Sperm Head Plasma Membrane Research Articles

Are isoforms of capacitating Na+ K+ -ATPase localized to sperm head rafts?

Capacitation begins in the sperm head plasma membrane (HPM). Membrane rafts could house signaling molecules, but although these specialized microdomains have been microscopically visualized in sperm heads, rafts have been isolated for study only from homogenized whole sperm or tails, never purified HPM. Sodium/potassium ATPase (Na+ K+ -ATPase) is a membrane-bound signaling protein that induces capacitation in bull sperm in response to the steroid hormone ouabain, and its subunit isoforms α1, α3, β1, β2, and β3 are known in HPM. This study hypothesized that rafts exist in the HPM of bull sperm, with Na+ K+ -ATPase subunit isoforms preferentially localized there. Western immunoblotting (WB) of HPM from fresh, uncapacitated bull sperm (n = 7 ejaculates), and detergent-resistant membranes isolated by density gradient centrifugation from this HPM, contained the raft-marker protein Flotillin-1; the non-raft fraction did not. HPM, raft, and non-raft contained all known Na+ K+ -ATPase isoforms including, for the first time, the previously unknown α2 isoform. Quantification (ImageQuant Software) found α3 and β1 were relatively dominant isoforms in the HPM raft. WB profiles of raft isoforms differed significantly from HPM and non-raft profiles, with unique banding patterns and amounts, hinting that the capacitation signaling in the now-identified HPM rafts may depend on unique sequences within the isoform structure.

Fertility-associated comprehensive proteomics of bovine sperm head plasma membrane

Fertility-associated comprehensive proteomics of bovine sperm head plasma membrane

Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step.

Developmental sperm contributions: fertilization and beyond

The objective of this review was to examine the role of the various spermatozoal components suspected of actively participating in early human development. The contributions of the fertilizing spermatozoon to the oocyte include, as a minimum, the delivery of the DNA/chromatin, a putative oocyte-activating factor (OAF), and a centriole. Recent data indicate that spermatozoa may also provide the zygote with a unique suite of paternal mRNAs; some transcripts might be crucial for early and late embryonic development and deficient delivery, or aberrant transcription might contribute to abnormal development and arrest. Clinical evidence from assisted reproduction suggests that failure to complete the fertilization process, syngamy, or early cleavage might be the result of an early paternal effect. It is speculated that an abnormal release of a putative OAF and/or dysfunctions of the centrosome and cytoskeletal apparatus may mediate these effects. On the other hand, a later paternal effect resulting in embryonic failure to achieve implantation, pregnancy loss, and/or developmental abnormalities resulting from "carried over" sublethal effects may be associated with sperm nuclear/chromatin defects, including the presence of aneuploidy, genetic anomalies, DNA damage, and possibly other causes. These findings highlight the need for continuous monitoring of clinical results.

Mechanisms underlying the micron‐scale segregation of sterols and GM1 in live mammalian sperm

We demonstrate for the first time that a stable, micron-scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, G(M1), localizes to this same domain in live sperm. Interestingly, the G(M1) undergoes an unexplained redistribution upon cell death. We now demonstrate that G(M1) is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing G(M1) in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the "acrosome reaction." Comparison of the dynamics of CTB-bound endogenous G(M1) and exogenous BODIPY-G(M1) in live murine sperm demonstrate that the sub-acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol- and G(M1)-enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or G(M1) that can be separated biochemically.

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: Functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.

ISOLATING NA+K+ATPASE IN BOAR (Sus scrofa) SPERMATOZOA MEMBRANES

Na+K+ATPase exists in the membranes of bull spermatozoa, concentrated in the head plasma membrane. In sperm, Na+K+ATPase actively transports ions contributing to the intracellular pH change in capacitation, and has very recently been implicated as a direct signalling molecule leading to tyrosine phosphorylation in capacitating bull sperm. The characteristics of Na+K+ATPase in boar sperm are unknown, and were the focus of this study. To determine the existence of Na+K+ATPase, and any preferential location of it within boar sperm, Western blot analyses were performed on whole sperm and purified boar sperm head plasma membrane (HPM). The HPM was isolated (n=8 ejaculates) by nitrogen cavitation and differential centrifugation. Its purity was confirmed by its significant enrichment in two plasma membrane marker enzymes (alkaline phosphatase 224 ± 18%, Mean ± SE; acid phosphatase 248 ± 65% vs activity in homogenised whole sperm); and by the significant diminution of activity of marker enzymes associated with the acrosome (acetylglucosaminidase 41.9 ± 8%) and mitochondria (succinate dehydrogenase; 3.21 ± 1%). Equal protein amounts from whole sperm (WS), HPM and sperm body membranes (SB; whole sperm minus HPM) from 3 ejaculates were analyzed by Western blotting for the alpha (isoforms 1 and 3), and beta (isoforms 1,2 and 3) subunits of Na+K+ATPase. Alpha 1 and 3, and beta 1 were less prevalent in HPM than in WS or SB, while alpha 2 and beta 3 were equally prominent in all fractions, based on densitometry of equal protein concentrations. Monoclonal antibodies for each subunit bound at the kD of the rat brain positive control, and several additional bands for each subunit; these are being investigated as multimers, glycosylated residues, and/or breakdown products. Immunofluorescence of the same monoclonal antibodies then delineated specific subunit locations on the sperm. Fresh sperm (n=4 ejaculates) on poly-LLysine slides at 4°C were permeabilized with Methanol or left intact, and incubated at 37°C with antibodies for each enzyme subunit, and compared to binding that occurred when all steps were at room temperature. The α3 subunit bound post-equatorially regardless of temperature; all other isoforms bound only at room temperature, indicating a high degree of temperature sensitivity. The antibodies to all beta subunits bound over the anterior acrosomes of permeabilised cells only, suggesting the antigens are on internal surface, while the alpha 1 bound to the anterior acrosome of intact cells, indicating an extracellular antigen. These results indicate that Na+K+ATPase exists in specific regions of boar spermatozoa membranes, and its configuration is temperature-sensitive. Funding from NSERC, OMAFRA, Bioniche and Semex Canada gratefully acknowledged. (poster)

Plasma membrane calcium ATPase is concentrated in the head of sea urchin spermatozoa

Plasma membrane Ca2+ATPases (PMCAs) export Ca2+ from cells in a highly regulated manner, providing fine-tuning to the maintenance of intracellular Ca2+ concentrations. There are few studies of PMCAs in spermatozoa, which is surprising considering the importance of this enzyme in all cell types. Here we describe the primary structure and localization of the PMCA of sea urchin spermatozoa (suPMCA). The suPMCA is 1,154 amino acids and has 56% identity and 76% similarity to all 4 human PMCA isoforms. The suPMCA shares the features of a typical PMCA, including domains for calmodulin binding, ATP binding, ATPase phosphorylation, and 10 putative transmembrane segments with two large cytoplasmic loops. Southern blots show that suPMCA is a single copy gene. Treatment of live sea urchin sperm with the PMCA inhibitor, 5-(-6)-carboxyeosin, results in elevations of intracellular Ca2+ and loss of flagellar motility. Immunoblotting and immunoflorescence show that suPMCA is concentrated in the sperm head plasma membrane. In previous work, we showed that a plasma membrane K+ dependent Na+/Ca2+ exchanger (suNCKX), which also keeps Ca2+ low in these cells, is concentrated in the sperm flagellum. Thus, the sperm head and flagellum localize different gene products, both functioning to keep intracellular Ca2+ low, while the sperm swims in seawater containing 10 mM Ca2+.

Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC.

This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

IN VITRO ACTION OF COBRA VENOM ON GOAT SPERMATOZOA ULTRASTRUCTURE BY TRANSMISSION AND SCANNING ELECTRON MICROSCOPY

Goat spermatozoa were incubated in vitro in Tris-citrate buffer, pH 7.2, containing 0, 40, 80, or 160 mug of Naja haje venom/mL buffer for 4 hours. During incubation, the percentages of sperm motility were decreased, while percentages of dead spermatozoa were increased in a time and concentration-dependent manner. The effect of venom concentrations on the ultrastructure of incubated spermatozoa was examined hourly by scanning (SEM) and transmission (TEM) electron microscopy. SEM results showed plasma membrane wrinkling at heads of some spermatozoa after 2 h incubation with 40mug venom. Most spermatozoa suffered membrane wrinkling after 4 h incubation. However, incubation with 80 mug venom caused membrane fractures in most sperm heads after 1 h incubation. The extent and depth of these fractures were increased after 2-3 h incubation. After 4 h incubation, plasma membrane focal erosion of many spermatozoa heads was common. Incubation with 160 mg venom induced sperm head swollen plasma membranes after 1 h incubation. Ruptured and disintegrated membranes were seen after 2 h; lysis and removal of external surface of spermatozoa head plasma membranes were recorded after 3-4 h incubation. TEM indicated slightly swollen areas on the sperm head plasma membrane, but showed normal nuclei, acrosomes, and tail regions after 2 h incubation in 40 mug cobra venom. The swollen areas were accompanied by sperm head membrane disintegration as well as membrane irregularities and distortion of tail mitochondrial cristae after 3-4 h incubation. However, incubation with 80 mug venom showed focal areas of membrane lysis and discontinuity in the sperm heads and tails increasing with incubation time. Severe axoneme and tail longitudinal fiber degeneration and increased numbers of distorted mitochondrial cristae were also observed after 3-4 h incubation. Spermatozoa incubation with 160 mug venom increased severity of plasma membrane dissolution, disintegration, and rupture. Axoneme disorganization and tail longitudinal fiber fusion were seen after 1-2 h; complete deformation of the motility system was recorded after 3-4 h incubation. Nuclei were normal, but acrosomes were partially damaged, and distorted mitochondrial cristae were increased after 4 h incubation. The results indicated that cobra venom induced a concentration and time-dependent alterations on spermatozoa plasma membrane as well as obvious deformation on the motility system and tail mitochondria, which are responsible for sperm viability and fertilizing ability.

Role and regulation of intracellular calcium in acrosomal exocytosis

Calcium influx is required for the mammalian sperm acrosome reaction, an exocytotic event occurring in the sperm head after binding to the egg. Prior to this binding, the spermatozoon undergo, in the female reproductive tract, a series of biochemical transformations, collectively called capacitation. The first event in capacitation is the elevation of intracellular calcium, bicarbonate and hydrogen peroxide, which collectively activate adenylyl cyclase to produce cyclic-AMP, which activates protein kinase A. During capacitation, there is an increase in the membrane-bound phospholipase C, and this binding is highly stimulated by adding epidermal growth factor to the cells. We suggest that zona-pellucida binds to at least two different receptors in the sperm head plasma membrane. One is a G i-coupled receptor that can activate phospholipase Cβ 1 and might regulate adenylyl cyclase to further enhance cyclic-AMP levels. The cyclic AMP activates protein kinase A to open a calcium channel in the outer acrosomal membrane, resulting in a relatively small rise in cytosolic calcium. This rise in Ca 2+ leads to activation of phospholipase Cγ, which is coupled to the second tyrosine kinase receptor. The products of phospholipase C activity, diacylglycerol and inositol-trisphosphate (IP 3), will lead to activation of protein kinase C (PKC) and IP 3-receptor. PKC will open a calcium channel in the plasma membrane and IP 3 will activate the calcium channel in the outer acrosomal membrane, leading to a higher increase in cytosolic calcium. In addition, the depletion of calcium in the acrosome will activate a store-operated Ca 2+ channel, resulting in a very fast increase in cytosolic calcium (300–500 nM), leading to membrane fusion and completing the acrosome reaction.

Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties.

In previous reports, it has been demonstrated that progesterone (P) stimulates capacitation, hyperactivation of human sperm motility and initiates the acrosome reaction (AR). This last effect has been related to the presence of non-genomic receptors for the steroid, localized on the sperm head plasma membrane. These receptors can be detected after treating spermatozoa with the non-permeable conjugate Progesterone - 3-(O-carboxymethyl) oxime: bovine serum albumin-fluorescein isothiocyanate (P-BSA-FITC). In the present study, the presence of progesterone receptors was determined in a selected sperm population with normal morphology and high progressive motility. In addition, other parameters such as the AR, hypo-osmotic swelling test, stability of chromatin and capacitating effect of P were evaluated. The percentage of P-BSA-FITC positive-spermatozoa present in the selected sperm population was higher than in total seminal spermatozoa. Furthermore, spermatozoa incubated with P showed a higher percentage motility and AR than did control spermatozoa. The HOS test indicated that membrane integrity of P-treated spermatozoa was not different to that found in the control sperm suspensions. Unexpectedly, the total sperm population treated with P showed a marked susceptibility to nuclear decondensation with reducing agents. According to these results, the selected sperm population of this study, able to respond to P, may be similar to that with good motility and normal morphology selected in the female reproductive tract, before fertilization.

Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida.

The recognition and binding of sperm cells to the zona pellucida (the extracellular matrix of the oocyte) are essential for fertilization and are believed to be species specific. Freshly ejaculated sperm cells do not bind to the zona pellucida. Physiologically this interaction is initiated after sperm activation in the female genital tract (capacitation) via a yet unknown mechanism, resulting in the binding of a receptor in the apical sperm plasma membrane to the zona pellucida. In order to mimic this biochemically, we isolated zona pellucida fragments from gilt ovaries to prepare an affinity column with the intact zona pellucida structure and loaded this column with solubilized apical plasma membranes of boar sperm cells before and after in vitro capacitation. With this technique we demonstrated that two plasma membrane proteins of capacitated boar sperm cells showed high affinity for zona pellucida fragments. Further analysis showed that these proteins were tyrosine phosphorylated. Plasma membrane proteins from freshly ejaculated sperm cells did not exhibit any zona pellucida binding proteins, likely because these proteins were not tyrosine phosphorylated.

Protein kinases in mammalian sperm capacitation and the acrosome reaction.

Binding to the zona pellucida of an egg stimulates the spermatozoon to undergo the acrosome reaction, a process that enables it to penetrate the egg. Before this binding, the spermatozoon undergoes a series of biochemical transformations in the female reproductive tract, collectively called capacitation. Only capacitated spermatozoa can bind to the zona pellucida and undergo the acrosome reaction. Protein kinases may be involved in the regulation of intracellular Ca2+ during capacitation and the acrosome reaction. The first event in capacitation is the increase in intracellular calcium, bicarbonate and hydrogen peroxide, which collectively activate adenylyl cyclase to produce cyclic AMP, which activates protein kinase A to phosphorylate certain proteins. During capacitation, there is an increase in membrane-bound phospholipase C, and this binding is highly stimulated by the addition of epidermal growth factor to the cells. The capacitated spermatozoon binds to the zona pellucida of the egg via specific receptors and it is suggested that the zona pellucida binds to at least two different receptors in the sperm head plasma membrane. One is a Gi-coupled receptor that can activate phospholipase Cbeta1 and may regulate adenylyl cyclase to further increase cyclic AMP concentrations. The cyclic AMP activates protein kinase A to open a calcium channel in the outer acrosomal membrane, resulting in a relatively small increase in cytosolic calcium. This increase in Ca2+ leads to activation of phospholipase Cgamma, which is coupled to the second tyrosine kinase receptor. The products of phosphatidyl-inositol bisphosphate hydrolysis by phospholipase C, diacylglycerol and inositol-trisphosphate, induce the activation of protein kinase C and a calcium channel in the outer acrosomal membrane, respectively. Protein kinase C opens a calcium channel in the plasma membrane and, together with the inositol-trisphosphate-activated calcium channel, leads to a second and higher increase in cytosolic calcium. In addition, the depletion of calcium in the acrosome activates a capacitative calcium entry mechanism in the plasma membrane, leading to a rapid increase in cytosolic calcium (300-500 nmol l(-1)). This increase in intracellular calcium concentration (and pH) leads to membrane fusion and the acrosome reaction.

Role of sperm head syndecan at fertilization in fish.

Fish sperm head plasma membranes have been demonstrated to contain syndecan (transmembrane heparan sulfate proteoglycan) immunofluorohistochemically and using immunoblot analysis and transglutaminase (TGase) by histochemistry. In order to examine the involvement of syndecan in fertilization, mature eggs were inseminated by direct mixing with untreated sperm or with sperm pretreated with an antiheparan sulfate (HS) antibody monoclonal (mAb), bovine serum albumin, human transferrin, or a TGase inhibitor (monodansylcadaverine, cystamine, and iodoacetamide). The fertilization rates of eggs inseminated with untreated and albumin-pretreated sperm were approximately 99.3% and 94.7%, respectively. Those of eggs pretreated with the anti-HS antibody and transferrin were 0% and 5.4%, respectively, whereas use of sperm pretreated with TGase inhibitors resulted in fertilization rates of approximately 13.2-17.8%. These results indicate that sperm head syndecan play an important role in fish sperm-egg contact and/or binding and that TGase inhibitors may reduce the fertilization rate by inhibiting sperm motility.

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa.

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.

Changes in lipid diffusibility in the hamster sperm head plasma membrane during capacitation in vivo and in vitro.

The technique of fluorescence recovery after photobleaching (FRAP) was employed on spermatozoa labeled with the fluorescent lipid analogue C14dil to provide two measures of lateral diffusion in the plane of the sperm plasma membrane during capacitation in vivo and in vitro: the diffusion coefficient (D) for C14dil and the fraction of C14dil that is free to diffuse (%R) within the domain. To evaluate changes in lipid diffusibility during capacitation in vivo, spermatozoa were recovered from the uterus within 30 min after ejaculation or from the oviduct at 2, 4, 6 and 8 hr after mating. To compare the changes which occur in vivo with those which occur during capacitation in vitro, caudal epididymal spermatozoa were incubated under capacitating or non-capacitating (control) conditions for 4 hr. Although transient changes in D occurred during the course of capacitation, there was no net change in D for either anterior (AH) or posterior head (PH) domains following capacitation in vitro or in vivo. Significant differences in the lipid diffusion coefficient between the two head domains were observed during the course of capacitation. A transient decrease in %R was observed for the AH domain during capacitation in vitro and incubation under control conditions, but no significant change in %R was observed in the AH domain during capacitation in vivo. A significant decline in %R of the PH domain was observed for spermatozoa during capacitation in vivo, in vitro and following incubation under non-capacitating conditions. These data indicate that the changes in the lipid diffusibility of the AH and PH domains which occur during capacitation in vivo exhibit both similarities and differences to those which occur during capacitation in vitro.

Voltage dependent calcium channels in mammalian spermatozoa.

Calcium influx is an absolute requirement for the physiological acrosome reaction in sperm from all sources examined, both invertebrate and mammalian. Pharmacological studies suggest that the major channel in the sperm head plasma membrane responsible for modulating calcium entry and intracellular ionized calcium levels could be either an L-type (a class of high voltage-activated) or a T-type (low voltage-activated) voltage-dependent calcium channel. Patch clamp analysis of calcium currents in immature spermatogenic cells demonstrates the presence of T-type currents. Therefore, an argument has been put forth that the acrosome reaction of ejaculated sperm is regulated by a T-type calcium channel. However, indirect analysis of calcium currents in mature sperm after transfer of ion channels to planar lipid bilayers detects three current types, including that similar, but not identical, to an L-type channel, but no T-type currents. Molecular cloning of the alpha-1 pore forming subunit of calcium channels expressed in the male reproductive tract and in ejaculated sperm has resolved this controversy, demonstrating the existence of only high voltage-activated channels. Further analysis of the alpha-1 subunit isoform from rat and human testis and sperm suggests that, as a result of alternate splicing, this L-type alpha-1 subunit could produce calcium currents that were T-like, e.g., transient, rapidly inactivating with slow deactivation. Multiple splice variants of this isoform were detected in human testis, suggesting a correlation with intra-individual variation in the ability of sperm to undergo an induced acrosome reaction and with male infertility. These variants could be developed as useful biomarkers for susceptibility to environmental and occupational toxicants. Knowledge of calcium channels structure will also contribute to design of new male contraceptives based on existing calcium channel antagonists.

Localization and role of sulfoglycolipid immobilizing protein 1 on the mouse sperm head.

Sulfoglycolipid immobilizing protein 1 (SLIP 1) is an evolutionally conserved sperm head plasma membrane protein (M(r) = 68 kDa) that binds to sulfogalactosylglycerolipid (SGG), the major sulfoglycolipid present in mammalian sperm. The purpose of this study was to characterize the initial localization and the immunoaggregated relocalization of SLIP1 on the mouse sperm head. Direct immunofluorescence (DF) of live sperm using FITC-antiSLIP1 Fab fragments and FITC-antiSLIP1 IgG indicated that SLIP1 was present in the postacrosomal region of the sperm head, although the intensity of immunostaining by FITC-antiSLIP1 IgG was greatest at the border between the postacrosomal region and the acrosome. Unlike that observed with FITC-antiSLIP1 Fab, DF using FITC-antiSLIP1 IgG indicated that SLIP1 was also present in the anterior tip of the sperm head convex ridge. Results from electron microscopic studies, using antiSLIP1 IgG followed by protein A-gold on live mouse sperm, were similar to the DF findings. In contrast, indirect immunofluorescence (IIF) of live mouse sperm using antiSLIP1 IgG and FITC-secondary antibody IgG detected SLIP1 in the sperm head convex ridge only. The IIF and DF results strongly suggest that these bivalent antibodies could induce the sperm antigen relocalization on live sperm heads. SLIP1 redistribution may be dependent on availability of excess SGG, the SLIP1 binding ligand, based on the observation that purified exogenous biotinylated SLIP1 bound to live mouse sperm at both the postacrosomal and convex ridge regions of the mouse sperm head. Immunoaggregation induced by the primary antiSLIP1 IgG or antiSLIP1 Fab with secondary antibody IgG did not cause the acrosome reaction, suggesting that SLIP1 is not involved in sperm signal transduction. Furthermore, postacrosomal SLIP1 was shown to be involved in zona binding, since sperm pretreated with antiSLIP1 Fab fragments (100 micrograms/ml) bound to the egg zona pellucida in vitro at approximately 35% of control levels.

Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction.

Progesterone initiation of te human sperm acrosome reaction (AR) includes stimulation of a transient Ca2+ influx and a transient Cl- efflux. A role for one or more plasma membrane receptors has been suggested, but, except for evidence supporting the involvement of a sperm GABAA-like receptor/Cl- channel, there is little information about possible receptor identity. Here, we attempt to identify plasma membrane progesterone receptors in human sperm using a mouse monoclonal antibody (mAb-C262) raised against the C-terminal steroid-binding domain of the human intracellular progesterone receptor. C-262 inhibited the progesterone-initiated AR in a dose-dependent manner. Maximum inhibition was 77% as detected by fluorescein isothiocyanate (FITC)-concanavalin A (conA). Motility was unaffected. A control mouse mAb (h-151) raised against the human estrogen receptor did not inhibit the progesterone-initiated AR. Western blotting with C-262, but not with h-151, detected a major sperm protein band of 50-52 kDa. In indirect immunofluorescence localization studies, live and ethanol-fixed uncapacitated sperm and fixed capacitated sperm incubated with C-262, but not with h-151, displayed fluorescence at the equatorial segment region of the sperm head plasma membrane. In spectrofluorometric studies using capacitated sperm loaded with the Ca2+ probe Fura-2 or the Cl- probe MEQ, C-262 but not h-151 inhibited both Ca2+ influx and Cl- efflux. These ion fluxes could be due to the binding of progesterone to two different receptor/channels or to its binding to one and cross talk with the other. Our results strongly support the involvement of sperm plasma membrane receptors in the progesterone-initiated AR and provide a candidate for one such receptor.