Differentiation of bovine preovulatory follicles during the follicular phase is associated with increases in messenger ribonucleic acid for cytochrome P450 side-chain cleavage, 3 beta-hydroxysteroid dehydrogenase, and P450 17 alpha-hydroxylase, but not P450 aromatase.

Abstract

In cattle, a dramatic increase in plasma estradiol occurs during the short 2- to 3-day follicular phase. The objective of this study was to investigate the molecular mechanisms that mediate this critical change, specifically whether increases in the steroidogenic ability of granulosa and thecal cells of the preovulatory follicle are associated with increases in the levels of messenger RNA (mRNA) for steroidogenic enzymes. Luteolysis and a follicular phase were induced cycling Holstein heifers (n=15) by injection of a luteolytic dose of prostaglandin F2 alpha (PGF 2 alpha) on day 6 or 7 of the estrous cycle (day 0 = estrus), and preovulatory follicles were obtained at three stages of differentiation (0, 12, or 24 h post-PGF2 alpha treatment). To assess developmental changes in steroidogenesis in vivo, estradiol and androstenedione were measured in follicular fluid and in culture medium after a 3-h incubation of granulosa and thecal cells in defined medium with or without gonadotropins. To determine whether changes in mRNA for steroidogenic enzymes are associated with changes in follicular steroidogenesis, levels of mRNA for cytochrome P450 side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P450 17 alpha-hydroxylase, and cytochrome P450 aromatase (P450arom) were measured in thecal and granulosa cells using ribonuclease protection assays. Concentrations of estradiol in follicular fluid were relatively high at time zero, increased significantly by 12 h, and increased further by 24 h post-PGF2 alpha treatment. However, the aromatizing activity of granulosa cells was high at the time of PGF2 alpha injection and did not increase significantly during the first 24 h after the initiation of luteolysis. The aromatizing activity of granulosa cells was reflected in levels of mRNA for P450arom, which was relatively abundant in granulosa cells obtained before luteolysis and did not increase further during the first 24 h of the follicular phase. Concentrations of androstenedione were virtually undetectable in follicular fluid at time zero and had increased dramatically by 12 and 24 h post-PGF2 alpha treatment. Similarly, thecal cells isolated at 24 h secreted 3-fold more androstenedione than cells isolated at the time of PGF2 alpha injection. Androstenedione production by thecal cells in response to LH was also markedly higher at 12 and 24 h than at the time of PGF2 alpha injection. Likewise; levels of mRNA for P450 17 alpha-hydroxylase increased significantly by 12 h post-PGF2 alpha treatment.(ABSTRACT TRUNCATED AT 400 WORDS)