Evidence for the role of a trypsin-like enzyme in the hamster sperm acrosome reaction.

Acrosome reactions occurring in vitro in hamster sperm capacitated by bovine follicular fluid were severly inhibited by four synthetic trypsin inhibitors and by Zn2+. Three polypeptide trypsin inhibitors and a synthetic chymotrypsin inhibitor did not inhibit the acrosome reaction, and Ca2+ overcame the inhibition by Zn2+. These results suggest that a trypsin-like enzyme (possibly acrosin) plays a role in the acrosome reaction.     

Studies related to progesterone-induced hamster sperm acrosome reaction

Experiments were designed to characterize the effect of progesterone on the hamster sperm acrosome reaction (AR). Progesterone stimulated exocytosis of previously capacitated spermatozoa in a dose-dependent manner Progesterone-3-(O-carboxymethyl)oxime:BSA conjugate also induced AR when added to capacitated sperm suspensions. EGTA and La³⁺, added 10 min before progesterone, completely abolished the steroid-stimulatory effect. Benzamidine, a trypsin inhibitor, also inhibited AR when added to sperm cells 10 min before progesterone. This effect was avoided when spermatozoa were treated with the Ca2+ ionophore ionomycin. Conversely, the H+ ionophore PCCP, or the Na⁺/K⁺ ionophore nigericin, did not prevent the effect of the inhibitor. Results suggest that progesterone acts on the hamster sperm plasma membrane to stimulate exocytosis, which requires external Ca2+ and presumably Ca2+ influx. In addition, a sperm trypsin like protease may be part of the mechanism by which progesterone stimulates AR. Since the ionomycin-induced AR does not require this proteolytic activity, the possible involvement of such an enzyme in the progesterone-stimulated Ca2+ influx necessary for the occurrence of AR is discussed. © 1996 Wiley-Liss, Inc.     

Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm

The glycosaminoglycans (GAGs) hyaluronic acid and heparin were added (10 micrograms and 100 micrograms/ml to golden hamster sperm suspensions previously incubated for 4.5 h under capacitating conditions. After additions, sperm were incubated for 5-15 min and acrosome reactions (AR) assayed in motile sperm by phase contrast microscopy. Hyaluronic acid and heparin significantly stimulated AR over control levels. Hyaluronic acid did not stimulate AR 15 min after addition to sperm previously incubated for only 2.5 h. Pre-incubation of hyaluronic acid with streptomyces hyaluronidase destroyed the ability of that GAG to stimulate the AR. These results indicate that GAGs (at least one of which, hyaluronic acid, is present in the oocyte cumulus oophorous) can rapidly stimulate the acrosome reaction in motile previously capacitated hamster sperm.     

Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction

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

Evidence suggesting a role for cyclic nucleotides in acrosome reactions of hamster sperm in vitro

There have been conflicting reports concerning the involvement of cyclic nucleotides in sperm capacitation. We have examined the effects of micromolar concentrations of dibutyryl cyclic AMP (Bt2cAMP) and of the phosphodiesterase inhibitors SQ20009 and ICI63,197 on hamster sperm incubated under in vitro capacitating conditions. Washed hamster sperm were incubated in a capacitation media containing bovine serum albumin, and a protein-free "motility-factor" from bovine adrenal cortex. Incubation for 3.5 hours was followed by addition of one of the compounds (0.1-10 microM) or control buffer. At the time of addition and after 30-120 minutes further incubation, sperm were examined by phase contrast microscopy. The final motility was similar to the initial motility (50-70%) and the same in incubation of controls or experimental compounds. Bt2cAMP, SQ20009, and ICI63,197 at these concentrations stimulated acrosome reactions to a statistically significant extent (P less than 0.005) compared to controls. Activation was stimulated to a varying degree by all three experimental compounds. These results suggest a role for cyclic nucleotides in capacitation and the acrosome reaction of hamster sperm.     

Hamster sperm glycine receptor: evidence for its presence and involvement in the acrosome reaction

Recent reports have provided evidence for the presence of amino acid neurotransmitter receptor/chloride channels in human and porcine spermatozoa and their involvement in the acrosome reaction (AR). In this work we investigated whether a glycine receptor (GlyR) was present in golden hamster sperm, and whether it had a role in the hamster AR. The neuronal GlyR agonist glycine, stimulated in a dose-dependent manner, the AR of hamster spermatozoa previously capacitated for at least 3 hr. This stimulation was completely inhibited by 50 microM (+)-bicuculline and by concentrations of strychnine as low as 10-50 nM; both agents are antagonists of neuronal GlyR when used at the concentrations reported in this study. beta-Alanine, another agonist of the neuronal GlyR, also stimulated the AR. The AR-stimulatory effect of this compound was completely abolished by 50 nM strychnine. The inhibitory effect of strychnine on the glycine-induced hamster sperm AR was completely overcome by subsequent treatment with the calcium ionophore ionomycin, demonstrating that the strychnine effect was specific for GlyR. Additional binding studies with (3)[H]-strychnine, the typical radioligand used to detect GlyR in several cells, demonstrated for the first time the presence of specific binding sites for strychnine in the hamster spermatozoa. Interestingly, binding increased during in vitro capacitation, particularly in those sperm suspensions showing high percentages of AR. Taken together these results strongly suggest the presence of a GlyR in the hamster spermatozoa, with a role in the AR when activated.     

Substructure of a cytoskeletal complex associated with the hamster sperm acrosome

Whole mount and thin section preparations of intact and selectively disrupted hamster spermatozoa revealed an organized array of cytoplasmic filaments associated with specific regions of the acrosome. The filaments were localized along the ventral surface of the spermatozoon and extended from its tip, distally to the anterior margin of the equatorial segment. Individual filaments were 11-13 nm in diameter and they were aligned parallel to one another to form a two-dimensional sheet oriented in the long axis of the spermatozoon. The filament complex adhered preferentially to the cytoplasmic surface of the outer acrosomal membrane rather than the plasma membrane. Examination of disrupted spermatozoa revealed that the distribution of this cytoskeletal assembly correlated with the distribution of a specific acrosomal matrix component. The possible role of this complex in the acrosome reaction or in the organization of acrosomal matrix domains is discussed.     

Zona-induced acrosome reaction of hamster spermatozoa

It is well established that the zonae pellucidae of mature unfertilized eggs have the ability to induce the acrosome reaction of capacitated spermatozoa. To determine if this capacity of the zona is species-specific, hamster spermatozoa were allowed to attach to the zonae of homologous and heterologous eggs and examined for the acrosome reaction. The zonae of eggs from six different species were tested and the zona of hamster egg was found to have the strongest capacity to induce the acrosome reaction of hamster spermatozoa, followed by human and rat zonae. The zonae and mouse, guinea pig, and domestic fowl eggs were incapable of inducing the acrosome reaction of hamster spermatozoa. The acrosome reaction-inducing ability of the hamster zona was found to increase during maturation in the ovary. The zona of mature unfertilized hamster eggs maintained their acrosome reaction-inducing ability even after aldehyde fixation or storage in a highly concentrated solution of ammonium sulfate.     

Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction

Recently, it has been demonstrated that mouse sperm contain a protein with properties similar to the inhibitory guanine nucleotide-binding regulatory protein, G_i (Kopf, G. S., Woolkalis, M. J., and Gerton, G. L. 1986. J. Biol. Chem., 261, 7327–7331). Since sperm-zona pellucida interaction represents a specialized form of intercellular communication and signal transduction we examined the role of the mouse sperm G_i-like protein in the zona pellucida-induced acrosome reaction using mechanically isolated, structurally intact zonae pellucidae. Sperm capacitated for 90 min in the presence of increasing concentrations of islet-activating protein (IAP) bind to the zona pellucida to a similar extent as control sperm incubated in the absence of this toxin. The zona pellucida-induced acrosome reaction, however, is inhibited in a concentration dependent manner by IAP, with half-maximal effects at 0.1-1.0 ng/ml IAP. IAP does not affect the ability of the sperm to become capacitated, but inhibits the cells from progressing into an intermediate stage prior to the completion of the acrosome reaction. When sperm are capacitated in the presence of 100 μM guanosine-5′-O-(3-thiotriphosphate) for 60 min prior to the addition of IAP during the final 30 min, the IAP-induced inhibition of the zona pellucida-induced acrosome reaction is abolished; capacitation in the presence of 100 μM guanosine-5′-O-(2-thiodiphosphate) does not abolish the inhibitory effects of IAP. The target of the IAP effect on intact sperm appears to be at the level of the G_i-like protein since IAP-catalyzed ³²P-ADP-ribosylation of the M_r = 41,000 substrate in detergent extracts of sperm is reduced when intact sperm are preincubated with IAP during capacitation. These data suggest that the mouse sperm G_i-like protein plays an intermediary role in the zona pellucida-induced acrosome reaction.     

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((?)δ⁹ THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [ H]CP-55,940 to sperm, defined as total binding displaced by (?)δ⁹ THC, was saturable: K_D 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (?)δ⁹ THC, and (+)δ⁹ THC. The rank order of potency to inhibit binding of [ H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (?)δ⁹THC > (+)δ⁹THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley-Liss, Inc.     

Effect of sperm diluents on the acrosome reaction in canine sperm

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.     

Zona pellucida-induced acrosome reaction in boar sperm

Induction of the acrosome reaction in boar sperm by the zona pellucida (ZP) was investigated. A modified cytochemical staining method for measuring the acrosome reaction in boar sperm gave equivalent results to those obtained with transmission electron microscopy. Isolated heat-solubilized ZP effectively induced the acrosome reaction in boar sperm at a concentration of 25 micrograms/ml. Electrophoretically purified ZP components were also tested for acrosome reaction-inducing activity; both the 55,000 and 90,000 components of the ZP were effective. The carbohydrate moiety of the 55,000 component was necessary for activity because the polypeptides derived by chemical deglycosylation of the two glycoproteins did not induce the acrosome reaction.     

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

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

The effects of products and inhibitors of arachidonic acid metabolism on the hamster sperm acrosome reaction

The mammalian sperm acrosome reaction (AR) is a fusion and fenestration of sperm head membranes which is essential for fertilization. Our earlier work demonstrated that arachidonic acid could stimulate the AR 15 min after addition to hamster sperm capacitated by incubation for 4.5 h. The present study was undertaken to determine whether inhibitors of arachidonic acid metabolism could affect the stimulation of the AR by arachidonic acid and whether products of its metabolism could stimulate the AR. Phenidone or nordihydroguaiaretic acid, inhibitors of both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, and docosahexaenoic acid, a cyclo-oxygenase pathway inhibitor, inhibited the AR induced by arachidonic acid. PGE2, a product of the cyclo-oxygenase pathway of arachidonic acid metabolism and 5- or 12-hydroxyeicosatetraenoic acid (HETEs) products of the lipoxygenase pathway, stimulated the AR when added to sperm capacitated by incubation for 4.5 h. Prostaglandins not derived from arachidonic were also tested: PGE1 stimulated the AR, but PGF1 alpha and PGA2 did not. We suggest that arachidonic acid metabolites produced by the sperm and by the female reproductive tract are important for the mammalian sperm AR.     

Evaluation of sperm acrosome reaction in the Asiatic elephant

This study focuses on the effect of chemicals on acrosome reaction in elephant spermatozoa. Semen was collected at the Washington Park Zoo in Portland, Oregon, from an 11-yr-old Asian elephant by artificial vagina (7 ejaculates) and transported to Mahidol University in Bangkok in extender at 4 to 5 degrees C within 24 to 28 h. A total of 500 x 10(6) sperm/mL was used for the control and for each of the 4 treatment groups: 1) cAMP (0.1 mM); 2) caffeine (0.1 mM); 3) Penicillamine hypotaurine and epinephine, PHE (penicillamine 2 mM, hypotaurine 1 mM, epinephine 1 mM); and 4) heparin (10 microg/mL) at 39 degrees C for 2 h. Aliquots were removed and the sperm viability, abnormal morphology, and acrosome status were evaluated by triple stain technique. Transmission electron microscopy (TEM) was used to observe changes of the sperm head membrane in all treatment groups. Trypan blue reliably stained dead spermatozoa, while rose Bengal stained only the spermatozoa with intact acrosomes. The concentration of dead sperm cells was similar in the 4 groups. The percentages of live acrosome-reacted spermatozoa in the control and in groups treated with caffeine, PHE, cAMP and heparin were 19.5 +/- 4.3, 38.1 +/- 4.0, 34.8 +/- 3.7, 29.8 +/- 0.8 and 28.0 +/- 4.2, respectively. The acrosome reaction rate was higher in the treatment groups than in the control (P<0.05). Caffeine and PHE caused significantly higher acrosome reaction of the sperm head than cAMP or heparin (P<0.05). The electron micrographs showed that the acrosome reaction occurred by the presence of apical vesiculation. The results indicated that 1) the triple stain technique allowed for evaluation of both viability and acrosome reaction simultaneously in elephant spermatozoa,2) acrosome reaction occurred at a high rate in all 3 treatment groups. 3) the effects of caffeine and PHE were significantly higher (P<0.05) than of cAMP and heparin, and 4) the data obtained from the triple stain technique corresponded to those from TEM.     

Stimulation of an exocytotic event, the hamster sperm acrosome reaction, by cis-unsaturated fatty acids

The influence of extracellular Mg2+ on Ca2+ ionophore (A23187 and ionomycin) induced secretion and changes in the cytosol pH of rabbit neutrophils suspended in Ca2+-free buffer has been investigated. While extracellular Ca2+ is obligatory for ionomycin induced secretion, we have defined conditions under which A23187 can induce secretion in Ca2+-free media. The different behaviour of these two Ca2+ ionophores is discussed on the basis of their different counter cation specificities.     

Potassium ion influx and Na+,K+-ATPase activity are required for the hamster sperm acrosome reaction

The role of a K+ ion influx and Na+,K+-ATPase activity in the hamster sperm acrosome reaction (AR) was examined, using a range of concentrations of K+,K+ ionophores and a Na+,K+-ATPase inhibitor. Washed epididymal hamster sperm, capacitated in vitro in an artificial medium containing 2 mM Ca2+, 147 mM Na+, and 3, 6, 12, 18, or 24 mM K+, began undergoing the AR after 3 h of incubation. Sperm incubated in low K+ (0.9 mM) failed to undergo the AR even after 5 h of incubation. Sperm in 0.9 mM K+ could be induced to undergo the AR when either K+ (12 mM) alone or K+ (12 mM) with 0.1 microM nigericin was added after 3.5 h of incubation. The addition of K+ alone stimulated the AR in 30 min, whereas nigericin plus K+ stimulated the AR 15 min after addition. Neither nigericin added alone (0.9 mM K+) nor nigericin plus 12 mM K+ added to a low Ca2+ (0.35 mM) system resulted in acrosome reactions. Valinomycin (1 nM) did not stimulate the AR when added together with K+ (3-24 mM) to sperm incubated in 0.9 mM K+ for 3.5 h but markedly decreased sperm motility. Micromolar levels of ouabain blocked the AR when added between t = 0--3 h to sperm incubated with 3-24 mM K+. Inhibition of AR by the addition of 1 microM ouabain to sperm incubated with 3 mM K+ was completely reversed by the addition of 0.1 microM nigericin at t = 3.5 h. These results suggest that Na+,K+-ATPase activity and the resulting K+ influx are important for the mammalian sperm AR. Some similarities between requirements for the hamster sperm AR and secretory granule exocytosis are discussed.     

Capacitation and the acrosome reaction in equine sperm.

During sexual reproduction, the sperm and oocyte must fuse before the production of a diploid zygote can proceed. In mammals such as equids, fusion depends critically on complex changes in the plasma membrane of the sperm and, not surprisingly, this membrane differs markedly from that of somatic cells. After leaving the testes, sperm cease to synthesize plasma membrane lipids or proteins, and vesicle-mediated transport stops. When the sperm reaches the female reproductive tract, it is activated by so-called capacitation factors that initiate a delicate reorientation and modification of molecules within the plasma membrane. These surface changes enable the sperm to bind to the extracellular matrix of the egg (zona pellucida ZP) and the zona then primes the sperm to initiate the acrosome reaction, an exocytotic event required for the sperm to penetrate the zona. This paper will review the processes that occur at the sperm plasma membrane before and during successful penetration of the equine ZP. It is noted that while several methods have been described for detecting changes that occur during capacitation and the acrosome reaction in bovine and porcine sperm, relatively little has been documented for equine sperm. Special attention will therefore be dedicated to recent attempts to develop and implement new assays for the detection of the capacitation status of live, acrosome-intact and motile equine sperm.