In vitro ovulation, prostaglandin synthesis, and proteolysis in isolated ovarian components of yellow perch (Perca flavescens): effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one and phorbol ester.

The present study investigated the effects of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 B-P) on (1) ovulation of isolated perch follicles in which the extrafollicular (EF) ovarian tissue had been removed; (2) prostaglandin F (PGF) and prostaglandin E (PGE) synthesis in EF tissue and intact (= follicles attached to EF tissue) and isolated follicles by radioimmunoassay and [14C]arachidonic acid incorporation; and (3) proteolysis in EF tissue and intact and isolated follicles by substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, the ovulatory and proteolytic effects of the phorbol ester, phorbol-12-myristate-13-acetate (PMA), and the calcium ionophore A23187 on 17,20B-P-stimulated follicles were also studied in the presence/absence of indomethacin (IM) and nordihydroguaiaretic acid (NDGA). Ultrastructural analyses revealed that the preparation of isolated follicles also removed the surface epithelium of the follicle. While 17,20B-P stimulated ovulation and an increase in PGF and PGE in incubates of intact perch follicles, it did not in incubates of isolated follicles. In contrast, PMA, A23187, and a combination of PMA and A23187 stimulated ovulation of these isolated follicles. PMA/A23187-induced ovulation could be blocked by NDGA but not IM, and two hydroxyeicosatetraenoic acids (11- and 15-HETEs) were capable of partially reversing the NDGA block. Incorporations with [14C]arachidonic acid revealed that the EF tissue had a significant potential to produce PGF; however, 17,20B-P did not stimulate an increase in PGF or PGE (measured by RIA) in incubates of EF tissue alone. In addition, neither ovulation nor an increase in prostaglandins was observed in cocultures of isolated follicles and EF tissue. One major protease (66 kDa) was observed in the medium during incubation of intact and isolated perch follicles. No protease activity was present in incubates of EF tissue alone. Protease activity in 17,20B-P-stimulated incubates of intact tissue was significantly higher than in steroid-stimulated incubates of isolated follicles. Protease activity increased in the medium during incubation with PMA or a combination of PMA and A23187. This activity was blocked by NDGA but not IM. The NDGA block was partially reversed by 11-HETE. The combined results suggest that there is an interaction of EF tissue and follicle that is necessary, particularly for the stimulation of ovulation and prostaglandin production. Further, the results with phorbol esters and NDGA suggest that the follicular control of ovulation in perch may involve protein kinase C acting through the production of a lipoxygenase product.