Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Sperm capacitation in vitro in the eld's deer

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.     

Hamster zonae pellucidae cannot induce physiological acrosome reactions in chemically capacitated hamster spermatozoa in the absence of albumin

Cauda epididymal hamster spermatozoa were capacitated with D-penicillamine in a chemically defined (protein-free) medium (= "chemical" capacitation). Hamster zonae pellucidae were incapable of inducing functional acrosome reactions in chemically capacitated hamster sperm in a protein-free medium during sperm-egg coincubation. The culture medium used throughout incubation was a modified Tyrode's solution containing 10 mM sodium lactate, 100 microM sodium pyruvate and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm motility was maintained in all media with PHE (20 microM penicillamine, 100 microM hypotaurine, and 1.0 microM epinephrine). Additional D-penicillamine (125 or 500 microM) or 3 mg/ml bovine serum albumin (high control: TALP-PVA) was used to capacitate sperm during preincubation at 1-2 X 10(6) sperm/ml for 4.0 h at 37 degrees C in 5% CO2 in air. Sperm were then coincubated (2 X 10(4) sperm/ml) with cumulus-free hamster eggs in TALP-PVA or TLP-PVA +/- additional D-penicillamine (total: 500 or 125 microM) for 1.5 or 6.0 h. Percent egg penetration was used as the definitive index of sperm capacitation and functional acrosome reactions. Chemically capacitated sperm did not penetrate eggs (0.0 +/- 0.0%) in the absence of albumin during 1.5 h of sperm-egg coincubation. When sperm were chemically capacitated with 125 microM or 500 microM D-penicillamine, then coincubated with eggs for 6.0 h in the absence of albumin, only 18.8 +/- 28.6% and 23.7 +/- 29.7%, respectively, of eggs were penetrated. Significantly (p less than 0.05) more eggs (67.7 +/- 22.4%) were penetrated when coincubated with chemically capacitated sperm for 1.5 h in medium containing albumin. These results demonstrate that zonae pellucidae of hamster eggs require the presence of albumin to efficiently induce functional acrosome reactions in sperm that are chemically capacitated with D-penicillamine.     

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.     

Baboon serum is superior to human or bovine serum albumin for baboon sperm capacitation and zona binding

Background  Baboon in vitro fertilization requires capacitated sperm in appropriate media. In this study, we compared the effect of baboon serum (Bas), human serum albumin (HSA) and bovine serum albumin (BSA) on baboon sperm capacitation.
Methods  Five males (n = 5) were electroejaculated and 43 oocytes retrieved from super-ovulated female baboons (n = 10). Each sperm sample was assessed for initial motility and concentration before and after swim-up. For swim-up, each sperm sample was incubated separately in Biggers–Whitten–Whittingham media containing either BaS, HSA, BSA or without protein supplementation (control). After swim-up, each sperm aliquot was incubated with two to three oocytes. The number of sperm bound to the zona was evaluated after overnight incubation.
Results  Sperm motility and zona binding was significantly higher after capacitation in media supplemented with BaS than in HSA or BSA or in media without protein supplementation ( P  < 0.05).
Conclusion  Baboon serum is superior to HSA or BSA for baboon sperm capacitation and zona binding.     

A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis

Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (P相似文献   

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.     

Development of ability to penetrate the cumulus oophorus by hamster spermatozoa capacitated in vitro,in relation to the timing of the acrosome reaction

Hamster spermatozoa were tested for their ability to penetrate the intact cumulus matrix at low sperm:egg ratios (approximately 3:1). Uncapacitated spermatozoa attached to the surface of the cumulus and could not penetrate. Spermatozoa capacitated in vitro began to be able to penetrate after about 2 hr of preincubation, coincidentally with the first appearance of hyperactivation and spontaneous acrosome reactions. In all, 628 in vitro incubated spermatozoa were evaluated on and in cumuli: 270 could penetrate, but only ten of these were judged to have intact, “unmodified” acrosomes. Almost all spermatozoa capable of penetrating showed optically “modified” and swelling acrosomal caps, and this confirms previous observations on cumulus penetration in vivo. Penetration appeared limited to a phase in capacitation prior to completion of the acrosome reaction, as spermatozoa that had lost the acrosomal cap penetrated poorly and showed reduced viability. Penetration of the cumulus was inhibited by the hyaluronidase inhibitor sodium aurothiomalate. Cumulus penetrating ability could result either from a change in surface properties of the sperm at capacitation, which renders them less “sticky” to the matrix, or from release or activation of a “cumulus lysin.” We conclude that the ability to enter the cumulus matrix coincides with physiological changes in spermatozoa that occur during a terminal phase of capacitation preceding complete loss of the acrosomal cap, and that initiation of this process in vivo must precede sperm-zona contact.     

Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa

This study aimed to demonstrate nitric oxide production by human spermatozoa and to characterize the interaction between nitric oxide and cAMP-related pathway in the control of human sperm capacitation and protein tyrosine phosphorylation. Spermatozoa were incubated in Tyrode's medium with or without bovine serum albumin (BSA), and nitric oxide was measured with the spin trap sodium N-methyl-D-glucamine dithiocarbamate. Under noncapacitating conditions, spermatozoa produced low levels of nitric oxide. However, under capacitating conditions, prominent nitric oxide adduct signals were obtained and a time-dependent increase of nitric oxide production was observed. When spermatozoa were incubated in Tyrode+BSA medium with nitric oxide-releasing compounds, intracellular cAMP concentrations increased to levels higher than those of spermatozoa incubated in Tyrode+BSA alone. In contrast, incubation with nitric oxide synthase inhibitors (N(G)-nitro-L-arginine methyl ester or N(G)-monomethyl L-arginine) decreased intracellular sperm cAMP concentrations. The inhibitory effect observed with N(G)-nitro-L-arginine methyl ester on capacitation and tyrosine phosphorylation of two sperm proteins (105, 81 kDa) was overcome by the presence of cAMP analogs or of a phosphodiesterase inhibitor. These results indicate that nitric oxide is produced by capacitating human spermatozoa and that it may act as a cellular messenger by modulating the cAMP pathway involved in capacitation and protein tyrosine phosphorylation.     

Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine,L-histidine,and L-cysteine in a protein-free culture medium

By using a chemically defined (protein-free) culture medium that supports sperm viability but not capacitation or the acrosome reaction, we have determined that hamster spermatozoa can be chemically capacitated in vitro by the divalent cation chelators D-penicilla-mine, L-histidine, and L-cysteine in the absence of bovine serum albumin (BSA). Washed cauda epididymal spermatozoa were preincubated (1–2 × 10⁶ sperm/ml) for 3, 4, or 6 hr at 37°C in 5% CO₂ in air. The basic culture medium used for sperm preincubation and for sperm:egg coincubation was a modified Tyrode's solution (protein-free) containing 10 mM sodium lactate, 100 μM sodium pyruvate, and 1.0 mg/ml polyvinylalcohol (TLP-PVA). Sperm viability was maintained in all preincubation and coincubation media with PHE (20 μM D-penicillamine, 100 μM hypotaurine, and 1.0 μM epinephrine). The low control sperm preincubation medium consisted of TLP-PVA. In some cases the high control preincubation medium also contained 3 mg/ml BSA (TALP-PVA). The experimental preincubation medium was TLP-PVA with additional D-penicillamine (125 or 500 μM), or L-histidine (10, 100, or 1,000 μM) or L-cysteine (25, 75, or 125 μM). After preincubation, sperm were coincubated (2 × 10⁴ sperm/ml) with cumulus-free hamster eggs in TALP-PVA ± additional D-penicillamine, L-histidine, or L-cysteine for 1.5 hr, fixed, and evaluated for percent egg penetration as an index of sperm capacitation. The results demonstrate that hamster spermatozoa can be chemically capacitated in vitro with D-penicillamine (500 μM: range of mean penetration values, 53.6%–84.3%), L-histidine (100 μM: range of mean values, 24.8%–56.3%) or L-cysteine (75 μM: 51.3%) in the absence of exogenous protein.     

Changes in the distribution of intramembranous particles and filipin-reactive membrane sterols during in vitro capacitation of golden hamster spermatozoa

Membrane alterations accompanying in vitro capacitation of hamster spermatozoa were examined using the freeze-fracture technique with or without use of filipin, a sterol-binding probe. In the spermatozoa prior to or at 10 min after start of incubation in capacitating medium, large (about 11 nm) and small (8–9 nm) intramembranous particles (IMPs) were present in the periacrosomal region of the sperm plasma membrane (PAPM). Filipin sterol complexes (FSCs) were densely (about 500/μ²) distributed in the PAPM prior to incubation. The density of FSCs in the PAPM was reduced by 70–80% of the original density by 2 hr of incubation. At the same time, small patches of IMP-free areas appeared in the plasma membrane above the equatorial and middle segments of the acrosome. By the end of 3 hr of incubation, the majority of small IMPs had disappeared from the PAPM. Remaining large and small IMPs tended to aggregate in the PAPM. During incubation in capacitation medium, “cords,” or linear arrangements of closely packed IMPs, appeared near the posterior ring of the sperm head. These observations strongly suggest that the acrosome reaction of the hamster spermatozoa is preceded by the removal (deletion) of filipin-reactive sterols (FRSs) and the disappearance of small IMPs from the lipid bilayer of PAPM.     

Capacitation and the acrosome reaction in squirrel monkey (Saimiri sciureus) spermatozoa evaluated by the chlortetracycline fluorescence assay

Capacitation and the acrosome reaction in squirrel monkey seminal spermatozoa diluted in Tyrode's medium (TALP) and TC-199 were monitored by a chlortetracycline (CTC) fluorescence assay. Four CTC patterns, similar to those found in human sperm, were readily characterized by fluorescent staining on the heads of the spermatozoa. The appearance of the capacitated (CP) pattern was dependent on the concentration of the bovine serum albumin. Acrosomal loss was observed in a maximum of 15% of the sperm in the populations studied here. Calcium ionophore A23187 (5 μM to 20μM) induced acrosomal loss in 60–70% of capacitated spermatozoa. However in freshly ejaculated sperm incubated under capacitating conditions or in spermatozoa incubated in Ca^{+ +}-free medium, A23187 failed to induce acrosomal loss. Furthermore, spermatozoa incubated in the presence of seminal plasma or spermatozoa obtained following a 1-hour “swim-up” procedure showed an identical timecourse of appearance of the CP pattern, indicating the lack of effect of seminal plasma on capacitation in the squirrel monkey.     

Effects of cumulus cells on sperm penetration of bovine oocytes in protein-free medium

The present study was conducted to examine the effects of cumulus cells on sperm capacitation, acrosome reaction and penetration of bovine oocytes in vitro in a protein-free medium. In vitro matured oocyte-cumulus complexes (OCCs) and denuded oocytes were co-incubated with spermatozoa in the medium with or without bovine serum albumin (BSA). Higher fertilization rates were obtained in the OCCs (92 and 89%, respectively) than denuded oocytes (57 and 6%, respectively) in the medium with or without BSA (P<0.01). Higher proportion of the denuded oocytes were fertilized in the medium with BSA (57%) than without BSA (6%; P<0.01). These results suggest that the cumulus cells are more effective for increasing fertilization rate than BSA (P<0.05). Both the percentages of capacitated and acrosome-reacted spermatozoa incubated for 4 h with isolated cumulus cells were not significantly different in the medium without cumulus cells in the presence or absence of BSA. The denuded oocytes were inseminated with isolated cumulus cells taken from OCCs matured with or without hormones, follicle stimulating hormone (FSH) and estradiol-17beta (E(2)), and from immature OCCs in a protein-free medium. Presence of the cumulus cells matured with hormones enhanced sperm penetration of denuded oocytes more effectively (81%) than either of the cells matured without hormones (41%) or the immature cells (26%; P<0.01). The conditioned medium of cumulus cells matured with hormones was not effective for sperm penetration of denuded oocytes (2%), while a high proportion (82%) of the oocytes were fertilized when they were inseminated with isolated cumulus cells (P<0.01). In conclusion, the presence of cumulus cells matured with FSH and E(2) was effective for sperm penetration but not for sperm capacitation or acrosome reaction.     

Identification of capacitation-associated phosphoproteins in porcine sperm electroporated with ATP-gamma-(32)P.

Our objectives were to incorporate ATP-gamma-(32)P into boar sperm to radiolabel endogenous phosphoproteins and compare phosphorylation patterns from sperm incubated in capacitating (CM) and non-capacitating conditions (NCM). Sperm were electroporated (1000 V/cm, 125 microF/cm, 65 Omega/cm, 0.3 msec) with ATP-gamma-(32)P which moderately decreased sperm viability (P < 0.01), but did not affect motility (P = 0.34) or the appearance of spontaneous acrosome reactions (P = 0.49). Sperm incubated in CM for 3 hr underwent capacitation, determined by the ability to undergo ionophore-induced acrosome reactions (P 0.05) and the 57 kDa phosphoprotein increased after capacitation (P /= 0.02). ATP-gamma-(32)P can, therefore, be incorporated into porcine sperm to radiolabel endogenous phosphoproteins, and the different profiles from sperm incubated in NCM versus CM suggest that capacitation is mediated by signaling events involving protein phosphorylation.     

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.     

Reactive oxygen species requirements for bovine sperm capacitation and acrosome reaction

Sperm capacitation is necessary for the fertilization of oocytes. During capacitation intracellular and membrane changes occur, that culminate with an exocytotic event called the acrosome reaction. The aim of this work was to study the participation of the superoxide anion (O2-.) and of hydrogen peroxide (H2O2) in the capacitation process and acrosome reaction in spermatozoa from cryopreserved bovine semen. Samples were capacitated with heparin or treated with the xanthine-xanthine oxidase-catalase system (X-XO-C) for the production of O2-. The percentage of capacitated spermatozoa was determined using the chlortetracycline (CTC) technique, by means of epifluorescence microscopy. Addition of X-XO-C to the incubation medium significantly induced capacitation (P < 0.05), but there were no differences with samples incubated with heparin. When the medium contained heparin or the X-XO-C, addition of superoxide dismutase (SOD, 0.5 mg/mL) significantly inhibited capacitation (P < 0.05). In samples treated with heparin and with diverse concentrations of H2O2 (10, 25, 50 and 250 microM) in the incubation medium, the percentage of capacitated spermatozoa was significantly reduced (P < 0.05); however, acrosome reaction was produced at concentrations of 10 and 25 microM H2O2. At concentrations greater than 25 microM H2O2 a deleterious effect was observed on sperm motility. From these results it may be inferred that O2-. is required in the capacitation process and that H2O2 may participate as an inductor of the acrosome reaction in spermatozoa from cryopreserved bovine semen.     

In vitro capacitation of stallion spermatozoa in calcium-free Tyrode's medium and penetration of zona-free hamster eggs

Capacitation of stallion spermatozoa in Tyrode's calcium-free (TCF) medium was assessed. Twelve gel-free ejaculates were collected. After removal from the seminal plasma, cells were washed three times with 0.85% saline containing 0.1% bovine serum albumin (BSA) and resuspended in TCF. Both washing and incubation media were adjusted to pH 8 and 300 to 310 mOsm. Final sperm concentration during incubation was 2 x 10(6) cells/ml. The diluted ejaculates were incubated for 2, 4, 6, 8 and 10 h at 37 degrees C in an atmosphere containing 5% CO(2). Acrosomes were stained with naphthol yellow and erythrocin B initially and after each incubation period and evaluated microscopically. Transmission electron microscopy was used to verify whether normal acrosome reaction was occurring or if cells were degenerating. Penetration of zona-free hamster oocytes was evaluated using 10(3) to 10(4) sperm/ml suspension and coincubating eggs for 3.5 to 4 h with sperm. Penetration tests were done for wash and incubation treatments and recorded positive when swollen sperm heads or male pronuclei were present. Incubation time affected acrosome integrity (P<0.001). Incubation for 8 to 10 h significantly improved acrosome reaction (P<0.001) and the percentage of reacted acrosomes increased sharply after 6 h of incubation (P<0.001). None of the washed sperm penetrated zona-free eggs at zero time, but sperm from all incubation treatments penetrated eggs. A peak penetration rate of 29.9% was observed at 8 h (P<0.001). Results indicate that under the conditions used, the requirement for Ca(++) in the medium for the process of capacitation and acrosome reaction can be substituted for by elevated pH.     

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.     

Induction of buffalo (Bubalus bubalis ) sperm capacitation and acrosome reaction in the excised reproductive tract of hamsters

A study was conducted on the induction of buffalo sperm capacitation and acrosome reaction in the excised reproductive tract of hamsters at the estrogen- and progesterone-dominated stages of estrus. The percentages of the maximum capacitation and acrosome reaction were significatly (P < 0.01) higher for spermatozoa incubated in the uterus with oviducts of estrogen dominated hamsters compared with those incubated in BWW medium in a test tube (64.6%, 60.2%; 16.2%, 14.7%). Buffalo spermatozoa incubated in the uterus and oviducts of progesterone-dominated hamsters showed significantly (P < 0.01) lower capacitation and acrosome reaction rates than those incubated in the uterus and oviducts of estrogen-dominated hamsters (34.8%, 34.3%: 64.6%, 60.2%). The percentage of capacitation and acrosome reaction in spermatozoa were significantly (P < 0.01) more when incubated in the uterus plus oviducts than without the oviduct irrespective of whether the reproduct tract of hamster was estrogen- or progesterone-dominated. The time for the onset of maximum capacitation and acrosome reaction was reduced from 12 to 10 h when the spermatozoa were incubated in the hamster reproductive tract rather than in BWW medium in test tubes. The significance of the results in relation to hormonal regulation of sperm capaciation and acrosome reaction are also discussed.