Running Title: Fsh-mediated Progestin Production

Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to the follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation is mediated by progestins, such as 17alpha,20beta-dihydroxypregn-4-en3-one (17,20beta-P), produced in response to the luteinizing hormone (Lh). In teleosts, the follicular synthesis of 17,20beta-P at the time of maturation is primarily due to the up-regulation of P450c17II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1) enzymes. Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages; and had a higher expression of cbr1 and the Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, the synthesis being sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription, and simultaneously downregulated the cyp17a1 and cyp19a1 steady-state mRNA levels, within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering the production of 17,20beta-P.