Recovery of penetration ability in protease-treated zona-free mouse eggs occurs coincident with recovery of a cell surface 94 kD protein.

Previous studies have demonstrated that protease treatment of zona-free mouse eggs impairs sperm-egg interaction (Boldt et al.: Biol Reprod 39:19-27, 1988) and causes modification of a 94 kD egg plasma membrane protein (Boldt et al., Gamete Res 23:91-101, 1989). In this report, the ability of eggs to recover penetration ability following protease treatment was examined. Zona-free mouse eggs were isolated and treated with either trypsin or chymotrypsin (1 mg/ml, 20 min), then cultured for 0, 3, or 6 hr before insemination. Eggs cultured for 3 or 6 hr displayed significantly higher penetration levels than eggs inseminated immediately after protease treatment, indicating a recovery of penetration ability during the 3 or 6 hr incubation period. The recovery of penetration ability was not blocked by inclusion of cyclohexamide (50 micrograms/ml) during the 3 or 6 hr culture period, indicating that protein synthesis was not required for recovery of fusion ability. Cell surface radiolabeling studies with 125I revealed that a 94 kD cell surface protein was lost immediately following trypsin or chymotrypsin treatment but was found on the egg surface after the 3 or 6 hr recovery period. Recovery of the 94 kD egg surface protein occurred in the presence of cyclohexamide, and metabolic radiolabeling studies with 35S-methionine confirmed that synthesis of a 94 kD protein was blocked by cyclohexamide. These results suggest that the recovery of penetration ability after protease treatment of zona-free eggs is due to recovery of the 94 kD cell surface protein, providing further evidence for the involvement of the 94 kD protein in sperm-egg interaction.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Premature cortical granule loss does not prevent sperm penetration of mouse eggs.

The role of cortical granules in the mouse egg's plasmalemma block to polyspermy was investigated by examining the effect of premature granule loss on egg fertility. Granule loss, quantitated by transmission electron microscopic examination, was induced in zona-free eggs by exposure to the divalent cation ionophore, A23187, or by mechanical removal of zonae. Egg exposure to ionophore led to the loss of approximately 50% of the egg's complement of granules in the absence of nuclear activation (parthenogenesis), while complete cortical granule loss accompanied the parthenogenetic activation seen in a limited population of mechanically stimulated eggs. Aged eggs underwent nuclear activation without a dramatic reduction in granule complements. The fertility of treated zona-free eggs was identical to that of controls, as measured by the percentage of eggs penetrated and by the mean number of sperm recovered per egg. Moreover, both ionophore-treated and aged eggs subsequently underwent a normal sperm-induced block response. Exposure of zona-intact eggs to ionophore was also without effect on egg fertility. These results indicate that cortical granules are not involved in the plasmalemma block to polyspermy in the mouse.     

An investigation of the latency period between sperm oolemmal adhesion and oocyte penetration

In clinical studies of the ability of capacitated human sperm to penetrate zona-free hamster eggs, we have previously observed that the ratio of oolemmal adherent to penetrating sperm varied between men. Sperm incorporation did not occur immediately following gamete adhesion and not all adherent sperm penetrated the egg. To further investigate this phenomenon, comparisons were made of the kinetics of gamete adhesion, membrane fusion, and sperm incorporation of capacitated mouse and human spermatozoa by zona-free hamster eggs and of mouse sperm by zona-free mouse and hamster eggs. Eggs were inseminated with either capacitated human or mouse sperm or combinations of both, washed out of sperm suspension after initial gamete adherence, and further incubated in sperm-free medium. Gamete membrane fusion was judged by dye transfer of Hoechst 33342 and sperm entry of the cortical ooplasm by observation of expanded sperm heads within acridine orange stained eggs. Oolemmal adherent mouse and human sperm fused with and penetrated zona-free hamster eggs at different times whether eggs were inseminated in parallel or with combinations of sperm of both species. Oolemmal adherent mouse sperm penetrated zona-free hamster eggs prior to their penetration of zona-free mouse eggs. Ultrastructural studies of zona-free human eggs inseminated with human sperm confirmed prior observations with hamster eggs that only acrosome-reacted human sperm adhere to the oolemma. These results have lead us to postulate that sperm entry into the egg may occur through a "zipper" mechanism involving the ligation of local gamete receptors similar to the incorporation of target particles by phagocytes and suggest that not all oolemmal adherent human sperm are capable of being incorporated although they have undergone an acrosome reaction.     

Enzymatic alteration of the ability of mouse egg plasma membrane to interact with sperm

The effect of a variety of proteolytic, glycosidic and lipid hydrolyzing enzymes on the ability of mouse egg plasma membrane to interact with sperm was evaluated in this study. Zona-free mouse eggs were exposed to enzymes at various concentrations, washed, and inseminated; the number of sperm attached to or having penetrated the egg plasma membrane was determined at 20 and 180 min post-insemination, respectively. The proteases trypsin and chymotrypsin caused concentration-dependent reductions in both sperm attachment and sperm penetration levels when eggs were incubated at enzyme concentrations ranging from 1- to 1000 micrograms/ml for 30 min prior to insemination. Time-course studies revealed significant inhibition of both sperm attachment and sperm penetration levels after treating zona-free eggs for 5 min at 1000 micrograms/ml of either trypsin or chymotrypsin. Several of the phospholipases tested, including phospholipases C, D, and A2, had no inhibitory effect on sperm penetration levels, with phospholipase C and A2 (100 micrograms/ml) causing inhibition of sperm attachment. Of the glycosidic enzymes evaluated, glucuronidase (1000 micrograms/ml) caused significant inhibition of sperm binding but not sperm penetration, and glucosidase, galactosidase, and neuraminidase had no effect on either sperm attachment or sperm penetration. These findings indicate that the ability of the mouse egg plasma membrane to fuse with sperm can be preferentially altered by treatment with proteases.     

Interspecies interaction of zona-free ova with spermatozoa in mouse,rat and hamster

Mouse, rat and hamster zona-free eggs were penetrated in vitro by spermatozoa of their own species and by alien spermatozoa of mouse, rat and hamster. The tested combinations showed very distinct differences in penetration ability. Mouse zona-free eggs were penetrated by spermatozoa of their own species only. Rat zona-free eggs were penetrated by their own and mouse spermatozoa. Hamster zona-free eggs were penetrated by their own, mouse and rat spermatozoa. Several proteolytic enzymes used for lysis of zona pellucida, time of sperm preincubation and sperm concentration did not affect sperm-egg interaction. It is concluded that the species specificity of egg plasma membrane in the rodents tested is probably based on some specific surface components.     

Distinct mechanisms underlie sperm-induced and protease-induced oolemma block to sperm penetration

Fertilization of a mouse egg results in modification of the cytoplasmatic membrane (oolemma) which makes fusion with additional sperm impossible. CD9 is a transmembrane protein reported to be responsible for gamete fusion. Since the molecular mechanism of zygote membrane modification after fertilization remains unknown, we were interested to check whether lack of CD9 is the reason for non-penetrability of zona-free zygotes. We wanted also to determine the effect of different methods of zona pellucida removal on the presence of CD9 on the surface of unfertilized eggs and their ability to be fertilized afterwards. We demonstrated that CD9 is present on the surface of both zygotes and parthenogenotes. We showed also that the treatment of eggs with pronase completely removes CD9 from the membrane of eggs making them infertile. Eggs treated with chymotrypsin and acid Tyrode still posses CD9 on their surface and remain fertile. The results of our experiments indicate that modification of the zygote oolemma does not involve a lack of CD9. We cannot exclude however, that the amount of CD9 decreases after fertilization. In addition, our studies indicate that the previously reported infertility of eggs treated with different proteases may result from the decrease or removal of CD9 and probably other proteins responsible for gamete fusion from the surface of eggs.     

Carbohydrate involvement in sperm-egg fusion in mice

The potential involvement of cell-surface carbohydrates in sperm-egg fusion in mice was evaluated in this study. Zona-free mouse eggs were inseminated in the presence of a variety of simple saccharides to determine if certain sugars would act as competitive inhibitors of sperm-egg fusion. Of the sugars tested, L-fucose, galactose, and N-acetylglucosamine caused the greatest inhibition of sperm penetration levels relative to controls. A number of complex saccharides or glycoproteins with differing carbohydrate structures, including fucoidan, ascophyllan, ovomucoid, ovalbumin, fetuin, asialofetuin, and chondroitin sulfate, were also tested as competitive inhibitors of fusion. Only the L-fucose containing saccharides fucoidan and ascophyllan caused significant inhibition of fusion at concentrations of 0.05-1.0 mg/ml and 0.1-5.0 mg/ml, respectively. None of the other compounds tested had any inhibitory effect on fusion when tested at concentrations up to 5 mg/ml. The effect of the inhibitory saccharides was not due to the presence of residual zona material on the surface of the zona-free eggs, since zona-free eggs did not bind an 125I-labeled antibody directed against the ZP3 protein of the mouse zona pellucida. Pretreatment of either sperm or eggs with fucoidan (1 mg/ml) for 60 min prior to insemination caused only small decreases in sperm penetration levels, indicating that fucoidan exerted its major inhibitory effect on fusion only when present during insemination. Treatment of sperm, but not zona-free eggs, with fucosidase prior to insemination caused significant reductions in sperm penetration levels. Other glycosidic enzymes, including glucosidase, galactosidase, and N-acetylglucosaminidase, had no inhibitory effect on the sperm. These data suggest that an L-fucose component of the sperm surface is involved in sperm-egg fusion in the mouse.     

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.     

Hamster oocyte penetration tests with oocytes frozen in propanediol: Comparison with non-frozen oocytes

Hamster oocytes were frozen using a 1,2-propanediol-sucrose procedure, which resulted in over 90% survival. After thawing and zona removal the oocytes were compared with non-frozen oocytes in a zona-free hamster egg test employing spermatozoa from human semen donors and suspected infertility patients. Similar data were obtained, indicating that propancdiol-sucrose frozen hamster eggs may be used in place of fresh eggs for convenience and to a void scheduling problems.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: II. A fertility assay for frozen-thawed semen

Frozen-thawed sperm from five bulls with fertility rates ranging from 48% to 77% were treated with seven concentrations of dilauroylphosphatidylcholine (PC12) liposomes to induce an acrosome reaction (AR) that enabled sperm to penetrate eggs. Treated sperm were incubated with liposomes for 7 min prior to insemination of zona-free hamster eggs in vitro. Sperm and eggs were incubated 3 hr at 39°C prior to fixation, staining, and examination for sperm penetration and nuclear decondensation. The percentage of motile sperm immediately after thawing as well as after treatment with liposomes had a low correlation with sire fertility (r = .39 and ?.63, respectively). The percentage of sperm exhibiting an AR was more highly correlated with fertility (r ? ?.85). Similar correlations were found between fertility and the penetration rates of zona-free hamster eggs or the total number of penetrating sperm. When data for two high and for two lower fertility buils were each grouped to increase information per data point the correlation between the PC12 concentration giving the maximum proportion of eggs penetrated and fertility was r = .92 (P ≤ .05). The correlation between the PC12 concentration producing the most total sperm penetrating the eggs and fertility r = .97 (P ≤ .05). It was concluded that PC12 liposomes induced an AR in bull sperm frozen-thawed in egg yolk extender. Frozen-thawed sperm from low fertility bulls require less PC12 to induce the AR and to penetrate zona-free hamster eggs than do sperm from higher fertility bulls. These differences in lipid requirements may help to provide a quick, direct laboratory assay method to estimate the fertility of frozen bull semen.     

Inhibition of in-vitro fertilization of intact and denuded hamster eggs by univalent anti-sperm antibodies.

Univalent (Fab) rabbit anti-hamster sperm antibodies added to an in-vitro fertilization system did not interfere with the sperm acrosome reaction or motility, but inhibited cumulus dispersion by the spermatozoa, sperm binding to and passage through the zona pellucida as well as sperm-egg fusion. Addition of the Fab preparations to the capacitated spermatozoa at various times before or up to 40-45 min after the sperm-egg mixing prevented penetration of spermatozoa through the zona pellucida. Detachment of the spermatozoa already bound as well as those partly inside the zona pellucida was achieved by a late addition of antibodies. In experiments with zona-free hamster eggs, addition of the Fab antibodies to the spermatozoa 10 min to 5 h before the introduction of unfertilized eggs reduced the rate of adhesion and fertilization to very low levels. These antibodies were not absorbed on hamster ovary, liver or kidney and had no direct effect on the fertilizability of zona-intact or zona-free eggs.     

The penetrability of rabbit ova treated with enzymes or anti-progesterone antibody: a probe into the nature of a mammalian fertilizin.

The acrosome reaction of rabbit spermatozoa, an essential prerequiste for penetration of the zona, occurs usually in the vicinity of the egg, suggesting that the rabbit may produce a factor akin to the 'fertilizin' of some invertebrates. Specific inactivation of such a factor should render eggs impenetrable and possibly point to the nature of a 'fertilizin' in mammals. Rabbit eggs with granulosa cells removed were treated for different periods with trypsin, chymotrypsin, neuraminidase or anti-progesterone antiserum, and then transferred alone, or together with control eggs (one group labelled with fluorescein isothiocyanate), to the oviducts of inseminated recipients. Three hours later the eggs were recovered and the experimental and control groups were compared for penetration of the vitellus and for numbers of spermatozoa within the perivitelline space or in the zona pellucida. None of these treatments affected the penetrability of the zona pellucida significantly since the number of spermatozoa within treated eggs in any one experiment was always comparable to that of untreated eggs exposed to the same fertilization environment. If there is a specific substance emanating from or present on the surface of the rabbit egg which induces the acrosome reaction, its activity seems unaffected by trypsin or chymotrypsin; the charged radicals of N-acetyl neuraminic acid or local concentrations of progesterone do not appear to be involved.     

Evidence suggesting a role for sperm metalloendoprotease activity in penetration of zona-free hamster eggs by human sperm

It has been reported that metalloendoprotease (MEP) activity is involved in somatic cell membrane fusion events and in the sea urchin sperm acrosome reaction (AR). MEP activity also has been demonstrated in human and other mammalian sperm. The present study was concerned with investigating whether a human sperm MEP is important in membrane events necessary for sperm egg fusion. Ejaculated human sperm were washed, capacitated in vitro, and preincubated with the competitive MEP inhibitors phosphoramidon (50 microM) or CBZ-L-phenylalanine (1 mM), with 100 microM diethylenetriaminepentaacetic acid (DTPA), a heavy metal chelator, or as controls, with the appropriate solvents. The AR was initiated in vitro with preovulatory human follicular fluid and the sperm washed to dilute inhibitors and then coincubated with zona-free golden hamster eggs (zonae and cumuli removed with trypsin and hyaluronidase, respectively). Eggs were washed after 0.5 h, and the number of sperm remaining bound was counted. After 2.5 h further incubation, the eggs were stained with acetolacmoid or acetoorcein and penetration was assayed by counting the number of decondensed sperm heads per egg (penetration index) and the percent of penetrated eggs. The inhibitor treatments did not decrease the percentage of penetrated eggs (range 80-90%), but a significant reduction in the penetration index was observed. Phosphoramidon reduced the penetration index by 45%, CBZ-L-phenylalanine by 57%, and DTPA by 56%. None of the inhibitors decreased the penetration index or the percentage of penetrated eggs when added directly to suspensions of acrosome-reacted sperm and zona-free eggs at the diluted levels that would have been present after washing inhibitor-treated sperm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.     

In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 mu M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2 57 ), 12 (7 58 ), 52 (31 60 ), and 86% (44 51 ) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2 2 ), 57 (4 7 ), 58 (18 31 ), and 34% (15 44 ), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11 49 ), and increased to 38% (21 55 ) at 5 h, to 46% (23 50 ) at 10 h, and to 56% (27 48 ) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.     

In vitro fertilization of two species of deer mouse eggs by homologous or heterologous sperm and penetration of laboratory mouse eggs by deer mouse sperm

Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Potassium dependence for sperm-egg fusion in mice

In this study, we examined the potassium requirements for sperm-egg fusion in mouse. Zona-free mouse eggs prepared by the method described by Boldt and Wolf were inseminated with capacitated sperm in culture media containing 0-6 mM extracellular K+, and scored for penetration. Penetration of zona-free eggs was dependent on extracellular K+, with no penetration observed under K(+)-free conditions. Media transfer experiments indicated that the lack of penetration observed was due to effects on fusion, and not on postpenetration events such as sperm head decondensation. To analyze whether the K+ effect was attributable to an effect on the sperm (i.e., occurrence of acrosome reactions), sperm were treated with the Ca2+ ionophore A23187 before insemination. Less than 5% of zona-free eggs were penetrated with ionophore-treated sperm under K(+)-free conditions, suggesting that K+ is required for fusion per se. Addition of ionophore to insemination cultures similarly did not overcome the block to fusion observed under K(+)-free conditions. The potassium channel blockers 4-aminopyridine (0.1-5 mM) and tetraethyl ammonium chloride (5-50 mM) had no inhibitory effect on fusion. These data indicate that extracellular K+ is required for sperm-egg fusion and that this requirement may not involve membrane K+ channels.