida was tested. The mean numbers of sperm bound per oocyte were significantly greater for capacitated sperm than for noncapacitated sperm. These results suggest that the deficient capacitation of sperm from t/t mice could be responsible for, or at least contribute to, their reduced ability to bind to the zona pellucida.

High-resolution mapping of sperm function defects in the t complex fourth inversion.

Structural variants of the mouse Chr 17-specific t complex, known as t haplotypes, express factors that alter the ability of sperm to carry out their roles in the normal fertilization process. In previous studies of males carrying heterospecific combinations of the t complex, we discovered a unique M. spretus/t haplotype phenotype of male sterility. In additional studies with mice carrying a series of M. spretus-M. m. domesticus recombinant Chr 17 homologs and a complete t haplotype (S-+/t), we monitored physiological aspects of sperm function to map a locus (Hst6) responsible for expression of the t-specific "curlicue" sperm flagellar curvature phenotype to 1 cM within the fourth inversion of the t complex. In the present report, we quantitatively analyze the in vitro capability of sperm from mice with similar S-+/t Chr 17 genotypes to fertilize zona pellucida-free mouse eggs. The results identify a locus, Stop1, mapping distal to Pim1, with acute effects on the ability of sperm to penetrate the oolemma. The data suggest that Stop1 is a complex locus consisting of at least two genetic elements, a proximal one overlapping the Hst6 locus, and another, distal to the Hst6 locus. Further quantitative analyses of the "curlicue" phenotype produced by sperm derived from these same animals indicate that expression of this chronic flagellar curvature phenotype also derives from at least two elements, both mapping within the Hst6 locus. Thus, these studies provide higher resolution mapping of the molecular basis of t haplotype-specific sperm dysfunction emanating from In(17)4.

Identification of the t complex-encoded cytoplasmic dynein light chain tctex1 in inner arm I1 supports the involvement of flagellar dyneins in meiotic drive.

The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell-specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.

Models for male infertility: the t haplotypes.

The t haplotypes are variant alleles of genes in the proximal region of mouse Chromosome 17, linked together by four inversions. While females carrying two t haplotypes are fertile, males are sterile. Their spermatozoa exhibit severe motility defects and are unable to penetrate zona pellucida-free oocytes. Spermatozoa from males carrying one t haplotype (t/+) exhibit mild motility deficits and a delay in penetration of the zona-free oocyte. The inversions of the t haplotypes contain several genes that cause or contribute to male sterility, at least some of which can be identified by analysis of mice carrying Mus spretus-Mus domesticus recombinant Chromosomes 17. The t haplotypes specify a number of sperm biochemical abnormalities, but these have not been related directly to defects in fertilization. In t/+ males, spermatozoa not bearing the t haplotype are defective in fertilization compared with t-bearing spermatozoa. The mechanism causing this is likely to involve haploid gene expression confined to the t-bearing spermatids. Since many genes situated in the region of the t haplotypes have human homologues, an understanding of t haplotype sterility in mice is expected to contribute significantly to our knowledge of the genetic basis for human sperm dysfunction.

Hst7: a male sterility mutation perturbing sperm motility, flagellar assembly, and mitochondrial sheath differentiation.

Hst7, a mouse hybrid sterility locus, has been mapped in close linkage to four other hybrid sterility loci, on proximal chromosome 17 within the t complex. When an allele (s) of Hst7 from the species Mus spretus is crossed into the Mus musculus domesticus (laboratory mouse) background, all male offspring are sterile. This occurs regardless of whether the Hst7 allele on the other chromosome 17 homolog is wild-type (+) or an allele (t) derived from the structurally variant homolog known as a t haplotype. Males of the Hst7 genotype s/+ produce sperm that, after release from the cauda epididymis, display moderate asthenospermia (straight line velocity = 49 +/- 4 microm/second, significantly lower than 102 +/- 7 microm/second for congenic wild-type controls) and normal morphology. However, males of the Hst7 genotype s/t produce sperm whose forward movement is below the detectable limit of the sperm motion analysis system. In addition, these sperm exhibit a variety of flagellar abnormalities, with about one third having normal heads attached to sacklike caudal regions. These sacks consist of membrane-delimited cytoplasm containing disorganized and/or misshapen axonemal elements. The remainder of the sperm from s/t mice have flagella with seemingly normal axonemes, although many exhibit enlarged areas of cytoplasm in their midpieces with extra layers of misaligned mitochondria. The s/t sperm mitochondria also display diffuse and vacuolated matrices reminiscent of meiotic germ cell and spermatid mitochondria. Observations of developing spermatids in the s/t testis reveal an unusual phenotype in which gaps of varying length occur in the mitochondrial wrapping of the midpiece. These data suggest that both the s and t alleles of Hst7 are defective alleles that contribute differentially to the severe asthenospermia phenotype and interact genetically to perturb flagellar development.

Properties and localization of a tyrosine phosphorylated form of hexokinase in mouse sperm.

Mouse sperm possess a phosphotyrosine-containing hexokinase type 1 (HK1) that is associated with the plasma membrane fraction of these cells (Kalab et al., 1994; J. Biol Chem 269:3810-3817). This apparent plasma membrane association appears unique, since somatic HK1 is normally cytoplasmic or bound to the outer mitochondrial membrane via contact sites with a voltage-dependent anion channel (porin) through a porin-binding domain. In male germ cells, three cDNA clones have been described that encode unique HK1 isoforms (HK1-sa, HK1-sb, HK1-sc) that do not contain porin binding domains (Mori et al., 1993: Biol Reprod 49:191-203). This suggests that these proteins might not be localized to the outer mitochondrial membrane and could have alternative functions in germ cells and/or sperm. We demonstrate in the mouse that male germ cells and sperm could potentially express four HK1 isoforms (HK1-sa, HK1-sb, HK1-sc, and the somatic HK1). At the protein level, at least one of the HK1 isoforms becomes phosphorylated on tyrosine residues during spermatogenesis. Treatment of sperm membrane fractions to dissociate the phosphotyrosine-containing HK1 (pY-mHK1) yields results demonstrating that pY-mHK1 has properties of an integral membrane protein. Indirect immunofluorescence using a monoclonal antibody to HK1 demonstrates specific staining both in the head and tail regions of sperm. Surface biotinylation of intact sperm followed by precipitation with either polyclonal HK1 antiserum or with avidin-Sepharose suggests that pY-mHK1 possesses an extracellular domain. These results suggest that mouse sperm contain at least one HK1 isoform that is present on the sperm head, has an extracellular domain, and behaves as an integral membrane protein.

Sperm from mice carrying two t haplotypes do not possess a tyrosine phosphorylated form of hexokinase.

Mouse sperm contain a tyrosine phosphorylated form of hexokinase type 1 (HK1; Kalab et al., 1994: J Biol Chem 269:3810-3817) that has properties consistent with an integral plasma membrane protein. Furthermore, this tyrosine phosphorylated form of HK1 has an extracellular domain and HK1 is localized to both the head and flagellum of nonpermeabilized cells (Visconti et al., 1995c). We have characterized HK1 in mature sperm from sterile tw32/tw5 mice (mutant sperm) that have defects in motility and sperm-egg interaction (Johnson et al., 1995: Dev Biol 168:138-149). Immunoprecipitation of mouse sperm extracts with an antiserum made against purified rat brain HK1 demonstrates the presence of HK1 in mutant sperm. Various biochemical and immunofluorescence assays indicate that at least a portion of the HK1 present in these cells is an integral membrane protein with an extracellular domain located on the sperm head and flagellum. However, immunoblot analysis with anti-phoshotyrosine antibodies demonstrates that HK1 in mutant sperm is not tyrosine phosphorylated. Northern blot and RT-PCR analysis does not indicate any obvious abnormalities in the transcription of somatic or germ cell-specific HK1 isoforms in mutant testes, and RFLP analysis of recombinant mice indicates that no genes specifying HK1 isoforms are located on chromosome 17. We have mapped the locus responsible for the lack of tyrosine phosphorylation of HK1 mutant sperm to the most proximal (to the centromere) of the four inversions within the t haplotype. A male sterility factor is located in this same inversion (Lyon, 1986: Cell 44:357-363). Since the mutant sperm are unable to complete fertilization, there could be a relationship between sterility and the lack of tyrosine phosphorylation of HK1 in these mutant sperm.

The cellular basis for interaction of sterility factors in the mouse t haplotype.

The t haplotypes are variant forms of the proximal one-third of chromosome 17 in the mouse. They contain four inversions (relative to the wildtype DNA) extending over most of this region and house a number of male sterility factors. Males carrying two complete t haplotypes (t/t) are sterile, as are males homozygous for S2, the sterility factor located in the most distal (relative to the centromere) inversion. Males homozygous for the sterility factor S1, located in the most proximal inversion, are not sterile; however, if such a male also is heterozygous for other sterility factors, then sterility results. It has been suggested therefore that homozygosity for S1 enhances the detrimental action of other sterility factors. Sperm from t/t males have severe motility defects and are unable to penetrate investment-free eggs, while sperm from fertile t/+ mice have less serious motility defects and exhibit a delay in penetration of investment-free eggs. To determine whether homozygosity for S1 enhances the cellular defects exhibited by sperm from mice heterozygous for other sterility factors, we compared the motility and egg-penetrating ability of sperm from fertile mice homozygous for S1 to that of sperm from mice carrying one complete t haplotype and one proximal or distal partial t haplotype. The data suggest that sperm from males carrying a proximal partial t haplotype and a complete t haplotype have serious defects in motility and penetration of the investment-free egg, and support the hypothesis that S1 enhances the detrimental effects of other sterility factors within the t haplotype.

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.

In the accompanying report (Visconti, P.E., Bailey, J.L., Moore, G.D., Pan, D., Olds-Clarke, P. and Kopf, G.S. (1995) Development, 121, 1129-1137) we demonstrated that the tyrosine phosphorylation of a subset of mouse sperm proteins of M(r) 40,000-120,000 was correlated with the capacitation state of the sperm. The mechanism by which protein tyrosine phosphorylation is regulated in sperm during this process is the subject of this report. Cauda epididymal sperm, when incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin do not display the capacitation-associated increases in protein tyrosine phosphorylation of this subset of proteins. This NaHCO3, CaCl2 or bovine serum albumin requirement for protein tyrosine phosphorylation can be completely overcome by the addition of biologically active, but not inactive, cAMP analogues. Addition of the active cAMP analogues to sperm incubated in media devoid of NaHCO3, CaCl2 or bovine serum albumin overcomes the inability of these media to support capacitation, as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. The effects of the cAMP analogues to enhance protein tyrosine phosphorylation and to promote capacitation appears to be at the level of the cAMP-dependent protein kinase (PKA), since two specific inhibitors of this enzyme (H-89 and Rp-cAMPS) block the capacitation-dependent increases in protein tyrosine phosphorylation in sperm incubated in media supporting capacitation. Capacitation, as assessed by the aforementioned endpoints, also appears to be inhibited by H-89 in a concentration-dependent manner. These results provide further evidence for the interrelationship between protein tyrosine phosphorylation and the appearance of the capacitated state in mouse sperm. They also demonstrate that both protein tyrosine phosphorylation and capacitation appear to be regulated by cAMP/PKA. Up-regulation of protein tyrosine phosphorylation by cAMP/PKA in sperm is, to our knowledge, the first demonstration of such an interrelationship between tyrosine kinase/phosphatase and PKA signaling pathways.

Sperm from mice carrying one or two t haplotypes are deficient in investment and oocyte penetration.

The t haplotypes, mutant forms of the proximal third of mouse chromosome 17 (the t complex), contain factors that contribute to defective sperm function in fertilization. Males carrying two t haplotypes (tx/ty mice) are sterile; their sperm have very poor motility and are unable to penetrate zona-free eggs. Although males carrying one t haplotype (t/+) are fertile, genetic evidence suggests that the sperm carrying the normal form of chromosome 17 (+t) are dysfunctional in fertilization, and some or all sperm have abnormal motility. Some of the same genetic factors that cause sterility in tx/ty males probably contribute to the dysfunction of +t sperm from t/+ males; however, it is unclear which steps in gamete interaction are defective in sperm from t/+ males, or whether the defects are similar to those observed in sperm from tx/ty males. We have developed a unique low sperm:egg ratio IVF assay for sperm function in fertilization. Using this assay, we have shown that tw5/+ sperm are less able than congenic +/+ sperm to penetrate the zona (probably due to their abnormal motility) and to penetrate the zona-free oocyte. Since tw5/tw32 sperm are unable to complete these same two steps in sperm-egg interaction, these specific deficits could be involved in both transmission ratio distortion and sterility. We have also shown that tw5/tw32 sperm are deficient in their ability to bind to the zona and to the oolemma. These results suggest that t haplotypes contain loci which affect a number of sperm functions and thus could be a rich source of genes important for sperm-egg interaction.

Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro.

Mammalian sperm exhibit characteristic motility changes associated with capacitation. Movement characteristics of bovine sperm incubated in noncapacitating (control, medium alone), capacitating (oviduct fluid, nonluteal, and luteal), or capacitating, acrosome reaction inducing (follicular fluid) conditions were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4 hours in a modified Tyrode's medium (control), 20 and 60% nonluteal (NL) or luteal (L) oviduct fluid (ODF), or 20 and 60% follicular fluid (FF). Relative to sperm incubated in control medium, motility of sperm treated with ODF or FF had increased linearity and vigorous motility. Sperm incubated in 60% ODF or FF showed a small decrease in mean trajectory/path straightness and velocity over time compared to 20% fluid treatments and control. Frequency distribution graphs were symmetric for 20% NL- and L-ODF treated sperm. However, 20% FF and 60% ODF and FF treatments had distributions skewed to the left, indicating smaller values for lateral head displacement (ALH) and curvilinear velocity (VCL). Median values for ALH and VCL were determined for control-treated sperm, and subtracted from individual sperm values for all treatments to estimate deviation from control, designated ALHc and VCLc. Three-dimensional plots of ALHc, VCLc and corresponding frequency indicated shifts in peak patterns for fluid-treated sperm compared to control sperm. Incubation in 20% ODF and FF resulted in peak shift for ALH and VCL values; yet, little change in peak position was observed in sperm incubated in 60% ODF and FF.(ABSTRACT TRUNCATED AT 250 WORDS)

Deficiency in fertilization by morphologically abnormal sperm produced by azh mutant mice.

Male mice homozygous for the azh mutation produce spermatozoa with abnormal head shapes and have significantly reduced fecundity, to between 5% and 10% that of wild-type or heterozygous mice. Several possible causes of this infertility were investigated. No gross endocrine disorders in azh/azh male mice were observed, and they exhibited apparently normal mating behavior. In addition, their sperm were motile, were capable of hyperactivated motility, and did not show premature acrosome reactions. However, quantitative analysis revealed slight but significant reductions in several motility parameters. Analysis of embryos following mating of azh/azh males with superovulated females indicated a reduction in the number of fertilized eggs compared to control matings. In vitro, spermatozoa from azh/azh mice failed to fertilize cumulus-intact/zona-intact and cumulus-free/zona-intact ova, although they successfully fertilized zona-free ova. These results indicate that the primary defect in fertility of azh/azh male mice is a result of sperm quality, likely, in sperm morphology, and is manifest at the level of interaction with the zona pellucida.

Factors that may regulate assembly of the mammalian sperm tail deduced from a mouse t complex mutation.

A unique pattern of aberrant sperm development occurs in laboratory mice (Mus domesticus) that have been made homozygous for an allele of an autosomal gene, Hst-6s, derived from another mouse species, Mus spretus. During the abnormal spermiogenic process of these sterile animals, the sperm tail fails to assemble normally, although a bud develops at the site of the centriole in early spermatids. The bud enlarges during sperm development and eventually contains elements of a mature sperm tail. Tubulin assembles primarily into single microtubules rather than doublets. These singlets form a circular array that is not oriented relative to the centriole. While dense fibers form in association with the singlet microtubules, the fibrous sheath components accumulate in a single irregular mass. These observations suggest that microtubule doublets are required for the organization of an axoneme. In addition, while dense fibers can form normally in association with singlet microtubules, the fibrous sheath requires the axoneme as a substrate for normal organization.

Hybrid sterility-6: a mouse t complex locus controlling sperm flagellar assembly and movement.

Mouse t haplotypes alter sperm differentiation, resulting in abnormal sperm movement and sterility. In previous studies, a locus responsible for hybrid sterility in the genus Mus,Hst-4, was mapped to the distal inversion of the t complex on chromosome 17. Here we report the identification and characterization of two additional hybrid sterility loci, Hst-5 and Hst-6, that map to the same inversion. We further show that an abnormality in sperm flagellar curvature deriving from interactions between t haplotypes and the M. spretus allele of Hst-6 is indistinguishable from one exhibited by sperm from mice carrying two t haplotypes. Additionally, we demonstrate that this latter phenotype maps to the distal inversion of t haplotypes. Morphological and functional studies of Hst-6 mutant sperm also imply that the product(s) of Hst-6 is a spermatogenic-specific protein, important for assembly and function of the sperm axoneme. Thus, Hst-6 provides direct access to the molecular basis of t haplotype-specific alterations in sperm function that emanate from the t complex distal inversion.