Ovarian aromatase cytochrome P-450 mRNA levels correlate with enzyme activity and serum estradiol levels in anestrous, pregnant and lactating rats.
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 [1 beta-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 approximately 2.8 pg/ml; LA approximately 0.2 pg/ml) and aromatase activity levels (IR approximately 0.8 pmol/h per mg protein; LA less than 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 (approximately 12.5 pg/ml) and aromatase activity (approximately 4.2 pmol/h per mg protein). During pregnancy, all three mRNA species were clearly detectable, but low serum E2 levels (approximately 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Lephart
E D
ED
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235.
Simpson
E R
ER
McPhaul
M J
MJ
D000780:Anestrus / Q000378:metabolism*; D000818:Animals; D001141:Aromatase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004958:Estradiol / Q000097:blood*; D005260:Female; D007774:Lactation / Q000378:metabolism*; D008969:Molecular Sequence Data; D010053:Ovary / Q000201:enzymology*; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D012333:RNA, Messenger; D004958:Estradiol; D001141:Aromatase