Zona-intact Eggs Research Articles

A Role of TMEM16E Carrying a Scrambling Domain in Sperm Motility.

Transmembrane protein 16E (TMEM16E) belongs to the TMEM16 family of proteins that have 10 transmembrane regions and appears to localize intracellularly. Although TMEM16E mutations cause bone fragility and muscular dystrophy in humans, its biochemical function is unknown. In the TMEM16 family, TMEM16A and -16B serve as Ca(2+)-dependent Cl(-) channels, while TMEM16C, -16D, -16F, -16G, and -16J support Ca(2+)-dependent phospholipid scrambling. Here, we show that TMEM16E carries a segment composed of 35 amino acids homologous to the scrambling domain in TMEM16F. When the corresponding segment of TMEM16A was replaced by this 35-amino-acid segment of TMEM16E, the chimeric molecule localized to the plasma membrane and supported Ca(2+)-dependent scrambling. We next established TMEM16E-deficient mice, which appeared to have normal skeletal muscle. However, fertility was decreased in the males. We found that TMEM16E was expressed in germ cells in early spermatogenesis and thereafter and localized to sperm tail. TMEM16E(-/-) sperm showed no apparent defect in morphology, beating, mitochondrial function, capacitation, or binding to zona pellucida. However, they showed reduced motility and inefficient fertilization of cumulus-free but zona-intact eggs in vitro. Our results suggest that TMEM16E may function as a phospholipid scramblase at inner membranes and that its defect affects sperm motility.

Prss37 Is Required for Male Fertility in the Mouse1

In order to understand the mechanisms of mammalian fertilization, studies using genetically manipulated animals have provided us with plenty of interesting and valuable information on the genetic factors affecting male fertility. In the present work, we demonstrate for the first time that Prss37, a previously uncharacterized putative trypsin-like serine protease, is required for male fertility. Prss37 is highly and exclusively expressed in the testis of adult mice, especially in the elongating spermatids during spermiogenesis, and almost vanishes in the mature sperm of mice. Mice deficient for Prss37 show male infertility, but their mating activity, spermatogenesis, sperm morphology, and motility remain unaffected. In vivo fertilization assays revealed that Prss37(-/-) mice exhibited a markedly decreased fertilization rate (2.3% vs. 70% of that in control mice) accompanied by the defect in sperm migration from uterus into oviduct. In vitro study further showed sperm were incapable of sperm-egg recognition/binding when zona-intact eggs were exposed to Prss37(-/-) sperm, in which mature Adam3 was completely undetectable. Interestingly, however, Prss37(-/-) sperm were able to fertilize cumulus-intact oocytes in vitro. These data clearly indicate that Prss37 deficiency causes the absence of mature Adam3 in sperm and a defect in sperm migration from uterus into oviduct, which mainly accounts for male infertility of Prss37-null mice, while the defect in sperm-zona binding seems irrelevant to the fertilizing ability of Prss37(-/-) sperm.

Na,K-ATPase alpha4 Isoform Is Critical for Sperm Motility and Fertility.

Active exchange of Na+ and K+ across the sperm plasma membrane is under the control of the Na,K-ATPase, an integral membrane enzyme, composed of catalytic alpha and glycosylated α subunits. Two molecular variants of the alpha subunit, the ubiquitous α1 and the sperm specific alpha4 coexist in the male gamete. These isoforms exhibit different biochemical properties; however, their function in sperm fertility is unknown. Here, we show that genetic deletion of alpha4 in mice causes complete male infertility. Sperm from these mice are unable to fertilize zona intact and zona free eggs in vitro. Sperm null in the alpha4 isoform show abnormal oocyte binding. Deletion of alpha4 produces severe reduction in sperm total, progressive and in a series of critical parameters of sperm motility. Moreover, alpha4 null sperm shows drastic reduction in the hyperactivation typical of sperm capacitation. In addition, absence of alpha4 causes a characteristic bend in the sperm flagellum, indicative of abnormal sperm ion regulation. Also, sperm devoid of alpha4 presents other alterations, including depolarization of the cell plasma membrane and increase in intracellular Na+ levels. Overall, this demonstrates the absolute requirement of alpha4 for sperm fertility and the inability of the alpha1 isoform to compensate for alpha4. Our findings reveal alpha4 as an attractive biomarker for male fertility and a novel target for male contraception. [Supported by NIH grants HD043044 and HD055763]. (poster)

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC −/− spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC −/− spermatozoa and are undetectable after sperm purification. HCO 3 − is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca 2+ entry into sAC −/− spermatozoa. Moreover, the delayed HCO 3 −-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC −/− spermatozoa. However, sAC −/− sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC −/− sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC −/− spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.

Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins

Capacitated acrosome-intact mouse spermatozoa bind to the egg’s extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor–ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars ( N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94–101]. To further elucidate the specificity of sperm–ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm–bead assay was carried out at 37 °C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm–bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor(s) that recognize and bind to the sugar residues.

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm.

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.

Binding of arylsulfatase A to mouse sperm inhibits gamete interaction and induces the acrosome reaction.

We have shown previously that male germ cell-specific sulfoglycolipid, sulfogalactosylglycerolipid (SGG), is involved in sperm-zona pellucida binding, and that SGG and its desulfating enzyme, arylsulfatase A (AS-A), coexist in the same sperm head area. However, AS-A exists at a markedly low level in sperm as compared to SGG (i.e., 1/400 of SGG molar concentration). In the present study, we investigated whether perturbation of this molar ratio would interfere with sperm-egg interaction. We demonstrated that purified AS-A bound to the mouse sperm surface through its high affinity with SGG. When capacitated, Percoll gradient-centrifuged mouse sperm were treated for 1 h with various concentrations of AS-A, their binding to zona-intact eggs was inhibited in a dose-dependent manner and reached the background level with 63 nM AS-A. This inhibition could be partially explained by an increase in premature acrosome reaction. The acrosome-reacted sperm population of the 63 nM AS-A-treated sperm sample was twice that of the control sample (treated with 63 nM ovalbumin) at 1 h (i.e., 32% vs. 15%) and rose to 53% at 2 h. This induction was presumably due to SGG aggregation attributed to AS-A, existing as a dimer at neutral pH, and could be mimicked by anti-SGG immunoglobulin (Ig) G/IgM + secondary IgG antibody. Drastic inhibition (75%) of in vivo fertilization was also observed in females inseminated with sperm suspension containing 630 nM AS-A as compared to the rate in females inseminated with sperm suspension included with 630 nM ovalbumin. Our results demonstrate a promising potential for AS-A as a nonhormonal, vaginal contraceptive.

Role of egg sulfolipidimmobilizing protein 1 on mouse sperm-egg plasma membrane binding.

We have shown that sperm sulfolipidimmobilizing protein 1 (SLIP1, molecular mass of 68 kDa), a sulfogalactosylglycerolipid (SGG)-binding protein, is significant in sperm-zona pellucida (ZP) interaction. The objective of this study was to localize SLIP1 on the egg and determine its role in gamete interaction. Immunofluorescence and immunoprotein A gold electron microscopy localized SLIP1 to the egg plasma membrane. In vitro gamete binding, using zona-free eggs preincubated with antiSLIP1 Fab before coincubation with sperm, showed a significant, dose-dependent decrease in sperm-egg plasma membrane binding. Similar results were obtained when affinity-purified antiSLIP1 IgG was used for egg pretreatment. The significance of egg SLIP1 in sperm-egg plasma membrane binding was further demonstrated by a decrease (36-52%) in in vitro fertilization when zona-intact eggs were pretreated with antiSLIP1 IgG. Since SLIP1 has been shown to bind SGG in vitro, we investigated the possibility that sperm SGG may participate in sperm-egg plasma membrane binding through egg SLIP1. Pretreatment of sperm with antiSGG Fab prior to coincubation with zona-free eggs resulted in a dose-dependent decrease in sperm-egg plasma membrane binding. Collectively, these findings strongly suggest a role for egg SLIP1 in sperm-egg plasma membrane interaction, which may be through its binding to sperm SGG.

Sequence divergence within the sperm-specific polypeptide TCTE1 is correlated with species-specific differences in sperm binding to zona-intact eggs.

The T-complex-associated testes-expressed (TCTE1) gene encodes a novel sperm cell-specific polypeptide (TCTE1) that is conserved across vertebrate species. TCTE1 is absolutely required for fertilization and is expressed in earlier stages of spermatogenesis. When the amino acid sequence of the TCTE1 gene product is compared among various mammalian species, a large, highly conserved domain is observed, along with a divergent domain encoding the 56-58 residues at the N terminus. In this study, the N-terminal regions of the TCTE1 polypeptide from three rodent species--mouse, gerbil, and rat--were compared. The results show that while the gerbil and mouse species are most distant in evolutionary terms, their TCTE1 homologs have not undergone significant divergence. In contrast, the N-terminal region of the rat TCTE1 homolog has evolved rapidly, a finding that indicates positive Darwinian selection. We have tested the correlation between TCTE1 divergence and heterospecific sperm-egg binding ability in the three species under study. Gerbil sperm bind to mouse eggs, while no significant binding is observed between rat sperm and mouse eggs. The results obtained support the hypothesis that the sperm-specific polypeptide TCTE1 may facilitate species-specific divergence of sperm function.

Sperm entry into fertilised mouse eggs.

We have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The 'perivitelline' block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.

In vitro fertilization of partially zona dissected or partially zona removed equine oocytes

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC−/− spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC−/− spermatozoa and are undetectable after sperm purification. HCO3− is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca2+ entry into sAC−/− spermatozoa. Moreover, the delayed HCO3−-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC−/− spermatozoa. However, sAC−/− sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC−/− sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC−/− spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.

Evidence for the presence of high-mannose/hybrid oligosaccharide chain(s) on the mouse ZP2 and ZP3.

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on plasma membranes of rat, mouse, hamster, and human spermatozoa [Tulsiani et al. J Cell Biol 1989; 109:1257; Biol Reprod 1990; 42:843]. Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro, suggesting that the sperm enzyme may have a receptor-like role in binding to the complementary molecules (presumably mannose-containing oligosaccharide [OS] chains) on the mouse zona pellucida (ZP) glycoconjugates [Cornwall et al. Biol Reprod 1991; 44:913]. In the studies reported here, we demonstrate the presence of high-mannose/hybrid-type OS on mouse zona components. Zona-intact eggs, prepared from superovulated mice, were radioiodinated, and the individual zona components (ZP1, ZP2, and ZP3) were isolated by electrophoresis followed by electroelution. The purified ZP components, when resolved by immobilized concanavalin A column chromatography, showed the following results: 1) Nearly all of the ZP1 applied to the immobilized lectin eluted in the column flow-through (effluent) fractions, and no radioactivity eluted with alpha-methyl mannoside, suggesting that ZP1 may not contain high-mannose/hybrid OS. 2) A significant amount of both ZP2 and ZP3 bound to the immobilized lectin, and nearly 16% and 8% of the two components, respectively, were repeatedly eluted with alpha-methyl mannoside.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium channel antagonists modulate the acrosome reaction but not capacitation in mouse spermatozoa.

Mouse spermatozoa require micromolar concentrations of calcium for capacitation but millimolar levels to initiate an acrosome reaction. Sperm suspensions were capacitated by incubation for 120 min in modified Tyrode's medium containing 90 microM-CaCl2 and then verapamil (0.5-50 microM) or nifedipine (0.1-100 nM), drugs shown to inhibit voltage-sensitive calcium channels in other cell types, was added before the introduction of 1.80 mM-CaCl2. Verapamil at 5-50 microM and nifedipine at 1-100 nM significantly inhibited the calcium-stimulated acrosome reaction response, relative to the drug-free control samples. The possibility that these compounds might inhibit calcium entry during capacitation was examined by incubating suspensions for 120 min in medium containing 90 microM-CaCl2 plus either 5 microM-verapamil or 1 nM-nifedipine, diluting to reduce drug concentration to one-tenth and then adding 1.80 mM-CaCl2. The considerable acrosome reaction response obtained indicated that spermatozoa had undergone capacitation and were able to respond to calcium, despite the continuous presence of calcium channel antagonist at a concentration able to inhibit the response at the end of capacitation. In-vitro fertilization studies indicated that both drugs significantly decreased ability of the spermatozoa to fertilize eggs, consistent with acrosome reaction data. However, results indicated that 50 microM-verapamil was able to induce initial stages of egg activation and thus prevent sperm-egg fusion in zona-intact eggs. The addition of verapamil or nifedipine to suspensions capacitated for 120 min in 1.80 mM-CaCl2 significantly potentiated the acrosome reaction response, compared with drug-free controls. Similar treatment of suspensions incubated for only 30 min, and hence only partly capacitated, failed to evoke a response. The potentiation of the acrosome reaction response by verapamil in cells capacitated in high calcium may indicate either retention, due to the action of antagonists, of released intracellular calcium stores, resulting in intracellular calcium concentrations above the threshold required to trigger the acrosome reaction or action of an activated guanine nucleotide binding (G) protein to produce an agonistic rather than an antagonistic response. These results suggest that calcium channels similar to those termed voltage-sensitive in other cell types may exist and play an important role in calcium movements at the end of capacitation, but not during earlier phases of capacitation.

Relationship between two types of mouse sperm surface sites that mediate binding of sperm to the zona pellucida.

There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.

A cell surface block to polyspermy occurs in golden hamster eggs

We have examined the frequency and fate of supernumerary sperm in the perivitelline space (PVS) of in vitro fertilized hamster eggs to determine if there is a cell surface block to polyspermy. The zona pellucida block to polyspermy is very effective since only one sperm penetrated the zona pellucida in 72.8% of the 876 fertilized eggs examined. Of the polypenetrated eggs, 41.6% had a supernumerary sperm within the PVS. The proportion of polypenetrated eggs with PVS sperm did not change when the duration of coincubation was increased from 3 to 6 hr. PVS sperm were found in 67% of the inseminations. From these data we conclude that there is a cell surface block to polyspermy in the hamster. To investigate the mechanism of the cell surface block, we used the Hoechst-transfer technique ( R. Hinkley, B. Wright, and J. Lynn, 1986, Dev. Biol. 118, 148–154) to monitor sperm-egg fusion. We first demonstrated that dye transfer from zona pellucida-free eggs to sperm only occurred when fusion was possible, i.e., in the presence of calcium, and that dye was transferred to all fused sperm. When cumulus-free, zona-intact eggs were preloaded with Hoechst dye and viewed 3 hr postinsemination, three classes of eggs with supernumerary sperm in the PVS were observed: eggs with only Hoechst-positive sperm (62%), eggs with only Hoechst-negative sperm (27%), and eggs with both a Hoechst-positive and a Hoechst-negative sperm (11%). Because of the limited time resolution of the Hoechst-transfer technique, the cell surface block could operate by preventing sperm fusion (Hoechst-negative), by the failure of the eggs to incorporate fused sperm (Hoechst-positive), and/or by the “unfusing” of fused sperm (Hoechst-positive and Hoechst-negative). We are unable at this time to differentiate between these mechanisms.

Characterization of the mouse sperm plasma membrane zona-binding site sensitive to trypsin inhibitors.

The first contact of mammalian gametes is the binding of the spermatozoon to the zona pellucida of the egg. Previous work has shown that binding of the spermatozoon to the zona in the mouse occurs prior to the acrosome reaction and that trypsin inhibitors block this initial binding. This suggests that the sperm surface contains a trypsinlike binding site that functions by an active site mechanism to effect initial zona binding. When suspensions of twice-washed spermatozoa were incubated with the serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (MUGB), the titrant was hydrolyzed at a rate of 8 pmoles/min-10(6) cells. MUGB was found to inhibit the binding of spermatozoa to the zona pellucida. The degree of inhibition and the rate of hydrolysis of MUGB by washed spermatozoa depend on the concentration of titrant, with half maximal effects at 13 microM and a linear correlation with r = 0.99. The analogous lysyl and arginyl trypsin substrates containing 7-amino-4-methylcoumarin as the fluorogenic leaving group were not hydrolyzed under the same conditions and did not inhibit zona binding. Both binding of sperm to zona-intact eggs and the hydrolysis of MUGB by sperm are inhibited by p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, and acid-solubilized zonae. The linear correlation coefficients of the inhibition of sperm binding and MUGB hydrolysis by these three substances are greater than 0.92. This "trypsinlike" sperm site is essential for sperm binding to the zona: its stereospecificity is unique in that it reacts with trypsin inhibitors but not with trypsin substrates.

Bicarbonate is essential for fertilization of mouse eggs: mouse sperm require it to undergo the acrosome reaction.

Fertilization of mouse eggs by capacitated mouse sperm occurs readily in vitro in a defined culture medium designated medium CM, derived from Krebs-Ringer bicarbonate medium by supplementation with glucose, lactate, pyruvate, and albumin. Medium CM contains 25 mM HCO-3 equilibrated with CO2 to pH 7.4. Replacement of the CO2/HCO-3 buffer with 25 mM N-hydroxyethylpiperazine-N-ethanesulfonate (HEPES) while otherwise maintaining pH, ionic strength, and composition identical to that of medium CM, yielded medium HM in which the percentage of in vitro fertilization observed was consistently zero. Addition of 10 mM HCO-3 to medium HM yielded medium HMB, in which the percentage of eggs fertilized was 70%, equal to that observed in medium CM. This percentage applies to cumulus-free, zona-intact eggs. With zona-free eggs, the percentage fertilization increased to 90% in media CM and HMB, but remained zero in medium HM. This result shows that HEPES does not inhibit fertilization while omission of CO2/HCO-3 blocks it completely. Capacitation of mouse sperm, as determined by chlortetracycline fluorescence assay, proceeded at the same rate in media CM and HMB, but was retarded in medium HM. Sufficient sperm became capacitated, however, that this could not be the block of fertilization. The acrosome reaction in sperm bound to isolated zonae pellucidae, as also determined by chlortetracycline fluorescence assay, occurred with the same time course in media CM and HMB, but did not occur in medium HM.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of fertility in vitro and in vivo after exposure of male mice to high environmental pressure

AbstractMale mice of the TO strain were exposed to 50 ATA helium (0.5 ATA oxygen) over 5 weeks and then assessed for fertility both in vitro and in vivo. Significantly lower values for acrosome loss and fertilization of zona intact eggs in vitro were obtained with spermatozoa from the pressure‐treated males. These results, coupled with the significant decrease in incidence and degree of polyspermy in zona‐free eggs, indicated that fewer competent cells were present after preincubation under capacitating conditions. Fewer motile cells were observed in most samples, along with a reduction in the incidence of hyperactivated motility in some. Testis weight was also significantly lower. Results of fertilization in vivo, after mating with untreated females, showed a consistent trend toward subfertility, with a lower pregnancy rate and a smaller litter size. These differences did not reach significance, however, unlike those obtained in earlier in vivo experiments with males of the BALB/c strain. The discrepancy between in vitro and in vivo results with TO males probably represents differences between unselected and selected sperm populations, while that observed in vivo between the two strains probably reflects the superior reproductive capacity of the TO strain.

Mouse sperm capacitation assessed by kinetics and morphology of fertilization in vitro.

Epididymal mouse spermatozoa were preincubated for periods of 5-120 min and then tested for their ability to penetrate freshly ovulated eggs synchronously and rapidly. When zona-intact eggs were used, only suspensions preincubated for 120 min gave consistently high rates of fertilization, but suspensions preincubated for 30 min were functionally equivalent to those incubated for 120 min when used with zona-free eggs; the only major observable differences were a 15-min lag in sperm-egg interaction and an increased incidence of asynchrony with multiple sperm penetrations. A morphological study of sperm-egg interactions using zona-intact eggs indicated that, within 35 min of gamete mixing, egg microvilli could be detected by SEM in association with the fertilizing sperm head. Using conventional light microscopic examination of fixed and stained preparations, initial stages of sperm head decondensation could be detected in the majority of eggs after 45-60 min and the process was essentially completed, with the egg at the telophase-second polar body stage of meiosis II, after 75 min. Similar kinetics were observed with sperm concentrations of 10(5) and 10(6)/ml. The time required for penetration by capacitated sperm suspensions is therefore relatively short and the most accurate information regarding state of capacitation and rate of sperm penetration can be obtained by choosing an appropriately short interval for sperm-egg interaction before assessment.

Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials

The plasma membrane of the rabbit egg allows only one sperm to enter the egg during fertilization, but the mechanism of this block to polyspermy is unknown. Electrophysiology and in vitro fertilization techniques were employed in this study to investigate the possibility that a voltage block to polyspermy exists in rabbit eggs. Ovulated zona-intact eggs had a mean membrane potential of −71 ± 2.1 mV (interior negative). A stereotypic response occurred 12–135 min following in vitro insemination in 19 of 40 eggs. Association of this stereotypic response with the appearance of pronuclei suggested that the electrical response was related to some interaction of gametes. This response consisted of a slow transient 8 ± 1.5 mV depolarization upon which were superimposed up to 36 repetitive diphasic insemination potentials. Each potential consisted of a brief 2.0 ± 0.44 mV hyperpolarization followed by a slow 2.5 ± 0.45 mV depolarization. The small amplitude of the stereotypic response when compared with the large variation of resting potentials suggested that the response was insufficient to block polyspermy by a mechanism dependent upon the magnitude of the rabbit egg membrane potential.