Acrosome Reaction In Mammalian Spermatozoa Research Articles

Acrosomal alkalization triggers Ca2+ release and acrosome reaction in mammalian spermatozoa.

The sperm acrosome reaction (AR), an essential event for mammalian fertilization, involves Ca2+ permeability changes leading to exocytosis of the acrosomal vesicle. The acrosome, an intracellular Ca2+ store whose luminal pH is acidic, contains hydrolytic enzymes. It is known that acrosomal pH (pHacr ) increases during capacitation and this correlates with spontaneous AR. Some AR inducers increase intracellular Ca2+ concentration ([Ca2+ ]i ) through Ca2+ release from internal stores, mainly the acrosome. Catsper, a sperm specific Ca2+ channel, has been suggested to participate in the AR. Curiously, Mibefradil and NNC55-0396, two CatSper blockers, themselves elevate [Ca2+ ]i by unknown mechanisms. Here we show that these compounds, as other weak bases, can elevate pHacr , trigger Ca2+ release from the acrosome, and induce the AR in both mouse and human sperm. To our surprise, μM concentrations of NNC55-0396 induced AR even in nominally Ca2+ free media. Our findings suggest that alkalization of the acrosome is critical step for Ca2+ release from the acrosome that leads to the acrosome reaction.

Papaverine-sensitive phosphodiesterase activity is measured in bovine spermatozoa.

Cyclic adenosine monophosphate (cAMP) plays a crucial role as a signaling molecule for capacitation, motility, and acrosome reaction in mammalian spermatozoa. It is well-known that cAMP degradation by phosphodiesterase (PDE) enzyme has a major impact on sperm functions. This study was undertaken to characterize cAMP-PDE activity in bovine spermatozoa. Total cAMP-PDE activity in cauda epididymal and ejaculated spermatozoa was 543.2±49.5 and 1252.6±86.5 fmoles/min/106 spermatozoa, respectively. Using different family-specific PDE inhibitors, we showed that in cauda epididymal and ejaculated spermatozoa, the major cAMP-PDE activity was papaverine-sensitive (44.5% and 57.5%, respectively, at 400nm, papaverine is a specific inhibitor of the PDE10 family). These data are supporting the functional presence of PDE10 in bovine spermatozoa and were further confirmed by western blot to be PDE10A. Using immunocytochemistry, we showed immunoreactive signal for PDE10A present on the post-acrosomal region of the head and on the flagella of ejaculated spermatozoa. Using papaverine, we showed that it promotes tyrosine phosphorylation of sperm proteins, phosphorylation of Erk1 and Erk2, and Ca2+ release from Ca2+ store. These results suggest that PDE10 is functionally present in bovine spermatozoa and is affecting different molecular events involved in capacitation, most probably by cAMP local regulation.

NAADP and the two-pore channel protein 1 participate in the acrosome reaction in mammalian spermatozoa

The functional relationship between the formation of hundreds of fusion pores during the acrosome reaction in spermatozoa and the mobilization of calcium from the acrosome has been determined only partially. Hence, the second messenger NAADP, promoting efflux of calcium from lysosome-like compartments and one of its potential molecular targets, the two-pore channel 1 (TPC1), were analyzed for its involvement in triggering the acrosome reaction using a TPCN1 gene-deficient mouse strain. The present study documents that TPC1 and NAADP-binding sites showed a colocalization at the acrosomal region and that treatment of spermatozoa with NAADP resulted in a loss of the acrosomal vesicle that showed typical properties described for TPCs: Registered responses were not detectable for its chemical analogue NADP and were blocked by the NAADP antagonist trans-Ned-19. In addition, two narrow bell-shaped dose-response curves were identified with maxima in either the nanomolar or low micromolar NAADP concentration range, where TPC1 was found to be responsible for activating the low affinity pathway. Our finding that two convergent NAADP-dependent pathways are operative in driving acrosomal exocytosis supports the concept that both NAADP-gated cascades match local NAADP concentrations with the efflux of acrosomal calcium, thereby ensuring complete fusion of the large acrosomal vesicle.

The Multi‐PDZ domain protein MUPP1 as a lipid raft‐associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.

The multi PDZ domain protein MUPP1: A scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The multi PDZ domain protein MUPP1: A scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

In guinea pig spermatozoa, the procaine-promoted synchronous acrosome reaction results in highly fertile cells exhibiting normal F-actin distribution

In guinea pig spermatozoa, procaine induces Ca 2+ independent hyperactivated motility suggestive of sperm capacitation. Nonetheless, in the presence of high extracellular Ca 2+, procaine increases cytoplasmic Ca 2+. We analyze the procaine effect on the acrosome reaction (AR) processes in guinea pig spermatozoa. Results indicated that: (i) in spermatozoa pre-incubated 5–30 min in MCM-PLG medium, procaine produced synchronous AR, (ii) the acrosome-reacted sperm number increased with the capacitation period before procaine treatment and with procaine concentration, (iii) acrosome reaction was blocked when Ca 2+ was omitted, (iv) plasma membrane–outer acrosomal membrane fusion started within 2 min after procaine treatment, (v) in acrosome-reacted spermatozoa, actin polymerization occurred and F-actin was located in the equatorial and post-acrosomal regions and (vi) procaine treatment resulted in highly fertile acrosome-reacted spermatozoa. This is the first report indicating that procaine promotes synchronic AR in mammalian spermatozoa. If procaine promotes premature AR of spermatozoa in vivo, it might be a factor for infertility in patients exposed to this local anesthetic.

Classic and Non-classic Progesterone Receptors are both Expressed in Human Spermatozoa

Progesterone is one of the physiological inducers of the acrosome reaction in mammalian spermatozoa. The receptor that responds to progesterone is not yet identified, and its properties differ in many aspects from the properties of the classic nuclear progesterone receptor, suggesting the participation of a novel or non-classic receptor. In this study, we investigated the expression of a novel progesterone-binding protein (hmPR1/PGMRC1) and its ortholog (hmPR2/PGMRC2), which have previously been identified in liver microsomes and are considered receptor candidates, along with the nuclear progesterone receptor. The purification procedure was optimized with special emphasis on the control of leukocyte contamination in single donor samples. The results indicate that all three proteins are expressed in human sperm, as transcripts have been detected in 46 %, 42 % and 37.5 % of individual samples, respectively (n = 24).

Molecular identification of Ca2+ channels in human sperm.

The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+) channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+) channels has been limited. Here we identified Ca(2+) channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+) channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+) channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H alpha1G alpha1E alpha1B alpha1C alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+) channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.

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

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.

Spatiotemporal Characterization of Intracellular Ca2+Rise during the Acrosome Reaction of Mammalian Spermatozoa Induced by Zona Pellucida

The mammalian sperm acrosome reaction (AR) is an essential event prior to sperm–egg fusion at fertilization, and it is primarily dependent on an increase in intracellular Ca2+concentration ([Ca2+]i). Spatiotemporal aspects of the [Ca2+]iincrease during the AR induced by solubilized zona pellucida (ZP) in hamster spermatozoa were precisely investigated with a Ca2+imaging technique using confocal laser scanning microscopy with two fluorescent Ca2+indicators. A rapid rise in [Ca2+]ioccurred immediately after the application of ZP solution through a micropipette. The rise was always initiated in the sperm head, even when the application was directed toward the tail. The elevated [Ca2+]iwas little attenuated during measurement for 30–40 s. Acrosomal exocytosis was detected as a sudden decrease of fluorescence in the acrosomal vesicle ∼20 s after the onset of the [Ca2+]irise. High-resolution imaging revealed that the [Ca2+]irise in the sperm head began at the region around the equatorial segment and spread over the posterior region of the head within 0.6 s, whereas Ca2+concentration in the acrosomal vesicle appeared to be unaltered. The [Ca2+]irise was completely abolished under Ca2+-free extracellular conditions, indicating that it is totally attributable to Ca2+influx. Nifedipine, an inhibitor of L-type Ca2+channels, did not affect the rising phase of the ZP-induced Ca2+response, but accelerated the decline of the [Ca2+]irise and inhibited acrosomal exocytosis. The present study provides implicative information about the spatial organization of functional molecules involved in the signal transduction in mammalian AR.

Identification of structural elements of the testis-specific voltage dependent calcium channel that potentially regulate its biophysical properties.

Calcium influx through voltage-dependent calcium channels regulates the physiological acrosome reaction of mammalian spermatozoa. Expression of the mRNA for these voltage-dependent calcium channels and its co-ordinated translation is initiated early in rat male germ line development and continues throughout spermatogenesis. Herein, we report the complete mRNA and deduced amino acid sequence of the alpha1c pore-forming subunit of the rat testis-specific L-type calcium channel. This subunit is transcribed from the alpha1c gene, which is also expressed in brain and cardiac muscle. The cardiac- and testis-specific isoforms of the alpha1c subunit are produced by alternate splicing of the same primary transcript. The testis-specific isoform differs from that of cardiac tissue at its amino terminus and in transmembrane segments IS6, IIIS2 and IVS3, which are also dihydropyridine binding sites. In somatic tissues, segments S2 and S3 regulate channel activation while the amino terminus and segment IS6 contribute to channel inactivation kinetics. The amino terminus and IS6 segment of the testis-specific alpha1c subunit are also expressed respectively, in the brain and in smooth muscle from lung where they alter the electrophysiological characteristics of the subunit to produce relatively slow inactivation kinetics. These findings provide a molecular explanation for the detection by others, by patch clamp analysis, of T-type calcium currents in immature spermatogenic cells and of atypical L-type calcium currents in mature spermatozoa.

Progesterone stimulates p42 extracellular signal-regulated kinase (p42erk) in human spermatozoa

Mitogen-activated protein kinases (MAPK), also known as extracellular signal-regulated kinases (ERKs) are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. Recently, we have demonstrated that ERKs are present in spermatozoa and are involved in the regulation of the process of capacitation. We report here the effect of progesterone, a well-known inducer of the acrosome reaction in mammalian spermatozoa, on the immunolocalization, phosphorylation and activity of ERKs in capacitated human spermatozoa. We demonstrated that short-term incubation of spermatozoa with progesterone induces phosphorylation and activation of ERKs, resulting in redistribution of the proteins from the post-acrosomal region to the equatorial segment within the sperm head. To investigate the role of ERKs on the biological effects of progesterone, we used the MAPK cascade inhibitor PD098059, which strongly inhibited progesterone-induced activation of ERK-2. This compound did not inhibit progesterone-induced acrosome reaction, although it prevented redistribution of the enzyme to the equatorial region of the sperm head. These results suggest that the two processes, although temporally related, are independent. In conclusion, we provide new insight into the signal transduction pathways involved in the non-genomic action of progesterone in spermatozoa and suggest a possible involvement of ERKs in the process of fertilization.

Localization of intracellular calcium and its possible role during spermatogenesis and sperm capacitation in the hamster

In guinea pig spermatozoa, procaine induces Ca2+ independent hyperactivated motility suggestive of sperm capacitation. Nonetheless, in the presence of high extracellular Ca2+, procaine increases cytoplasmic Ca2+. We analyze the procaine effect on the acrosome reaction (AR) processes in guinea pig spermatozoa. Results indicated that: (i) in spermatozoa pre-incubated 5–30 min in MCM-PLG medium, procaine produced synchronous AR, (ii) the acrosome-reacted sperm number increased with the capacitation period before procaine treatment and with procaine concentration, (iii) acrosome reaction was blocked when Ca2+ was omitted, (iv) plasma membrane–outer acrosomal membrane fusion started within 2 min after procaine treatment, (v) in acrosome-reacted spermatozoa, actin polymerization occurred and F-actin was located in the equatorial and post-acrosomal regions and (vi) procaine treatment resulted in highly fertile acrosome-reacted spermatozoa. This is the first report indicating that procaine promotes synchronic AR in mammalian spermatozoa. If procaine promotes premature AR of spermatozoa in vivo, it might be a factor for infertility in patients exposed to this local anesthetic.

The motility and fertility of golden hamster sperm cultured in BSA-free medium

Before fertilization, capacitation and the acrosome reaction in mammalian spermatozoa must be completed. The motility and fertility of hamster sperm were examined in four kinds of modified Tyrode's solution with or without bovine serum albumin (BSA). Since the presence or absence of polyvinylalcohol (PVA) in the media was another variable, its effect on the sperm motility and fertility was also studied. Sperm were incubated in four different media for up to 6 h at 37.5°C. After 4 h of incubation in the media containing BSA alone or BSA and PVA, sperm were hyperactivated, showing a high sperm motility index (SMI) and were able to fertilize more than 80% of eggs. However, their fertility rapidly decreased during further incubation. In contrast, sperm in the medium containing PVA and no BSA showed low SMI scores after 4 h. However, during the following 2-h period, the SMI progres-sively increased and sperm were hyperactivated. Furthermore, the hyperactivated sperm in the PVA containing medium were able to effectively fertilize eggs. Our results indicate that hamster sperm can be capacitated in BSA-free medium and that capacitation occurs much more slowly in such a medium. We suggest that PVA is a reasonable alternative to BSA in in vitro fertilization and that this slowly progressing system may be a good model for studying various steps in sperm activation.

Role of protein kinase C in the acrosome reaction of mammalian spermatozoa

Mammalian spermatozoa undergo a Ca(2+)-dependent exocytotic event before fertilization which is known as the acrosome reaction. The process of exocytosis in several cell systems is mediated by a protein kinase C (PKC)-catalysed phosphorylation. Addition of phorbol 12-myristate-13-acetate or the membrane-permeant diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which are potent activators of PKC, to bovine spermatozoa resulted in stimulation of the acrosome reaction. This stimulation was inhibited by low concentrations (50% inhibition at 0.7 nM) of the PKC inhibitor staurosporine. PKC specific activity in bovine spermatozoa is extremely low in comparison with other cells; however, it is comparable with the activity found in human spermatozoa. Immunohistochemical analysis using anti-PKC antibodies revealed staining in the equatorial segment, the post-acrosomal region and the upper region of the head. We propose that PKC is involved in the mammalian sperm acrosome reaction.

Role of lipoxygenase in the mechanism of acrosome reaction in mammalian spermatozoa

The acrosome reaction (AR) in bull spermatozoa was induced by the Ca 2+-ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca 2+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 μM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20, 40 or 80 μg/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 μg/ml, respectively. Under identical conditions, 100 μM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 15-HETE, the classical product of 15-lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15-HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa.

Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.

Cellular and Molecular Biology of Capacitation and Acrosome Reaction in Mammalian Spermatozoa

Publisher Summary This chapter reviews the information available on different aspects of capacitation and acrosome reaction (AR) and proposes a tentative but unified view of the mechanisms involved in these phenomena. The capacitation is achieved in the female reproductive tract; it is also accomplished outside the female reproductive tract in numerous in vitro studies using well-defined media in several mammalian species. There is reasonable consensus that capacitation involves no morphological change in spermatozoa that can be discerned, even under the electron microscope; it does facilitate the occurrence of a distant morphological event in spermatozoa known as AR. AR involves point fusions between the outer acrosomal membrane and overlying plasmalemma leading to the release of acrosomal contents or enzymes required at the site of fertilization. The capacitation involves two important changes at the molecular level in spermatozoa: (1) sperm surface alteration and/or intramembranal molecular mobility to facilitate Ca 2+ influx and (2) changes in sperm energetics through alterations in oxygen uptake and glucose utilization manifested in the form of changes in pattern of flagellar beat.

Neuraminidase induces capacitation and acrosome reaction in mammalian spermatozoa.

The treatment of epididymal spermatozoa of guinea pig and ejaculated spermatozoa of rabbit with neuraminidase from Arthrobacter ureafaciens induced significant acrosome reaction while the neuraminidase from Cl. perfringens failed to do so. The addition of the neuraminidase inhibitors kept the enzyme induced acrosome reaction to the control level. The zona-free hamster ova test showed that the treatment of spermatozoa with Arthrobacter neuraminidase rendered 82% of the guinea pig and 69% of the rabbit spermatozoa capable of fertilization. Thus, neuraminidase seems to enhance the rate of acrosome reaction by first capacitating spermatozoa in vitro.

In vitro fertilization of banteng ( [formula omitted][formula omitted]): A case report

Before fertilization, capacitation and the acrosome reaction in mammalian spermatozoa must be completed. The motility and fertility of hamster sperm were examined in four kinds of modified Tyrode's solution with or without bovine serum albumin (BSA). Since the presence or absence of polyvinylalcohol (PVA) in the media was another variable, its effect on the sperm motility and fertility was also studied. Sperm were incubated in four different media for up to 6 h at 37.5°C. After 4 h of incubation in the media containing BSA alone or BSA and PVA, sperm were hyperactivated, showing a high sperm motility index (SMI) and were able to fertilize more than 80% of eggs. However, their fertility rapidly decreased during further incubation. In contrast, sperm in the medium containing PVA and no BSA showed low SMI scores after 4 h. However, during the following 2-h period, the SMI progres-sively increased and sperm were hyperactivated. Furthermore, the hyperactivated sperm in the PVA containing medium were able to effectively fertilize eggs. Our results indicate that hamster sperm can be capacitated in BSA-free medium and that capacitation occurs much more slowly in such a medium. We suggest that PVA is a reasonable alternative to BSA in in vitro fertilization and that this slowly progressing system may be a good model for studying various steps in sperm activation.