Pisum sativum agglutinin used as an acrosomal stain of porcine and caprine sperm

Fluorescein isothiocyanate-labelled pisum sativum agglutinin (FITC-PSA) was evaluated as an acrosomal stain for porcine and caprine sperm that had previously been stained with Hoechst 33258 to assess cell viability. The FITC-PSA procedure was as accurate as other procedures in assessing acrosomal presence or absence on either fresh or liquid-stored porcine sperm. Approximately half of the incubated porcine sperm with acrosomal loss maintained membrane impermeability to the Hoechst 33258; these were potentially viable acrosome-reacted sperm. The FITC-PSA procedure was significantly correlated with the assessment of acrosomal status of cryopreserved caprine sperm by transmission electron microscopy (TEM). However, TEM results indicated a higher percentage of caprine sperm with acrosomal loss. Ability to penetrate zona-free hamster ova was not highly correlated with the percentage of viable acrosome-reacted porcine or caprine sperm. The FITC-PSA procedure provides an estimate of viable acrosome reactions and may be a useful tool in the evaluation of sperm fertility.     

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.     

Changes in exposed membrane proteins during in vitro capacitation of boar sperm

Exposed plasma membrane proteins were labeled with 125I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.     

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.     

Macaque sperm fusion with zona-free hamster oocytes

Abstract: Semen from six cynomolgus macaques was washed twice by centrifugation in BWW media and resuspended in 1:2 (vol:vol) of Tes-Tris-buffered eggyolk extract (EXT) diluent and BWW (EXT-BWW). Caffeine and dbcAMP were added to induce capacitation. Sperm were treated with calcium ionophore, A23187, followed by the addition of EXT to recover motility, then washed into BWW. The percent motile sperm was similar in ionophore-treated and control sperm suspensions, but motility of treated sperm decreased significantly by 20 min after treatment. The percentage of viable, acrosome-reacted sperm was 56.3% following ionophore treatment versus 4.0% in control suspensions. When ionophore-treated sperm were coincubated with zona-free hamster oocytes, 51 % of oocytes had evidence of sperm fusion and no oocytes were penetrated after incubation with control sperm. These results are consistent with the hypothesis that macaque sperm are capable of fusion with zona-free hamster oocytes if sperm are viable and acrosome-reacted.     

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.     

Development of a zona-free hamster ova bioassay for goat sperm

In vitro conditions for a zona-free hamster ova bioassay of caprine sperm fertility were assessed. Washing the cryopreserved sperm by dilution and centrifugation resulted in greater ability to penetrate zona-free hamster ova than allowing the sperm to swim-up into medium. A 12-h preincubation of sperm prior to insemination of the zona-free hamster ova was optimal. Sperm had greater ability to penetrate the zona-free hamster ova when incubated in a Tris-buffered medium than when incubated in Ham's F-10 (95 vs 2%, P<0.001), although motility was not well maintained in the Tris-buffered medium. Ten million sperm/ml was sufficient for maximum penetration. The ability to penetrate zona-free hamster ova was positively but not significantly correlated with the sperm head ultrastructure, suggesting the two techniques may assess different aspects of sperm fertility.     

Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Effect of dilauroylphosphatidylcholine on the acrosome reaction and subsequent penetration of bull spermatozoa into zona-free hamster eggs

Incubation of bull sperm with liposomes made with phosphatidylcholine (PC) containing fatty acyl chains of either 10 (PC10) or 12 (PC12) carbons resulted in greater than 90% of the sperm exhibiting an acrosome reaction (AR) within 15 min. Liposomes of PC10 rapidly destroyed sperm motility while PC12 acrosome-reacted sperm remained motile for several h. Liposomes of PC with greater than or equal to 14-carbon fatty acyl chains had no effect on the AR or motility of sperm. The AR was not induced by lysophospholipids, because lysophospholipids were not detected in the PC liposomes, and the AR did not occur when lysophospholipids were tested at the same concentration as PC12. The concentration of PC12 necessary to induce maximal numbers of acrosome-reacted sperm varied with the concentration of sperm. The effect of PC12 on sperm also varied with the ratio of live to dead sperm in a sample. When 3 X 10(6) bull sperm/ml were treated with 0, 10, 20, and 30 microM PC12 for 7 min prior to addition to zona-free hamster eggs, 6, 6, 98, and 77% of the eggs were penetrated, respectively. Lipid concentrations of 0 microM and 10 microM did not affect the AR, whereas higher levels induced the AR in sperm. This procedure can quickly provide acrosome-reacted bull sperm for use with various in vitro fertilization procedures and for assessment of male fertility.     

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.     

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.     

Promotion of capacitation of guinea pig spermatozoa by the membrane mobility agent,A2C,and inhibition by the disulfide-reducing agent,DTT

The membrane mobility agent A₂C accelerates the onset of the acrosome reaction of guinea pig spermatozoa by promoting capacitation. Spermatozoa incubated in a suspension of A₂C particles in Ca²⁺-free medium for one hour undergo a synchronous, rapid acrosome reaction upon the addition of Ca²⁺. These acrosome-reacted spermatozoa are capable of fertilization as assessed by their ability to penetrate (fuse with) zona-free hamster eggs. The disulfide-reducing agent, dithiothreitol (DTT) inhibits A₂C-mediated capacitation. It also blocks fertilization of zone-free eggs by acrosome-reacted spermatozoa by preventing attachment of the spermatozoa to the egg plasma membrane. The mode of A₂C action on spermatozoa is compared to that of A₂C-induced fusion in somatic cells. The similarity of the molecular events in the sperm membrane during capacitation and the acrosome reaction to these in other fusion events is pointed out. Inhibition of capacitation by DTT points to the importance of membrane and/or submembrane proteins and thiol groups in this process. Oxidation of sperm membrane SH groups may play an important role in in vivo capacitation.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

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.     

In vitro capacitation of Siberian tiger spermatozoa

Three experiments were designed to determine optimum conditions for capacitation of Siberian tiger (Panthera tigris altaica) sperm in vitro using the zona-free hamster egg sperm penetration assay (SPA) as a verification of capacitation. Sperm collected from a 9-year-old captive Siberian tiger were subjected to different in vitro washing conditions, preincubation times, and temperatures to induce capacitation. Sperm were able to penetrate zona-free hamster ova after 2 hours preincubation at 37°C but not at time 0. Preincubation at room temperature was not sufficient to prepare sperm for fertilization. The presence of seminal plasma during the 2-hour, 37°C preincubation did not affect the ability of tiger sperm to penetrate zona-free hamster eggs. The SPA can provide a means for evaluation of in vitro capacitation of Siberian tiger sperm.     

Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrode's medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.