Abstract
We have previously shown that pertussis-toxin-sensitive inhibitory guanine-nucleotide-binding-regulatory proteins (G proteins) are involved in the signal transduction of steroidal maturation-inducing hormone (MIH) of rainbow trout (Oncorhynchus mykiss) oocytes, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) [Yoshikuni, M. & Nagahama, Y. (1994) Dev. Biol. 166, 615-622]. In this study, we obtained five different cDNA fragments of G protein alpha subunits from medaka (Oryzias 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)). Using a newly developed extraction method for medaka oocyte RNA, we demonstrated that oocytes expressed both G(i alpha a) and G(i alpha c), but not G(i alpha b). Full-length cDNA 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 sequences of G(i alpha a) and G(i alpha c) exhibited significant similarity with G(i alpha1) and G(i alpha2) of other species, respectively. Both 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 pertussis toxin. G(o alpha), another G protein that is ADP-ribosylated by pertussis toxin, was not detected in oocytes, although it was expressed in brain tissue. Western blot analyses using a specific antibody against G(i alpha1) and G(i alpha2) subunit proteins revealed that in both medaka and rainbow trout G(i alpha) subunit protein (40 kDa) contents were abundant in plasma membranes of postvitellogenic immature oocytes, decreased in mature oocytes, and were absent in ovulated eggs. Furthermore, specific 17alpha,20beta-DP binding to plasma membranes was higher 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 17alpha,20beta-DP receptors during oocyte maturation of fish oocytes.
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