Identification of PLCγ-Dependent and -Independent Events during Fertilization of Sea Urchin Eggs

AbstractAt fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+action potential, Ca2+release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cγ (PLCγ), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCγ as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCγ and (1) Ca2+green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCγ produced a concentration-dependent inhibition of Ca2+release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCγSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCγSH2 domains was further indicated by the finding that microinjection of PLCγSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCγSH2 fusion protein. These results indicate that PLCγ plays a key role in several fertilization events in sea urchin eggs, including Ca2+release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCγ activation or Ca2+increase

Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone

AbstractThe meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2–7E). Npr2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3+/+ and Gpr3−/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG