Steroidogenic cytochrome P450 enzyme messenger ribonucleic acids and follicular fluid steroids in individual follicles during preovulatory maturation in the pig

Abstract

Changes in follicular concentrations of steroidogenic cytochrome P450 enzyme mRNAs were determined during preovulatory maturation. RNA was isolated from 59 individual follicles dissected from 18 pigs during altrenogest- synchronized preovulatory follicular maturation: at Day 1 (pre-follicular phase), Day 3 (early follicular phase), Day 5 (mid-follicular phase), and Day 7 (late follicular phase, 24-36) h after the onset of the LH surge). Follicular fluid was aspirated for steroid RIA. RNA was also isolated from pooled granulosa cells, theca tissue, and luteal tissue. RNA was analyzed by Northern and slot-blot procedures using cDNA probes to human aromatase cytochrome P450 (P450(arom)), porcine 17α-hydroxylase cytochrome P450 (P450(17α)), and porcine cholesterol side-chain cleavage cytochrome P450 (P450(scc)). P450(arom) mRNA was expressed in both granulosa and theca interna cells but was not detectable in luteal tissue from Day 13 of the estrous cycle. Follicular P450(arom) mRNA concentration tended to increase between Days 1 and 5 and decreased (p ≤ 0.05) by 92% between Days 5 and 7. Follicular fluid estradiol-17β concentration increased 17-fold between Days 1 and 5 and then decreased (p ≤ 0.05) by 96% between Days 5 and 7. P450(17α) mRNA was present in theca interna but was not detected in granulosa cells or luteal tissue. Follicular P450(17α) mRNA concentrations did not differ significantly among days, but the content per follicle increased (p < 0.05) between Days 1 and 5 and decreased (p < 0.05) between Days 5 and 7. P450(scc) mRNA was detected in granulosa cells, theca interna, and luteal tissue; but its concentration was 4- to 5-fold greater in luteal than in follicular tissue. Follicular P450(scc) mRNA concentration increased (p < 0.05) between Days 3 and 5 and decreased (p ≤ 0.05) by 69% between Days 5 and 7. Follicular fluid progesterone concentration increased (p < 0.05) progressively between Days 1 and 7. P450(scc) mRNA concentration in newly formed luteal tissue recovered from one Day 7 pig was 8-fold greater than that in follicles recovered on Day 7. We conclude that changes in follicular estradiol-17β production could be explained, in part, by changes in the pattern of P450(arom) mRNA expression, which increased until Day 5 and then decreased after the preovulatory LH surge. In contrast, progesterone production increased throughout preovulatory maturation in spite of a transient decrease in P450(scc) mRNA concentration after the LH surge.