Role for endocytosis of a constitutively active GPCR (GPR185) in releasing vertebrate oocyte meiotic arrest

Abstract

Vertebrate oocytes are naturally arrested at prophase of meiosis I for sustained periods of time before resuming meiosis in a process called oocyte maturation that prepares the egg for fertilization. Members of the constitutively active GPR3/6/12 family of G-protein coupled receptors represent important mediators of meiotic arrest. In the frog oocyte the GPR3/12 homolog GPRx (renamed GPR185) has been shown to sustain meiotic arrest by increasing intracellular cAMP levels through GαSβγ. Here we show that GPRx is enriched at the cell membrane (~80%), recycles through an endosomal compartment at steady state, and loses its ability to signal once trapped intracellularly. Progesterone-mediated oocyte maturation is associated with significant internalization of both endogenous and overexpressed GPRx. Furthermore, a GPRx mutant that does not internalize in response to progesterone is significantly more efficient than wild-type GPRx at blocking oocyte maturation. Collectively our results argue that internalization of the constitutively active GPRx is important to release oocyte meiotic arrest.

Xenopus oocyte prophase I meiotic arrest is released independently from a decrease in cAMP levels or PKA activity

“…PKA inhibits the maturation signaling pathway through at least two points: mRNA translation and Cdc25C activation (Duckworth et al, 2002;Matten et al, 1994), consistent with the idea that it is acting as a safety mechanism to eliminate spontaneous maturation in the absence of a positive signal. Although challenging the accepted dogma, our model is supported by earlier studies (Gelerstein et al, 1988;Nader et al, 2014;Noh and Han, 1998;Schmitt and Nebreda, 2002) and the yinyang balance between the mPRβ and cAMP pathways effectively explains the discrepancies in the literature.…”

“…Surprisingly, knockdown of GPR185 is not sufficient to induce oocyte maturation (Deng et al, 2008;Ríos-Cardona et al, 2008), arguing that other GPCRs are involved in maintaining meiotic arrest. We have previously shown that (1) blocking exocytosis, while maintaining endocytosis, releases meiotic arrest, (2) P4 results in the internalization of GPR185 and (3) a GPR185 mutant that does not internalize is more effective at maintaining meiotic arrest (ElJouni et al, 2007;Nader et al, 2014). These data argue that P4 induces internalization of GPR185 and potentially other GPCRs that are involved in maintaining high levels of cAMP and PKA and thus meiotic arrest.…”

“…However, the early cAMP drop was not sufficient to induce maturation, because exposure to P4 from 15 s to 10 min was not sufficient to mature oocytes; rather, oocytes required a minimum of 30 min exposure to P4 to induce significant oocyte maturation (Nader et al, 2014). However, oocytes might require an integrated sustained lower level of cAMP to inhibit PKA activity and relieve meiotic arrest.…”

“…Furthermore, some studies found that a decrease in cAMP levels was not sufficient to induce spontaneous maturation, and that an increase in cAMP accelerates maturation (Gelerstein et al, 1988;Noh and Han, 1998). Consistently, exposing oocytes to P4 for <30 min induced a decrease in cAMP but did not induce oocyte maturation, which required ≥30 min incubation with P4 (Nader et al, 2014). Hence, there is strong support for the cAMP-PKA axis maintaining the prolonged oocyte prophase I meiotic arrest.…”

“…Whereas some studies detect a 10-60% drop in cAMP levels from 15 s up to 6 h after P4 addition (Bravo et al, 1978;Cicirelli and Smith, 1985;Gelerstein et al, 1988;Maller et al, 1979;Mulner et al, 1979;Nader et al, 2014;Sadler and Maller, 1981;SchorderetSlatkine et al, 1982), others failed to detect any changes in cAMP levels in response to P4 (Noh and Han, 1998;O'Connor and Smith, 1976;Schutter et al, 1975;Thibier et al, 1982). Furthermore, some studies found that a decrease in cAMP levels was not sufficient to induce spontaneous maturation, and that an increase in cAMP accelerates maturation (Gelerstein et al, 1988;Noh and Han, 1998).…”