Estrogen signaling characteristics of Atlantic croaker G protein-coupled receptor 30 (GPR30) and evidence it is involved in maintenance of oocyte meiotic arrest

Human G protein-coupled receptor 30 (GPR30) mediates estradiol-17 beta (E2) activation of adenylyl cyclase in breast cancer cells and displays E2 binding typical of membrane estrogen receptors (mERs). We identified a mER in Atlantic croaker ovaries with characteristics similar to those of human GPR30. To confirm the proposed role of GPR30 as a mER in this distantly related vertebrate group, we cloned GPR30 from croaker ovaries and examined its distribution, steroid binding, and signaling characteristics. Western blot analysis showed the GPR30 protein (similar to 40 kDa) is expressed on the plasma membranes of croaker oocytes and HEK293 cells stably transfected with GPR30 cDNA. Plasma membranes prepared from croaker GPR30-transfected cells displayed high-affinity, limited-capacity, and displaceable binding specific for estrogens, characteristic of mERs. Consistent with previous findings with human GPR30, estrogen treatment of plasma membranes from both croaker ovaries and GPR30-transfected cells caused activation of a stimulatory G protein (Gs) resulting in increased cAMP production. Treatment with E2 as well as G-1, a specific GPR30 ligand, significantly reduced both spontaneous and progestin-induced maturation of both croaker and zebrafish oocytes in vitro, suggesting a possible involvement of GPR30 in maintaining oocyte meiotic arrest in these species. Injection of antisense oligonucleotides to GPR30 into zebrafish oocytes blocked the inhibitory effects of estrogen on oocyte maturation, confirming a role for GPR30 in the control of meiotic arrest. These findings further support our previous suggestion that GPR30 is a vertebrate mER. In addition, the results suggest GRP30 may play a critical role in regulating reentry into the meiotic cell cycle in fish oocytes.