Inhibition of mos-induced Oocyte Maturation by Protein Kinase A

The relationship between the mos protooncogene protein and cAMP-dependent protein kinase (PKA) during the maturation of Xenopus oocytes was investigated. Microinjection of the PKA catalytic subunit (PKA0 into Xenopus oocytes inhibited oocyte maturation induced by the mos product but did not markedly affect the autophosphorylation activity of injected mos protein. By contrast, PKA~ did not inhibit maturation promoting factor (MPF) activation or germinal vesicle breakdown (GVBD) that was initiated by injecting crude MPF preparations. In addition, inhibiting endogenous PKA activity by microinjecting the PKA regulatory subunit (PKAr) induced oocyte maturation that was dependent upon the presence of the endogenous mos product. Moreover, PKA~ potentiated mos protein-induced MPF activation in the absence of progesterone and protein synthesis. These data are consistent with the hypothesis that progesterone-induced release from G2/M is regulated via PKA~ and that PKA~ negatively regulates a downstream target that is positively regulated by mos. F ULLY grown Xenopus oocytes are arrested in prophase of meiosis I and are induced to mature upon exposure to progesterone (29). Synthesis of the mos protooncogene product, pp39-", is required for the activation of maturation-promoting factor (MPF) I, an activity responsible for coordinating the biochemical events of meiosis I and 1I (9, 22, 39, 41). Injecting the mos product into a two-cell embryo arrests the injected cell at metaphase, which led to the identification of mos as an active component of cytostatic factor (CSF) (41). CSF is a calcium-sensitive activity believed to be responsible for the arrest of an unfertilized egg at metaphase of meiosis II and for the stabilization of MPF (29, 31, 32, 41). The observation that mos is synthesized prior to MPF activation during meiosis I, led us to propose that mos is an ~initiator" and is required to activate MPF from pre-MPF (40). Furthermore, pp39 ~o' is required at all stages during oocyte maturation (9, 22). mos RNA or protein can initiate MPF activation when microinjected into fully grown oocytes (16, 40, 50). Recently, using recombinant mos protein (MBPmosx,), we have shown that the protooncogene product is both necessary and sufficient to initiate meiosis I (50). However, injected oocytes do not progress to meiosis iI in the absence of protein synthesis (50), even though mos is required during this period (9, 22). This suggests that additional proDr. Daar's present address is Biological Response Mtxlifiers Program, NCIFrederick Cancer Research and Development Center, P.O. Box B, Frederick, MD 21702. 1. Abbreviations used in this paper: CSE cytostatic factor; GVBD, germ vesicle breakdown; MBS, modified Barth solution; MPF, maturation promoting factor; PKA, protein kinase; PKI, PKA inhibitory poptide. teins synthesized de novo are required for meiosis H and CSF arrest (50). Progesterone markedly enhances MBP-mos~-induced germ vesicle breakdown (GVBD) in the absence of protein synthesis (50), suggesting that the hormone removes a biochemical block to MPF activation that pre-exists in the oocyte. This system provided a means for testing the biochemical events involved in oocyte maturation in the absence of protein synthesis requirements, cAMP-dependent protein kinase (PICA) has been implicated as a negative regulator of GriM transition and it is generally believed that progesterone stimulation of oocytes causes a transient decrease in cAMP levels. This, in turn, leads to a decrease in PKA activity and results in the dephosphorylation of a presumptive maturation-inhibiting phosphoprotein (44). While the role of cAMP and PKA in oocyte maturation is unclear (44) the following observations support the above hypothesis: progesterone inhibits adenylate cyclase activity in frog oocytes (13, 20, 34, 36); cAMP levels decrease during oocyte maturation in several organisms including frog (24, 25, 42), starfish (30), and mammals (43); the injection of phosphodiesterase induces maturation (7; Foerder, C. A., T. J. Martins, J. A. Beavo, and E. G. Krebs. 1982. J. Cell Biol. 95:304a), while inhibitors of phosphodiesterase prevent maturation (7, 37, 43); activators of adenylate cyclase cause an increase in cAMP levels, resulting in inhibition of Xenopus and mammalian oocyte maturation (17, 34, 49); and the injection of either the PKA regulatory subunit (PKA~), which binds to and inactivates the catalytic subunit, or the PKA inhibitory peptide (PKI) induces maturation (26), while the catalytic subunit of PKA (PKA0 inhibits oocyte maturation in amphibians (26) and mammals (7). Since progesterone influences the PKA pathway and The Journal of Cell Biology, Volume 120, Number 5, March 1993 1197-1202 1197 on July 7, 2017 jcb.rress.org D ow nladed fom potentiates mos-induced MPF activation in the absence of protein synthesis, we examined the effects of the PKA subunits on mos function. We show that the PKAc does not affect MPF-induced maturation, but markedly inhibits MBPmos~=-induced GVBD. By contrast, in the absence of protein synthesis the regulatory subunit (PKA~) potentiates MPB-mos ~ promotion of GVBD almost as efficiently as progesterone. Materials and Methods