Acrosomal Exocytosis In Human Sperm Research Articles

The pair ceramide 1-phosphate/ceramide kinase regulates intracellular calcium and progesterone-induced human sperm acrosomal exocytosis.

Before fertilization, spermatozoa must undergo calcium-regulated acrosome exocytosis in response to physiological stimuli such as progesterone and zona pellucida. Our laboratory has elucidated the signaling cascades accomplished by different sphingolipids during human sperm acrosomal exocytosis. Recently, we established that ceramide increases intracellular calcium by activating various channels and stimulating the acrosome reaction. However, whether ceramide induces exocytosis on its own, activation of the ceramide kinase/ceramide 1-phosphate (CERK/C1P) pathway or both is still an unsolved issue. Here, we demonstrate that C1P addition induces exocytosis in intact, capacitated human sperm. Real-time imaging in single-cell and calcium measurements in sperm population showed that C1P needs extracellular calcium to induce [Ca2+]i increase. The sphingolipid triggered the cation influx through voltage-operated calcium (VOC) and store-operated calcium (SOC) channels. However, it requires calcium efflux from internal stores through inositol 3-phosphate receptors (IP3R) and ryanodine receptors (RyR) to achieve calcium rise and the acrosome reaction. We report the presence of the CERK in human spermatozoa, the enzyme that catalyzes C1P synthesis. Furthermore, CERK exhibited calcium-stimulated enzymatic activity during the acrosome reaction. Exocytosis assays using a CERK inhibitor demonstrated that ceramide induces acrosomal exocytosis, mainly due to C1P synthesis. Strikingly, progesterone required CERK activity to induce intracellular calcium increase and acrosome exocytosis. This is the first report, implicating the bioactive sphingolipid C1P in the physiological progesterone pathway leading to the sperm acrosome reaction.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm.

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.

Different Approaches to Record Human Sperm Exocytosis.

Acrosome reaction is an exocytic process that enables a sperm to penetrate the zona pellucida and fertilize an egg. The process involves the fenestration and vesiculation of the sperm plasma membrane and outer acrosomal membrane, releasing the acrosomal content. Given the importance of the acrosome secretion in fertilization, many different methods have been developed to detect the acrosome reaction of sperm. In this chapter, we describe detailed practical procedures to assess the acrosomal status of human spermatozoa. To do this, we resorted to light optical and epifluorescence microscopy, flow cytometry, and transmission electron microscopy. We also itemize the protocol for real-time measurements of the acrosome reaction by confocal microscopy. Further, we discuss the level of complexity, costs, and the reasons why a researcher should choose each technique.This chapter is designed to provide the user with sufficient background to measure acrosomal exocytosis in human sperm.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm.

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l−1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02–0.1 μmol l−1), whereas higher concentrations (>5 μmol l−1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis.

Does dynamin regulate human sperm acrosomal exocytosis? Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. N/A. This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.

Munc18-1 Controls SNARE Protein Complex Assembly during Human Sperm Acrosomal Exocytosis

The spermatozoon is a very specialized cell capable of carrying out a limited set of functions with high efficiency. Sperm are then excellent model cells to dissect fundamental processes such as regulated exocytosis. The secretion of the single dense-core granule of mammalian spermatozoa relies on the same highly conserved molecules and goes through the same stages as exocytosis in other types of cells. In this study, we describe the presence of Munc18-1 in human sperm and show that this protein has an essential role in acrosomal exocytosis. We observed that inactivation of endogenous Munc18-1 with a specific antibody precluded the stabilization of trans-SNARE complexes and inhibited acrosomal exocytosis. Addition of recombinant Munc18-1 blocked secretion by sequestering monomeric syntaxin, an effect that was rescued by α-soluble NSF attachment protein. By electron microscopy, we observed that both the anti-Munc18-1 antibody and recombinant Munc18-1 inhibited the docking of the acrosome to the plasma membrane. In conclusion, our results indicate that Munc18-1 plays a key role in the dynamics of trans-SNARE complex assembly and/or stabilization, a process that is necessary for the docking of the outer acrosomal membrane to the plasma membrane and subsequent fusion pore opening.

Secretory Phospholipase A2 Group IID Is Involved in Progesterone‐Induced Acrosomal Exocytosis of Human Spermatozoa

Phospholipase A2 (PLA(2)) plays a major role during acrosomal exocytosis (AE) in mammalian spermatozoa, but the identity of PLA(2) subtypes present in spermatozoa remains elusive. This study explored whether secretory PLA(2) Group IID (sPLA(2)-IID) isoform is present in human spermatozoa and whether it is involved in AE. Localization and expression of sPLA(2)-IID in human spermatozoa were explored by immunofluorescence staining and Western blot analysis. Occurrence of AE was evaluated by triple staining, and arachidonic acid (AA) levels were quantified by gas chromatography-mass spectrometry. Sperm motion parameters and hyperactivation were analyzed by computer-assisted sperm analysis. sPLA(2)-IID was localized in the postacrosomal region of the head and the midpiece of tail in human sperm. A 16-kd protein band was detected by Western blotting in sperm extracts. Progesterone-induced AE was significantly inhibited in a concentration-dependent manner using a sPLA(2)-IID neutralizing antibody. The increase in AA levels seen during progesterone-stimulated exocytosis was significantly abrogated by the antibody. The sPLA(2)-IID antibody significantly inhibited hyperactivation, sperm curvilinear velocity, and amplitude of lateral head displacement, but it did not affect the proportion of motile sperm. In conclusion, sPLA(2)-IID is present at the head and midpiece in the human sperm, and activation of such sPLA(2)-IID seems to be involved in AE. Therefore, sPLA(2)-IID isoform plays a functional role during the AE in human sperm.

Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calcium-dependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis.

Sphingosine 1-Phosphate and Sphingosine Kinase Are Involved in a Novel Signaling Pathway Leading to Acrosomal Exocytosis

Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. Here we report that this lipid triggers acrosomal exocytosis in human sperm by a mechanism involving a G(i)-coupled receptor. Real-time imaging showed a remarkable increase of cytosolic calcium upon activation with sphingosine 1-phosphate and pharmacological experiments indicate that the process requires extracellular calcium influx through voltage and store-operated calcium channels and efflux from intracellular stores through inositol 1,4,5-trisphosphate-sensitive calcium channels. Sphingosine 1-phosphate-induced exocytosis requires phospholipase C and protein kinase C activation. We investigated possible sources of the lipid. Western blot indicates that sphingosine kinase 1 is present in spermatozoa. Indirect immunofluorescence showed that phorbol ester, a potent protein kinase C activator that can also trigger acrosomal exocytosis, redistributes sphingosine kinase 1 to the acrosomal region. Functional assays showed that phorbol ester-induced exocytosis depends on the activation of sphingosine kinase 1. Furthermore, incorporation of (32)P to sphingosine demonstrates that cells treated with the phorbol ester increase their sphingosine kinase activity that yields sphingosine 1-phosphate. We present here the first evidence indicating that human spermatozoa produce sphingosine 1-phosphate when challenged with an exocytic stimulus. These observations point to a new role of sphingosine 1-phosphate in a signaling cascade that facilitates acrosome reaction providing some clues about novel lipid molecules involved in exocytosis.

Dynamic resolution of acrosomal exocytosis in human sperm

An essential step in mammalian fertilisation is the sperm acrosome reaction (AR) - exocytosis of a single large vesicle (the acrosome) that surrounds the nucleus at the apical sperm head. The acrosomal and plasma membranes fuse, resulting in both the release of factors that might facilitate penetration of the zona pellucida (which invests the egg) and the externalisation of membrane components required for gamete fusion. Exocytosis in somatic cells is a rapid process - typically complete within milliseconds - yet acrosomal enzymes are required throughout zona penetration - a period of minutes. Here, we present the first studies of this crucial and complex event occurring in real-time in individual live sperm using time-lapse fluorescence microscopy. Simultaneous imaging of separate probes for acrosomal content and inner acrosomal membrane show that rapid membrane fusion, initiated at the cell apex, is followed by exceptionally slow dispersal of acrosomal content (up to 12 minutes). Cells that lose their acrosome prematurely (spontaneous AR), compromising their ability to penetrate the egg vestments, are those that are already subject to a loss of motility and viability. Cells undergoing stimulus-induced AR (progesterone or A23187) remain viable, with a proportion remaining motile (progesterone). These findings suggest that the AR is a highly adapted form of exocytosis.

Biological Effects of Recombinant Human Zona Pellucida Proteins on Sperm Function1

The initial interaction between gametes takes place at the level of the sperm surface and the zona pellucida (ZP), the extracellular matrix of the egg in mammals. Successful fertilization requires the proper molecular recognition of the ZP by the sperm. Recently, human ZP was demonstrated to be composed of four proteins: ZP1, ZP2, ZP3, and ZP4. The goals of this study were to determine the effects of recombinant human ZP2, ZP3, and ZP4 on human sperm acrosomal exocytosis and sperm motility. Exposure of sperm to ZP proteins, alone or in combination, promoted acrosomal exocytosis in a time-dependent manner. This effect occurred in parallel with a considerable decrease in progressive motility, coincident with an increase in nonprogressive sperm motility. An analysis of kinetic parameters of ZP-treated sperm demonstrated that a characteristic motility pattern could be defined by values of curvilinear velocity > 63.9 mum/s and linearity <or= 15.5%. A strong correlation between curvilinear velocity and the amplitude of lateral head displacement was also observed. The incidence of sperm having these particular kinetic parameters increased after exposure to ZP proteins. These studies of two processes involved in sperm penetration through the ZP confirm that zona glycoproteins promote acrosomal exocytosis and now establish an additional role for these components as modifiers of sperm motility.

Lack of voltage-dependent calcium channel opening during the calcium influx induced by progesterone in human sperm. Effect of calcium channel deactivation and inactivation.

Progesterone induces calcium influx and acrosomal exocytosis in human sperm. Pharmacologic evidence suggests that voltage-dependent calcium channels (VDCCs) are involved. In this study, membrane potential (Vm) and intracellular calcium concentration ([Ca(2+)](i)) were monitored simultaneously to assess the effect of VDCC gating on the calcium influx triggered by progesterone. Holding the Vm to values that maintained VDCCs in a deactivated (-71 mV) closed state inhibited the calcium influx induced by progesterone by approximately 40%. At this Vm, the acrosomal reaction induced by progesterone, but not by A23187, was inhibited. However, when the Vm was held at -15 mV (which maintains VDCCs in an inactivated closed state), the progesterone-induced calcium influx was stimulated. Furthermore, the progesterone and voltage-dependent calcium influxes were additive. These findings indicate that progesterone does not produce VDCC gating in human sperm.

The cAMP-dependent kinase pathway and human sperm acrosomal exocytosis.

The human sperm acrosome reaction (AR) appears to be analogous to various somatic cell exocytotic events. The AR can be induced in vitro by naturally occurring and synthetic compounds, such as, human periovulatory follicular fluid (hFF) and calcium ionophore A23187. The events that culminate in the AR appear to involve at least two second messenger pathways. One pathway involves the generation of the second messenger adenosine 3':5'-cyclic monophosphate (cAMP) by the amplifying enzyme adenylate cyclase and leading to the activation of cAMP-dependent kinase (PKA). The effect of PKA stimulators, such as, forskolin and cAMP analogues, on AR was tested and they were found to stimulate the AR. Inhibitors of specific components in the PKA pathway, e.g. adenosine analogues and PKA inhibitors, induced dose-dependent reductions in the AR. Furthermore, naturally occurring agonists, including, hFF and solubilized human zona pellucida (sZP), in combination with PKA inhibitors, led to a significantly lower AR. Collectively, these data provide strong support for the role of the PKA pathway in the AR.

A role for diacylglycerol in human sperm acrosomal exocytosis.

Using human spermatozoa stimulated with either progesterone or the Ca2+ ionophore A23187 to undergo acrosomal exocytosis, we have investigated potential pathways for generation of diacylglycerol (DAG) and have examined the possibility that DAG plays an important role in the exocytotic response. Both treatments resulted in rapid and considerable generation of DAG, followed by a limited rise in phosphatidic acid (PA). Further experiments indicated that phospholipase C (PLC) activity is important in this generation of DAG, but phospholipase D activity probably is not. In addition, polyphosphoinositide-specific phosphoinositidase C activation and hydrolysis, of phosphatidylinositol 4,5-bisphosphate appears to be a necessary prerequisite for activation of the PLC pathway. Finally the DAG formed appears to be important in acrosomal exocytosis: (i) blocking DAG metabolism with a DAG kinase inhibitor resulted in both increased endogenous levels of DAG and a significantly increased exocytotic response in stimulated cells and (ii) exogenous DAG induced exocytosis in capacitated spermatozoa whereas PA did not. Taken together, these results suggest that DAG plays a key role in events leading to membrane fusion during human sperm acrosomal exocytosis stimulated by natural agonists.

Progesterone action on the human sperm surface is potentiated by an egg-associated acrosin activator

The progesterone-induced Ca 2+ influx and acrosomal exocytosis in human sperm are recently described examples of non-genomic steroid action on the cell surface. These progesterone effects are known to be inhibited by synthetic protease inhibitors. In this study we tested a hypothesis that a physiological activator of the sperm protease acrosin modulates the sperm response to progesterone. It was found that the activator augments the amplitude of the progesterone-induced Ca 2+ transient and accelerates the progesterone-induced acrosomal exocytosis. These observations suggest a physiological significance of the modulation of nongenomic steroid effects by protease regulators.