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The spermatozoa of the musk shrew, Suncus murinus, have a fan-like giant acrosome with a diameter of approximately 20 mm. The aim of this study was to investigate how this giant acrosome is constructed in the musk shrew spermatid and, in particular, how the Golgi apparatus involved in acrosome formation behaves. The behaviour of the Golgi apparatus was monitored by confocal laser scanning microscopy with antibody against a Golgi-associated Rab6 small GTPase. In the early Golgi phase, small Golgi units, the Golgi satellites, localized as a large aggregate in the juxtanuclear cytoplasm. As acrosome formation progressed, the Golgi satellites gradually dispersed, associated with proacrosomal vesicles and an acrosomal vesicle, and finally became distributed as multiple small units over the whole surface of an acrosomal cap in the round spermatid. The mode of acrosome formation in musk shrews was distinctly different from that in rats and mice, in which the Golgi apparatus remains as a single unit throughout acrosome formation. In musk shrews, the proacrosomal vesicles formed successively by the Golgi satellites coalesced, one after another, into a potential acrosomal vesicle. This process may result in further enlargement of the acrosome. The results of the present study indicate that Golgi satellites are necessary for the biogenesis and development of the giant acrosome in musk shrew spermatozoa.

This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.

The ability of a single electric pulse to mimic a block against sperm penetration in bovine oocytes matured in vitro was investigated. Confocal laser scanning microscopy detected a global loss of spots, presumed to be cortical granules, stained with Lens culinaris agglutinin, in pulsed oocytes. Transmission electron microscopy revealed that cortical granule exocytosis occurred within 1 min of stimulation and the number of remaining cortical granules was significantly reduced in all pulsed oocytes. The ability of pulsed oocytes to undergo fertilization in vitro was also affected, as only 31% of the pulsed oocytes were penetrated compared with 87% in the control group. Since incidences of penetration in pulsed oocytes (31%), and of polyspermy in control oocytes (18%) did not differ and were highly correlated (P = 0.009) among trials (n = 15), the induced block is considered to be comparable with the natural block triggered by a spermatozoon. The increased resistance of the zona pellucida to pronase E observed in pulsed oocytes suggests that the induced block depends, at least partly, on modifications of zona pellucida glycoproteins. Finally, the majority (66%) of pulsed, penetrated oocytes did not form male pronuclei, probably as a consequence of asynchrony between the formation of female pronucleus and sperm penetration. The reduced ability of the cytoplasm to induce the formation of a male pronucleus was accompanied by a fall in histone H1 kinase activity to basal values by 3 h after stimulation. These results demonstrate that a single electric pulse can induce a block against sperm penetration similar to that of the spermatozoon itself.

In the musk shrew, Suncus murinus, the behaviour of the cumulus-egg complex and its interaction with spermatozoa were unusual in several respects. The cumulus oophorus was ovulated about 15.5 h after mating or treatment with hCG as a hyaluronidase-insensitive matrix-free ball of cells which remained for relatively long periods of about 14 h around fertilized, and for about 24 h around unfertilized eggs. As a probable function of the small number of up to about 10 or 20 spermatozoa that generally reached the oviduct ampulla from isthmic crypts, there was often a delay of up to 10 h after ovulation before most eggs were penetrated. Soon after ovulation, however, the corona radiata retreated progressively from the zona pellucida, creating a closed perizonal space within the cumulus oophorus. Usually, most spermatozoa that did reach the ampulla were found within a cumulus and generally within that perizonal space. However, whereas the acrosome was intact among the few free ampullary spermatozoa, and in those adhering to the zona of cumulus-free eggs after delayed mating, all spermatozoa seen moving within the cumulus or adhering to the zona of unfertilized eggs had shed the giant acrosome. In accord with current observations in other shrews, the cumulus in Suncus may therefore function not only to sequester spermatozoa, but also as an essential mediator of fertilization-probably by inducing the acrosome reaction. In the absence of the acrosomal carapace that expresses the zona receptors in most mammals, fertilizing Suncus spermatozoa could use an unusual array of barbs on the exposed perforatorium to attach to the zona pellucida.

Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups (81-88%) and 54-74% of embryos formed normal late stage fetuses.

The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.

Spermatozoa of the field vole (Microtus montebelli) that bound to the zona pellucida of field vole oocytes underwent the acrosome reaction before passing through it. In contrast, vole spermatozoa that bound to the zonae of mouse and hamster oocytes penetrated the zonae without any sign of the acrosome reaction. The presence or absence of proteinase/hyaluronidase inhibitors in the medium did not make any difference to zona penetration by acrosome-intact vole spermatozoa. These observations suggest that field vole spermatozoa can cross mouse and hamster zonae mechanically without assistance from zona-hydrolysing enzymes.

Motility, acrosome reaction and oocyte fertilizing ability were assessed for bull spermatozoa after incubation in regional (isthmic or ampullary), bovine oviductal fluid, pooled by stage of the oestrous cycle. Oviductal fluids collected daily from isthmic and ampullary cannulae implanted in the same oviduct were divided into pools, representing two oestrous cycle stages, based on daily serum progesterone concentrations. Ejaculated bull spermatozoa were incubated for 0-6 h in each type of oviductal fluid. Incubation in isthmic oviductal fluid collected during the nonluteal stage, including oestrus and ovulation, decreased overall sperm motility (from 71.7% motile spermatozoa to 34.0%) and both path (78 microns s-1 versus 86-89 microns s-1) and progressive (74 microns s-1 versus 83-85 microns s-1) velocities of spermatozoa motion. Spermatozoa incubated in isthmic, non-luteal oviductal fluid had a higher rate and extent of sperm acrosome reaction (213% of control versus 136-161% of control by 2 h incubation) compared with spermatozoa incubated in other oviductal fluid types. However, incubation in nonluteal ampullary fluid increased the number of spermatozoa, which were both acrosome reacted and live, and able to fertilize bovine ova (88.7% fertilized versus 75-81%). Glycosaminoglycan concentrations were similar among types of oviductal fluid (0.77-0.88 mg ml-1). These findings indicate that oviductal fluid differentially affects sperm function, depending on the oviduct region and the stage of the oestrous cycle at which the fluid was obtained.

Mouse caput spermatozoa are considered immature and thus unable to fertilize oocytes. In this study, we determined whether washing mouse caput spermatozoa increased their ability to acrosome react in response to a physiological stimulus. The results obtained showed that mouse caput spermatozoa incubated in Earles' modified medium containing calcium chloride and supplemented with BSA and pyruvate for 1 h at 37 degrees C and then washed acrosome reacted in response to both solubilized zonae and immunoaggregation of a zona binding site. In addition, the material removed from caput spermatozoa by washing blocked induced acrosome reactions of cauda spermatozoa. The data indicate that mouse caput epididymal spermatozoa, if incubated and washed, can undergo physiological acrosome reactions.

Acrosomal loss was induced in marsupial spermatozoa by an intermediate of the phosphoinositide pathway. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8; 100 mumol l-1) induced acrosomal loss in 70% of brushtail possum (Trichosurus vulpecula) spermatozoa and in 80% of tammar wallaby (Macropus eugenii) spermatozoa. The DiC8-induced acrosomal loss was not enhanced by co-incubation with calcium ionophore A23187 and occurred in Ca(2+)-free medium and in the presence of the calcium chelator EGTA (3 mmol l-1). There was no evidence of uptake of 45Ca2+ during the DiC8-induced acrosomal loss. Inhibitors of protein kinase C [1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine] and phospholipase A2 [dexamethasone] did not effect DiC8-induced acrosomal loss in wallaby spermatozoa. The phorbol ester, phorbol 12-myristate 13-acetate, at a concentration of 10 mumol l-1 had no effect on possum spermatozoa and induced acrosomal loss in only 6% of wallaby spermatozoa. It appears that the DiC8-induced acrosome reaction is not mediated by activation of the phosphoinositide pathway and that extracellular calcium is not required for the membrane fusion event. As acrosomal loss was seen only at relatively high concentrations of diacylglycerol (> 50 mumol l-1) and there is no evidence of involvement of other phosphoinositide intermediates or analogues, it is likely that its role is as a direct membrane fusogen.

Mouse sperm capacitation in vitro, leading to hyperactivated motility, acrosomal exocytosis and rapid fertilization, takes approximately 120 min in a medium containing sufficient Ca2+. During that period, spermatozoa incubated in 45Ca2+ exhibited a biphasic pattern of Ca2+ uptake, with the first and lower peak occurring from 10 to 50 min and the second and higher peak from 60 to 90 min. When the exogenously supplied glucose was reduced from 5.56 mmol l-1 to 5.56 mumol l-1, the latter supporting capacitation but not fertilization, only the first peak of 45Ca2+ uptake was observed. Increasing the glucose to a millimolar concentration produced a second peak of uptake. We therefore propose that the first phase of 45Ca2+ uptake is associated with capacitation and the second phase with acrosomal exocytosis, which are both necessary prerequisites for fertilization. In micromolar glucose the rate of 45Ca2+ uptake during the first 30 min was 47% higher than in millimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca2+ concentration ([Ca2+]i) and hence accelerate capacitation. This hypothesis was confirmed by demonstrating both significantly accelerated transition from the uncapacitated F pattern of chlortetracycline (CTC) fluorescence to the capacitated B and AR patterns and significantly higher fertility in vitro in suspensions preincubated for 30 min in micromolar glucose, compared with those maintained continuously in millimolar glucose. These results suggest that an ATP-dependent mechanism, for example a Ca(2+)-ATPase, may be involved in maintaining a low [Ca2+]i. In micromolar glucose, available ATP would be limited and hence the ATPase activity would decline, allowing [Ca2+]i to rise.(ABSTRACT TRUNCATED AT 250 WORDS)

The proportion of human spermatozoa from 28 ejaculates to lose their acrosomes during cryopreservation was measured and correlated with the number that became immotile or lost the integrity of their plasma membrane. The ability of washed spermatozoa to acrosome react in response to A23187 before and after cryopreservation was compared. Motility was assessed by time-lapse photography; intact acrosomes were stained with fluorescein conjugated Pisum sativum agglutinin and dead spermatozoa were stained with bisbenzimide (H33258). Twenty-four per cent of spermatozoa lost their acrosomes during freezing and thawing, but the number that did so was not correlated with the number that became immotile or non-viable. Frozen spermatozoa exhibited fewer spontaneous acrosome reactions than did fresh spermatozoa (5 versus 13% after 4 h), but they responded to A23187 in a similar way. Although frozen spermatozoa were significantly more likely to die during the incubation, the data do not suggest that degenerative acrosome loss had a major influence on the results. In the hamster egg test frozen-thawed spermatozoa achieved more penetrations than did fresh spermatozoa when stimulated with 0 or 1 mumol A23187 l-1 but considerably fewer when stimulated with 4 mumol A23187 l-1. The following conclusions were made. First, cryopreservation damage to the acrosome, the plasma membrane and the flagellum can occur independently. Second, acrosome function is maintained after cryopreservation as long as the organelle remains mechanically intact. Third, some spermatozoa that lose their acrosomes during cryopreservation remain viable and can fuse with zona-free hamster eggs.

At least some of the spermatozoa that remain motile following cryopreservation have sustained sublethal damage that reduces their functional capacity in vivo. Although it is believed that acrosomal damage is partly responsible for impaired sperm function in vivo, direct evidence for this hypothesis is lacking because spermatozoa have not been collected from the female reproductive tract for evaluation. In the study reported here, cervical mucus was collected from women 24 h after artificial insemination by cervical cup. For both cryopreserved and nonfrozen inseminates, spermatozoa within the cervical mucus and spermatozoa that migrated out of mucus into culture medium (t = 1 h) were viable and had intact acrosomes. However, although nonfrozen spermatozoa did not initially respond to induction of the acrosome reaction with follicular fluid, a significant proportion of cryopreserved spermatozoa did respond. These results demonstrate that cryopreservation increases the acrosomal lability of spermatozoa residing in the female reproductive tract. An in vitro test was developed to detect this form of cryodamage. Sperm-free mucus was collected before insemination and spermatozoa from the inseminate were allowed to swim into this column of mucus in vitro. Spermatozoa recovered from this mucus sample were compared with spermatozoa from the paired sample collected from the cervix 24 h later. This in vitro test could detect acrosomal lability in cryopreserved semen samples, and this approach may prove valuable for studying sublethal cryodamage to the acrosome.

The effect of exogenous platelet-activating factor (PAF) on human spermatozoa was examined by monitoring acrosomal status and hamster oocyte penetration. Induction of the acrosome response by PAF was dose, and time, dependent and required capacitated spermatozoa. Addition of 10(-14) mol PAF l-1 enhanced the acrosome reaction eightfold compared with controls treated with human serum albumin (HSA). Similar concentrations of lyso-PAF failed to induce acrosomal loss and preincubation of spermatozoa with CV3988, an inhibitor of PAF, prevented PAF induction of the acrosome reaction. PAF significantly increased the number of zona-free hamster oocytes penetrated compared with controls (9.8 +/- 0.5 of 25 oocytes were penetrated by control spermatozoa compared with 23.3 +/- 0.8 out of 25 oocytes penetrated after incubation of spermatozoa with 10(-14) mol PAF l-1; 93% of all oocytes were penetrated by at least one spermatozoon following incubation with PAF), and also increased the number of decondensed spermatozoa found per egg during the sperm penetration assay (from 1.7 +/- 0.3 spermatozoa/egg with control spermatozoa to 3.3 +/- 0.5 spermatozoa/egg with PAF-treated spermatozoa). PAF-induced increases in acrosome reaction and sperm penetration assay values were similar to effects obtained with human follicular fluid and were calcium dependent. Induction of the acrosome reaction by physiological concentrations of PAF appeared to be morphologically similar to the response induced by follicular fluid as assessed by electron microscopy.

Mouse spermatozoa require extracellular Na+ for both capacitation and acrosomal exocytosis, but the minimum concentrations differ widely: > 1 < or = 25 mmol Na+ l-1 will support capacitation, but > 125 mmol Na+ l-1 is needed for acrosomal exocytosis in capacitated cells. Our conclusions are based on evidence obtained from sperm cells preincubated in iso-osmotic media with differing concentrations of Na+ and then analysed for occurrence of the acrosome reaction, capacitation-related changes in chlortetracycline (CTC) fluorescence and in vitro fertilization. The modified Tyrode's medium used as the control medium in these experiments contained 150 mmol Na+ l-1 and supported full sperm function. At least some of the Na+ needs to be internalized to promote the functional changes, as evidenced by the ability of the monovalent cation ionophore monensin to accelerate capacitation and trigger acrosomal exocytosis in control medium. However, in low Na+ (25 mmol l-1) medium, monensin could only modulate the transition to the capacitated state, assessed with CTC, indicating that higher concentrations of extracellular Na+ are required for initiation of acrosomal exocytosis. We suggest that changes in the composition of the female reproductive tract fluids serve to control expression of sperm functional potential. Before ovulation in the mouse, sufficient Na+ and Ca2+ are present to promote capacitation. However, the Na+ concentration is marginal for support of acrosomal exocytosis and the relatively high K+ reinforces an inhibition of exocytosis. At ovulation, the release of follicular fluid would increase the Na+ and decrease the K+ concentrations, thereby permitting full expression of fertilizing potential. Possible mechanisms that might be involved in the Na(+)-related responses, including a Na(+)-Ca2+ exchanger, a Na(+)-K+ ATPase and a Na(+)-H+ exchanger, were also investigated. If a Na(+)-Ca2+ exchanger has a role to play, it is not during capacitation per se. Incubation of sperm cells in high Na+, low Ca2+ (90 mumol CaCl2 l-1) medium that supports capacitation, followed by introduction of monensin, which would have promoted an influx of Na+ and could have, in turn, activated a Na+ out, Ca2+ in response, did not accelerate transition to the capacitated state (B pattern of CTC fluorescence). In contrast, it is possible that a Na(+)-K+ ATPase may play a role during capacitation. Incubation of suspensions in control medium plus ouabain, which would inhibit the ATPase, significantly accelerated the transition from the incapacitated to the capacitated state, although it did not trigger acrosomal exocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

A monoclonal antibody (J-23) to the 15 kDa component on the sperm head, the acceptor, which functions in zona binding, was shown to induce the acrosome reaction in capacitated cells, but not in fresh cells. The antibody recognized its epitope in the acrosomal cap region of fresh spermatozoa and in the equatorial region on washed and capacitated spermatozoa. However, equatorial expression did not depend on the acrosome reaction, since washing fresh spermatozoa increased the percentage with equatorial fluorescence, but did not increase the percentage with reacted acrosomes. The data indicate that the acrosome reaction can be induced in capacitated spermatozoa in the absence of zona glycoproteins.

When western blots of human sperm proteins solubilized by acid extraction (presumably mainly acrosomal proteins) or by sodium dodecyl sulfate (SDS) were probed with biotin-conjugated Pisum sativum agglutinin (PSA), distinct sets of proteins were labelled in both preparations. When smears of human spermatozoa were treated with methanol either for 30 s or for 15 min and then exposed to FITC-conjugated PSA, the resulting fluorescence pattern essentially depended on the time of methanol treatment. With the longer treatment, fewer spermatozoa showed selective acrosomal labelling and more were labelled uniformly throughout, without a clear predilection for a single sperm region. With the shorter time of methanol treatment, the poorly topographically differentiated, whole-cell labelling was typical of dead spermatozoa as confirmed by a close correlation between the percentages of spermatozoa showing this type of labelling and of those stained supravitally with Hoechst 33258. The preferential whole-cell labelling of dead spermatozoa with PSA is considered to be due to increased availability of the nonacrosomal set of PSA-reactive sites in dead spermatozoa after a short treatment with methanol, whereas this treatment is probably not sufficient to expose most of these sites when applied to living spermatozoa. The simplicity of the staining protocol makes this method feasible in routine work in a number of clinical and research applications.

Boar spermatozoa acquired resistance to cold shock immediately after exposure to 2.0 mmol butylated hydroxytoluene (BHT) l-1 when Beltsville thawing solution was used as a basic diluent, as judged by motility (the proportion of motile spermatozoa) and acrosomal integrity. The concentration of BHT could be reduced to 0.2 mmol l-1 without decreasing the protective action. However, motility was altered in the presence of greater than 0.15 mmol BHT l-1. Beltsville freezing 5 (BF5) diluent was more effective than Beltsville thawing solution in protecting spermatozoa from cold shock, but addition of BHT to BF5 diluent did not affect the motility and acrosomal morphology of spermatozoa before or after cold shock. Dilution of BHT-treated spermatozoa with BF5 diluent did not restore motility and did not afford further protection against cold shock; it was detrimental to spermatozoa treated with 2 mmol BHT l-1 for greater than 15 min. Egg yolk or lecithin had a detrimental effect. When spermatozoa were treated with 0.05-0.10 mmol BHT l-1 before slow cooling to 5 degrees C, the progressive motility and acrosomal integrity were maintained better after storage for 6 days than in untreated spermatozoa.

Concentrations of the intracellular Ca(2+)-mediator calmodulin (CaM), were measured by radioimmunoassay during heparin-induced capacitation of bull spermatozoa. Heparin reduced sperm CaM concentrations in a dose-dependent manner corresponding with an increase in in-vitro fertilization rates. Such reductions were observed after heparin treatment for 4-6 h, which is in agreement with the length of the capacitation period in bulls and was concomitant with an increase in CaM concentration in the incubation medium, suggesting translocation of CaM from the spermatozoa to the surrounding milieu. This CaM translocation was inhibited partly by the protease inhibitor benzamidine, suggesting a role for the sperm protease in this process.

The epididymis was reflected unilaterally or bilaterally to the abdomen in adult hamsters, leaving normally functioning testes in the scrotum. In unilateral cases, spermatozoa taken from the abdominal cauda, 1 month or more post-operatively, underwent a reversal of head agglutination and dispersed earlier, and underwent hyperactivation and fertilized cumulus-free eggs about 30-45 min sooner than did spermatozoa from the contralateral scrotal cauda. In addition, spermatozoa from the abdominal cauda began to undergo a spontaneous acrosome reaction 30-45 min earlier and to a greater extent than in control spermatozoa. Finally, in females mated at or soon after ovulation, spermatozoa ejaculated by bilaterally cryptepididymal males fertilized eggs 30-45 min before those from normal males. Other females mated to bilaterally cryptepididymal males gave birth to normal litters. The results are considered in terms of the possibility that temperature-sensitive sperm-binding macromolecules, which may be involved in sperm storage in the cauda epididymis, could be one determinant of the need for capacitation.

Ejaculated spermatozoa from brush-tailed possums and tammar wallabies were washed by a 'swim up' procedure into Hanks Balanced Salt Solution (HBSS), and then exposed to test solutions. Spermatozoa were incubated at 33 degrees C, or room temperature when long-term sperm survival (greater than 10 h) was required. Exposure of spermatozoa to calcium ionophore A23187, cyclic nucleotides, phosphoinositide pathway intermediates, lysophospholipids, trypsin or 'capacitating' high ionic-strength medium (380 mosmol) followed by 3% bovine serum albumin for periods up to 24 h did not induce acrosomal loss. However, there were major changes within the acrosome: large numbers of empty membrane-bound vesicles were formed, the electron density of the acrosomal matrix decreased and the acrosome swelled slightly. The origin of the vesicles is unclear but the acrosomal membranes and the plasma membrane remained intact.

Three egg-yolk diluents, which have been used successfully in cryopreservation of human spermatozoa, were compared for their ability to protect macaque semen against cryodamage. TEST (Tes + Tris + egg yolk), TEST with 20% skim milk (TSM), and egg yolk-citrate (EYC), each with 3 or 5% glycerol were compared using 12 ejaculates from 6 male cynomolgus macaques. Computer-aided analysis of sperm motion was used to determine the percentage motility (%M), curvilinear velocity (VCL), and linearity (LIN) of spermatozoa after thawing. The supravital stain Hoechst 33258 and a fluoresceinated pea lectin were used to determine the % of viable spermatozoa with intact acrosomes. TSM and TEST were superior to EYC in terms of % M and of % viable, acrosome-intact spermatozoa. TSM and TEST produced equivalent VCL and LIN values, while EYC had clearly reduced VCL and LIN. There were no interactions between diluent and glycerol level. The 3% glycerol level gave superior results to 5% glycerol for %M. EYC, which is widely used for cryopreservation of human spermatozoa, was not suitable for cynomolgus monkey semen. Artificial insemination with semen cryopreserved in TSM resulted in a healthy, full-term infant.

Large pieces of human cumulus oophorus were exposed for 20-30 min to washed spermatozoa or to spermatozoa recovered after a swim-up procedure, and then fixed for electron microscopy. Spermatozoa of both populations penetrated deeply into the cumulus within that time, and none of 48 observed clearly had undergone an acrosome reaction (AR). As measured by fluorescence microscopy, an AR rate of 12% in spermatozoa obtained at 4 h following a swim-up increased to about 25% in samples incubated in culture dishes for approximately 20 h. However, this latter AR rate was no different in the presence or absence of a cumulus/oocyte complex, and was only moderately greater in 50% follicular fluid. Nor was it affected to any degree by the absence of calcium or by a low (26 degrees C) temperature, both of which are regulators of the physiological AR in other species. By contrast, a clear dose-related enhancement of the AR by the calcium ionophore A23187 was almost completely Ca2(+)-dependent. We conclude that the human cumulus oophorus does not rapidly induce an AR in spermatozoa capacitated in vitro and, unlike the situation in some other mammals, that washed human spermatozoa do not first require a period of capacitation in order to penetrate it. The results also point to the likelihood that ARs monitored in free-swimming human spermatozoa are for the most part spurious or artefactual, and they show that in-vitro AR rates in such populations do not parallel their fertilizing ability.

The human acrosome reaction was induced with the calcium ionophore A23187 and the proportion of reacted spermatozoa was determined with fluorescein-labelled Pisum sativum agglutinin. Human oocytes that failed to fertilize in vitro were used to test binding of spermatozoa to the zona pellucida (ZP) and oolemma. Differential labelling of spermatozoa with fluorescein and rhodamine was used to control for variability in the oocytes. Spermatozoa labelled with one fluorochrome were treated with A23187 and mixed with equal numbers of motile untreated control spermatozoa labelled with the other fluorochrome. The mixture was incubated with zona-intact and zona-free oocytes for 4 h. The sperm-ZP binding ratio of test to control spermatozoa was significantly decreased with increasing proportions of acrosome reacted spermatozoa. In contrast, the sperm-oolemma binding ratio was significantly increased with A23187 treatment. This suggests that acrosome-reacted spermatozoa do not bind to the human ZP.

Earlier studies demonstrated that epididymal mouse spermatozoa have a surface-associated factor which inhibits fertilizing ability in a reversible manner. The factor can be removed from uncapacitated spermatozoa by gentle centrifugation, resulting in immediately highly fertile gametes, and it can be added back to capacitated spermatozoa, resulting in poorly fertile cells in which the acrosome reaction has been blocked. Using such inhibition of in-vitro fertilizing ability as an assay, we have carried out experiments to characterize the factor. It appears to be an anionic polypeptide with Mr of approximately 40,000 (according to its behaviour on gel filtration). It is stable to heating at 100 degrees C for 15 min and is not destroyed by proteases at pH 8.0, yet inhibitory activity decreases during sperm incubation in capacitating conditions and is also destroyed in partially purified preparations by endogenous enzyme action during incubation at pH 5.0. Activity is not adsorbed to either concanavalin A-agarose or wheat-germ agglutinin-agarose, suggesting that terminal mannose and N-acetylglucosamine residues are not abundant. The factor causes rapid changes in the patterns of chlortetracycline fluorescence seen on sperm heads, a parameter used to assess the capacitated state. Removal of the factor from uncapacitated cells results in a shift to a predominance of capacitated patterns, while the addition of crude or partially purified factor to capacitated cells inhibits the acrosome reaction and causes a shift to the uncapacitated pattern in acrosome-intact spermatozoa. The factor therefore behaves as a decapacitation factor. However, it appears to differ from other characterized decapacitation factors in terms both of molecular size and of abundance of mannose and N-acetylglucosamine residues.

Female guinea-pigs were naturally or artificially inseminated before or after ovulation and the distribution of spermatozoa in the oviducts and the time of sperm penetration into the eggs were determined. When animals were inseminated before ovulation, the spermatozoa stayed in the distal half of the oviduct until about the time of ovulation. Only a very few spermatozoa were present in the proximal half of the oviduct at the time of ovulation, but these were sufficient to effect fertilization. When animals were inseminated after ovulation, the spermatozoa ascended the oviduct faster than when animals were inseminated before ovulation, and fertilization commenced in 4 hr. Regardless of the time of insemination, the spermatozoa participating in fertilization appeared to undergo the acrosome reaction after they reached the proximal part of the oviduct or when they were very near the eggs.

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

Current concepts of the macromolecular organization of membrances and of ways in which membrane organization may change during membrane fusion are considered briefly. The properties of the sperm membrane at different phases of its development up to fertilization are then examined in the light of this more general model.

Significant differences in the frequency of spontaneous and induced acrosome reactions (identified by electron microscopy) were detected between 5 individual sperm samples. Maximal stimulation of the acrosome reaction was achieved with ionophore A23187, no differences being detected between the two preincubation times used (5 and 15 h). The cumulus oophorus and follicular fluid caused a similar increase in the proportion of acrosome-reacted spermatozoa after 5 h of preincubation. It is concluded that specific products of the cumulus and/or granulosa cells may contribute to the acrosome reaction-inducing activity of human follicular fluid.

Albumin was required specifically for penetration of the zona pellucida (less than 10% of eggs fertilized in the absence of albumin), but was not required for capacitation. A similar rate of capacitation was observed in the presence of albumin at concentrations ranging from 30 to 1 mg/ml, while a slightly slower rate was observed in the presence of 0.25 and 0.1 mg albumin/ml. In the absence of albumin, capacitation occurred at a rate which lagged behind that of the albumin-incubated counterparts by about 30 min; once capacitated, the addition of albumin promoted rapid sperm penetration. In albumin-free media (+/- the macromolecule PVA), sperm motility was frequently reduced, with fewer cells exhibiting hyperactivated motility, but improvements were observed after introduction of albumin. Acrosome loss was significantly lower in the absence of albumin, but within 5 min of its addition at concentrations ranging from 30 to 0.1 mg/ml to capacitated sperm suspensions, acrosome loss was stimulated and reached levels similar to those seen in control samples. Therefore, albumin can trigger the acrosome reaction in capacitated spermatozoa. It appears to act by assisting in the removal of a surface-associated inhibitory component, the presence of which stabilizes the sperm membranes and inhibits the acrosome reaction.

The acrosome reaction was induced by jelly coat factors, nigericin, or elevated pH. When spermatozoa were preincubated for 5 min in sea water maintained at pH 7.9 in the presence of 1 mM-phenylmethylsulphonyl-fluoride (PMSF), 1 mM-benzamidine, 0.1 mM-1-chloro-3-tosyl-amido-7-amino-2-heptanone (TLCK), 5 mM-diisopropylphosphofluoridate (DFP) or 5 mM-DFP that was previously hydrolysed, only DFP or its hydrolysis product(s) prevented formation of the acrosomal filament induced by jelly coat factors. When incubation with inhibitors was extended to 2 h only TLCK and its hydrolysis products inhibited the jelly-induced acrosome reaction. Only DFP significantly inhibited the acrosome reaction induced by elevated pH (9.0). Nigericin induced acrosome reactions in the presence of DFP or TLCK. These findings do not support the concept of an active role for acrosin in development of an acrosome reaction.

The substrates carbobenzyloxyserylleucylamide, carbobenzyloxyglycylleucylamide and carbobenzyloxyglycylphenylalanylamide were used as potential competitive inhibitors of endogenous metalloendoprotease activity. When the acrosome reaction was elicited by a potential physiological stimulus, human follicular fluid, each of the substrates (1-1.5 mM) inhibited exocytosis. Carbobenzyloxyserylleucylamide also inhibited the acrosome reaction when exocytosis was stimulated using the calcium ionophore ionomycin, but carbobenzyloxyglycylleucylamide was not inhibitory and carbobenzyloxyglycylphenylalanylamide actually enhanced exocytosis under these conditions. Experiments using the fluorescent indicator fura-2 revealed that the increase in intracellular, free calcium stimulated by follicular fluid in human spermatozoa was depressed by carbobenzyloxyglycylphenylalanylamide but not by carbobenzyloxyserylleucylamide. The peptide carbobenzyloxyglycylglycylamide, which is not a substrate for metalloendoproteases, had no effect on the acrosome reaction, whether stimulated by follicular fluid or ionomycin. While the results with carbobenzyloxyserylleucylamide suggest a possible involvement of a metalloendoprotease in the human sperm acrosome reaction, our other results demonstrate that these carbobenzyloxy peptides have complex effects on the process of exocytosis in human spermatozoa, and suggest caution in interpretation of data obtained using such peptides on intact cells.

Particles found in bovine seminal vesicle secretion were enriched by centrifugation. They varied in size and morphology and contained Mg2+,Ca2+-activated ATPase, aminopeptidase A, alanyl aminopeptidase, gamma-glutamyl transpeptidase and dipeptidyl peptidase IV activities. Hyperactivation of sperm motility and the acrosome reaction were induced by these particles in epididymal spermatozoa suspended in a modified Ringer medium. The hyperactivation, analysed with a microscopic slide test, started within minutes of exposure to membrane particles and continued for 3-4 h, during which time spermatozoa underwent the acrosome reaction. Acrosome staining, phase-contrast microscopy and transmission electron microscopy revealed that the acrosome reaction started within 60 min at 37 degrees C and affected up to 80% of spermatozoa in 4 h. These membrane particles differed from those reported previously in other species in enzyme composition, function and organ of origin.

Follicular fluid samples from rabbits, cats, pigs, women and cows had acrosome reaction-inducing activity (ARIA) on rabbit spermatozoa as determined by differential staining after incubation with these fluids. Activity was retained after dialysis and at least 50% was found to be labile when heated to 56 degrees C for as little as 20 min. The induction of the acrosome reaction by bovine follicular fluid showed a dependence on the concentration of follicular fluid and spermatozoa and on calcium ions, and had a pH optimum of approximately 8. Enzyme treatments showed that proteases destroyed the ARIA and this activity was completely blocked by treatment of bovine follicular fluid with goat anti-bovine antiserum. Electron microscope observations indicated the similarity of the reactions observed to that occurring in vivo. It is concluded that the rabbit acrosome reaction inducing-activity of bovine follicular fluid is a serum component(s), probably a protein(s) of high molecular weight.

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

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.

Three autoantigens, S, P and T, have been isolated from epididymal spermatozoa of guinea-pigs. S and P are soluble antigens present on the acrosomal membranes and acrosome matrix. T antigen, a family of several membrane glycoproteins, is distributed over the whole cytoplasmic membrane and on the external acrosomal membrane. Specific antibodies were used to localize the antigens on capacitated and acrosome-reacted spermatozoa and to define their role in acrosome reaction. After capacitation S antigen appeared on the head surface and there was an apparent clustering of T antigen. Both phenomena were prevented by the presence of specific antibodies in the capacitating medium. The acrosome reaction was inhibited both by anti-T and anti-S antibodies as well as by Fab fragments of the same antibodies. The localization of S, P and T antigens on the inner acrosomal membrane after the acrosome reaction suggests that they could also play a role in subsequent steps of fertilization.

When guinea-pig spermatozoa were suspended in a minimal culture medium (MCM-PL), 2-deoxy-D-glucose (100 microM) and 2-amino-2-deoxy-D-glucose (100 microM) were potent inhibitors of the acrosome reaction without affecting the sperm motility, whereas the N-acetyl derivative 2-acetamido-2-deoxy-D-glucose (2 mM) had no inhibitory effect. The addition of D-glucose (2 mM) partly inhibited the percentage acrosome reaction of spermatozoa suspended in Medium MCM-PL, but DL-alpha-glycerophosphate (2 mM) and myo-inositol (2 mM) had no effect. In addition, DL-alpha-glycerophosphate (2 mM) did not overcome the inhibitory effect of 2-deoxy-D-glucose on the sperm acrosome reaction. The inhibitory action of 2-deoxy-D-glucose (100 microM) on the sperm acrosome reaction assessed after a 3-h incubation was irreversible and was only completely effective if the sugar was added within 30 min of the start of incubation. When spermatozoa suspended in Medium MCM-PL were treated with 2-deoxy-D-glucose (100-200 microM) for an extended incubation up to 6 h, the inhibitory effect of 2-deoxy-D-glucose was partly overcome. Spermatozoa treated with 2-deoxy-D-glucose had significantly reduced concentrations of ATP after incubation for 2 h in Ca2+-free media, compared with the ATP concentrations of spermatozoa preincubated for 2 h in Ca2+-free media that supported acrosome reactions. The addition of Ca2+ (5 mM) caused a rapid decrease in ATP concentrations of spermatozoa suspended in Medium MCM-PL, while the addition of the monovalent ionophore monensin (50 microM) and Ca2+ stimulated sperm acrosome reactions as well as an additional decline in the sperm ATP concentrations. However, monensin (50 microM) in the absence of Ca2+ caused only a slight decline in the sperm ATP concentrations over the 15-min incubation period. The depletion of the sperm ATP concentrations by 2-deoxy-D-glucose may retard completion of the capacitation process and the resultant acrosome reaction.

The substrate requirements for capacitation of epididymal mouse spermatozoa, initiation of the acrosome reaction and support of fertilization of mouse eggs in vitro were examined by assessing not only fertilization rates, but also the stages of egg activation and sperm head decondensation in fertilized eggs to monitor the temporal aspects of these processes. Although early events of capacitation did not require exogenous substrates, the fertilization process was effectively blocked at the terminal stages of capacitation in the absence of a glycolysable sugar, and addition of glucose was obligatory to initiate both the acrosome reaction and the whiplash motility associated with fertilizing ability. Once the spermatozoa had been primed by glucose, however, the removal of exogenous glucose did not block fertilization. The need for oxidative metabolism was excluded because successful fertilization could be achieved with glucose as the sole exogenous substrate under strictly anaerobic conditions or in the presence of 2.5 microM-oligomycin. We suggest, therefore, that epididymal mouse spermatozoa can be almost completely capacitated in the absence of metabolic processes simply by release into substrate-free medium. They require exposure to glucose to permit induction of the acrosome reaction and motility changes, necessary prerequisites for fertilization; such exposure is followed by rapid and synchronous penetration of eggs.

Ejaculated spermatozoa were rendered immotile by incubation for 8 h at 37 degrees C in 2.9% sodium citrate. Immotile spermatozoa (5 x 10(6)) were surgically inseminated into the uterine lumen of does and samples were recovered from the uterus 5, 15, 30 and 60 min after insemination. Incubation in utero led to a resumption of progressive motility and induced head-to-head agglutination. Motility and head-to-head agglutination were highest (64 and 70% respectively) at 5 min, and declined (48 and 49%) by 60 min. The percentage of spermatozoa with an intact acrosome did not change during the in-utero incubation. When spermatozoa from a single ejaculate were evaluated in different females there was significant variation (P less than 0.01) in the reinitiation of motility and agglutination. Most agglutinated spermatozoa (greater than 96%) had an intact acrosomal membrane while acrosomal integrity of single spermatozoa differed greatly among females. We conclude that the agglutinated (motile with intact acrosomes) and non-agglutinated (usually immotile and with disrupted acrosomes) represent different populations within the uterine lumen.

Guinea-pig spermatozoa were induced to undergo an acrosome reaction with the ionophore A23187. A time course for the activation of protease activity was established. Acid treatment of fully activated spermatozoa at pH 2.35 for 30 min exposed additional activity. This was attributed to the acid dissociation of a protein inhibitor from acrosin. The acrosin content of fully activated and acid-dissociated sperm extracts was measured using a sensitive active-site titrant for serine proteases. The number of acrosin molecules per spermatozoon, calculated on the basis of the sperm count, was approximately 2 x 10(6), of which half were available without dissociation of the inhibitor.

GB24 is a mouse monoclonal antibody raised against human trophoblast microvilli, which recognizes an antigenic determinant on the acrosomal region of the human sperm head. By indirect immunofluorescence, reactivity of GB24 could not be detected on freshly ejaculated spermatozoa but was strongly positive after sperm permeabilization with acetone. On viable, motile spermatozoa, reactivity appeared after induction of the acrosomal reaction with the calcium ionophore A23187. These results suggest that the antigen recognized by GB24 is present on the inner acrosomal membrane. A quantitative evaluation assay of the acrosome reaction on viable spermatozoa by flow cytometry using GB24 and indirect immunofluorescence is proposed.

Guinea-pig spermatozoa, incubated in a minimal culture medium (MCM-PL) containing 150 mM-Na+ and no added K+, capacitate within 2 h and respond to added Ca2+ with maximal percentage of acrosome reactions. When spermatozoa, initially capacitated in Medium MCM-PL, were washed and resuspended in saline-based media, the motile spermatozoa showed acrosome reactions only in response to added Ca2+ at an extracellular pH greater than 7.4. In contrast, resuspension of capacitated spermatozoa into isotonic sucrose or choline-based media containing no added Na+ and K+ (pH 7.8) did not support acrosome reactions in response to the addition of Ca2+. Examination under the electron microscope showed that these spermatozoa responded to added Ca2+ with swelling or cavitation of the acrosomal matrix but fusion of the plasma membrane and acrosomal membrane did not occur. Treatment of spermatozoa with monensin, a monovalent cationic ionophore, induced rapid acrosome reactions in the presence of extracellular Ca2+ and Na+. In contrast, spermatozoa incubated for 3 h in a sucrose-based Na+-deficient medium or Medium MCM-PL containing 10 mM-KCl failed to give a significant percentage of acrosome reactions in response to the addition of Ca2+, but the addition of ouabain (0.1 mM) prevented the K+ inhibition. Amiloride, a sodium channel blocker in various other tissues, retarded the development of acrosome reactions of spermatozoa incubated in Medium MCM-PL. Based on these data, it appears that an increase in intracellular Na+ has a functional role in acquisition of guinea-pig sperm fertilizing ability.

Spermatozoa were collected from the cauda epididymidis of golden hamsters and guinea-pigs, and the acrosome reaction was induced in vitro. Movement characteristics of the spermatozoa were assessed with high-speed cinemicrography. Before the initiation of the acrosome reaction (preactivated spermatozoa), sperm movement in both species was characterized by progressive swimming by regular flagellar waves of moderate amplitude and relative high frequency. After the acrosome reaction (activated spermatozoa), sperm movement in both species was not progressive, and was characterized by whiplash-like flagellar undulations of significantly (P less than 0.05) higher amplitude and lower frequency. Calculation of the hydrodynamic power output by a new theory indicated that no significant change occurred after activation.

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.

The effect of human follicular fluid (FF) on the incidence of spontaneous acrosome reactions (AR) in human spermatozoa was examined over a 24-25 h period using electron microscopy. Suspensions of motile spermatozoa were prepared by a swim-up method in Earle's medium, known to support in-vitro fertilization. After adjusting the concentration to 10 x 10(6) cells/ml, suspensions were diluted 1:1 with medium (control) or FF, the latter giving a final concentration of 50% FF. In addition, at 5 h and 24 h an aliquant of the control suspension was removed, diluted 1:1 with FF and incubated for 1 h; the three suspensions were examined at 6 h and 25 h. Continuous exposure to 50% FF stimulated the AR, the effect being significant (P less than 0.001) at 25 h. However, the 1-h short exposure of spermatozoa to FF did not produce an increase in AR, even after 24 h preincubation. In a separate series of experiments, the effect of continuous incubation for 24 h in increasing concentrations of FF was investigated. A significant linear dose-dependent effect on the AR was observed with all concentrations assessed (P less than 0.01 for 12.5% FF and P less than 0.001 for 25, 50, 75 and 100% FF, compared with FF-free control). Therefore, human FF can stimulate the AR, but only after a continuous exposure to FF. A short exposure to FF, even after 24 h preincubation, does not trigger an increased AR response.