On the role of glucose in capacitation and acrosomal reaction of guinea pig sperm

In order to determine whether metabolizable sugars delayed capacitation of guinea pig spermatozoa, these cells were pre-incubated in Tyrode's pyruvate lactate glucose medium (T-PLG) or Tyrode's glucose solution (T-G). They were then transferred to minimal culture medium containing pyruvate and lactate (MCM-PL) and the occurrence of acrosomal reactions (AR) was determined by light microscopic observations of wet mount aliquots. The percentage of acrosomal reactions was quantitated in fixed samples and occurrence of a true AR was confirmed by electron microscopy. Activated acrosome-reacted spermatozoa were observed within 5 min when cells were transferred to MCM-PL solution, after preincubating them for 60–120 min either in T-PLG or T-G media. By 15 min in MCM-PL the percentage of acrosome-reacted spermatozoa reached values similar to those obtained in cells pre-incubated from the beginning in MCM-PL medium (P > 0.05 in both) but significantly different from T-PLG and T-G controls (P < 0.0005 in both). The acrosomal reaction was external calcium dependent and independent of the Tyrode's media pH ranging from 7.2 to 8.0. The results obtained suggested that capacitation occurred in T-PLG and that it was not delayed by glucose; the results also suggested that capacitation could occur within a short time with glucose as the only exogenous substrate, but that the acrosome reaction could have been arrested by a glucose metabolite. Data are presented which suggest that intracellular levels of glucose-6-phosphate (as 2-deoxyglucose-6-phosphate)could play a key role in the expression of the acrosome reaction in sperm already able to perform it. A new hypothesis is suggested for the development of the fertilizing potential of guinea pig sperm when in the female genital tract.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

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.     

Changes in guinea pig sperm intracellular sodium and potassium content during capacitation and treatment with monovalent ionophores

Guinea pig spermatozoa were collected from the caudae epididymides in various isotonic solutions and the intracellular sodium and potassium content was determined by atomic absorption spectroscopy. The sperm intracellular Na and K content was found to be influenced by large variations in the extracellular concentrations of these ions. Treatment of spermatozoa suspended in a saline-based solution with the monovalent ionophores monensin or nigericin caused an approximate 2-fold increase in the intracellular Na content and a 3–6 fold decrease in the intracellular K content. Incubation of the spermatozoa in a K⁺-free minimal culture medium (MCM-PL) at a pH of 7.6 or 8.3 for 2 hr caused an approximate 2-fold increase in the sperm intracellular Na content and a 5-fold decrease in the intracellular K content. The motile spermatozoa incubated for 2 hr at pH 7.6 showed less than 5% acrosome reactions, compared with 30–40% acrosome reactions after incubation at pH 8.3, in response to the addition of 5 mM Ca²⁺. Changes in the sperm intracellular elemental composition during culture in vitro, which may lead to an acrosome reaction, are discussed.     

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.     

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.     

Dithiothreitol,a disulfide-reducing agent,inhibits capacitation,acrosome reaction,and interaction with eggs by guinea pig spermatozoa

Capacitation of guinea pig spermatozoa in vitro was inhibited by the disulfide-reducing agent dithiothreitol (DTT). Even a brief treatment with DTT inhibited capacitation unless an oxidizing agent (glutathione disulfide) was present in the posttreatment medium. Precapacitated spermatozoa were unable to undergo the acrosome reaction in the presence of DTT, indicating that this reagent also blocks the acrosome reaction. Acrosome-reacted spermatozoa were incapable of attaching to and penetrating the zona pellucida in the presence of DTT. Even when acrosome-reacted spermatozoa were directly brought to the surface of zona-free eggs, they were unable to bind to and fuse with the egg plasma membrane so long as DTT was present in the medium. These observations suggest that the tertiary and quaternary structures of sperm surface proteins regulated by their thioldisulfide status are of critical importance in the physiology and function of spermatozoa preliminary to and in the process of fertilization.     

Cytochemical localization of membrane-bound Mg2+-dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome-reacting guinea-pig spermatozoa was examined cytochemically using the Wachstein-Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg²⁺-dependent and inhibited by both Ca²⁺ and SH-blocking agents. This Mg²⁺-dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome-reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg²⁺-dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca²⁺ is one of the important biochemical events involved in the acrosome reaction.     

Effects of various lipids on the acrosome reaction and fertilizing capacity of guinea pig spermatozoa with special reference to the possible involvement of lysophospholipids in the acrosome reaction

The effects of lipids on the survival, acrosome reaction, and fertilizing capacity of guinea pig spermatozoa were studied by incubating the spermatozoa in media containing various concentrations of the lipids. Lipids tested were: phosphatidyl-choline (PC), -ethanolamine (PE), -inositol (PI), -serine (PS), sphingomyelin (S), cholesterol (C), lysophosphatidyl-choline (LC), -ethanolamine (LE), -inositol (LI), -serine (LS), and glyceryl monooleate (M). When spermatozoa were incubated in a regular medium (containing 2 mM Ca²⁺) with M, the majority underwent the acrosome reaction within 1 hour. None of the other lipids were as effective as M, and some were totally ineffective under the same conditions. However, when spermatozoa were preincubated in Ca²⁺-free medium containing LC, LE, or LI, they gained the ability to undergo the acrosome reaction. One hour of preincubation in Ca²⁺-free medium with LC, LE, or LI was enough to render the vast majority of spermatozoa capable of undergoing the acrosome reaction in response to Ca²⁺. The optimum concentrations for LC, LE, and LI were approximately 85 μg/ml, 210 μg/ml, and 140 μg/ml, respectively. Spermatozoa that had undergone the acrosome reaction by pretreatment with LC, LE, or LI remained actively motile and were capable of fertilizing eggs. LS was totally ineffective in rendering the spermatozoa capable of undergoing the acrosome reaction, and in fact it inhibited the acrosome reaction by itself and also inhibited the LC-, LE-, or LI-mediated acrosome reaction. LS did not prevent acrosome-reacted spermatozoa from penetrating the zona pellucida, but did prevent sperm-egg fusion. Based on these findings, it is suggested that lysophospholipids are intricately involved in the sperm acrosome reaction and perhaps in sperm-egg fusion.     

Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa

The majority of the spermatozoa precapacitated in Ca²⁺-free medium underwent the acrosome raction rapidly when they were transferred to Ca²⁺-containing medium. The presence of Na⁺ and Ca²⁺ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca²⁺ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na⁺, K⁺, Rb⁺, and Cs⁺ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na⁺. Li⁺ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na⁺ medium (no Ca²⁺) and then to Ca²⁺ medium (no Na⁺). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na⁺) and Ca²⁺ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.     

The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro

Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High-speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non-rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.     

Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa

Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode’s complete medium with heparin (TCMH; a capacitating medium containing Ca²⁺, NaHCO₃ and heparin), Tyrode’s complete medium heparin-free (TCM; a medium containing just Ca²⁺ and NaHCO₃) or Tyrode’s basal medium (TBM; a non-capacitating medium free of Ca²⁺, NaHCO₃ and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=−0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Effect of cholesterol and other sterols on human sperm acrosomal responsiveness

Human sperm become responsive to inducers of the acrosome reaction when they are washed free of seminal plasma and incubated in an appropriate medium. Previous work has shown that cholesterol-enriched medium prevents sperm from becoming responsive to the inducer, progesterone. Sperm that were incubated 24 hr in cholesterol-enriched medium and then treated with progesterone showed no evidence of membrane fusion, indicating that cholesterol acts at a stage before the earliest morphological change. Inhibition of acrosomal responsiveness by cholesterol was reversible. Among other sterols reported in mammalian sperm, desmosterol and cholesterol sulfate also inhibited sperm from becoming responsive, but cholesterol palmitate did not. Our results support a model in which sperm unesterified cholesterol, or a molecule in equilibrium with it, suppresses acrosomal responsiveness. Cholesterol-enriched medium also prevented sperm from becoming responsive to the calcium/proton exchanging ionophore, ionomycin, suggesting that cholesterol's effect may be, at least in part, at a point in the signal transduction pathway subsequent to the rise in intracellular-free calcium. © 1996 Wiley-Liss, Inc.     

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.     

GABA和孕酮对人及豚鼠精子的体外获能作用

为了探讨γ－氨基丁酸（ＧＡＢＡ）是否参与人及豚鼠精子体外获能的调节,将生育男子和豚鼠清子分别悬浮于ＢＷＷ和低Ｃａ＾２＋最小获能培养基（ＬＣａ＾２＋－ＭＣＭ）中,加入ＧＡＢＡ、孕酮（Ｐ４）、ＧＡＢＡＡ受体激动剂及其拮抗剂,在５％ＣＯ２孵箱３８．５℃培养２ｈ。然后用ｉｏｎｏｐｈｏｒｅ Ａ２３１８７激发精子顶体反应（ＡＲ）和超激活运动（ＨＡＭ）。以精子与金霉素（ＣＴＣ）荧光结合类型、ＡＲ和ＨＡＭ为指标来     

Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions

The kinetics of spontaneous and induced acrosomal loss have been studied in human sperm incubated in capacitating and noncapacitating media. Acrosomal status was quantitated using indirect immunofluorescence with a monoclonal antibody. The response of sperm to induction by calcium ionophores was time dependent reaching a maximum after 6 hours of incubation under capacitating conditions. The inducible population slowly decreased in size through the balance of a 24-hour incubation. The time-dependent development of ionophore responsiveness by sperm exposed to capacitating conditions corroborates the idea that only capacitated cells can respond to undergo acrosomal loss in response to ionophore. In contrast, only a small, constant percentage of sperm incubated under noncapacitating conditions responded to ionophore. Substitution experiments involving the addition or deletion of human serum albumin suggest that albumin is not absolutely required for capacitation but is essential for the maintenance of motility. Polyvinyl alcohol can be substituted for serum albumin, but it does not support capacitation or motility as well as HSA. These studies may provide a basis for optimizing capacitating conditions for human sperm in vitro as well as for diagnosing fertility or fertility potential based on measurements of spontaneous and ionophore induced acrosomal loss under defined culture conditions.