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Biology of Reproduction 61, 76-84 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Roles of Bicarbonate, cAMP, and Protein Tyrosine Phosphorylation on Capacitation and the Spontaneous Acrosome Reaction of Hamster Sperm1

Pablo E. Visconti2,a, J. Stewart-Savageb, Aida Blascoc, Licia Battagliac, Patricia Mirandac, Gregory S. Kopfa, and Jorge G. Tezónc

a Center for Research on Reproduction & Women's Health, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6080 b Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148-2960 c Instituto de Biología y Medicina Experimental (IBYME), Obligado 2490, (1428) Buenos Aires, Argentina

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 µM, and with an EC50 of 100 µM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 µM dibutyryl cAMP (dbcAMP), alone or together with 100 µM 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.

1 This work was supported by grants from the NIH (HD06274; HD33052; HD22732; HD34811) and support from the Special Program of Research, Development and Research Training in Human Reproduction (WHO Project 97095), and Consejo de Investigaciones Científicas Y Técnicas of Argentina (CONICET).

2 Correspondence: Pablo E. Visconti, Center for Research on Reproduction and Women's Health, John Morgan Building, Room 314, University of Pennsylvania Medical Center, Philadelphia, PA 19104–6080. FAX: 215 349 5118; visconti{at}mail.med.upenn.edu




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