Levels of messenger ribonucleic acid for cytochrome P450 17 alpha-hydroxylase and P450 aromatase in preovulatory...

During the transition from the follicular to the luteal phase, follicular steroid secretion shifts from predominantly 17 beta-estradiol and androgen production before the LH surge to decreased androgen and estrogen and increased progesterone production after the LH surge. Our objective was to determine if changes in 17 beta-estradiol and androgen production by the preovulatory follicle are effected via changes in mRNA levels for the steroidogenic enzymes cytochrome P450 17 alpha-hydroxylase/C17, C20-lyase (P450-17 alpha) and cytochrome P450 aromatase (P450arom). Heifers were injected with prostaglandin F2 alpha (PGF2 alpha) during the luteal phase to initiate luteolysis and a follicular phase. Preovulatory follicles were obtained at two times before the LH surge, in the early follicular phase (24 h after PGF2 alpha injection) and in the midfollicular phase (48 h after PGF2 alpha), and at one time in the late follicular phase, after the LH surge but before ovulation (20 h after the onset of estrus, about 20 h after the LH surge). Granulosa cells and theca interna were isolated from preovulatory follicles obtained during the early follicular phase and cultured with or without ovine LH (100 ng/ml; n = 4 follicles isolated 24 h after PGF2 alpha). Total RNA extracted from granulosa cells and theca interna, either immediately after cell isolation during the early (n = 5), mid (n = 4)-, or late follicular phase (n = 5) or after 24 or 72 h of culture, was subjected to Northern analysis for P450-17 alpha and P450arom mRNA. Autoradiographs were scanned densitometrically, and values were adjusted for loading and transfer efficiency. P450-17 alpha mRNA was highly abundant in theca interna before the LH surge, but decreased by 96% after the LH surge (P < 0.001). In contrast, P450-17 alpha mRNA in the granulosa cell fraction was very low at all three times of follicle isolation and most likely was due to a slight contamination of the granulosa cell preparation with theca interna. P450arom mRNA was abundant in granulosa cells isolated in the early and midfollicular phase, but decreased by 94% after the LH surge (P < 0.001). In contrast, P450arom mRNA was undetectable in theca interna. Levels of androstenedione and 17 beta-estradiol in follicular fluid were high during the early and midfollicular phase and decreased dramatically after the LH surge (P < 0.05 and P < 0.001, respectively). P450-17 alpha and P450arom mRNA were undetectable after 24 h of culture in the presence or absence of LH and thereafter.(ABSTRACT TRUNCATED AT 400 WORDS)