Multiple effects of seminal plasma on the acrosome reaction of human sperm

Mammalian sperm do not respond to inducers of the acrosome reaction immediately after ejaculation. They become responsive after they are removed from seminal plasma and incubated in an appropriate medium. We tested the effects of seminal plasma on the development of acrosomal responsiveness. Washed human sperm incubated 24 hr in vitro with 10% (v/v) seminal plasma did not complete an acrosome reaction when exposed to human follicular fluid, progesterone, or ionomycin. Seminal plasma did not reduce sperm viability or motility. Electron microscopy of sperm incubated 24 hr with 5% seminal plasma and then treated with progesterone revealed no sign of membrane fusion or other changes that are associated with the acrosome reaction. During a 12-hr incubation, seminal plasma was 50% effective at inhibiting the acrosomal response to progesterone when diluted 821 ± 112 foid (mean ±SD, n = 3). Sperm that were incubated with seminal plasma for 24 hr and then washed free of the seminal plasma became acrosomally responsive over the following 24 hr, at a rate similar to that of sperm not incubated with seminal plasma in vitro. When sperm were incubated 6 hr without seminal plasma and then seminal plasma was added, the sperm population transiently became more responsive to progesterone, and then became unresponsive. During incubation in vitro, the ability of sperm to have an augmented response to a mixture of seminal plasma plus progesterone developed slightly earlier and more rapidly than ability to respond to progesterone alone. When sperm were incubated 24 hr without seminal plasma, a few acrosome reacted in response to the addition of seminal plasma alone. Therefore, depending on how it is applied, seminal plasma can prevent or reverse the development of acrosomal responsiveness, and it can enhance or induce the acrosome reaction. © 1993 Wiley-Liss, Inc.     

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.     

Cholesterol efflux promotes acrosome reaction in goat spermatozoa

Cholesterol efflux and membrane destabilization play an important role in sperm capacitation and membrane fusion in the acrosome reaction (AR). In this study we establish the effect of cholesterol removal from spermatozoa on acrosomal responsiveness. Mature goat spermatozoa were incubated in BSA-free medium in the presence of beta-cyclodextrin (betaCD) as cholesterol acceptor. After incubation with 8 mM betaCD, 50-60% of cholesterol was released from sperm membranes with no loss in the phospholipid content, and 35% of AR was induced. However, when 30% of cholesterol was lost, this moderate cholesterol decrease was unable to initiate AR. Cholesterol desorption was very rapid, following an exponential kinetics with a half-time of around 10 min, which is in contrast with the slow sigmoidal kinetics of acrosomal responsiveness: around 2 h was required for maximal AR. Our results suggest that cholesterol efflux has a direct influence on the onset of the AR, that is, merely removing cholesterol would trigger the AR.     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4–5 hours at 37°C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.     

Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction

This study describes investigations of the importance of intraacrosomal pH in the hamster sperm acrosome reaction (AR). Washed cauda epididymal sperm were capacitated in vitro in a medium containing 2 mM Ca2+, 144 mM Na+, and 3 mM K+. Such sperm underwent a significant increase in the number of AR within 10 min after the addition of the Mg2+-ATPase (adenosine triphosphatase) inhibitors DCCD (20 microM) or NBD-Cl (10 microM) or the proton ionophore FCCP (6 micrograms/ml) at 3.5 hr of incubation or after addition of HN4Cl (3 mM) at 4 hr of incubation. Addition of the mitochondrial electron transport inhibitor rotenone (2.5 microM) at 3.5 hr or of NaCl (3 mM) or KCl (3 mM) at 4 hr did not stimulate AR over control levels, suggesting that the stimulation of AR by the other compounds was not directly due to depletion of acrosomal adenosine triphosphate (ATP) or alteration of the acrosomal transmembrane potential. The AR also was not stimulated by either DCCD or FCCP added prior to 3 hr of incubation of sperm, whereas both compounds were increasingly effective at stimulating AR with increasing length of preincubation of sperm before the addition of the test compounds. The intraacrosomal pH of sperm incubated in low [K+] (0.6-0.9 mM) for 3.5 hr rose by at least one pH unit (as measured with the fluorescent dye 9-aminoacridine) within 15-30 min after raising extracellular [K+] to 4.2-4.5 mM. The pH rise occurred even in the presence of the Ca2+-chelator EGTA (2 mM). Either FCCP (8 micrograms/ml) or DCCD (20 microM), but not rotenone (2.5 microM), plus K+ (3.6 mM), raised the intraacrosomal pH of sperm incubated for 3 hr in low [K+] within 10 min after addition. No pH rise occurred in the absence of additional K+. These results demonstrate that the intraacrosomal pH of the hamster sperm becomes more alkaline in a process not requiring high concentrations of external Ca2+, but requiring K+. The results of this and previous studies lead us to suggest here that the intraacrosomal pH rise may be mediated via a change in K+ and H+ permeability of sperm head membranes, which allows K+ influx and H+ efflux, and via inhibition of an acrosomal Mg2+-ATPase proton pump. We propose that the permeability changes and the consequent alkalinization of the acrosomal interior are important steps in late capacitation and/or the mammalian AR.     

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.     

A new procedure for rapidly scoring acrosome reactions of human sperm

Because the acrosome of human sperm is too small to be directly visualized by phase-contrast microscopy, acrosome reactions (that is loss of the acrosome) are generally not evaluated in studies of human sperm capacitation and fertilization. Nevertheless, it would be useful in such studies to have a technique for easily identifying and quantitating acrosome-reacted sperm. In this paper, we describe a method for labeling the human sperm acrosome with fluorescein-conjugated Ricinus communis agglutinin-60 (FITC-RCA); we show that in sperm without acrosomal caps, FITC-RCA labeling occurs either not at all or only in the equatorial segment of the acrosome. To determine if the absence of FITC-RCA labeling in the acrosomal cap region gives a reliable estimate of acrosome reactions, washed sperm or sperm incubated in a capacitating medium (BWW) were divided into two groups, which were then fixed for FITC-RCA labeling or transmission electron microscopy. Counts of acrosome reactions made by each method were similar, and we observed an increase in the percentage of reactions following incubation in BWW. We conclude that the FITC-TCA labeling technique is a reliable method for accurately scoring the percentage of acrosome-reacted human sperm.     

Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation

We previously targeted EGFP (a mutant of green fluorescent protein) to the lumen of the mouse sperm acrosome and reported the time course of EGFP release during the acrosome reaction. In the study reported here, we estimated the pH within the mouse sperm acrosome utilizing the pH-dependent nature of EGFP fluorescence. The average intra-acrosomal pH was estimated to be 5.3 +/- 0.1 immediately after sperm preparation, gradually increasing to 6.2 +/- 0.3 during 120 min of incubation in TYH media suitable for capacitation. Spontaneous acrosome reactions were noted to increase concomitantly with acrosomal alkalinization during incubation. We also demonstrated that acrosomal antigens detected by monoclonal antibodies MN7 and MC41 did not dissolve following the acrosome reaction in pH 5.3 media, but dissolved at pH 6.2. These data suggest that acrosomal alkalinization during incubation conducive for sperm capacitation may function to alter acrosomal contents and prepare them for release during the acrosome reaction.     

Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro,in relation to the timing of the acrosome reaction

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.     

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.     

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.     

The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The t1/2 of this process was 5-10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an ED50 and a maximally effective dosage of 20 and 50 ng protein/microliter, respectively. Zonae pellucidae caused up to a 48-fold increase in the apparent initial rate and a 3- to 4-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida.     

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 human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.     

The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

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

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

Competition between zonae pellucidae and a proteinase inhibitor for sperm binding.

Murine sperm bind a proteinase inhibitor of seminal vesicle origin at ejaculation. The inhibitor binds in the acrosomal region of the sperm head and is removed during in utero or in vitro incubation. Adding inhibitor to sperm reduces their ability to bind zonae, while adding the purified inhibitor binding site to cumulus-free, zona-intact oocytes reduces the ability of the oocytes to bind sperm. Immuno-aggregation of the inhibitor binding site results in exocytosis of the acrosome. These observations suggest that the inhibitor binding site may participate in zona binding and the acrosome reaction. If the inhibitor binding site binds both the zona and the seminal inhibitor, then these components should compete with each other for that site on the sperm. We show that purified seminal inhibitor, as well as other proteinase inhibitors, block zona-induced acrosome reactions. Likewise, zona glycopeptides block inhibitor/anti-inhibitor-induced acrosome reactions in a concentration-dependent fashion. The inhibitor/anti-inhibitor-induced acrosome reaction is sensitive to pertussis toxin and proteinase inhibitor and thus is similar to zona-induced reactions. These findings support the suggestion that the trypsin inhibitor binding site on the head of the sperm functions to insure sperm-zona binding and induction of the acrosome reaction.