Showing papers by "Pablo E. Visconti published in 1999"

Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation

Abstract: Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO(3) are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO(4) inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-beta-cyclodextrin or OH-propyl-beta-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation.

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

Abstract: 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 35 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

Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation

Abstract: Sperm adhesion to egg zonae pellucidae initiates sperm acrosome reactions, an exocytotic event that is an early step during fertilization. Previously, it was suggested that zona pellucida-evoked Ca2+ entry into sperm through low voltage-activated Ca2+ channels is an essential step in acrosome reactions, based on the inhibitory effects of Ca2+ channel antagonists. However, analysis of this channel is limited by the inability to apply electrophysiological methods directly to sperm. In this report, optical methods of determining membrane potential and internal Ca2+ levels were used to demonstrate that (i) contact with zonae pellucidae activates a transient Ca2+ response in sperm that has a time course and antagonist sensitivity anticipated of low voltage-activated Ca2+ channels; (ii) these channels are unavailable for opening in uncapacitated sperm because of voltage-dependent, steady state inactivation; (iii) membrane hyperpolarization during sperm capacitation is sufficient to recruit channels into a closed state, from which they are available for opening during fertilization; and (iv) channel conductance state may be a factor in determines the efficacy with which channel antagonists inhibit fertilization. This study provides evidence for the activation of sperm Ca2+ channels during gamete adhesion and offers a mechanism that may account for aspects of the regulation of sperm fertility during capacitation through the control of channel availability. Finally, these results suggest that channel conductance state may be a central feature in the design of channel antagonists that inhibit sperm function.

Capacitation of the Mammalian Spermatozoon

Abstract: Publisher Summary The acquisition of progressive motility and fertilization competence are acquired in many mammals during transit through the epididymis but complete fertilization capacity in vivo is conferred during residence in the female reproductive tract. The molecular and physiological events comprising this extratesticular maturational process that lead to the fertilization-competent state are referred to collectively as sperm capacitation. Sperm do not have the ability to fertilize eggs but require a limited time of residence in the female tract in order for sperm to gain the capacity for fertilization. The chapter provides a framework with which to develop new approaches to regulate fertility. It focuses on some of the recent developments in the field and provides a discussion of future experimental approaches to understand this important sperm maturational event. Capacitation has been defined as the time interval between sperm deposition in the female reproductive tract during natural mating and the time during which fertilization occurs.