Differentiation of ovarian theca-interstitial cells in vitro: regulation of 17 alpha-hydroxylase messenger ribonucleic acid expression by luteinizing hormone and insulin-like growth factor-I.
Insulin-like growth factor-I (IGF-I) has been shown to synergistically augment LH-stimulated androgen biosynthesis in ovarian theca-interstitial cells (TIC). Additional evidence suggests that IGF-I may play a role in stimulating TIC differentiation during early follicle development. The purpose of the present studies was to examine the role of IGF-I in TIC differentiation by determining the effects of IGF-I on P450(17 alpha) mRNA expression in TIC differentiating in vitro. TIC isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation were cultured with and without LH and IGF-I for up to 6 days. At various times, cytoplasmic RNA was extracted from the TIC, and P450(17 alpha) mRNA was measured by a specific assay, using reverse transcription and the polymerase chain reaction. LH stimulated a dose-related increase in P450(17 alpha) mRNA, with an ED50 comparable to that for androsterone biosynthesis (33.0 +/- 3.8 ng/ml), but significantly less than the ED50 for cAMP accumulation (385 +/- 0.1 ng/ml). IGF-I alone did not stimulate P450(17 alpha) mRNA, but in the presence of LH (100 ng/ml) stimulated a dose-related (ED50, 4.1 +/- 1.6 ng/ml) increase (3-fold) in P450(17 alpha) mRNA. IGF-I did not alter the ED50 for LH stimulation of P450(17 alpha) mRNA (36.85 +/- 1.1 ng/ml). Detailed time-course studies revealed that IGF-I did not alter the 20-h lag phase before LH caused an increase in TIC androsterone biosynthesis; however, IGF-I stimulated a more rapid increase in androsterone than LH alone. In the presence of LH alone, P450(17 alpha) mRNA levels remained low for approximately 18 h, then increased rapidly to maximum levels by 30 h where they were maintained through 48 h. Concomitant treatment with LH plus IGF-I did not alter the 18-h lag phase, but P450(17 alpha) mRNA levels increased to approximately 2-fold higher levels than with LH alone. The results of our studies demonstrate that IGF-I increases the expression of P450(17 alpha) mRNA in TIC and support the hypothesis that IGF-I may play a role in stimulating thecal differentiation in developing follicles.
Magoffin
D A
DA
Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center/University of California School of Medicine, Los Angeles 90048.
Weitsman
S R
SR
D000199:Actins / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D007986:Luteinizing Hormone / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics*; D013799:Theca Cells / Q000166:cytology* / Q000378:metabolism
D000199:Actins; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D007986:Luteinizing Hormone; D013254:Steroid 17-alpha-Hydroxylase