Cumulus-intact Eggs Research Articles

Lack of NWC protein (c11orf74 homolog) in murine spermatogenesis results in reduced sperm competitiveness and impaired ability to fertilize egg cells in vitro.

NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that NWC knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of NWC knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm.

SPACA7 Is a Novel Male Germ Cell-Specific Protein Localized to the Sperm Acrosome That Is Involved in Fertilization in Mice1

Sperm acrosome associated 7 (SPACA7) is a novel protein of unknown function with no homology to any known protein. Spaca7 transcripts are detected only in testis and predict a 158-residue mature polypeptide with one potential N-glycosylation site and no cysteines. Orthologs are present in various species, including mice and humans. We developed a polyclonal antibody to mouse SPACA7 to study its expression and function. Western blotting and immunofluorescence microscopy detected SPACA7 only in testis, and it was detected in testis starting at Postnatal Day 21 and into adulthood. Immunofluorescence staining of testicular germ cells detected weak SPACA7 expression as early as zygotene spermatocytes. Higher expression was observed in round spermatids, where SPACA7 was localized to a perinuclear spot adjacent to the Golgi and to the acrosome of elongating spermatids and spermatozoa. Immunogold electron microscopy demonstrated that SPACA7 is localized within the proacrosomal granule of round spermatids and the acrosome of spermatozoa. Finally, we showed that SPACA7 was retained within the acrosome of epididymal sperm and was released upon the acrosome reaction. To assess if SPACA7 was involved in fertilization, in vitro fertilization assays in the presence of anti-SPACA7 IgG were performed. Anti-SPACA7 inhibited fertilization of cumulus-intact eggs and prominently delayed cumulus dispersal. However, anti-SPACA7 did not inhibit fertilization of cumulus-free eggs. Our findings indicate that release of SPACA7 from the acrosome accelerates cumulus dispersal and facilitates fertilization via unknown mechanisms. This study is the first to document the expression of endogenous SPACA7 and a function for this novel acrosomal protein.

Ca 2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Ca(2+) ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30-45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3(-), a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca(2+) increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.

Sperm defects in mice lacking a functional Niemann–Pick C1 protein

The Niemann-Pick C1 (NPC1) gene encodes for a multiple membrane spanning protein, which regulates the trafficking of low-density lipoprotein-mediated endocytosed cholesterol. Mutation of the human NPC1 gene causes Niemann-Pick type C (NPC) disease. The Npc1(NIH) mice, a model of human NPC disease, bear a spontaneous mutation of the Npc1 gene, and are infertile. In this study, we have performed sperm analysis to search for the cause of male infertility in the Npc1(NIH) mouse. The number of cauda sperms in Npc1(-/-) mice was decreased roughly three-and-half-fold of that in wild-type mice. The decreased sperm number in Npc1(-/-) mice is due, at least in part, to partial arrest of spermatogenesis in the testes, as revealed by histological analysis. Compared to wild-type sperm, Npc1(-/-) sperm displayed a high frequency of morphological abnormalities, including tailless heads and aberrant heads. In the in vitro fertilization (IVF) assay using cumulus-intact eggs, Npc1(-/-) sperm failed to produce two-cell embryos. In the IVF assay where zona-free eggs were used, Npc1(-/-) sperm bound normally but could not fuse with the egg. Further analysis indicated that Npc1(-/-) sperms are drastically impaired in the binding to the egg zona pellucida, only 14% of the level of wild-type sperm. Moreover, on Npc1(-/-) cauda sperm, one-third of the total cyritestin protein was not proteolytically processed, while fertilin beta was processed normally. Taken together, these results demonstrate that there are multiple defects in sperms from mice lacking a functional NPC1 protein, and these observed sperm defects may result in sterility.

Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme That Catalyzes Post-translational Protein Tyrosine O-Sulfation, Causes Male Infertility

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.

Effect of Cumulus Maturity on Sperm Penetration in the Golden Hamster

The influence of the cumulus on gamete interaction was investigated in naturally and precociously ovulated eggs from superstimulated (eCG) hamsters. Inseminations were performed in which the absolute gamete ratio (sperm/egg) was held constant, but the relative gamete ratio (sperm x egg-1 x ml-1) was varied 10-fold. Sperm penetration to the perivitelline space was significantly higher at the higher relative gamete ratio, indicating that there is no chemoattractant in the cumulus and that gamete interaction in cumulus-intact eggs is a function of collision frequency, which varies according to the relative gamete ratio. The level of successful gamete interaction as a function of the relative gamete ratio was determined for naturally and precociously ovulated eggs. The level of sperm penetration reached saturation at a relative gamete ratio of 10(3.3) for naturally ovulated eggs and 10(4.0) sperm x egg-1 x ml-1 for precociously ovulated eggs. At suboptimal relative gamete ratios, there was no difference in the level of penetration between partially and fully capacitated sperm. These results indicate that the larger, more mature cumulus of naturally ovulated eggs is successfully penetrated at lower collision frequencies than the denser, less mature cumulus of precociously ovulated eggs. To determine whether the maturity of the cumulus affects the timing of sperm penetration, cumulus-free and cumulus-intact eggs were inseminated at their optimal relative gamete ratios. The rate of sperm penetration of partially capacitated sperm was not different between cumulus-free and cumulus-intact eggs, regardless of ovulation method. However, the period of time between insemination and when half the eggs were penetrated (T50) was longer in cumulus-intact eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol inhibits human and hamster sperm penetration of eggs.

The effect of alcohol on the fertilizing ability of both human and hamster spermatozoa was examined by an in vitro fertilization assay using hamster ova. Spermatozoa were incubated in capacitating media for 3 hr (hamster sperm) and 4 hr (human sperm). Hamster ova were inseminated with preincubated sperm and were examined after 2 to 3 hr. Ethanol was added to the capacitating media at concentrations of 25, 50, 100, 200, and 400 mg%. Fertilization of zona-free hamster eggs by human spermatozoa was reduced from 49.6% in no alcohol to 16.7% in 400 mg% ethanol. Fertilization of hamster eggs by hamster sperm revealed a reduction from 63.6% to 33.7% in cumulus-intact eggs and from 65.8% to 10.8% in cumulus-free eggs in the presence of ethanol at 400 mg%. Hamster sperm acrosome reaction was reduced from 47% to 12%. When these hamster sperm with reduced acrosome reaction were placed with zona-free hamster eggs, the 100% fertilization rate was not reduced; however, the fertilization index, which reflects the number of swelling sperm heads per egg, was reduced from 8.5 to 1.8. This suggests that as little as 12% of the sperm with an acrosome reaction is sufficient to fertilize 100% of the zona-free eggs. If ethanol was added to the insemination media only, there was no inhibition of fertilization by human sperm or hamster sperm that had been previously capacitated in an ethanol-free media. Removal of the ethanol from the preincubated sperm produced fertilization at control levels; thus the inhibitory effect is reversible. These results indicate that ethanol may affect fertilization by an inhibition of the capacitation and/or acrosome reaction process.

Both taurine and albumin support mouse sperm motility and fertilizing ability in vitro but there is no obligatory requirement for taurine.

When mouse spermatozoa were washed immediately upon release from the epididymis, preincubated for up to 120 min in PVA-containing, albumin-free medium and assessed for their ability to fertilize cumulus-intact eggs in vitro, they were poorly fertile in comparison with their unwashed counterparts in the same medium. Fertilizing ability could be significantly improved by introducing taurine or albumin or by washing a second time at the end of preincubation. The most effective treatment was provided by the continuous presence of low concentrations (0.05-0.1 mg/ml) of BSA, similar to the amount of albumin detected in the supernatants removed during washing. There was no evidence that acrosome loss was inhibited by washing; rather, it was enhanced by the removal of a surface component which inhibits the acrosome reaction. The presence of taurine did not further increase this response. Motility, reduced in washed suspensions, was improved by the presence of taurine or albumin and experimental results suggest that this was a major factor in the improvement of fertilizing ability after introduction of these compounds. Although taurine, hypotaurine and albumin were all found in the sperm washings and thus would be present in unwashed, fertile samples, low concentrations of albumin were able to maintain full fertilizing ability. Therefore, unlike hamster spermatozoa, mouse spermatozoa would not appear to have an obligatory requirement for a motility stimulating factor such as taurine.

Mouse sperm capacitation in vitro involves loss of a surface-associated inhibitory component.

The increasing fertility of epididymal mouse sperm suspensions as preincubation time is extended is accompanied by the inactivation or destruction of an inhibitory component. Alternatively, precocious removal of the component, achieved by centrifugation, leads to significant improvement in fertilizing ability. Suspensions were preincubated for a total of 120 min, with aliquants being removed at 5, 30 and 120 min. By gently washing samples and resuspending in fresh medium, the poor fertility of unwashed 5- and 30-min suspensions was increased such that 30-min washed samples did not differ significantly from fully capacitated, highly fertile 120-min unwashed control samples. When the supernatants obtained during washing of uncapacitated suspensions (5 and 30 min preincubation) were added to capacitated (120 min preincubation) populations, fertilization of cumulus-intact eggs was markedly and significantly inhibited, although fertilization of zona-free eggs was unaffected. In contrast, supernatants from capacitated suspensions were not inhibitory. When suspensions were preincubated in Ca2+-free media, both washing and exposure to hyperosmolal conditions improved fertilizing ability after addition of exogenous Ca2+, although not to the extent seen in control samples. Removal of the inhibitory component therefore increased the response of spermatozoa to Ca2+. The component was shown to be cell-associated and to inhibit the acrosome reaction in capacitated suspensions. Finally, the inhibition was shown to be reversible, with further incubation of inhibited suspensions restoring the original fertility.

Potassium ions modulate expression of mouse sperm fertilizing ability, acrosome reaction and hyperactivated motility in vitro.

In K+-free medium, epididymal sperm suspensions, whether washed free of epididymally-derived K+ or not, were unable to penetrate washed cumulus masses; some penetration of zona-free eggs was obtained with unwashed sperm suspensions, while washed samples were generally non-fertilizing. Within 5 min of K+ introduction, however, spermatozoa were able to fertilize intact eggs rapidly and synchronously, indicating that K+ was not required for capacitation. Measurements of extracellular K+ concentrations in these experiments indicate that 0.1-0.15 mM-K+ is sufficient to support sperm: egg fusion, but concentrations greater than 0.15 mM are required for penetration of cumulus-intact eggs. When medium of normal osmolality (318 mosmol) but elevated K+/Na+ ratio (27.7 mM/125 mM) was compared with control medium (2.7/150), the former promoted lower rates of penetration after both 30 and 120 min preincubation (8 and 10%, respectively) than those obtained with control medium (45 and 95%). Upon reduction to the ratio in control media, however, the fertilizing potential of these suspensions was equivalent to control samples: relatively slow and asynchronous penetration after 30 min preincubation (50%) and rapid, synchronous penetration after 120 min (92%). Thus there was no evidence of a shortening of sperm capacitation time, but rather a suppression of fertilizing potential in the presence of elevated K+. Uterine sperm samples recovered shortly after mating gave similar results when tested in these media 30 and 120 min after release from the male tract. Preincubation of epididymal samples in high K+ (27.7 mM) hyperosmolal media (368 mosmol) for 30 min significantly shortened sperm capacitation as shown by rapid penetration of intact eggs (94%) after reduction in osmolality, but this appeared to be a non-specific effect; high Na+ (175 mM) hyperosmolal medium had a similar effect (98% of eggs fertilized). Acrosome loss and hyperactivated motility were significantly lower in media with very low or very high K+ concentrations but, after alteration to control medium values, increased to levels similar to those obtained with control samples. It is proposed that the relatively high K+ concentrations found in female tract fluids (approximately 20-30 mM) may serve to modulate fertilizing potential of spermatozoa in vivo.

Anti‐Mouse Sperm Antiserum

Rabbit anti‐mouse sperm (αMS) Fab fragments have been prepared, which are capable of inhibiting spermzona interaction (both binding and penetration) and sperm‐egg fusion in vitro. In cumulusintact eggs, αMS Fab did not inhibit zona penetration, but did inhibit sperm‐egg fusion, implying that separate and distinguishable antigens are involved in these two fertilization events. Preliminary identification of the antigens recognized by αMS IgG, using a gel overlay technique, revealed four major antigens of 200 Kd, 55–63 Kd, 37 Kd, and 33 Kd, and three minor antigens of 110 Kd, 48 Kd, and 40 Kd.