Effect of pH on the development of acrosomal responsiveness of human sperm.

Human sperm incubated in vitro gradually become responsive to inducers of the acrosome reaction. The roles of constituents of the incubation medium are not well understood. These experiments tested the effect of the extracellular pH on sperm acrosomal responsiveness. Sperm were incubated 24 h in media with pH varying between 6.7 and 7.6 and then exposed to progesterone to determine the number of sperm that had become acrosomally responsive. The number of responsive sperm was very low following incubation at pH 6.7-7.0, and increased with the pH over the range 7.0-7.6. Sperm incubated at low pH were not damaged as assessed by motility or viability, and if they were transferred to medium of high pH for 8 h, the inhibition of acrosomal responsiveness was reversed. Inhibition of acrosomal responsiveness at low pH was not due to impaired loss of sperm cholesterol, but was correlated with a reduced intracellular pH. The inhibition of acrosomal responsiveness by media of low pH may result from preventing the normal capacitation-related rise in intracellular pH.

Sphingomyelin modulates capacitation of human sperm in vitro.

Ejaculated mammalian sperm must mature (capacitate) before they can undergo acrosomal exocytosis and fertilize an egg. Loss of sperm sterols is an early step in capacitation. Because sphingomyelin slows cholesterol efflux from other cells, the role of sphingomyelin in capacitation was tested. Human sperm were exposed to sphingomyelinase and then incubated for as long as 24 h. The ability of sperm to acrosome-react in response to progesterone was tested to measure capacitation. Sphingomyelinase-treated sperm became responsive to progesterone approximately 10 h earlier than control sperm. Sphingomyelinase also increased spontaneous acrosomal exocytosis. The effects of sphingomyelinase were accompanied by accelerated losses of the inhibitory sterols, cholesterol and desmosterol. To test whether sphingomyelinase-generated ceramide might promote capacitation, sperm were incubated for 8 h with the cell-permeable ceramide N:-hexanoylsphingosine (25 microM) or with solvent. Ceramide increased the incidence of progesterone-responsive sperm and, at later times, spontaneously reacted sperm. N:-Hexanoylsphinganine, an inactive control ceramide, had no effect. These results suggest that sphingomyelin in the sperm influences the rate of capacitation by slowing the loss of sterols, and that exogenous sphingomyelinase accelerates capacitation by speeding the loss of sterols and by generating ceramide.

Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm.

Human sperm incubated in vitro gradually become capable of acrosome-reacting in response to the agonist, progesterone (P4). Loss of unesterified cholesterol is an obligatory step in the development of acrosomal responsiveness. These experiments tested the ability of methyl-beta-cyclodextrin (MbetaCD) to accelerate sperm cholesterol loss and the development of acrosomal responsiveness. Incubating sperm 30 min in MbetaCD (2.5-10 mM) decreased sperm cholesterol by as much as 89% in a dose-dependent fashion. MbetaCD caused some sperm (maximum of 16% following treatment with 5 mM MbetaCD) to become responsive to P4, and it caused a dose-dependent increase in spontaneous acrosome reactions. The number of responsive sperm increased in the first 3 hr following their removal from MbetaCD. Continuing incubation to 24 hr increased the numbers of spontaneously reacted sperm and dead sperm, but not P4-responsive sperm. It appears, therefore, that up to 3 hr are required for the full expression of P4-responsiveness in cholesterol-depleted sperm. The observed effects of MbetaCD are due to its cholesterol-depleting properties, because including sufficient cholesterol with MbetaCD to reduce the loss of sperm cholesterol inhibited the effects of MbetaCD on cell viability, spontaneous acrosome reactions, and responsiveness to P4. MbetaCD accelerates the appearance of the functional stages that sperm normally pass through during incubation in vitro, reinforcing the view that cholesterol loss is an important determinant of the rate at which sperm become acrosomally responsive.

Prostasome fraction of human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist progesterone.

Seminal plasma prevents human sperm from becoming acrosomally responsive. These experiments tested the idea that the inhibitory activity of seminal plasma is contained in the particulate prostasome fraction. Most of the inhibitory activity was sedimentable (105,000 g, 2 h) and the majority of the recovered activity was in the prostasome fraction. The recovery of inhibitory activity in the prostasome fraction (42% of the activity in unfractionated seminal plasma) was similar to the recovery of cholesterol in that fraction (41%), consistent with cholesterol's role as the major inhibitor in seminal plasma. To test whether components of the prostasome fraction bind to sperm, the prostasome fraction was made fluorescent with fluorescein isothiocyanate or with the acetoxymethyl ester of 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. No labeled material was seen to bind to sperm, suggesting that exchange of cholesterol between prostasomes and sperm takes place through the aqueous phase or at the time of vesicle-sperm collision.

Control of human sperm intracellular pH by cholesterol and its relationship to the response of the acrosome to progesterone.

When incubated in vitro, human sperm gradually become capable of acrosome-reacting in response to the agonist progesterone. Loss of unesterified cholesterol is required for sperm to become responsive to progesterone, but how cholesterol regulates acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm cholesterol leads to a rise in the intracellular pH (pH(i)) that makes the sperm responsive to progesterone. pH(i) was measured using BCECF (2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein) in freshly ejaculated sperm (T0 sperm) and in sperm incubated in vitro overnight (T24 sperm). During incubation, pH(i) increased from 6.94 +/- 0.03 to 7.08 +/- 0.01 (mean +/- SEM, n = 4, p < 0.01). Incubating sperm 24 h in medium supplemented with 1 microM cholesterol to prevent loss of sperm cholesterol suppressed the rise of pH(i) (T24C sperm, pH(i) = 6.96 +/- 0.03, n = 4, p = 0.64 compared to T0 sperm). To test whether their lower pH(i) prevents T24C sperm from reacting, we treated T24C sperm with the alkalinizing agents trimethylamine chloride (TMA) or NH4Cl. These agents did cause T24C sperm to respond to progesterone in a dose-dependent fashion, but they also caused a similar increase in the number of reacting T24 sperm. These agents probably do not reverse the inhibiting effects of high cholesterol but rather make responsive a subpopulation of sperm that is present regardless of the cholesterol content. NH4Cl and TMA did not make T0 sperm responsive to progesterone. The acidifying agent sodium propionate did not diminish the response of T24 sperm to progesterone. In summary, pH(i) increases during incubation in vitro in a cholesterol-dependent fashion. Elevated pH(i) alone is probably not sufficient to make sperm acrosomally responsive.

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.

Unesterified cholesterol content of human sperm regulates the response of the acrosome to the agonist, progesterone.

Human sperm become responsive to inducers of the acrosome reaction if they are washed free of seminal plasma and incubated in an appropriate medium. We tested the hypothesis that sperm must lose cholesterol during incubation in order to become responsive to the agonist, progesterone. Freshly ejaculated sperm contained 2.92 +/- 0.202 nmol unesterified cholesterol/10(7) sperm (mean +/- SEM, n = 18). When incubated for 24 h in vitro, sperm suspensions lost 29 +/- 6% of their free cholesterol (n = 23). Sperm lost cholesterol slightly faster than they became acrosomally responsive. Adding cholesterol to the medium prevented sperm from losing cholesterol and from becoming responsive. Varying the cholesterol content of the medium had similar effects on loss of sperm cholesterol (ED50 = 406 nM) and acrosomal responsiveness (ED50 = 388 nM). Incubating sperm with a 1:150 dilution of seminal plasma (containing 5.18 microM cholesterol) also prevented sperm from losing cholesterol and from becoming responsive. Incubating sperm 24 h in medium containing 0.5 mg/ml phosphatidylcholine increased the amount of cholesterol lost and the number of sperm that became responsive. Our results support a model in which sperm unesterified cholesterol (or a molecule in equilibrium with it) suppresses acrosomal responsiveness. Sperm must lose unesterified cholesterol to become responsive to progesterone.

Human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist, progesterone: cholesterol is the major inhibitor.

Seminal plasma inhibits human sperm from developing the ability to undergo the acrosome reaction. The inhibitory activity was identified as that of cholesterol on the basis of its solubility in organic solvents, its chromatographic behavior (adsorption, thin-layer, and gas chromatography), and its mass spectrum. Contrary to findings in other reports, no evidence for inhibitory proteins or peptides was found, and spermine was not an effective inhibitor. The inhibitory activity of untreated seminal plasma from individual ejaculates was highly correlated with the cholesterol content of the ejaculates (r = 0.96), suggesting that the amount of cholesterol determines the inhibitory activity of unfractionated seminal plasma. The inhibitory activity of unfractionated seminal plasma was significantly less, relative to the cholesterol content, than the activity of pure cholesterol, which is consistent with the idea that there are components in seminal plasma that partially counter the effect of cholesterol by promoting the development of acrosomal responsiveness.

Phosphatidylcholine enhances the acrosomal responsiveness of human sperm.

Supplementing bovine serum albumin-containing medium with phosphatidylcholine (PC) accelerated the in vitro development of human sperm acrosomal responsiveness. Responsiveness was assessed by exposing the sperm to progesterone. The maximum effect was produced by incubation with 100 micrograms PC/ml, which resulted in 40% (23-56%) (mean, 95% confidence limits) of the sperm becoming responsive to progesterone at 24 hours, compared to 23% (10-40%) of control sperm. Enhancement was apparent after as little as 6 hours of incubation in vitro, and the number of responsive sperm was still increasing at the last time point tested (30 hours). PC had no apparent ill effects; it did not alter the percentage of motile sperm or the percentage of sperm stained with the supravital stain, Hoechst 33258. Enhanced responsiveness required prolonged incubation in PC, because PC was not effective when it was only applied at the same time as progesterone. Lysophosphatidylcholine did not enhance acrosomal responsiveness when used at concentrations from 10 ng/ml to 100 micrograms/ml, indicating that the effect of PC was not due to trace amounts of lysophosphatidylcholine. PC also increased the response of sperm to the Ca2+/H(+)-exchanging ionophore, ionomycin, suggesting that PC modifies an event that is coincident with or subsequent to the rise in intracellular free Ca2+ that is triggered by progesterone.

Assessing acrosomal status of bovine sperm using fluoresceinated lectins.

Binding of 12 lectins to bull sperm was analyzed to select a lectin that bound preferentially to the acrosomal region. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) were suitably specific for intracellular, acrosome-associated glycoconjugates. Peanut agglutinin exhibited almost no detectable binding to sperm surface receptors, but intense binding to the area of the acrosome anterior to the equatorial segment. In contrast, PSA bound intensely to anterior and equatorial acrosomal regions, and weakly to the other regions of the sperm. Acrosomal labeling by both lectins decreased when sperm were induced to acrosome-react with calcium ionophore. To determine if these lectins could be used to assess acrosomal status, we compared the percentage of acrosome-reacted sperm that were detected by staining with naphthol yellow and erythrosin B with the percentage that were detected by lectin labeling. The incidence of reacted sperm detected by PSA labeling was not significantly different from that detected by naphthol yellow/ erythrosin B (P = 0.46). The incidence of reacted sperm detected by PNA was correlated with the incidence detected by naphthol yellow/erythrosin B, but was significantly lower (P = 0.003). We conclude that labeling permeabilized sperm with fluoresceinated PSA can serve as a rapid assay for acrosomal status.

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 fold (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.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cryopreservation on human sperm acrosomes.

Total acrosin activity and acrosomal status were determined before and after cryopreserving human spermatozoa. Three different cryopreservation protocols were used. Both acrosin activity and the incidence of intact acrosomes decreased during cryopreservation. The magnitudes of the decreases were weakly but significantly correlated (r = 0.29, P less than 0.05), suggesting that acrosomal loss contributed to the decrease in acrosin activity. The effects of the three cryopreservation protocols were not significantly different. Motility decreased more (average 43%) than did the percentage of spermatozoa with intact acrosomes (27%) and the total acrosin activity (24%). These measurements suggested that acrosomal damage may have been secondary to cell death. This hypothesis was tested by determining the acrosomal status of spermatozoa that survived cryopreservation. Spermatozoa that were motile after thawing averaged 96% acrosome-intact; their acrosin activity, however, was significantly less than that of motile, unfrozen spermatozoa. These observations support the idea that the acrosomal loss due to cryopreservation is associated with cell death but also demonstrate decreased total acrosin activity of the acrosome-intact spermatozoa that survive cryopreservation.

A modified and improved assay for sperm amidase activity.

An assay for sperm amidase activity has been modified so that many samples can be measured rapidly, making it more suitable for use in a clinical setting. The modified assay gave the same results (P greater than 0.05) as its lengthier parent assay (Kennedy et al, 1989). The measured enzyme activity was proportional to the number of sperm assayed over the range tested (3.5 x 10(5) to 2.1 x 10(6) sperm). The within-assay coefficient of variation was 7.8 +/- 1.8% (average +/- SE, n = 12), and the between-assay coefficient of variation was 7.2 +/- 1.2% (n = 3). The sensitivity was 7.6 microIU/well.

Regional binding of human anti-sperm antibodies assessed by indirect immunofluorescence.

Indirect immunofluorescence (IIF) can be a powerful tool for determining the site on spermatozoa to which antibodies bind. Human sera that contain anti-sperm antibodies are often of low titre, and may contain antibodies directed against both intracellular and surface antigens. We have developed an IIF protocol that helps to distinguish intracellular from surface labelling. The two types of labelling were differentiated by exposing the spermatozoa to Hoechst 33258, a nuclear stain of low membrane permeability, to tag the spermatozoa that had disrupted membranes. Surface labelling detected in this fashion was patchy. It was much more uniform if the spermatozoa were fixed in paraformaldehyde, or if a univalent, Fab fragment was used as the second antibody. Thus, it is likely that most of the patchy appearance is due to the bivalent second antibody cross-linking mobile antigen-antibody complexes. For some sera, patching was so pronounced that it appeared to remove the label from portions of the sperm surface, giving a misleading picture of the regions to which the antibodies were directed. Fourteen sera were used in IIF and none of them labelled spermatozoa solely on the head or on the tail.

Correlation of acrosomal status and sperm performance in the sperm penetration assay.

The sperm of some infertile men are unable to penetrate zona pellucida-free hamster oocytes but gain that ability after treatment with human follicular fluid (hFF). We asked whether altered incidences of acrosome reacted sperm explained these observations. Patient sperm failing to penetrate oocytes had fewer acrosome reactions than did healthy males, but the percentage reacted was not correlated with oocyte penetration. Sperm incubated 3 hours, then exposed to hFF, exhibited increased penetrations for 7 of 10 males, without an increase in percentage reacted sperm. Sperm incubated 22 hours before hFF treatment had penetrating ability enhanced 250- to 1000-fold, but the percentage reacted increased only sixfold. We conclude that factors other than the percentage reacted sperm are the major determinants of penetration capacity.

The physiology of sperm recovered from the human cervix: acrosomal status and response to inducers of the acrosome reaction.

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)

Methods for evaluating the acrosomal status of mammalian sperm.

A full understanding of the acrosome reaction is central to understanding sperm function. Acrosomal status can be determined on living, motile sperm in only a few mammalian species. For other species, many light microscopic methods have been developed, including colored stains for bright-field microscopy, and probes for fluorescence microscopy. We review the existing methods and the criteria that should be considered in the choice of an assay.

A new procedure for determining acrosomal status of very small numbers of human sperm.

The acrosome of human sperm cannot be easily distinguished by light microscopy. Although several techniques are now available to label the acrosomal region of human sperm and report acrosomal status, they generally require large numbers of sperm. We describe here a new procedure in which sperm are collected and treated on small-pore filters. The acrosomal region is then labeled using fluoresceinated lectin. The main advantage of this method is that it enables the study of the acrosomal status of sperm in samples with very low sperm concentration.