Corticotropin-Releasing Hormone/Urocortin-Receptor Actions Modulate Cell Numbers and Progesterone Production by Primate Luteinizing Granulosa Cells In Vitro.

Abstract

Based on studies detecting corticotropin-releasing hormone (CRH), urocortin1 and 2 (UCN1 and 2) and their receptors R1/R2 in the developing corpus luteum in primates, we hypothesized that this local system modulates luteal structure-function. Therefore, experiments were designed to determine if ligands for CRH receptor 1 (R1: CRH, urocortin or UCN) and receptor 2 (R2: UCN, UCN2) influence progesterone (P) production by primate luteinizing granulosa cells (LGCs). Cells [n=4 preparations/experiment (Exp)]were obtained by follicle aspiration from rhesus monkeys after controlled ovarian stimulation and cultured for 5 days in fibronectin-coated wells containing chemically-defined medium. LGCs were incubated in the presence or absence of 100 ng/ml recombinant hLH (NIDDK), plus 10-6 M CRH, UCN or UCN2 (Exp 1) or 10-610-7 M CRHR1 (Antalarmin) or CRHR2 ( Astressin2B) antagonists (Exp2). Medium was collected daily and assayed for P. On day 5, DNA content per well was determined by crystal violet assay. Compared to controls (medium alone), DNA content was 2-fold (p<0.05) greater in the presence of LH. Therefore P levels were normalized to DNA content per well. In controls, P levels decreased between day 1 and 2, to low levels at 3-5 days (p<0.05). In the presence of LH, P levels increased between day 1 and 3, with peak levels sustained on day 4 (P<0.05). In Exp1, CRH ligands alone did not alter DNA content, but levels in the presence of both LH and CRH or UCN2 were greater (p<0.05) than those in LH alone. Both UCN and UCN2 decreased (p<0.05) P levels after day 1, such that the typical decline in P levels between day 1 and 5 was eliminated (p>0.10). In the presence of LH, CRH ligands displayed modest effects, e.g., UCN2 tended to reduce P levels after 1 to 5 days (p<0.05). In Exp2, CRHR1 and CRHR2 antagonists at 10-6 but not 10-7 M, increased DNA content 2-fold (p<0.05), but only in the presence of LH. CRHR1, but not CRHR2, antagonist at 10-6 M decreased P levels compared to controls or 10-7 M doses; by day 5, P levels with 10-6 M CRHR1 antagonist were near baseline (p<0.05). However P levels in the presence of LH plus either antagonist were not different from those in LH alone. The current results suggest that, in the presence of LH, both CRHR1 and R2 ligands modulate cell survival in vitro. However, CRH may promote P production, whereas UCN and UCN2 inhibit P production by LGCs in vitro, perhaps via CRHR1 versus CRHR2, respectively. Research supported by U54-HD18185 (Proj 3); D43TW00688 (MC); RR00163 (poster)