RESUMO
Sperm must undergo the acrosome reaction (AR) in order to fertilize the egg. In sea urchins, this reaction is triggered by the egg jelly (EJ) which, upon binding to its sperm receptor, induces increases in the ion permeability of the plasma membrane and changes in protein phosphorylation. Here, we demonstrated that the sperm expresses ROCK (approximately 135kDa), which is a serine/threonine protein kinase. ROCK localized, as RhoGTPase (Rho), in the acrosomal region, midpiece and flagellum. H-1152, a ROCK antagonist, inhibited the two cellular processes defining the AR: the acrosomal exocytosis and the actin polymerization. The ionophores nigericin and A23187 reversed the AR inhibition induced by H-1152, suggesting that ROCK functions at the level of the EJ-induced ion fluxes. Accordingly, H-1152 blocked 70% the intracellular alkalinization induced by EJ. These results indicate that EJ activates a Na+-H+ exchanger (NHE) in the sperm through a Rho/ROCK-dependent signaling pathway that culminates in the AR.
Assuntos
Reação Acrossômica/fisiologia , Ouriços-do-Mar/metabolismo , Espermatozoides/fisiologia , Quinases Associadas a rho/metabolismo , Animais , Células Cultivadas , Homeostase/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Quinases Associadas a rho/químicaRESUMO
At fertilization, the sea urchin egg undergoes an internal pH (pHi) increase mediated by a Na+ -H+ exchanger. We used antibodies against the mammalian antiporters NHE1 and NHE3 to characterize this exchanger. In unfertilized eggs, only anti-NHE3 cross-reacted specifically with a protein of 81-kDa, which localized to the plasma membrane and cortical granules. Cytochalasin D, C3 exotoxin (blocker of RhoGTPase function), and Y-27632 (inhibitor of Rho-kinase) prevented the pHi change in fertilized eggs. These inhibitors blocked the first cleavage division of the embryo, but not the cortical granule exocytosis. Thus, the sea urchin egg has an epithelial NHE3-like Na+ -H+ exchanger which can be responsible for the pHi change at fertilization. Determinants of this pHi change can be: (i) the increase of exchangers in the plasma membrane (via cortical granule exocytosis) and (ii) Rho, Rho-kinase, and optimal organization of the actin cytoskeleton as regulators, among others, of the intrinsic activity of the exchanger.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Óvulo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ouriços-do-Mar/citologia , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Citocalasina D/farmacologia , Exotoxinas/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Piridinas/farmacologia , Espermatozoides/efeitos dos fármacos , Quinases Associadas a rhoRESUMO
Cortical granules are secretory vesicles of the egg that play a fundamental role in preventing polyspermy at fertilization. In the sea urchin egg, they localize directly beneath the plasma membrane forming a compact monolayer and, upon fertilization, undergo a Ca(2+)-dependent exocytosis. Cortical granules form during early oogenesis and, during maturation, translocate from the cytosol to the oocyte cortex in a microfilament-mediated process. We tested the hypothesis that these cortical granule dynamics were regulated by Rho, a GTPase of the Ras superfamily. We observed that Rho is synthesized early in oogenesis, mainly in a soluble form. At the end of maturation, however, Rho associates with cortical granules. Inhibition of Rho with the C3 transferase from C. botulinum blocks cortical granule translocation and microfilaments undergo a significant disorganization. A similar effect is observed by GGTI-286, a geranylgeranyl transferase inhibitor, suggesting that the association of Rho with the cortical granules is indispensable for its function. In contrast, the anchorage of the cortical granules in the cortex, as well as their fusion at fertilization, are Rho-independent processes. We conclude that Rho association with the cortical granules is a critical regulatory step in their translocation to the egg cortex.
Assuntos
Leucina/análogos & derivados , Oócitos/crescimento & desenvolvimento , Ouriços-do-Mar/crescimento & desenvolvimento , Vesículas Secretórias/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , ADP Ribose Transferases/farmacologia , Citoesqueleto de Actina/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Toxinas Botulínicas/farmacologia , Imunoquímica , Leucina/farmacologia , Meiose , Oócitos/enzimologia , Oócitos/metabolismo , Oogênese , Ouriços-do-Mar/enzimologia , Ouriços-do-Mar/metabolismoRESUMO
Fertilization of the sea urchin egg triggers a Ca(2+)-dependent cortical granule exocytosis and cytoskeletal reorganization, both of which are accompanied by an accelerated protein synthesis. The signaling mechanisms leading to these events are not completely understood. The possible role of Rho GTPases in sea urchin egg activation was studied using the Clostridium botulinum C3 exotoxin, which specifically ADP-ribosylates Rho proteins and inactivates them. We observed that incubation of eggs with C3 resulted in in situ ADP-ribosylation of Rho. Following fertilization, C3-treated eggs were capable of performing cortical granule exocytosis but not the first cytokinesis. C3 caused in both unfertilized eggs and early embryos alterations in the state of actin polymerization and inhibition of the spindle formation. Moreover, C3 diminished markedly the rate of protein synthesis. These findings suggested that Rho is involved in regulating the acceleration of protein synthesis that accompanies the egg activation by sperm.
Assuntos
Biossíntese de Proteínas , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Toxinas Botulínicas/farmacologia , Cálcio/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Exocitose , Fertilização , Microscopia de Fluorescência , Ouriços-do-Mar , Transdução de Sinais , Frações Subcelulares/metabolismo , Fatores de Tempo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.