| Estradiol is required for the survival of the rat corpus luteum (CL) throughout pregnancy and is produced throughout gestation by the CL itself. In early pregnancy, estradiol is produced de novo from progesterone, however during the second half of pregnancy, the CL loses this capability. This is compensated by the fetal placenta, which at this stage begins to secrete androstenedione which the CL can convert to estradiol. The steroidogenic enzymes involved in the production of estradiol by the rat CL have not been characterized, and this study sought to examine the changes occurring at a molecular level which allowed for this change in the mechanism of estradiol biosynthesis.;We found that PRL receptor-associated protein (PRAP), a luteal protein originally cloned in our lab, is a novel isoform of 17betaHSD responsible for the final step of estradiol biosynthesis in the rat CL. Transfection studies in vitro found that the association of PRAP with the PRL receptor allowed PRL to induce the phosphorylation of PRAP on tyrosine residues, and that this involved the tyrosine kinase JAK2. Through RT-PCR, the mRNA expression of PRAP/17betaHSD and P450c17, another enzyme involved in estradiol synthesis, were found to change in the CL during pregnancy, and their differential expression patterns appear to be crucial to the change in estradiol production in the rat CL. The expression of both of these steroidogenic enzymes is regulated by both luteinizing hormone (LH) and estradiol over the course of gestation, as determined by in vivo experiments. We cloned and characterized the PRAP promoter, and demonstrated that two Sp1 binding sites appear to be involved in regulation of its basal expression. In a rat luteal cell line, cAMP, a second messenger utilized by LH in the rat corpus luteum, could inhibit PRAP promoter activity and stimulate P450c17 promoter activity, mimicking the effects of LH on the mRNA expression of these two enzymes in the rat corpus luteum. In EMSA experiments, using control and hCG-treated luteal nuclear extracts, a NF-Y binding site in the PRAP promoter was shown to bind NF-Y in control rats, while it did not bind when treated with hCG. This mechanism was not used by cAMP in the rat luteal cell line, suggesting that LH could inhibit PRAP transcription through two pathways, one involving cAMP and a second which leads to prevention of NF-Y binding to the PRAP promoter. |
