Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa.

Progesterone induced a rapid, long-lasting, dose-dependent increase of intracellular free calcium concentration ([Ca2+]i) in human sperm capacitated overnight. This effect was not counteracted by the cytosolic progesterone receptor antagonist RU486 (1 mumol/L) nor by the GABA-A receptor antagonists bicuculline (10 mumol/L) and picrotoxin (50 mumol/L). Also, the rank order of potency of several progestative steroids on [Ca2+]i differed from that previously reported for uterine intracellular progesterone receptor or for P-GABA interaction in the central nervous system, indicating a different pathway for progesterone stimulation of human sperm. Modifications of basal and progesterone-stimulated [Ca2+]i during sperm capacitation were also studied. A progressive, parallel increase of basal and progesterone-stimulated [Ca2+]i in capacitating spermatozoa was found. In particular, progesterone-stimulated [Ca2+]i increased from a basal concentration of 147% +/- 17% at 10 minutes to 327% +/- 65% after 120 minutes of incubation in capacitating medium. This increase was well correlated with basal [Ca2+]i (r = 0.93). In contrast, basal and progesterone-stimulated [Ca2+]i concentrations were constantly low in spermatozoa incubated in noncapacitating medium. In capacitated spermatozoa, initial responsiveness to progesterone and basal [Ca2+]i was higher than in capacitating and noncapacitated samples, and remained constant throughout the duration of the experiment. The progressive, parallel increase of [Ca2+]i and response to progesterone observed during in vitro capacitation of human spermatozoa might be physiologically relevant in vivo during capacitation of sperm in the female genital tract.     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

Summary.  Bovine spermatozoa from frozenthawed semen are sensitive to lipid peroxidation. Vitamin E protects sperm membrane against oxidative damage. Sperm capacitation produces structural changes on the plasma membrane. Reactive oxygen species could be involved in the capacitation process. The aim of this work was to study the influence of natural antioxidants on the plasma membrane and the influence of reactive oxygen species during bovine sperm capacitation. Sperm samples were frozen in a standard diluent, with and without vitamin E (1 mg ml^-1). Heparin (60 μg ml^-1) was used as a sperm capacitation inductor. Sperm capacitation was evaluated by chlorotetracycline assay. Lipid peroxidation was determined by the 2-thiobarbituric acid assay. A diminution of thiobarbituric acid reactive substances was observed in sperm samples frozen with vitamin E ( P < 0.05). The addition of vitamin E to the freezing diluent had no effect on the capacitated pattern ( P > 0.05).
When vitamin E and vitamin E + vitamin C were added to the capacitation medium, a significant decrease in the percentage of capacitated spermatozoa ( P < 0.05) was observed in both cases. The addition of superoxide dismutase (0.1 mg ml^-1) or H₂O₂ (50 μM) in the incubation medium, decreased the percentage of capacitated spermatozoa ( P < 0.05). Vitamin E protects the plasma membrane against lipid peroxidation during sperm capacitation, and the presence of superoxide anion would be necessary for frozen-thawed bull sperm capacitation.     

The cyclic GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates human sperm motility and capacitation but not acrosome reaction

Capacitation is the series of transformations that spermatozoa undergo in the female genital tract in order to bind to the zona pellucida, initiate the acrosome reaction, and fertilize an egg. Cyclic adenosine monophosphate (cAMP) plays an important role in this process and its levels are regulated by 2 key enzymes, adenylyl cyclase and cyclic nucleotide phosphodiesterase (PDE), the latter being involved in cAMP degradation. Evidence was provided for the involvement of PDE in sperm motility and capacitation. Of the 10 gene families of PDE that exist in mammalian tissues, the calcium-calmodulin-dependent (type 1) and the cAMP-specific (type 4) have been found in human spermatozoa. Using sildenafil, we investigated a highly potent cyclic guanosine monophosphate (cGMP)-specific PDE (type 5) inhibitor and whether this PDE is present in human spermatozoa and is involved in sperm functions. Sildenafil inhibited PDE activity of Percoll-washed spermatozoa with an IC50 of 97+/-3 and 33+/-3 microM when cAMP and cGMP, respectively, were used as substrates. Because the IC50 of sildenafil obtained for PDE type 5 is much lower (2 to 6 nM) than that obtained with sperm PDE, the data suggest that PDE type 5 represents only a small fraction of the whole PDE activity of spermatozoa. Sildenafil causes dose-dependent increases in sperm cAMP levels and capacitation, which are associated with an increase in the levels of tyrosine phosphorylation of 2 fibrous sheath proteins (p105/81). Sperm velocity, amplitude of lateral head displacement, and hyperactivation were increased at 30-180 minutes. Sildenafil did not trigger the acrosome reaction in capacitated spermatozoa. These results suggest that under our experimental conditions, sildenafil triggers human sperm motility and capacitation, probably via its inhibitory action on PDE activity other than type 5 with a resultant rise in cAMP levels.     

Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine

Identification of norepinephrine (NE) within the microenvironment of the bovine oviduct suggests a potential role for catecholamines in the events surrounding fertilization. Previous studies have shown that the catecholamines capacitate and induce the acrosome reaction in spermatozoa from several species. The current project was undertaken to investigate the role of catecholamines in bovine sperm capacitation and the acrosome reaction. Freshly ejaculated bovine spermatozoa were incubated in NE (0-1000 ng/mL) and induced to acrosome-react with lysophosphatidylcholine (LPC). Additionally, spermatozoa capacitated with heparin were incubated with NE (0-1000 ng/mL) to assess its ability to induce the acrosome-reaction in capacitated spermatozoa. Concentrations of NE were chosen on the basis of physiological concentrations previously determined for bovine oviductal fluid. NE at concentrations of 10 and 20 ng/mL capacitated bovine spermatozoa after 2 hours of incubation. Additionally, spermatozoa incubated for 2 hours with heparin were induced to acrosome-react with 10 and 20 ng/mL NE. Interestingly, higher concentrations of NE inhibited both capacitation and the acrosome reaction. Incubating spermatozoa with dopamine or epinephrine did not result in capacitation or the acrosome reaction, suggesting that the action of NE was specific to that catecholamine. The ability of NE to capacitate or induce the acrosome reaction appears to be dependent on the presence of another membrane-destabilizing factor. Although adrenergic receptors have not been identified on spermatozoa from any species, the action of NE on spermatozoa may be a receptor-mediated event. This study suggests a possible function for oviductal catecholamines in sperm preparation prior to fertilization.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.     

Effect of spermine on sperm capacitation of guinea pig in vitro.

Spermine (Sp) 10(-5) mM had vigorous activity of guinea pig spermatozoa, while it completely abolished sperm forward motility (SFM) at a concentration of 10(-3) mM. There appeared to be a dose relationship to inhibition to motility. 2-Difluoromethylornithine 10 mM antagonized the Sp-induced inhibition of SFM after 3 h of incubation. Capacitation of a guinea pig sperm was inhibited by Sp in a concentration-dependent manner. The majority of acrosome-reacted sperm did not display hyperactivated motility. Precapacitated sperm were able to undergo the acrosome reaction (AR) in the presence of Sp. Moreover, Sp-mediated inhibition of capacitation was a reversible process. Once sperm capacitation was completed, Sp no longer inhibited AR. Before capacitation, the content of Sp in spermatozoa was 4.5 +/- 0.5 micrograms/5 x 10(7) cells, whereas in case of capacitated spermatozoa it was significantly decreased (2.1 +/- 0.4 micrograms/5 x 10(7) cells). The penetration of spermatozoa into the zona-free hamster eggs in the presence of Sp was markedly decreased, but it did not affect the fertilizability of ova as compared to the control. These results suggest that Sp may be an inhibitory agent of sperm capacitation in guinea pig in vitro, and it may also be involved in the modulation of capacitation.     

Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) spermatozoa

Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37 degrees C (5% CO(2) in air) for 2 hours in a modified Biggers-Whitten-Whittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO(3). Caffeine (1 mmol/L) and dbcAMP (1.2 mmol/L) were added to the medium, and incubation was performed for an additional 30 minutes. Sperm motility was determined by computer-assisted sperm analysis, and membrane lipid order and sperm viability was determined by flow cytometry with merocyanine (2.7 micromol/L) and Yo-Pro-1 (25 nmol/L), respectively. Tyrosine phosphorylation of proteins in sperm tail was immunohistochemically examined by means of anti-phosphotyrosine (alpha-PY; clone 4G10). Capacitation resulted in a significant increase in the amplitude of lateral head displacement and beat cross frequency (P < .005) and a significant decrease in linearity and straightness in capacitated spermatozoa (P < .005), compared with control spermatozoa, which suggests the expression of hyperactivated motility. Animals in which capacitation was induced had a significant increase in the number of spermatozoa showing tyrosine phosphorylation of tail proteins (P < .0001) and a significant increase in the intensity of merocyanine fluorescence (P < .0001), compared with control animals. The observed decrease in membrane lipid order after capacitation was induced was not associated with surface exposure of phosphatidylserine, as determined by flow cytometry with annexin V-Alexa Fluor 488. Merocyanine may be a useful tool for investigating the role of the plasma membrane on capacitation and other cytotoxic events in macaque spermatozoa.     

Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L-arginine and regulated through a cAMP/PKA-independent pathway

The aim of the present study was to determine the effect of L-arginine on nitric oxide (NO*) synthesis, capacitation and protein tyrosine phosphorylation in buffalo spermatozoa. Ejaculated buffalo spermatozoa were capacitated in the absence or presence of heparin, or L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) for 6 h. Capacitating spermatozoa generated NO* both spontaneously and following stimulation with L-arginine and L-NAME quenched such L-arginine-induced NO* production. Immunolocalization of NOS suggested for existence of constitutive NOS in buffalo spermatozoa. L-Arginine (10 mm) was found to be a potent capacitating agent and addition of L-NAME to the incubation media attenuated both L-arginine and heparin-induced capacitation and suggested that NO* is involved in the capacitation of buffalo spermatozoa. Two sperm proteins of M(r) 38 000 (p38) and 20 000 (p20) were tyrosine phosphorylated extensively by both heparin and L-arginine. Of these, the tyrosine phosphorylation of p38 was insensitive to both induction by cAMP agonists as well as inhibition by a protein kinase A (PKA) inhibitor. Further, most of these L-arginine-induced tyrosine phosphorylated proteins were localized to the midpiece and principal piece regions of flagellum of capacitated spermatozoa and suggested that sperm flagellum takes active part during capacitation. These results indicated that L-arginine induces capacitation of buffalo spermatozoa through NO* synthesis and tyrosine phosphorylation of specific sperm proteins involving a pathway independent of cAMP/PKA.     

Modulation of human sperm function by follicular fluid

Human follicular fluid (hFF), present in the ampullary environment, can reduce the number of sperm bound to the zona pellucida. The aim of the present study was to investigate the effects of follicular fluid on sperm function. The presence of 50% v/v follicular fluid resulted in a significant reduction in the number of bound spermatozoa with respect to control medium (12.7 +/- 5.5 sp HZ(-1) versus 24.6 +/- 5.7 sp HZ(-1), P = 0.03) as measured by the hemizona binding assay. This reduction in zona binding capacity was not associated with a loss of sperm viability, motility or a premature acrosomal reaction. When capacitated spermatozoa were previously exposed 1 h to follicular fluid, a significant reduction in the number of alpha-d-mannose binding sites on sperm head was detected (23.7 +/- 3.1% versus 15.5 +/- 2.4%, P < 0.05). In addition, sperm fertilizing capacity (assessed as the acrosome reaction to ionophore challenge score) in the presence of follicular fluid was also diminished (38.0 +/- 4.8% versus 22.6 +/- 4.9%, P < 0.01). No modification in the pattern of protein tyrosine phosphorylation which occurs during capacitation was observed in the presence of the fluid. Taken together, the results indicate that the decrease in sperm zona-binding capacity observed in the presence of hFF was related to a lower number of sperm containing alpha-d-mannose receptors.     

Epididymal protein CRISP1 plays different roles during the fertilization process

Rat epididymal CRISP1, the first described member of the evolutionarily conserved Cysteine-RIch Secretory Protein (CRISP) family, is expressed in the proximal regions of the epididymis and associates with the sperm during epididymal transit. Evidence indicates the existence of 2 populations of CRISP1 in spermatozoa: a major one, loosely bound, which is released during capacitation and, therefore, proposed as a decapacitating factor; and a minor one, strongly associated with spermatozoa that remains on the cells after capacitation and is proposed to participate in gamete interaction. Originally localized to the dorsal region of capacitated sperm, CRISP1 migrates to the equatorial segment with capacitation and acrosome reaction. Consistent with these localizations, in vitro fertilization experiments support the involvement of CRISP1 in the first step of sperm-zona pellucida (ZP) interaction and subsequent gamete fusion through its interaction with egg-complementary sites. The potential roles of CRISP1 in capacitation and fertilization have been further supported by the finding that capacitated spermatozoa from CRISP1 "knockout" animals exhibit low levels of protein tyrosine phosphorylation and have an impaired ability to fertilize zona-intact and zona-free eggs in vitro. Moreover, recent evidence from mutant spermatozoa reveals that CRISP1 mediates the stage of sperm binding to the ZP. Altogether, these observations support the view that CRISP1 is a multifunctional protein playing different roles during fertilization through its different associations with and localizations on spermatozoa. We believe these results contribute to a better understanding of the molecular mechanisms involved in both the fertilization process and the acquisition of sperm-fertilizing ability that occurs during epididymal maturation.     

Effect of human sperm exposure to progesterone on sperm-oocyte fusion and sperm-zona pellucida binding under various experimental conditions

In this study, the effect of human sperm exposure to progesterone on sperm/oocyte fusion, using the hamster egg penetration test, and on sperm/zona pellucida (ZP) binding, using the hemizona assay, was investigated under various experimental conditions. A brief exposure of human spermatozoa to progesterone exerted a stimulatory effect on sperm/oocyte fusion which was dose-dependent, capacitation-dependent, influenced by the source of serum albumin in capacitating medium, and was higher than that produced by the exposure to progesterone from the onset of capacitation. The exposure of capacitated spermatozoa to progesterone during 20 min-spermatozoa/ZP-coincubation produced an enhancement of ZP-binding, which was not significantly influenced by the source of serum albumin in capacitating medium. A significantly lower ZP-binding was exhibited by spermatozoa exposed to progesterone from the beginning of capacitation. These results indicate that progesterone exerts a stimulatory effect on human sperm's fertilizing ability, which occurs mainly in post-capacitation events directly involved in sperm/oocyte fusion and in ZP-binding. Conditions optimizing these effects are provided. They should be taken into account in the standardization of experimental and clinical studies designed to evaluate the response of human spermatozoa to progesterone.     

Alterations in sperm protein phosphorylation in male infertility

Protein phosphorylation is involved in sperm capacitation, so the effect of protein phosphatase inhibitors on the capacitation of spermatozoa of males with unexplained infertility was investigated. d-mannose ligand specific receptor expression in fresh, living spermatozoa, capacitated or treated with calyculin A (an inhibitor of protein phosphatases 1 and 2A), was studied in three groups of men: pre-vasectomy (fertile) males, males in couples with male infertility, and males in couples with infertility of unknown aetiology. Flow cytometry showed significant differences between infertile couples with a male factor and fertile couples (P < 0.05), both after capacitation and after treatment with calyculin A. In the group of couples with infertility of unknown aetiology (n = 15), d-mannose receptor expression was diminished in six cases after classical capacitation. However, when the spermatozoa of these six men were treated with calyculin A, five showed an increased specific d-mannose receptor expression. From these results it is suggested that in vitro treatment of spermatozoa with inhibitors of protein phosphatases may be of great value in some cases of unexplained infertility.     

Calcium requirement and time course of capacitation of goat spermatozoa assessed by chlortetracycline assay

Summary We standardized chlortetracycline fluorescent assay for studies of calcium requirement and time course of capacitation of goat spermatozoa. Three distinct fluorescent patterns were easily detected in goat spermatozoa incubated under capacitating conditions. Categorised according to nomenclature reported earlier, these are: ‘F’ with bright fluorescence in the post-acrosomal region, characteristic of uncapacitated acrosomal-intact cells; ‘B’ with bright fluorescence on the anterior portion of the head and dark band in the postacrosomal region, characteristic of capacitated, acrosome-intact cells; ‘AR’ with lack of fluorescence on the head characteristic of acro-some-reacted cells. A close correspondence was observed when the results of CTC assay were compared with those obtained by transmission electron microscopy. Goat spermatozoa were not capacitated when calcium was omitted from the medium and 80% had CTC fluorescence of ‘F’ type. The size of ‘B’ cell population increased with increase in calcium concentration; at 1.0 mmol 1^-1 a peak representing 65–70% capacitated cells accumulated in 4 h. At higher concentrations, ‘AR’ cells were found along with ‘B’ cells and the two cell types were in equal proportions at 1.71 mmol 1^-1. Time course studies revealed a 2 h incubation period at 1.0 mmol 1^-1 and 1 h at 2 mmol 1^-1 calcium concentration before transformation of ‘F’ cells to ‘B’ cells was noticed. However, at no time were ‘AR’ cells found exclusively pointing to an equilibrium between the two sperm populations. Goat spermatozoa were also not capacitated when phosphate was omitted from the medium. Permeant anions (NO₃^?, SCN^?), permeant weak acid (HCO₃^?) and organic phosphates (β-glycerophosphate, glucose-6-phosphate) were unable to replace phosphate. The reason for their failure for the incidence of capacitation was traced to low uptake of calcium by goat spermatozoa. In the presence of phosphate, a 6–8-fold increase was measured over the calcium uptake when phosphate was omitted (2–4 nmol 1^?1 10⁸ cells^?1). Mersalyl inhibited the calcium uptake by goat spermatozoa as well as its capacitation most likely by inhibiting the calcium phosphate transporter located in the sperm plasma membrane.     

Relationship between the proportion of capacitated spermatozoa present in frozen-thawed bull semen and fertility with artificial insemination

The objective of the present study was to confirm the presence of prematurely capacitated spermatozoa in frozen-thawed bull semen and to investigate the relationship of premature capacitation to the fertility of the respective semen. Twenty batches of frozen semen from young AI bulls of the Swedish Red and White breed with known fertility (expressed as 56-day non-return rates; 56 d-NRR) were tested using a Chlortetracycline (CTC) assay to assess capacitation status in frozen-thawed spermatozoa. The status of capacitation, as evidenced in this experiment, was further tested based on the hypothesis that capacitated spermatozoa present in frozen-thawed semen should undergo the acrosome reaction (AR) on co-incubation with homologous zona pellucida (ZP) glycoproteins. The percentage (mean +/- SEM) of uncapacitated, capacitated and acrosome-reacted spermatozoa in the frozen-thawed semen (n = 20) were 49.3 +/- 11.9, 36.3 +/- 8.3 and 14.2 +/- 11.9, respectively. On co-incubation with ZP, there was a significant increase (p = 0.001) in the proportion of spermatozoa undergoing the AR compared to the control with a concurrent decrease in the proportion of capacitated spermatozoa, suggesting that a proportion of capacitated spermatozoa were undergoing the AR. The proportion of viable, uncapacitated spermatozoa present in the frozen-thawed semen was correlated to the 56 d-NRR (n = 20, r = 0.5, p = 0.03). In conclusion, a proportion of spermatozoa in frozen-thawed semen was capacitated and the proportion of viable, uncapacitated spermatozoa present in semen was positively correlated to fertility.     

Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.     

Differential activities of malate and isocitrate NAD(P)-dependent dehydrogenases are involved in the induction of capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm catabolic processes produce energy for capacitation and acrosome reaction induction required for oocyte fertilization. The aim was to determine metabolic enzymes' activities and their participation in the supply of energy and generation of the redox state to acquire fertilizing capacity. Capacitation was induced with heparin and quercetin, and the acrosome reaction with progesterone. Enzymatic activities were determined spectrophotometrically. The chlortetracycline and differential-interferential contrast microscopy/tryptan blue techniques were used to evaluate capacitation and acrosome reaction, acrosomal integrity and sperm viability respectively. A 2 : 1 and 3 : 1 ratio were obtained for isocitrate dehydrogenase (IDH)-NADP/NAD and malate dehydrogenase (MDH)-NADP/NAD activities respectively. MDH-NADP activity remained constant with different treatments, unlike MDH-NAD activity, which diminished with both capacitation inducers and in acrosome-reacted spermatozoa previously treated with heparin (P < 0.05). IDH-NADP decreased its activity 50% in spermatozoa capacitated with heparin and acrosome reacted with progesterone (P < 0.05). Capacitation and acrosome reaction processes induced with heparin and progesterone, respectively, involve a differential oxidative metabolism, with the participation of MDH-NAD(P) and IDH-NAD(P) enzymes, whose activities would be linked to the malate-aspartate, lactate-pyruvate and isocitrate cytosolic-mitochondrial shuttles. These enzymes play a major role in supplying reduction equivalents and/or energy required for capacitation and acrosome reaction in cryopreserved bovine spermatozoa.     

Evidence for the involvement of sperm angiotensin converting enzyme in fertilization

Recently it has been observed that ejaculated human sperm possess high angiotensin converting enzyme (ACE) activity and that this enzyme is released during the process of capacitation. This observation raises the possibility that ACE may be involved in the fertilization process. To verify this hypothesis, we tested the effects of a potent ACE inhibitor, Captopril, on acrosome reaction induced by capacitating medium (3.5% HSA-added BWW) and on ability of human capacitated spermatozoa to penetrate zona-free hamster oocytes. Addition of Captopril (100 nmol l-1) modified neither sperm motility nor viability at any time considered, but significantly reduced the acrosome reaction percentages of sperm incubated in capacitating medium. Furthermore, Captopril significantly reduced the percentage of penetrated oocytes. The mean penetration rates both in the absence and presence of Captopril were 65.5 +/- 4.9% and 26.9 +/- 2.3% (P less than 0.001) respectively. These findings provide evidence that sperm release of ACE during capacitation may have a physiological role in the regulation of the mechanisms that allow sperm acrosome reaction and thus fertilizability.     

Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction.

The acrosome reaction of spermatozoa may be analogous to various somatic cell exocytotic events that incorporate cascade reactions. One such cascade system involves the hydrolysis of a membrane-bound phospholipid; generation of the intracellular second messenger, diacylglycerol; and activation of protein kinase C, followed by the phosphorylation of a number of intracellular proteins. Stimulators of protein kinase C, phorbol diesters and synthetic diacylglycerols, were evaluated to determine if this system functions in the human sperm acrosome reaction. Phorbol 12-myristate 13-acetate and 4 beta-phorbol 12,13-didecanoate caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa. Conversely, an inactive phorbol diester had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. The synthetic diacylglycerols, 1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-dioleoyl-sn-glycerol caused a significant (P less than 0.01) increase in the acrosome reaction of capacitated spermatozoa, and to a similar extent as the phorbol diesters. A nonactivating isomer of 1,2-dioleoyl-sn-glycerol, 1,3-diolein, had no significant (P greater than 0.05) stimulatory effect on the acrosome reaction. Protein kinase C activation is a diacylglycerol-dependent and Ca2(+)-dependent process, and stimulation of the acrosome reaction by 1,2-dioctanoyl-sn-glycerol required the presence of calcium ions in the capacitation medium. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), prevented the diacylglycerol-induced acrosome reaction (P less than 0.01). These results support the hypothesis that protein kinase C, via activation by the intracellular second messenger diacylglycerol, has a role in the human sperm acrosome reaction.