The ionophore-induced acrosome reaction differs structurally from the spontaneous acrosome reaction.

The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-induced vesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation.     

Physiological inducers of the acrosome reaction

This article reviews recent studies on physiological inducers of the acrosome reaction in starfish. Upon encountering the jelly coat of eggs, starfish sperm undergo the acrosome reaction in response to a cooperation of three jelly components: a sulfated glycoprotein named acrosome reaction-inducing substance (ARIS), a group of steroidal saponins named Co-ARIS, and an oligopeptide presumably having an activity to increase the intracellular pH of sperm. ARIS induces the acrosome reaction in high Ca²⁺ or high pH sea water. In normal sea water, both ARIS and Co-ARIS are required for the induction. In addition to ARIS and Co-ARIS, a third jelly component, the oligopeptide, is necessary to mimic the full capacity of the jelly coat to induce the acrosome reaction. ARIS and Co-ARIS cooperatively increase the intracellular Ca²⁺ by stimulating Ca²⁺ channels, while the oligopeptide increases the intracellular pH by stimulating Na⁺/H⁺ exchange systems. When sperm meet the eggs, both changes are simultaneously achieved in them and thus they undergo the acrosome reaction.     

Modulation of spermatozoon acrosome reaction

Spermatozoon acrosome reaction is an exocytotic event of the utmost importance for the development of mammalian fertilisation. Current evidence shows that the triggering of the acrosome reaction (AR) could be regulated by the action of diverse compounds, namely, metabolites, neurotransmitters and hormones. The aim of the present review is to describe the modulating effects of several compounds that have been classified as inductors or inhibitors of acrosome reaction. Among AR inductors, it is necessary to mention progesterone, angiotensin II, atrial natriuretic peptide, cathecolamines, insulin, leptin, relaxin and other hormones. Regarding the inhibitors, oestradiol and epidermal growth factor are among the substances that retard AR. It is worth mentioning that gamma-aminobutyric acid, a neurotransmitter known to be an inhibitor in the central nervous system, has been shown to induce AR. The multiple hormones located in the fluids of the female reproductive tract are also likely to act as subtle regulators of AR, constituting a fundamental aspect for the development of successful fertilisation. Finally, it is necessary to emphasise that the study of regulation exerted by hormones and other compounds on AR is essential for further understanding of mammalian reproductive biology, especially spermatozoon physiology.     

The acrosome reaction in human spermatozoa

During gamete interaction, sperm acrosome reaction (AR) induced by oocyte investment is a prerequisite event for the spermatozoa to pass through the zona pellucida (ZP), fuse with and penetrate the oocyte. Progesterone (P4), secreted by cumulus cells, is an important cofactor for the occurrence of this exocytosis event. The AR results from the fusion between outer acrosomal and plasma membranes, leading to inner acrosomal membrane exposure. Binding of agonists, P4 or ZP3 glycoprotein, to plasma membrane sperm receptors activates intraspermatic signals and enzymatic pathways involved in the AR. Among the proteins or glycoproteins described as potential sperm receptors for ZP, Gi/Go protein-coupled and tyrosine kinase receptors have been described. Sperm receptors for P4 are poorly characterized, except a putative GABA(A)-like receptor. ZP- and P4-promoted AR is mediated by an obligatory intracellular calcium increase, appearing first at the acrosome equatorial segment and spreading throughout the head. The plasma membrane channels involved in calcium entry are operated by a plasma membrane depolarization and protein phosphorylations mediated by protein kinase C and tyrosine kinase protein. Part of the calcium increase could also be due to intracellular store release through IP3- and nucleotide (cAMP)-gated channels. Besides adenylate cyclase and phospholipase C activations, intracellular calcium increase also stimulates PLA2 activity and actin depolymerization, leading to membrane fusion. Evaluation of AR by staining or fluorescent probes can be useful to predict fertilization success and to direct the therapeutic strategy in male infertility.     

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.     

Human oocyte-cumulus complexes stimulate the human acrosome reaction

Oocyte-cumulus complexes were obtained, after induced ovulation, from infertile patients participating in an in-vitro fertilization programme. About 6 h after retrieval and depending on the expansion of the cumulus, 100,000 motile spermatozoa, prepared by a migration-centrifugation method, were added. After 14-18 h incubation at 37 degrees C, oocytes were examined for signs of fertilization (pronuclei and polar body formation) and then removed; spermatozoa remaining in the incubation medium were fixed for transmission electron microscopy. To provide an adequate number of cells for observation, spermatozoa from a minimum of 3-5 oocytes from the same patient were pooled. When sufficient spermatozoa were available after insemination, the remainder of the suspension was incubated at 37 degrees C and fixed along with the corresponding oocyte-incubated sample. In all, 32 sperm samples were assessed and fertilized oocytes were obtained with 29 of these. In the 24 samples in which greater than 100 spermatozoa (mean of 192) could be assessed, 32% of spermatozoa had initiated or completed the acrosome reaction. In the 15 of these 24 samples for which oocyte-free controls were available, 31% of cells were reacting or reacted, compared with 15% of cells (P less than 0.001) in the controls. In the remaining 8 samples, incubated with oocyte-cumulus complexes, less than 100 but greater than or equal to 20 spermatozoa (mean of 42) were assessed and again 32% of spermatozoa were reacted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the acrosome reaction in human spermatozoa

Large pieces of human cumulus oophorus were exposed for 20-30 min to washed spermatozoa or to spermatozoa recovered after a swim-up procedure, and then fixed for electron microscopy. Spermatozoa of both populations penetrated deeply into the cumulus within that time, and none of 48 observed clearly had undergone an acrosome reaction (AR). As measured by fluorescence microscopy, an AR rate of 12% in spermatozoa obtained at 4 h following a swim-up increased to about 25% in samples incubated in culture dishes for approximately 20 h. However, this latter AR rate was no different in the presence or absence of a cumulus/oocyte complex, and was only moderately greater in 50% follicular fluid. Nor was it affected to any degree by the absence of calcium or by a low (26 degrees C) temperature, both of which are regulators of the physiological AR in other species. By contrast, a clear dose-related enhancement of the AR by the calcium ionophore A23187 was almost completely Ca2(+)-dependent. We conclude that the human cumulus oophorus does not rapidly induce an AR in spermatozoa capacitated in vitro and, unlike the situation in some other mammals, that washed human spermatozoa do not first require a period of capacitation in order to penetrate it. The results also point to the likelihood that ARs monitored in free-swimming human spermatozoa are for the most part spurious or artefactual, and they show that in-vitro AR rates in such populations do not parallel their fertilizing ability.     

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.     

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.     

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.     

Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn^−/− males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn^−/− males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn^−/− mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.     

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.     

Potentiation of the cGMP-induced guinea pig acrosome reaction by zinc

The addition of 8 bromo cGMP (10 mM) immediately (approximately 2 min) upon suspension of Guinea pig sperm in glucose-free BWW medium induces a significant number of acrosome reactions within minutes. The simultaneous addition of micromolar concentrations of ZnCl2 (25-1000 microM) potentiates the cGMP effects. Mid-micromolar concentrations were able to dramatically increase the percentage of acrosome reactions by a factor of 6.5 over 8 bromo cGMP alone. The induction of acrosome reactions was dependent upon external Ca++, and it is suggested that in this species Zn++ functions by affecting cyclic nucleotide metabolism and/or Ca++ flux.     

Inhibition of the human sperm acrosome reaction by proteinase inhibitors

The analogue of the second messenger cAMP, dibutyryl cAMP (dbcAMP), was shown to induce the human sperm acrosome reaction to the same extent as calcium ionophore A23187, providing preliminary evidence for the involvement of the adenylate cydase system in the acrosome reaction (AR) of human spermatozoa. Using the human synchronous acrosome reaction system, proteinase inhibitors were tested for their effect on the dbcAMP-induced human sperm acrosome reaction. The proteinase inhibitor 4′-acctamidophenyl4-guanidinoben-zoate (AGB), an inhibitor of proacrosin activation and of acrosin, when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, significantly (P < 0.01) inhibited the acrosome reaction at final concentrations of 1 × 10^?4 M to 1 × 10^?6 M in comparison to dbcAMP treatment alone. At concentrations less than 1 × 10^?6 M, no significant inhibitory effect was seen. Similarly, para-aminobenzamidine (pAB), also an inhibitor of proacrosin activation and of acrosin, significantly (P < 0.01) inhibited the dbcAMP-induced acrosome reaction at final concentrations of 1 × 10-4 M to I × 10-6 M when added at either the onset of incubation or to capacitated spermatozoa, 5 min prior to stimulation by dbcAMP, in comparison to stimulation by dbcAMP alone. However, at concentrations less than 1 × 10^?6 M, no significant (P > 0.05) inhibitory effect was seen. These results indicate that a serine proteinase, most likely acrosin, has a role in the human sperm acrosome reaction and suggest that the enzyme functions after the involvement of the adenylate cyclase system.     

Observations on the acrosome reaction of human sperm in vitro

Human sperm were incubated in vitro in serum or the defined medium TMPA and were periodically assessed for acrosome reactions using two new methods of assay. The first method, FITC-RCA labeling, was previously shown to be valid for estimating the percentage of normal acrosome reactions of human sperm. The second method, a triple staining technique, is shown in this study to give results comparable to those obtained with FITC-RCA labeling. The percentage of acrosome-reacted sperm was determined at 0, 2.5, 5, and 7 hr of incubation. In both media, some sperm had reacted by 2.5 hr; a maximum percentage of reactions occurred between 5 and 7 hr. The maximum percentage never exceeded 20–25%, which represents only one-third of the live sperm, ie, those potentially able to undergo normal acrosome reactions. It will be important in future studies to determine if this low-peak percentage is due to the fact that: (1) Commonly used culture media are suboptimal or (2) only about 25% of the sperm in a human ejaculate are capable of undergoing normal acrosome reactions.     

Effects of metalloendoprotease substrates on the human sperm acrosome reaction

The substrates carbobenzyloxyserylleucylamide, carbobenzyloxyglycylleucylamide and carbobenzyloxyglycylphenylalanylamide were used as potential competitive inhibitors of endogenous metalloendoprotease activity. When the acrosome reaction was elicited by a potential physiological stimulus, human follicular fluid, each of the substrates (1-1.5 mM) inhibited exocytosis. Carbobenzyloxyserylleucylamide also inhibited the acrosome reaction when exocytosis was stimulated using the calcium ionophore ionomycin, but carbobenzyloxyglycylleucylamide was not inhibitory and carbobenzyloxyglycylphenylalanylamide actually enhanced exocytosis under these conditions. Experiments using the fluorescent indicator fura-2 revealed that the increase in intracellular, free calcium stimulated by follicular fluid in human spermatozoa was depressed by carbobenzyloxyglycylphenylalanylamide but not by carbobenzyloxyserylleucylamide. The peptide carbobenzyloxyglycylglycylamide, which is not a substrate for metalloendoproteases, had no effect on the acrosome reaction, whether stimulated by follicular fluid or ionomycin. While the results with carbobenzyloxyserylleucylamide suggest a possible involvement of a metalloendoprotease in the human sperm acrosome reaction, our other results demonstrate that these carbobenzyloxy peptides have complex effects on the process of exocytosis in human spermatozoa, and suggest caution in interpretation of data obtained using such peptides on intact cells.     

Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction

Gamete recognition in the mouse is mediated by galactosyltransferase (GalTase) on the sperm surface, which binds to its appropriate glycoside substrate in the egg zona pellucida (Lopez, L. C., E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur, 1985, J. Cell Biol., 101:1501-1510). GalTase has been localized by indirect immunofluorescence to the dorsal surface of the anterior sperm head overlying the intact acrosome. Sperm binding to the zona pellucida triggers induction of the acrosome reaction, an exocytotic event that results in vesiculation and release of the outer acrosomal and overlying plasma membranes. Consequently, we examined the fate of sperm surface GalTase after the acrosome reaction. Contrary to our expectations, surface GalTase is not lost during the acrosome reaction despite the loss of its membrane domain. Rather, double-label indirect immunofluorescence assays show that GalTase is redistributed to the lateral surface of the sperm, coincident with the acrosome reaction. This apparent redistribution of GalTase was confirmed by direct enzymatic assays, which show that 90% of sperm GalTase activity is retained during the acrosome reaction. No GalTase activity is detectable on plasma membrane vesicles released during the acrosome reaction. In contrast, removal of plasma membranes by nitrogen cavitation releases GalTase activity from the sperm surface, showing that GalTase redistribution requires a physiological acrosome reaction. The selective redistribution of GalTase to a new membrane domain from one that is lost during the acrosome reaction suggests that GalTase is repositioned for some additional function after initial sperm-zona binding.