Showing papers on "Acrosome reaction published in 1999"

Ion Channels in Sperm Physiology

Abstract: Fertilization is a matter of life or death. In animals of sexual reproduction, the appropriate communication between mature and competent male and female gametes determines the generation of a new individual. Ion channels are key elements in the dialogue between sperm, its environment, and the egg. Components from the outer layer of the egg induce ion permeability changes in sperm that regulate sperm motility, chemotaxis, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize protein and difficult to study electrophysiologically. Thus understanding how sperm ion channels participate in fertilization requires combining planar bilayer techniques, in vivo measurements of membrane potential, intracellular Ca2+ and intracellular pH using fluorescent probes, patch-clamp recordings, and molecular cloning and heterologous expression. Spermatogenic cells are larger than sperm and synthesize the ion channels that will end up in mature sperm. Correlating the presence and cellular distribution of various ion channels with their functional status at different stages of spermatogenesis is contributing to understand their participation in differentiation and in sperm physiology. The multi-faceted approach being used to unravel sperm ion channel function and regulation is yielding valuable information about the finely orchestrated events that lead to sperm activation, induction of the acrosome reaction, and in the end to the miracle of life.

Infections in the male genital tract and reactive oxygen species

Abstract: In the male genital tract, reactive oxygen species (ROS) are generated by spermatozoa and leukocytes including neutrophils and macrophages. ROS are involved in the regulation of sperm functions such as capacitation and the acrosome reaction. Infections lead to an excessive ROS production, resulting in an 'oxidative burst' from neutrophils/macrophages as a first-line defence mechanism. This is modulated by several cytokines and the pro-oxidant mechanisms of bacteria and viruses. At the site of an infection, the degree of activation of leukocytes, i.e. the amount of ROS produced, and the available antioxidative systems determine whether spermatozoa are damaged or not. During an infection, an imbalance of pro- and antioxidants favouring the former results in oxidative stress which impairs the sperm functions mentioned, as well as motility and fertilization. ROS produced during infections of the testis and epididymis are especially harmful to spermatozoa due to the longer contact time and the lack of antioxidant protection. In the final ejaculate, only very high numbers of ROS-producing leukocytes are detrimental to sperm functions. An infectious injury involving ROS in the prostate gland, seminal vesicles or epididymis could impair sperm functions indirectly. Pro- and antioxidative properties of therapeutics are currently receiving more attention as part of anti-infectious therapies. At present, there are many unresolved questions concerning the exact role of ROS during infections of the male genital tract because of the difficulty of specifically assessing the site of generation and the short-lived effects of ROS. New techniques may enable specific studies to fill this gap in the near future.

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Abstract: Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 35 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium This delay was not observed in the presence of dbcAMP and IBMX Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation

Simple histochemical stain for acrosomes on sperm from several species.

Abstract: The acrosome reaction is an exocytotic 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 contents. Many different methods have been developed to detect the acrosomal status of sperm. These techniques are sometimes complicated, costly, and can be used on only a few species. The aim of this study was to develop an efficient and inexpensive method to assess the acrosomal status of sperm from a variety of species. We prepared and fixed sperm from humans, cattle, swine, rabbits, guinea pigs, and mice and stained them with Coomassie G-250. The acrosomes were stained intensely blue in color. Following capacitation, some sperm were incubated for 1 hr with 10 μM calcium ionophore A23187 to induce the acrosome reaction. They were also stained with Coomassie G-250. Ionophore-treated sperm lacked Coomassie staining over the acrosomal region. Differential interference contrast (DIC), bright field microscopy or Pisum sativum agglutinin staining confirmed that the acrosomes of sperm from these species were reacted in response to calcium ionophore treatment and the acrosome reaction frequencies matched results with Coomassie staining. These results demonstrate that the acrosomal status of mammalian sperm from several species can be determined easily and reliably using this simple Coomassie Blue G-250 staining method. Mol. Reprod. Dev. 52:445–449, 1999. © 1999 Wiley-Liss, Inc.

Identification and characterization of a novel functional estrogen receptor on human sperm membrane that interferes with progesterone effects.

Abstract: The presence of a novel functional estrogen receptor on the human sperm surface has been demonstrated by using different experimental approaches. Ligand blot analysis of sperm lysates, using peroxidase-conjugated estradiol as probe, identified a specific estradiol-binding protein of approximately 29-kDa apparent molecular mass. The same protein band was also revealed by using alphaH222 antibody, which is directed against the steroid binding domain of the genomic estrogen receptor. The biological effects of estrogen receptor were investigated by analyzing calcium fluxes, tyrosine phosphorylation, and acrosome reaction (AR) in response to 17beta-estradiol (17betaE2) and by measuring the steroid influence on calcium and AR in responses to progesterone (P), a well-known physiological stimulus for human spermatozoa. Our results demonstrate that 17betaE2 induces a rapid and sustained increase of intracellular calcium concentrations ([Ca2+]i). This effect is totally dependent on the presence of extracellular calcium, because it is completely abolished in a calcium-depleted medium. The dose-response curve for calcium increase to 17betaE2 is biphasic with a first component in the nanomolar range (effective concentration 50 = 0.60 +/- 0.12 nmol/L) and a second component in the micromolar range (EC50 = 3.80 +/- 0.26 micromol/L). 17BetaE2 stimulates tyrosine phosphorylation of several sperm proteins, including the 29-kDa protein band, and determines a reduction of calcium response to P, finally resulting in inhibition of P-stimulated sperm AR. Conversely, no direct effect of 17betaE2 is observed on AR. 17BetaE2 effects on calcium are clearly mediated by a membrane receptor, because they are reproduced by the membrane-impermeable conjugate of the hormone BSA-E2 and reduced by sperm preincubation with alphaH222 antibody. Taken together, our results clearly show the presence of a functional surface estrogen receptor, of 29 kDa, on human spermatozoa. This receptor may play a role in the modulation of nongenomic action of P in these cells during the process of fertilization.

Isolation and Molecular Characterization of AKAP110, a Novel, Sperm-Specific Protein Kinase A-Anchoring Protein

Abstract: Agents that increase intracellular cAMP are potent stimulators of sperm motility. Anchoring inhibitor peptides, designed to disrupt the interaction of the cAMP-dependent protein kinase A (PKA) with A kinase-anchoring proteins (AKAPs), are potent inhibitors of sperm motility. These data suggest that PKA anchoring is a key biochemical mechanism controlling motility. We now report the isolation, identification, cloning, and characterization of AKAP110, the predominant AKAP detected in sperm lysates. AKAP110 cDNA was isolated and sequenced from mouse, bovine, and human testis libraries. Using truncated mutants, the RII-binding domain was identified. Alignment of the RII-binding domain on AKAP110 to those from other AKAPs reveals that AKAPs contain eight functionally conserved positions within an amphipathic helix structure that are responsible for RII interaction. Northern analysis of eight different tissues detected AKAP110 only in the testis, and in situ hybridization analysis detected AKAP110 only in round spermatids, suggesting that AKAP110 is a protein found only in male germ cells. Sperm cells contain both RI, located primarily in the acrosomal region of the head, and RII, located exclusively in the tail, regulatory subunits of PKA. Immunocytochemical analysis detected AKAP110 in the acrosomal region of the sperm head and along the entire length of the principal piece. These data suggest that AKAP110 shares compartments with both RI and RII isoforms of PKA and may function as a regulator of both motility- and head-associated functions such as capacitation and the acrosome reaction.

Identification of a Human Homologue of the Sea Urchin Receptor for Egg jelly: A Polycystic Kidney Disease-Like Protein

Abstract: Previous studies have shown sequence similarity between a region of the autosomal dominant polycystic kidney disease (ADPKD) protein, polycystin-1 and a sea urchin sperm glycoprotein involved in fertilization, the receptor for egg jelly (suREJ). We have analysed sequence databases for novel genes encoding PKD/REJ-like proteins and found a significant region of homology to a large open reading frame in genomic sequence from human chromosome 22. Northern analysis showed that this is a functional gene [termed the polycystic kidney disease and receptor for egg jelly related gene ( PKDREJ )], but unlike polycystin-1, has a very restricted expression pattern; the approximately 8 kb transcript was found exclusively in testis, coincident with the timing of sperm maturation. The PKDREJ transcript was cloned by screening a testis cDNA library and RT-PCR which revealed a 7660 bp mRNA terminating with a 900 bp 3'UTR and a polyA tail. Comparison with genomic sequence showed that PKDREJ is intronless; sequencing the mouse orthologue revealed a similar structure. The predicted human PKDREJ protein has 2253 amino acids (calculated molecular mass 255 kDa) and sequence similarity over approximately 2000 amino acids with polycystin-1, corresponding to the predicted membrane associated region and the area of homology ( approximately 1000 amino acids) with the suREJ protein (the REJ module). The suREJ protein binds the glycoprotein coat of the egg (egg jelly), triggering the acrosome reaction, which transforms the sperm into a fusogenic cell. The sequence similarity and expression pattern suggests that PKDREJ is a mammalian equivalent of the suREJ protein and therefore may have a central role in human fertilization.

Protein kinases in mammalian sperm capacitation and the acrosome reaction.

Abstract: 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.

Nitric Oxide Regulates Human Sperm Capacitation and Protein-Tyrosine Phosphorylation In Vitro

Abstract: The aim of the present study was to investigate whether the generation of nitric oxide by human spermatozoa is associated with human sperm capacitation and with the tyrosine phosphorylation of sperm proteins. Human spermatozoa were capacitated in the presence or absence of nitric oxide-releasing compounds or nitric oxide synthase inhibitors, and then the percentage of acrosome loss induced by human follicular fluid or by calcium ionophore was determined. The presence of the nitric oxide-releasing compounds primed spermatozoa to respond earlier to human follicular fluid whereas nitric oxide synthase inhibitors decreased the percentage of acrosome reaction. Moreover, nitric oxide modulated tyrosine phosphorylation of sperm proteins. A tight correlation between capacitation and tyrosine phosphorylation regulated by nitric oxide was observed. Results indicate that nitric oxide is involved in human sperm capacitation and emphasize the importance of oxidoreduction reactions in the fine control of sperm physiology.

Structure of the sulfated alpha-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus : patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition

Abstract: The egg jelly coats of sea urchins contains sulfated polysaccharides responsible for inducing the sperm acrosome reaction which is an obligatory event for sperm binding to, and fusion with, the egg. Here, we extend our study to the sea urchin Strongylocentrotus franciscanus. The egg jelly of this species contains a homofucan composed of 2- O -sulfated, 3-linked units which is the simplest structure ever reported for a sulfated fucan. This polysaccharide was compared with other sulfated alpha-L-fucans as inducers of acrosome reaction in conspecific and heterospecific sperm. Although all these fucans are linear polymers composed of 3-linked alpha-L-fucopyranosyl units, they differ in the proportions of 2-O- and 4-O-sulfation. The reactivity of the sperm of each species is more sensitive to the egg jelly sulfated fucan found in their own species. The reactivity of the sperm does not correlate with the charge density of the fucan, but with the proportion of 2-O- and 4-O-sulfation. The pattern of sulfation may be an important feature for recognition of fucans by the sperm receptor contributing to the species-specificity of fertilization.

Intracellular Ca2+-Mg2+-ATPase Regulates Calcium Influx and Acrosomal Exocytosis in Bull and Ram Spermatozoa

Abstract: Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca 2+ -Mg 2+ -ATPase (Ca 2+ pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca 2+ , since incubation of cells in the absence of added Ca 2+ or in the presence of the calcium channel blocker, La 3+ , completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca 2+ concentration ([Ca 2+ ] i ), and this increase was inhibited when extracellular Ca 2+ was chelated by EGTA, indicating that this rise in Ca 2+ is derived from the external medium. This rise of [Ca 2+ ] i took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP 3 -R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP 3 -R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca 2+ elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca 2+ store. We suggest here that the cytosolic Ca 2+ is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca 2+ pump and that the accumulated Ca 2+ is released from the acrosome via an IP 3 -gated calcium channel. The ability of thapsigargin to increase [Ca 2+ ] i could be due to depletion of Ca 2+ in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca 2+ ] i in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca 2+ ] i during capacitation and the AR.

Progesterone-Mediated Calcium Influx and Acrosome Reaction of Human Spermatozoa: Pharmacological Investigation of T-Type Calcium Channels

Abstract: The mechanisms of the progesterone (P4)-activated Ca21 influx and the relationship between the intracellular free Ca21 concentration ([Ca21]i) and the acrosome reaction (AR) were investigated in this study. We compared the [Ca21]i of uncapacitated and capacitated human sperm populations in response to P4 stimulation; characterized the effects of the pharmacological agents pimozide and mibefradil, inhibitors of T-type voltage-operated calcium channels (VOCCT), on the P4-activated Ca21 influx; and determined the effects of these drugs on the P4-initiated AR. Since pimozide can also inhibit calmodulin-dependent enzymes, we examined the effects of the calmodulin antagonist, calmidazolium, on the above-mentioned events. The basal [Ca21]i and the amplitude of the P4-activated Ca21 influx were significantly (p , 0.05) higher in capacitated sperm populations. Also, in capacitated sperm populations, all three pharmacological agents significantly (p , 0.05) inhibited the P4-activated Ca21 influx (IC50): calmidazolium (0.7 mM) . pimozide (8 mM) . mibefradil (11 mM). By contrast, the effects of these drugs on the P4-initiated AR were varied: pimozide (10 and 20 mM) significantly (p , 0.05) increased the percentage of AR spermatozoa, calmidazolium was without effect, and mibefradil (20 mM) significantly (p , 0.05) inhibited the AR. These disparate results do not allow us to reach any definitive conclusion concerning the role of a sperm VOCCT in the mechanism of the P4-initiated AR. However, the differences between the [Ca21]i and AR effects, in particular the inverse relationship in the case of pimozide, suggest a dissociation between the amplitude of the P4-stimulated Ca21 signal and the downstream biological effect of that signal, the AR.

Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa.

Abstract: It is now widely accepted that the higher levels of reactive oxygen species (ROS) produced by damaged or deficient spermatozoa are associated with a loss of motility and a decreased capacity for sperm-oocyte fusion. Furthermore, earlier studies show, under physiological conditions, that some ROS may be involved in capacitation and hyperactivation of human spermatozoa. We measured ROS levels, acrosome reaction (AR) and acrosin activity (AA) in semen samples from suspected subfertile men to reveal the influence of ROS on AR and AA of human spermatozoa. Semen samples were obtained from 60 patients. Samples with > or = 1 x 10(6) leukocytes/mL were excluded from the study. ROS production was determined using a chemiluminescence technique. AR was determined using a triple stain technique. The percentage of acrosome-reacted spermatozoa after low temperature induction of the AR (test value), and the inducibility of AR (= the difference between the test value and the control), were calculated. The AA was analysed by determining the proteolytic potential of spermatozoa on gelatin plates. The mean halo diameter and percentage of halo formation in each sample were measured as AA parameters. Scatter plots of ROS levels and AR parameters showed that the percentage of acrosome reacted spermatozoa and AR inducibility were better in samples with low rather than high ROS levels. On the other hand, there were no apparent similarities between ROS and the AA parameters. Therefore, the percentage of acrosome-reacted spermatozoa and AR inducibility were significantly higher in the low than in the high ROS group (p = 0.028, p = 0.0001, respectively). In addition, there was no significant difference in AA parameters between groups. These findings suggest that lower ROS in semen may have a role in AR but excessive ROS may exert a negative influence on AR, while ROS in semen has no relationship to AA.

Heparin and High-Density Lipoprotein Mediate Bovine Sperm Capacitation by Different Mechanisms

Abstract: Capacitation is an important process in bovine sperm maturation and is an obligatory step prior to fertilization. Two capacitating agents, namely heparin and high-density lipoprotein (HDL), have been shown to induce sperm capacitation. A family of major proteins of bovine seminal plasma designated BSP-A1/A2, BSP-A3, and BSP-30 kDa (collectively called BSP proteins) bind to the sperm surface upon ejaculation via their membrane choline phospholipids. Our previous studies with bovine epididymal sperm showed that BSP proteins potentiate sperm capacitation induced by heparin and HDL. This study was undertaken to clarify the mechanism of capacitation induced by heparin and HDL in the presence of BSP proteins. Washed bovine ejaculated sperm were incubated with heparin (12 microg/ml) or HDL (10-160 microg/ml) in the presence of polyclonal antibodies against purified BSP proteins (anti-BSP proteins). The percentage of capacitated sperm was evaluated after the induction of the acrosome reaction (AR) with lysophosphatidylcholine. When sperm were incubated for 5 h with heparin and anti-BSP proteins (40 microg/ml), the AR level was not significantly different from control levels (16. 8 +/- 0.9% vs. 12.9 +/- 0.9%). In contrast, incubation of sperm for 8 h with HDL and anti-BSP proteins did not inhibit the AR (42.4 +/- 1.1% vs. 17.1 +/- 1.6 for the control samples). We also investigated the effect of heparin and HDL on protein tyrosine phosphorylation associated with capacitation. The tyrosine phosphorylation of a group of proteins was increased in the presence of heparin. However, HDL did not significantly stimulate protein phosphorylation. The increase in phosphorylation was correlated with an increase in the AR after the incubation with heparin but not with HDL. These results indicate that heparin and HDL mediate capacitation via different mechanisms.

The Role of Carbohydrates in the Induction of the Acrosome Reaction in Mouse Spermatozoa

Abstract: Capacitated acrosome-intact mouse spermatozoa bind to the egg's zona pellucida in a receptor-ligand-mediated manner. Mouse zona pellucida 3 (mZP3) is a glycoprotein that functions as a primary ligand and inducer of the acrosome reaction (AR). Multiple sugar residues on mZP3 are thought to be recognized by complementary sugar binding enzymes (glycosidases or glycosyltransferases) or sugar binding lectin-like proteins on the sperm surface. To elucidate the nature of the sugar residues involved in sperm-egg recognition, several neoglycoproteins (ngps) were tested for their ability to induce the AR. Ngps are synthetic glycoproteins with a known monosaccharide conjugated to BSA. Capacitated mouse spermatozoa were treated in the absence or presence of several concentrations of ngps. A significantly greater number of spermatozoa underwent the AR in the presence of mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA than in their absence. Glucose-BSA or galactose-BSA had no effect on the AR. Inclusion of millimolar concentrations of unconjugated sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) neither induced the AR nor blocked induction of the AR by ngps. These results demonstrate that some sugar residues can induce the AR, but only when conjugated to a protein backbone. Glucosaminyl-BSA (but not mannosyl-BSA or galactosaminyl-BSA) was a substrate for sperm-surface galactosyltransferase (GT), an enzyme thought to function as a receptor by binding to complementary glucosaminyl residues on mZP3. These data suggest a possible interaction between protein-conjugated glucosaminyl residues and sperm GT in the induction of the AR.

Detection of partial and complete acrosome reaction in human spermatozoa: which inducers and probes to use?

Abstract: The acrosome reaction (AR), an essential step for achieving mammalian fertilization, was recently introduced as a means of clinical evaluation of male fertility. However, most of the available techniques for acrosomal status assessment (except those employing electron microscopy) do not define whether the measurements represent partial or complete AR. We, therefore, performed a crossover investigation of the types of inducers and probes required for detecting partial or complete AR in human spermatozoa. The acrosomal status before and after stimulation with four AR inducers was evaluated after incubation fo r3hi ncapacitating conditions. We used a fluorescence-activated cell sorter with fluorescein isothiocyanate-conjugated monoclonal antibody CD46 (FITC‐CD46) targeting the inner acrosomal membrane for detecting a complete AR, and fluorescein isothiocyanate‐Pisum sativum agglutinin (FITC‐PSA) targeting the acrosomal content for detection of both partial and complete AR. Without stimulation or following stimulation with progesterone, follicular fluid (FF) or phorbol myristate ester (PMA), the AR could be detected with FITC‐PSA but not with FITC‐CD46. Following stimulation with the calcium ionophore A23187, the AR could be detected by both FITC‐PSA and FITC‐CD46. These results suggest that spontaneous AR as well as AR induced by progesterone, PMA and FF are partial. In contrast, the AR induced by A23187 is total, i.e. both partial and complete. These findings are valuable for both research and clinical purposes and are a step towards an international agreement on a standard test for human sperm AR, for which there is an urgent need.

Characterization of human zona pellucida glycoproteins

Abstract: The human egg may only be fertilized by one spermatozoon to prevent polyploidy. In most mammals, the primary block to polyspermy occurs at the zona pellucida (ZP). Little is known of the human ZP and the changes occurring following fertilization to prevent polyploidy. Using antibodies directed against synthetic peptides predicted from the human ZP2 and ZP3 cDNA, we identified ZP3 as a 53-60 kDa glycoprotein and ZP2 as a 90-110 kDa glycoprotein in prophase-I oocytes. Characterization of the ZP from metaphase II arrested eggs (inseminated-unfertilized and fertilized-uncleaved), shows no visible modification of ZP3, but demonstrates that ZP2 undergoes limited proteolysis in the amino terminal domain, to a 60-73 kDa species, denoted ZP2p, which remains linked to the proteolysed fragments by intramolecular disulphide bonds. A lack of ZP2 proteolytic activity in acrosomal supernatants is consistent with an oocyte origin for the protease. The ZP2-specific protease may be released during cortical granule exocytosis which occurs during meiotic maturation and following sperm-egg fusion as part of the block to polyspermy. Since mouse ZP2 acts as a secondary sperm receptor, it is possible that intact ZP2 binds a secondary egg binding protein, whereas cleaved ZP2 does not, suggesting a possible mechanism for the block to polyspermy.

Expression of Recombinant Human Zona Pellucida Protein 2 and Its Binding Capacity to Spermatozoa

Abstract: The human zona pellucida (ZP) is composed of three major glycoproteins: ZP1, ZP2, and ZP3. The aim of this study was to clarify the role of ZP2 by focusing on the polypeptide structure. We produced in Escherichia coli a recombinant human ZP2 protein (rec-hZP2) corresponding to amino acid sequence 1-206 of the mature protein. The final yield of rec-hZP2 protein was 80 microg/ml Luria Broth medium. After 2-h incubation of human spermatozoa with rec-hZP2 in vitro, an immunofluorescent study indicated that rec-hZP2 bound only to acrosome-reacted spermatozoa. The binding site migrated from the acrosome to the midpiece of the spermatozoa. Rabbit and mouse antisera produced against rec-hZP2 stained native human ZP in the immunofluorescent study, and significantly blocked human sperm binding and penetration into human ZP as compared to control values. The N-terminal polypeptide portion of human ZP2 was shown to contain a binding site for acrosome-reacted spermatozoa and to play an important role in secondary sperm binding and penetration into the ZP.

No evidence for killer sperm or other selective interactions between human spermatozoa in ejaculates of different males in vitro

Abstract: This study examines one of the possible mechanisms of sperm competition, i.e. the kamikaze sperm hypothesis. This hypothesis states that sperm from different males interact to incapacitate each other in a variety of ways. We used ejaculates from human donors to compare mixes of semen in vitro from the same or different males. We measured the following parameters: (i) the degree of sperm aggregation, velocity and proportion of morphologically normal sperm after 1 and 3 h incubation in undiluted semen samples, (ii) the proportion of viable sperm plus the same parameters as in (i) in 'swim-up' sperm suspensions after 1 and 3 h incubation, (iii) the degree of self and non-self sperm aggregation using fluorescent dyes to distinguish the sperm of different males, and (iv) the extent of sperm capacitation and acrosome-reacted sperm in mixtures of sperm from the same and different males. We observed very few significant changes in sperm aggregation or performance in mixtures of sperm from different males compared with mixtures from the same male and none that were consistent with previously reported findings. The incapacitation of rival sperm therefore seems an unlikely mechanism of sperm competition in humans.

Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do?

Abstract: Evidence from pharmacological studies suggests that induction of the acrosome reaction of mammalian spermatozoa by solubilized zona pellucida, and possibly by progesterone, is dependent upon Ca2+ influx through voltage-operated Ca2+ channels. Studies on Ca2+ accumulation and membrane potential in ligand-stimulated or artificially depolarized spermatozoa support such a conclusion. Electrophysiological studies on rodent spermatogenic cells have revealed the presence of a 'T' type voltage-operated Ca2+ current. This current has pharmacological attributes consistent with those of the putative channel responsible for Ca2+ influx mediating the acrosome reaction. However, use of molecular techniques to study human and rodent testis and spermatogenic cells has detected the presence of three different voltage-operated Ca2+ channel subunits. One of these (alpha lE) may generate T-currents, though this is currently disputed. Voltage-operated Ca2+ channel structure and the relationship between channel subunit expression and the characteristics of consequent Ca2+ currents is briefly reviewed. The nature and function of T-channel-mediated Ca2+ influx is examined in the context of the time-course of ligand- and depolarization-induced elevation of [Ca2+]i in mammalian spermatozoa. It is likely that a secondary Ca2+ response (mobilization of stored Ca2+ or activation of a second Ca(2+)-influx pathway) is required for the acrosome reaction. Evidence for the existence and participation of various candidates is discussed (including voltage-operated Ca2+ channels, which may be functionally expressed only in mature spermatozoa), the available evidence favouring a secondary Ca(2+)-influx pathway. Immediate priorities for future research in this area are proposed.

Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20.

Abstract: The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilization. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 micrograms/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2-3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomolgus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

Effect of methyl‐β‐cyclodextrin on the acrosomal responsiveness of human sperm

Abstract: 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.