Hormonal Regulation, Tissue Distribution, and Content of Aromatase Cytochrome P450 Messenger Ribonucleic Acid and Enzyme in Rat Ovarian Follicles and Corpora Lutea: Relationship to Estradiol Biosynthesis*

Abstract

The following study was undertaken to compare the content of aromatase cytochrome P450 (P450arom) mRNA with the content of the enzyme in rat ovarian tissues and to relate these changes with estradiol biosynthesis by follicles and corpora lutea isolated throughout pregnancy. A deoxyoligonucleotide (62 mer) probe derived from an amino acid sequence of purified human placental P450arom was used to screen a rat granulosa cell λgtll cDNA expression library. Seven cDNA clones, ranging in size from 0.6–2.0 kilobases (kb), were identified and plaque purified. In vitro translation using mRNA that had been selected by hybridization to a 1.2-kb rat P450arom cDNA insert yielded an ³⁵S-labeled translation product that bound antihuman aromatase immunoglobulin and comigrated with purified human placental aromatase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thus verifying that the clones do encode for P450arom. Using the 1.2-kb cDNA insert as a radiolabeled probe, the hormonal regulation, tissue distribution, content, and size of mRNA for P450arom were analyzed. Filter hybridization assays demonstrated that P450arom mRNA was low in small antral (SA) follicles, increased 16-fold in preovulatory (PO) follicles, and reached a peak in granulosa cells within 1 h after an ovulatory dose of hCG. In the corpus luteum of pregnancy, P450arom mRNA content was low on day 4, and increased 3-fold on days 7–11 and 10-fold on days 15–19 of gestation. P450arom mRNA then decreased on days 21 and 23, the day of parturition. Northern analyses of RNA from PO follicles and corpora lutea revealed three bands of P450arom mRNA that were 3.3, 2.6, and 1.9 kb in size. Immunoblots of soluble cell extracts of SA, PO, and luteinizing (PO plus hCG) follicles and corpora lutea of pregnancy demonstrated that aromatase enzyme was low in SA follicles, increased 1.5- to 3-fold in PO follicles, and decreased within 3–5 h after an ovulatory dose of hCG. Changes in the content of P450arom enzyme in luteal cells during pregnancy exhibited a pattern similar to that observed for P450arom mRNA. In contrast, changes in estradiol biosynthesis by follicles and corpora lutea were not directly related to the contents of P450arom mRNA and enzyme. For example, although corpora lutea isolated on days 15–21 of gestation contain the highest amount of P450arom mRNA and enzyme, these tissues did not produce the most estradiol when incubated for 5 h at 37 C in the presence of aromatizable androgen substrate. Rather, PO follicles and corpora lutea isolated on days 22 and 23 of gestation exhibited marked increases in estradiol synthesis despite lower contents of the enzyme. When hCG was administered to rats on days 8–9 or 13–14 of gestation to mimic the postpartum rise in endogenous LH, estradiol synthesis by follicles incubated in the presence of 300 ng/ml testosterone increased from 6 ng/mg-5 h (day 13; no hCG) to 220 ng/mg-5 h (day 15; post-hCG). In corpora lutea isolated from the ovaries of these same hCGtreated rats, aromatase activity was lower (1.5 ng/mg-5 h) and showed little or no change in response to 1.5, 3.0, or 6.0 IU hCG. The content of P450arom enzyme and mRNA in corpora lutea also remained unaffected by the hCG treatments. These observations indicate that the contents of P450arom mRNA and enzyme in rat ovarian follicles and corpora lutea are regulated by different hormonal factors and that the capacity of follicles and corpora lutea to synthesize estradiol is not strictly related to the changes in the contents of P450arom mRNA and enzyme contained within these tissues (Endocrinology122: 1426–1436,1988)