Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20α-dihydroprogesterone, a product of 20α-hydroxysteroid dehydrogenase (20α-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20α-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450scc) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2α (PGF2α) was added 7–8 days after seeding. In prolactin-treated cells, PGF2α reduced steroidogenesis after 4–45 h, and at 45 h total progestins and P450scc mRNA were reduced by 45%. At 8–45 h PGF2α reduced the percent progesterone out of total progestins, and at 45 h 20α-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2α had little effect on total progestins or 20α-HSD mRNA but doubled P450scc mRNA. Phospholipase C activity was stimulated by PGF2α regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2α; the finding that without prolactin PGF2α has an alternative set of actions could help in identifying the signaling pathways of PGF2α responsible for its luteolytic effects.
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