chlortetracycline and oxophenylarsine

In the present study, the viability, intracellular pH (pHi), cAMP ([cAMP]i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the 'B' pattern of chlortetracycline binding, increased pHi, [cAMP]i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca2+ and cAMP levels. Intracellular pH was increased while both Ca2+ and [cAMP]i were decreased. Physiological concentrations of H2O2 increased [cAMP]i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca2+ concentration, whereas the acrosome reaction was correlated with pHi and Ca2+ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations.

Topics: 1-Methyl-3-isobutylxanthine; Acrosome Reaction; Animals; Arsenicals; Calcium; Cattle; Cell Survival; Chlortetracycline; Cyclic AMP; Exocytosis; Heparin; Hydrogen Peroxide; Hydrogen-Ion Concentration; Male; Phosphodiesterase Inhibitors; Phosphotyrosine; Protein Tyrosine Phosphatases; Reproductive Techniques; Signal Transduction; Sperm Capacitation; Spermatozoa; Time Factors; Vanadates

Although semen cryopreservation is widely and commonly used in the bovine breeding industry, half the spermatozoa do not survive and most of those that do survive undergo numerous physiological changes that affect their fertilising ability. The aim of the present study was to determine how cryopreservation affects the intracellular events involved in sperm capacitation and acrosome reaction. Immediately after thawing and washing, almost 50% of spermatozoa were capacitated and more than 20% had lost their acrosome. The sperm cAMP concentration was lower than that in freshly ejaculated spermatozoa, but the cytosolic pH (pHcyt) was in the expected range. The free cytosolic Ca2+ concentration ([Ca2+]cyt) was higher than in fresh spermatozoa and cryopreserved spermatozoa had internally stored Ca2+. Phenylarsine oxide increased pHcyt and both cytosolic and stored Ca2+ concentrations, whereas orthovanadate enhanced acrosome loss and protein tyrosine phosphorylation (P-Tyr). Heparin increased the percentage of spermatozoa expressing the B (capacitated) chlortetracycline binding pattern, pHcyt, P-Tyr and Ca2+ storage. Moreover, positive correlations exist between capacitation, cAMP, P-Tyr and stored Ca2+, whereas the acrosome reaction is positively correlated with pHcyt and [Ca2+]cyt. These results demonstrate that sperm regulatory mechanisms may be affected by the cryopreservation procedure, but frozen-thawed sperm can still regulate their capacitation and acrosome reaction signalling pathways.

Topics: 1-Methyl-3-isobutylxanthine; Acrosome Reaction; Animals; Arsenicals; Calcium; Cattle; Cell Survival; Chlortetracycline; Cryopreservation; Cyclic AMP; Exocytosis; Heparin; Hydrogen Peroxide; Hydrogen-Ion Concentration; Male; Phosphodiesterase Inhibitors; Phosphotyrosine; Protein Tyrosine Phosphatases; Reproductive Techniques; Semen Preservation; Signal Transduction; Sperm Capacitation; Sperm Motility; Spermatozoa; Time Factors; Vanadates