Mitogen-activated protein (MAP) kinase has been reported to be activated during oocyte meiotic maturation in a variety of mammalian species. However, the mechanism(s) responsible for MAP kinase activation and the consequence of its premature activation during gonadotropin-induced oocyte meiotic resumption have not been examined. The present experiments were conducted to investigate the possible role of MAP kinase in FSH-induced and spontaneous oocyte meiotic resumption in the mouse. MAP kinase kinase (MAPKK, MEK) inhibitor, PD98059 or U0126, produced a dose-dependent inhibitory effect on both FSH-induced oocyte meiotic resumption and MAP kinase activation in the oocytes. However, the same inhibitor did not block spontaneous meiotic resumption of either denuded or cumulus cell-enclosed mouse oocytes, despite the activity of MAP kinase being totally inhibited. Immunoblotting the oocytes and the cumulus cells with the anti-active MAP kinase antibody showed that MAP kinase activity in the oocytes was detected at 8 h of FSH treatment, prior to germinal vesicle breakdown and increased as maturation progressed in the following culture period. In the cumulus cells, MAP kinase was activated even faster, its activity was detected at 1 h of FSH stimulation and increased gradually until 8 h of FSH treatment, then decreased and diminished after 12 h of FSH action. These data demonstrated that the MEK-MAP kinase pathway is implicated in FSH-induced but not spontaneous oocyte meiotic resumption.
At the time of fertilization mammalian spermatozoa undergo a Ca2+-dependent exocytotic event, which is known as the acrosome reaction (AR). We describe here that EGF-receptor (EGFR) is localized in the head of bull spermatozoa and that epidermal growth factor (EGF) can induce the occurrence of the AR in its typical dose-dependent manner. Previously we showed that protem kinase C (PKC) is involved in the cascade leading to AR in bull spermatozoa. Here, we show that PKC is involved in the mechanism in which EGF exerts its effect on AR. These findings together with our results which show inhibition of AR by tyrosme-phosphorylation inhibitors, indicate that ejaculated bull sperm contain a typical 170-kDa EGFR which is active in the mechanism leading to AR.
The presence of ATP in the genital tract fluid of mammals provokes questions regarding its function in the fertilization process. We investigated the effect of extracellular ATP (ATPe) on the activation of bovine spermatozoa. A signal transduction mechanism for ATP involving the receptor-mediated release of second messengers is described. Treatment of spermatozoa with ATP, uridine triphosphate (UTP), or 2-methylthio-ATP resulted in a concentration-dependent increase of acrosomal exocytosis, whereas treatment with either AMP or adenosine induced little exocytosis. This suggested that the receptor involved is of the P2 and not the P1 type. Several lines of evidence also suggest that the ATP purinoceptor is of the P2y and not the P2x type. First, the acrosome reaction was induced by the P2y-agonists ATP, UTP, or 2-methylthio-ATP, but no effects were shown by the P2x-agonists alpha,beta-methylene-ATP or beta,gamma-methylene-ATP. Second, ATP-induced acrosomal exocytosis was inhibited by the P2y antagonists, but not by the P2x antagonists. Third, enhanced Ca2+ uptake into the cells was observed with ATP and 2-methylthio-ATP, but not with beta,gamma-methylene-ATP. Additionally, ATP induced elevation of intracellular Ca2+ and cAMP, and the effect on cAMP was predominantly enhanced by including Ca2+ and the Ca2+-ionophore A23187 in the incubation medium. Extracellular ATP also activates protein kinase Calpha (PKCalpha), and the acrosome reaction, stimulated by ATPe, is inhibited by a PKC-specific inhibitor. In summary, we suggest that ATPe activates the P2 purinoceptor that elevates [Ca2+]i, which leads to PKCalpha activation and culminates in acrosomal exocytosis.
Ejaculated mammalian spermatozoa must reside in the female genital tract for some time before gaining the ability to fertilize the egg. During this time, spermatozoa undergo some physiological changes that collectively are called capacitation. Capacitation of mammalian spermatozoa is a prerequisite for acrosome reaction, which is an exocytotic event occurring before fertilization. The specific biophysical and biochemical changes that accompany sperm capacitation and the agonists inducing acrosome reaction are not fully understood. Using SDS-gel electrophoresis and immunoblotting, we demonstrate the existence of a class of angiotensin receptors (AT1) in bovine spermatozoa. In capacitated sperm, we show that angiotensin II (ANG II) AT1 receptors are localized in the head and tail, whereas in noncapacitated cells the receptors are localized in the tail only. We find that ANG II markedly stimulates acrosomal exocytosis of capacitated bovine spermatozoa in vitro in a concentration range of 0.1–10 nM. No effect of ANG II was found in noncapacitated cells. The ability of ANG II to stimulate the acrosome reaction depends on the presence of calcium ions in the incubation medium. The ANG II-induced acrosome reaction was markedly inhibited by a selective AT1 receptor antagonist, losartan (DUP 753). PD-123319, a selective antagonist of the ANG II AT2 receptor, had no effect on the ANG II-induced acrosome reaction. Thus ANG II via activation of AT1 receptors may play a regulatory role in the induction of the acrosome reaction.
Protein kinase C (PKC) has been implicated in the sperm acrosome reaction. In the present study, we demonstrate induction of the acrosome reaction and activation of sperm PKCalpha by lysophosphatidic acid (LPA), which is known to induce signal transduction cascades in many cell types via binding to specific cell-surface receptors. Under conditions by which LPA activates PKCalpha, there is significant stimulation of the acrosome reaction, which is inhibited by PKC inhibitors. Protein kinase Calpha belongs to the Ca(2+)-dependent classical PKC family of isoforms, and indeed we show that its activation depends upon the presence of Ca(2+) in the incubation medium. Protein kinase Calpha is a known regulator of phospholipase D (PLD). We investigated the possible regulatory relationships between PKCalpha and PLD1. Using specific antibodies against PLD1, we demonstrate for the first time its presence in bovine sperm. Furthermore, PLD1 coimmunoprecipitates with PKCalpha and the PKCalpha-PLD1 complex decomposes after treatment of the cells with LPA or 12-O:-tetradecanoyl phorbol-13-acetate, resulting in the translocation of PKCalpha to the plasma membrane and translocation of PLD1 to the particulate fraction. A possible bilateral regulation of PKCalpha and PLD1 activation during the sperm acrosome reaction is suggested.
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