INHIBITORY GUANINE-NUCLEOTIDE-BINDING-REGULATORY PROTEIN ALPHA-SUBUNITS IN MEDAKA (ORYZIAS-LATIPES) OOCYTES - CDNA CLONING AND DECREASED EXPRESSION OF PROTEINS DURING OOCYTE MATURATION

We have previously shown that pertussis-toxin-sensitive inhibitory gua nine-nucleotide-binding-regulatory proteins (G proteins) are involved in the signal transduction of steroidal maturation-inducing hormone (M IH) of rainbow trout (Oncorhynchus mykiss) oocytes, 17 alpha,20 beta-d ihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP) [Yoshikuni, M. & Nagaha ma, Y. (1994) Dev. Biol. 166, 615-622]. In this study, we obtained fiv e different cDNA fragments of G protein a subunits from medaka (Oryzia s latipes) intact ovarian follicles (three subtypes of G(i alpha), G(i alpha a), G(i alpha b) and G(i alpha c); two subtypes of G(s alpha), G(s alpha d), and G(s alpha e)). U-sing a newly developed extraction m ethod for medaka oocyte RNA, we demonstrated that oocytes expressed bo th G(i alpha c), and G(i alpha c), but not G(i alpha b). Full-length c DNA clones for G(i alpha a) and G(i alpha c), were then isolated from a medaka ovarian follicle cDNA library. The predicted amino acid seque nces of G(i alpha a), and G(i alpha c) exhibited significant similarit y with G(i alpha 1) and G(i alpha 2) Of Other species, respectively. B oth G(i alpha a) and G(i alpha c) possessed a specific Cys residue in the C-terminal region that was the site for ADP-ribosylation by pertus sis toxin. G(o alpha), another G protein that is ADP-ribosylated by pe rtussis toxin, was not detected in oocytes, although it was expressed in brain tissue. Western blot analyses using a specific antibody again st G(i alpha 1) and G(i alpha 2) subunit proteins revealed that in bot h medaka and rainbow trout G(i alpha) subunit protein (40 kDa) content s were abundant in plasma membranes of postvitellogenic immature oocyt es, decreased in mature oocytes, and were absent in ovulated eggs. Fur thermore, specific 17 alpha,20P-DP binding to plasma membranes was hig her in postvitellogenic immature oocytes than in ovulated eggs. Taken together, these results suggest that G(i alpha a) and/or G(i alpha c) may be involved in the transduction of the signal from 17 alpha,20 bet a-DP receptors during oocyte maturation of fish oocytes.