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

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

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).     

Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn^−/− males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn^−/− males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn^−/− mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.     

Effects of platelet activating factor on capacitation and acrosome reaction in mouse spermatozoa

Platelet activating factor (PAF) plays an important role in mammalian reproduction. The aim of this study was to investigate the effects of PAF on capacitation and acrosome reaction of mouse spermatozoa by chlortetracycline (CTC) fluorescence assay and coomassie blue staining. The percentage of capacitated mouse spermatozoa was increased (P < 0.05) by incubation with 50 ng/ml PAF for 20-120 min. The peak response occurred between 80 to 100 min of exposure to PAF. In contrast, the effects of PAF on acrosome reaction may be not receptor-mediated since lyso-PAF had the same effects. Ionophore A23187 stimulated an increase in acrosome-reacted spermatozoa of PAF-treated spermatozoa, but not of lyso-PAF-treated ones. These results suggest that PAF mainly acts on sperm capacitation.     

Neuraminidase induces capacitation and acrosome reaction in mammalian spermatozoa

The treatment of epididymal spermatozoa of guinea pig and ejaculated spermatozoa of rabbit with neuraminidase from Arthrobacter ureafaciens induced significant acrosome reaction while the neuraminidase from Cl. perfringens failed to do so. The addition of the neuraminidase inhibitors kept the enzyme induced acrosome reaction to the control level. The zona-free hamster ova test showed that the treatment of spermatozoa with Arthrobacter neuraminidase rendered 82% of the guinea pig and 69% of the rabbit spermatozoa capable of fertilization. Thus, neuraminidase seems to enhance the rate of acrosome reaction by first capacitating spermatozoa in vitro.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Strontium supports human sperm capacitation but not follicular fluid-induced acrosome reaction.

The ability of strontium (Sr(2+)) to replace calcium (Ca(2+)) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca(2+)- or Sr(2+)-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr(2+) were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca(2+), sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca(2+). The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca(2+)- or Sr(2+)-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca(2+) present in Sr(2+) medium. From these results, it can be concluded that Sr(2+) can replace Ca(2+) in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Extended exposure to follicular fluid is required for significant stimulation of the acrosome reaction in human spermatozoa

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.     

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.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

Changes in the distribution of lectin receptors during capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx modifications are known to occur during capacitation and the acrosome reaction (AR). These changes are very important for gamete recognition and fertilization in mammals but are not fully understood. The purpose of this study was to determine the distribution of surface carbohydrates in boar spermatozoa during capacitation and the AR. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. Thirty-nine ejaculates from fertile boars of various breeds were analyzed. N-Acetylglucosamine and sialic acid, mannose and fucose residues were detected by fluorescence microscopy and flow cytometry using FITC-conjugated lectins. Triticum vulgaris agglutinin (WGA) bound on the head and tail of fresh sperm, and fluorescence intensity (FI) decreased in capacitated sperm (6751 to 5621 fluorescence units (FU), P<0.05), and decreased further in acrosome-reacted sperm (5240 FU, P<0.05). Concanavalia ensiformis agglutinin (Con-A) bound homogeneously on the head and the midpiece of fresh sperm with a FI of 5335 FU, and increased in capacitated sperm (5957 FU, P<0.05) mainly on the acrosomal region. In acrosome-reacted sperm, fluorescence was concentrated on the border of the acrosomal region (5608 FU, P<0.05). It was not possible to detect Ulex europaeus agglutinin (UEA) by fluorescence microscopy. However, flow cytometry revealed UEA receptors (187 FU), with a nonsignificant decreased number in capacitated (142 FU) and AR sperm (142 FU). Labeling patterns were similar in all breeds. Sperm glycocalyx modifications observed in this study provide insights to the molecular modifications accompanying capacitation and the AR. This kind of study could improve the diagnosis of reproductive problems of subfertile boars and males of other species.     

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome loss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these responses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine-containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln-FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

Effects of the purified IGF-I complex on the capacitation and acrosome reaction of rabbit spermatozoa

A protein complex containing IGF-I, purified from rabbit seminal plasma, was used to investigate its effects on the capacitation and acrosome reaction of rabbit spermatozoa. Uncapacitated sperm (Pattern F), capacitated sperm (Pattern B), and acrosome-reacted sperm (Pattern AR) were determined by CTC staining, and the results were validated by PSA-staining. The addition of the IGF-I complex to the capacitative medium directed the spermatozoa to spontaneous acrosome reaction. On the other hand, IGF-I complex, added to capacitated spermatozoa, acted as inducer of the acrosome reaction. Results of IVF experiments showed high rates of fertilization with capacitated spermatozoa, acrosome-reacted by either A23187 or IGF I complex, whereas significantly lower rates were obtained with spermatozoa capacitated in the presence of IGF-I complex.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.