Effects of platelet activating factor on the motility and acrosome reaction of bovine spermatozoa

The effects of platelet activating factor (PAF) on motility and the acrosome reaction of ejaculated bull spermatozoa were evaluated. Washed spermatozoa (30 x 10(6)/ml) were incubated (39 degrees C) for up to 2 h with 10 to 200 muM PAF in a modified Tyrode's solution (pH 7.4) containing 3 mg/ml bovine serum albumin. Sperm motility was evaluated subjectively and by computer-assisted semen analysis. Percent acrosome-reacted spermatozoa was quantified microscopically from fixed smears following Giemsa staining. Percent fertilization by PAF-treated spermatozoa was determined using in vitro-matured bovine ova. Percent sperm motility decreased with >/= 50 muM PAF, while the rate of motility loss increased with PAF concentration (P<0.001). Percent acrosome reactions increased with PAF concentration during incubation (P<0.001). Acrosomal loss was rapid and complete with 200 muM PAF. At concentrations between 80 to 120 muM PAF, bull spermatozoa underwent acrosome reactions without a rapid loss of motility and penetrated in vitro-matured bovine ova at a rate comparable to that of heparin-capacitated spermatozoa (68 versus 54%, respectively). Incubation of bull spermatozoa with 10 to 50 muM PAF for 45 min had no effect on percent progressive motility, sperm velocity or other motility parameters. These results indicate that PAF can be used to induce acrosome reactions in bull spermatozoa and to promote in vitro fertilization of bovine ova. Under the conditions used in this study, PAF did not stimulate bovine sperm motility.     

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).     

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.     

Calcium channel antagonists modulate the acrosome reaction but not capacitation in mouse spermatozoa

Mouse spermatozoa require micromolar concentrations of calcium for capacitation but millimolar levels to initiate an acrosome reaction. Sperm suspensions were capacitated by incubation for 120 min in modified Tyrode's medium containing 90 microM-CaCl2 and then verapamil (0.5-50 microM) or nifedipine (0.1-100 nM), drugs shown to inhibit voltage-sensitive calcium channels in other cell types, was added before the introduction of 1.80 mM-CaCl2. Verapamil at 5-50 microM and nifedipine at 1-100 nM significantly inhibited the calcium-stimulated acrosome reaction response, relative to the drug-free control samples. The possibility that these compounds might inhibit calcium entry during capacitation was examined by incubating suspensions for 120 min in medium containing 90 microM-CaCl2 plus either 5 microM-verapamil or 1 nM-nifedipine, diluting to reduce drug concentration to one-tenth and then adding 1.80 mM-CaCl2. The considerable acrosome reaction response obtained indicated that spermatozoa had undergone capacitation and were able to respond to calcium, despite the continuous presence of calcium channel antagonist at a concentration able to inhibit the response at the end of capacitation. In-vitro fertilization studies indicated that both drugs significantly decreased ability of the spermatozoa to fertilize eggs, consistent with acrosome reaction data. However, results indicated that 50 microM-verapamil was able to induce initial stages of egg activation and thus prevent sperm-egg fusion in zona-intact eggs. The addition of verapamil or nifedipine to suspensions capacitated for 120 min in 1.80 mM-CaCl2 significantly potentiated the acrosome reaction response, compared with drug-free controls. Similar treatment of suspensions incubated for only 30 min, and hence only partly capacitated, failed to evoke a response. The potentiation of the acrosome reaction response by verapamil in cells capacitated in high calcium may indicate either retention, due to the action of antagonists, of released intracellular calcium stores, resulting in intracellular calcium concentrations above the threshold required to trigger the acrosome reaction or action of an activated guanine nucleotide binding (G) protein to produce an agonistic rather than an antagonistic response. These results suggest that calcium channels similar to those termed voltage-sensitive in other cell types may exist and play an important role in calcium movements at the end of capacitation, but not during earlier phases of capacitation.     

Effect of platelet activating factor on the motility and acrosome reaction of buffalo (Bubalus bubalis ) spermatozoa

Platelet activating factor (PAF; 1-0-alkyl-2 acetyl-sn-glycerol-3 phosphocholine) has been shown to have a wide range of biological activities. In this study, PAF was used to induce acrosome reactions in fresh as well as frozen-thawed buffalo spermatozoa at different incubation periods and PAF levels. As the period of incubation increased, there was a gradual decrease in motility and increase in acrosome reaction in both fresh and frozen-thawed spermatozoa. With increasing PAF levels, the motility of fresh spermatozoa decreased and acrosome reaction increased whereas in frozen-thawed semen, motility remained almost constant, and the increase in acrosome reaction was not pronounced. Differences in motility and acrosome reaction among different bulls, types of semen, periods of incubation and PAF levels were significant (P < 0.01). A PAF level of 100 microM and an incubation period of 15 min were found to be optimum for inducing acrosome reaction in buffalo spermatozoa, since at this combination acrosome reaction increased significantly (P < 0.01) over that of the control without much loss of motility.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

Effects of the purified IGF-I complex on the capacitation and acrosome reaction of rabbit spermatozoa

A protein complex containing IGF-I, purified from rabbit seminal plasma, was used to investigate its effects on the capacitation and acrosome reaction of rabbit spermatozoa. Uncapacitated sperm (Pattern F), capacitated sperm (Pattern B), and acrosome-reacted sperm (Pattern AR) were determined by CTC staining, and the results were validated by PSA-staining. The addition of the IGF-I complex to the capacitative medium directed the spermatozoa to spontaneous acrosome reaction. On the other hand, IGF-I complex, added to capacitated spermatozoa, acted as inducer of the acrosome reaction. Results of IVF experiments showed high rates of fertilization with capacitated spermatozoa, acrosome-reacted by either A23187 or IGF I complex, whereas significantly lower rates were obtained with spermatozoa capacitated in the presence of IGF-I complex.     

Albumin is required to support the acrosome reaction but not capacitation in mouse spermatozoa in vitro

Albumin was required specifically for penetration of the zona pellucida (less than 10% of eggs fertilized in the absence of albumin), but was not required for capacitation. A similar rate of capacitation was observed in the presence of albumin at concentrations ranging from 30 to 1 mg/ml, while a slightly slower rate was observed in the presence of 0.25 and 0.1 mg albumin/ml. In the absence of albumin, capacitation occurred at a rate which lagged behind that of the albumin-incubated counterparts by about 30 min; once capacitated, the addition of albumin promoted rapid sperm penetration. In albumin-free media (+/- the macromolecule PVA), sperm motility was frequently reduced, with fewer cells exhibiting hyperactivated motility, but improvements were observed after introduction of albumin. Acrosome loss was significantly lower in the absence of albumin, but within 5 min of its addition at concentrations ranging from 30 to 0.1 mg/ml to capacitated sperm suspensions, acrosome loss was stimulated and reached levels similar to those seen in control samples. Therefore, albumin can trigger the acrosome reaction in capacitated spermatozoa. It appears to act by assisting in the removal of a surface-associated inhibitory component, the presence of which stabilizes the sperm membranes and inhibits the acrosome reaction.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa

The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca²⁺) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris–citrate–fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 μg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF–EY). In egg yolks and the TCF–EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca²⁺ by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF–EY. One half of each TCF- and TCF–EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 °C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. Results: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca²⁺ concentrations in egg yolks were 524.8 ± 131.4 ng/g, 13.9 ± 2.03 mg/g and 1.27 ± 0.17 mg/g, respectively. In the TCF–EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1 mg/g. In TCF–semen, at D1, motility and viability were significantly higher than in TCF–EY-samples (p < 0.05), however at D4, no significant differences were detectable. Further, in TCF–semen, percentages of spermatozoa with intact membranes decreased significantly (p < 0.05) and capacitated spermatozoa increased (p < 0.05), which was not seen in TCF–EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF–EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Changes in the distribution of lectin receptors during capacitation and acrosome reaction in boar spermatozoa

Sperm glycocalyx modifications are known to occur during capacitation and the acrosome reaction (AR). These changes are very important for gamete recognition and fertilization in mammals but are not fully understood. The purpose of this study was to determine the distribution of surface carbohydrates in boar spermatozoa during capacitation and the AR. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. Thirty-nine ejaculates from fertile boars of various breeds were analyzed. N-Acetylglucosamine and sialic acid, mannose and fucose residues were detected by fluorescence microscopy and flow cytometry using FITC-conjugated lectins. Triticum vulgaris agglutinin (WGA) bound on the head and tail of fresh sperm, and fluorescence intensity (FI) decreased in capacitated sperm (6751 to 5621 fluorescence units (FU), P<0.05), and decreased further in acrosome-reacted sperm (5240 FU, P<0.05). Concanavalia ensiformis agglutinin (Con-A) bound homogeneously on the head and the midpiece of fresh sperm with a FI of 5335 FU, and increased in capacitated sperm (5957 FU, P<0.05) mainly on the acrosomal region. In acrosome-reacted sperm, fluorescence was concentrated on the border of the acrosomal region (5608 FU, P<0.05). It was not possible to detect Ulex europaeus agglutinin (UEA) by fluorescence microscopy. However, flow cytometry revealed UEA receptors (187 FU), with a nonsignificant decreased number in capacitated (142 FU) and AR sperm (142 FU). Labeling patterns were similar in all breeds. Sperm glycocalyx modifications observed in this study provide insights to the molecular modifications accompanying capacitation and the AR. This kind of study could improve the diagnosis of reproductive problems of subfertile boars and males of other species.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

Effects of angiotensin II on the acrosome reaction in equine spermatozoa

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.     

An investigation of capacitation and the acrosome reaction in dog spermatozoa using a dual fluorescent staining technique

The processes of capacitation and acrosomal exocytosis of dog spermatozoa in vitro have yet to be fully investigated. Firstly, we investigated the effectiveness of a technique for staining dog spermatozoa with the fluorescent labels Hoechst 33258 and chlortetracycline. A modified fluorescence microscopy staining method was shown to be effective for the assessment of both viability and functional status in this species. Secondly, the presence of the ionophore A23187 in culture medium was shown to promote capacitation and the acrosome reaction of dog spermatozoa. We have therefore established that this dual fluorescent staining method can be used for monitoring these events in the dog, and it may be useful in future studies of optimal in vitro culture conditions.     

FSCB phosphorylation in mouse spermatozoa capacitation

It is generally accepted that spermatozoa capacitation is associated with protein kinase A-mediated tyrosine phosphorylation. In our previous study, we identified the fibrous sheath CABYR binding protein (FSCB), which was phosphorylated by PKA. However, the phosphorylation status of FSCB protein during spermatozoa capacitation should be further investigated. To this aim, in this study, we found that phosphorylation of this 270-kDa protein occurred as early as 1 min after mouse spermatozoa capacitation, which increased over time and remained stable after 60 min. Immunoprecipitation assays demonstrated that the tyrosine and Ser/Thr phosphorylation of FSCB occurred during spermatozoa capacitation. The extent of phosphorylation and was closely associated with the PKA activity and spermatozoa motility characteristics. FSCB phosphorylation could be induced by PKA agonist DB-cAMP, but was blocked by PKA antagonist H-89.Therefore, FSCB contributes to spermatozoa capacitation in a tyrosine-phosphorylated format, which may help in further elucidating the molecular mechanism of spermatozoa capacitation.     

Effect of progesterone on bovine sperm capacitation and acrosome reaction

Progesterone (P) appears to stimulate sperm capacitation and/or induce the acrosome reaction (AR) in some species. In bovine, it is now well established that the BSP-A1/-A2 proteins (the major proteins of bovine seminal plasma) promote sperm capacitation. In this study, we investigated the effect of P on bovine sperm cholesterol efflux, capacitation, and the AR. Labeled bovine epididymal sperm were incubated (0-6 h) with different concentrations of P (0.01-10 microg/ml) in the presence or absence of BSP-A1/-A2 proteins (capacitating conditions). At different time intervals, aliquots of sperm were taken to determine the sperm cholesterol efflux, sperm capacitation (AR induced by lysophosphatidylcholine, lyso-PC), and sperm AR. The results show that the presence of P in the media did not affect the membrane cholesterol efflux potential of the BSP-A1/-A2 proteins. P alone did not stimulate the AR with or without lyso-PC unless the epididymal sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2). When washed ejaculated sperm were continuously incubated with P, the P did not stimulate AR. However, when ejaculated sperm were preincubated (6 h) with heparin (capacitation medium) and then incubated 15 min with P (2 microg/ml), the percentage of AR obtained was similar to that obtained with lyso-PC. The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 microg/ml of P. These results demonstrate a potential role of P in bovine sperm AR but not in capacitation.     

Involvement of cholesterol, calcium and progesterone in the induction of capacitation and acrosome reaction of mammalian spermatozoa

The aim of this paper was to review the effects of some important substances involved in the induction of sperm plasma membrane changes referred to as acrosome reaction, namely cholesterol (C), calcium (Ca(2+)) and progesterone (P(4)). For this purpose, mechanisms of capacitation and acrosome reaction (AR) as well as the processes, C, Ca(2+) and P(4) are involved in, are described. Subsequently, to get a better insight into possible beneficial and detrimental effects on sperm function, the occurrence of these molecules in semen extenders is discussed.