Studies on Mouse Oocyte Meiotic Arrest and Maturation

Mammalian oocytes enter meiosis during embryonic development and arrest at prophase I until a preovulatory surge of lutenizing hormone (LH) from the pituitary stimulates meiotic resumption. Meiotic arrest is maintained by high levels of cAMP generated in the oocyte by the constitutively active receptor, GPR3. It is unknown how GPR3 activity in the oocyte is regulated and whether the receptor is desensitized and endocytosed by GPCR kinase (GRK)-induced phosphorylation and β-arrestin recruitment. In the mouse oocyte, endocytic inhibition increased cAMP levels and inhibited spontaneous maturation. We hypothesize that GPR3 signals from the cell surface and inhibiting endocytosis causes GPR3 to accumulate at the cell surface and increase cAMP levels. In order to directly compare the localization and signaling of GPR3, we extended our studies in HEK293 cells. Endocytic inhibition increased cell surface-localized GPR3 and cAMP levels while overexpression of GRK2 and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. Mutation of potential phosphorylation sites in the third intracellular loop dramatically increased cAMP production compared to WT; however, these sites are not targeted by GRK2 for desensitization. Mutation of serines in the C-terminus did not change cAMP levels but did decrease the membrane localization of GPR3. We conclude that GPR3 signals at the cell membrane and does not continue to signal following internalization, although placement at the plasma membrane may not be required for cAMP production and GPR3 may be able to signal prior to membrane insertion. Further studies are required to determine if GPR3 is phosphorylated and targeted for regulation by other kinases or G protein-coupled interacting proteins