Ovarian aromatase cytochrome P-450 mRNA levels correlate with enzyme activity and serum estradiol levels in anestrous, pregnant and lactating rats

Abstract

We examined the changes in P-450AROM mRNA, aromatase enzyme activity and serum estradiol levels (E2) in anestrous, pregnant mare's serum gonadotropin (PMSG)-treated immature, pregnant, and lactating rats to determine if: (a) the various mRNA species encoding P-450AROM in rat ovarian tissue are differentially expressed during different hormonal states, and (b) a positive relationship exists between P-450AROM mRNA and enzymatic activity in rat ovarian tissue and serum estradiol levels from the same animals.Utilizing three different cDNAs encoding rat P-450AROM, levels of P-450AROM mRNA were determined by RNA blot analysis and scanning densitometry. Probe 1, a 5' probe, detects all three P-450AROM mRNA species in rat ovarian tissue (i.e. at 1.7, 2.2 and 2.7 kb). Probe 2 contains an unspliced intronic sequence in place of the heme-binding domain at its 3' terminus and thus the mRNA detected by this probe must encode a nonfunctional aromatase protein. Only the two smaller (i.e. nonfunctional) mRNA species at 1.7 and 2.2 kb are detected by probe 2. Probe 3 contains the heme-binding region and hybridizes to principally the largest mRNA transcript at 2.7 kb (but hybridizes also to a 1.7 kb mRNA transcript). Aromatase enzyme activity was measured by using a saturating concentration of [1β-3H] testosterone as substrate in the [3H]water-release assay while serum estradiol levels were determined by radioimmunoassay.In immature rats (IR) or lactating animals (LA) P-450AROM mRNA was not detectable along with low serum estradiol (IR ≈ 2.8 pg/ml; LA ≈ 0.2 pg/ml) and aromatase activity levels (IR ≈ 0.8 pmol/h per mg protein; LA < 0.5 pmol/h per mg protein). Anestrous animals treated with 5 IU of PMSG resulted in a clear increase (24 h later) in P-450AROM mRNA levels, in concert with a 4-fold increase in serum E2 (≈ 12.5 pg/ml) and aromatase activity (≈ 4.2 pmol/h per mg protein). During pregnancy, all three mRNA species were clearly detectable, but low serum E2 levels (≈ 0.6 pmol/ml) and P-450AROM mRNA abundance were observed at 3 days of gestation (DG). Levels of all three P-450AROM mRNA species increased markedly at 15 and 18 DG; thereafter, the levels declined at 20 DG and further decreased at 22 DG. However, regardless of the probe utilized (probe 1, 2 or 3) in the RNA blot analyses, the mRNA transcripts detected by each probe were expressed in a concerted fashion with respect to abundance and pattern. Aromatase activity at 15 DG was high (≈3.0 pmol/h per mg protein) and remained at relatively high levels until 20 DG (≈4.0 pmol/h per mg protein), thereafter the activity decreased perinatally, at 22 DG (≈1.8 pmol/h per mg protein). Estradiol levels during late gestation (at 15 DG) were at high levels (≈9.0 pg/ml) and continued to increase to 18.0 pg/ml until 19 DG, thereafter, E2 levels decreased at 20 and 22 DG (≈7.0 to 8.0 pg/ml). A correlation calculated between the normalized P-450AROM mRNA levels (using probe 3) and aromatase activities revealed a strong correspondence between the P-450AROM mRNA pattern of abundance and enzyme activity in anestrous, pregnant and lactating rats, r = 0.93, p = 0.002. Also a significant positive correlation between ovarian aromatase activity and serum E2 levels was obtained, r = 0.88, p = 0.01. These findings suggest that as many as four different species of P-450AROM mRNA in ovarian tissue appear to be expressed in a concerted fashion regardless of the hormonal state or developmental stage of the animal, and (b) in anestrous, pregnant and lactating rats, a significant positive correlation exists between aromatase activity and serum E2 levels which are regulated, in part, by the levels of P-450AROM mRNA encoding the aromatase enzyme.