DIFFERENTIATION OF BOVINE PREOVULATORY FOLLICLES DURING THE FOLLICULAR PHASE IS ASSOCIATED WITH INCREASES IN MESSENGER-RIBONUCLEIC-ACID FORCYTOCHROME-P450 SIDE-CHAIN CLEAVAGE, 3-BETA-HYDROXYSTEROID DEHYDROGENASE, AND P450 17-ALPHA-HYDROXYLASE, BUT NOT P450 AROMATASE

In cattle, a dramatic increase in plasma estradiol occurs during the s hort 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 granu losa and thecal cells of the preovulatory follicle are associated with increases in the levels of messenger RNA (mRNA) for steroidogenic enz ymes. Luteolysis and a follicular phase mere induced in cycling Holste in heifers (n = 15) by injection of a luteolytic dose of prostaglandin F-2 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 d ifferentiation (0, 12, or 24 h post-PGF(2 alpha) treatment). To assess developmental changes in steroidogenesis in vivo, estradiol and andro stenedione were measured in follicular fluid and in culture medium aft er a 3-h incubation of granulosa and thecal cells in defined medium wi th or without gonadotropins. To determine whether changes in mRNA for steroidogenic enzymes are associated with changes in follicular steroi dogenesis, levels of mRNA for cytochrome P450 sidechain cleavage (P450 scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P45 0 17 alpha-hydroxylase, and cytochrome P450 aromatase (P450arom) were measured in thecal and granulosa cells using ribonuclease protection a ssays. Concentrations of estradiol in follicular fluid were relatively high at time zero, increased significantly by 12 h, and increased fur ther by 24 h post PGF(2 alpha) treatment. However, the aromatizing act ivity of granulosa cells was high at the time of PGF(2 alpha) injectio n and did not increase significantly during the first 24 h after the i nitiation of luteolysis. The aromatizing activity of granulosa cells w as reflected in levels of mRNA for P450arom, which was relatively abun dant in granulosa cells obtained before luteolysis and did not increas e further during the first 24 h of the follicular phase. Concentration s of androstenedione were virtually undetectable in follicular fluid a t time zero and had increased dramatically by 12 and 24 h post-PGF(2 a lpha) treatment. Similarly, thecal cells isolated at 24 h secreted 3-f old more androstenedione than cells isolated at the time of PGF(2 alph a) injection. Androstenedione production by thecal cells in response t o LH was also markedly higher at 12 and 24 h than at the time of PGF(2 alpha) injection. Likewise, levels of mRNA for P450 17 alpha-hydroxyl ase increased significantly by 12 h post-PGF(2 alpha) treatment. The s tage of follicular development also significantly affected levels of m RNA for P450scc and 3 beta HSD, which were relatively low in both thec al and granulosa cells at the time of PGF(2 alpha) injection, but incr eased significantly during the first 24 h of the follicular phase. In summary, levels of mRNA for P450scc and 3 beta HSD in both granulosa a nd thecal cells and for P450 17 alpha-hydroxylase in thecal cells incr eased within the first 24 h of follicular differentiation during the f ollicular phase. Therefore, the increase in estradiol production durin g the follicular phase may be due primarily to increases in the availa bility of the aromatizable substrate, whereas changes in levels of mRN A for P450arom and the aromatase system may play only a minor role.