Denis A. Magoffin

Overexpression of theca-cell messenger RNA in polycystic ovary syndrome does not correlate with polymorphisms in the cholesterol side-chain cleavage and 17α-hydroxylase/C17-20 lyase promoters

OBJECTIVEnTo determine whether overexpression of CYP17 or CYP11A messenger (m)RNA in theca cells from polycystic ovaries is related to polymorphic regions in the gene promoters that may increase transcription.nnnDESIGNnCase-control study.nnnSETTINGnResearch institute.nnnPATIENT(S)nFifty-one women with PCOS and 280 regularly cycling controls underwent genotyping. Thecal cells were obtained from 23 women with PCOS and 51 controls.nnnMAIN OUTCOME MEASURE(S)nOvarian tissue was obtained from women with PCOS undergoing wedge resection for treatment of their infertility and from controls undergoing ovariectomy for indications unrelated to the study. Expression of mRNA in theca cells was measured by using competitive reverse transcriptase polymerase chain reaction. Genotype analysis for polymorphisms in the CYP11A and CYP17 promoters was performed by using polymerase chain reaction.nnnRESULT(S)nAlthough expression of CYP11A and CYP17 mRNA was higher in women with PCOS, no significant dose effects of CYP11A or CYP17 alleles were observed with respect to serum testosterone; follicular fluid androstenedione, estradiol, and androstenedione-to-estradiol ratio; or CYP11A or CYP17 mRNA expression.nnnCONCLUSION(S)nOverexpression of CYP17 and CYP11A mRNA in theca cells from polycystic ovaries is explained by polymorphic differences in the gene promoters.

Effect of insulin-like growth factor-I on cholesterol side-chain cleavage cytochrome P450 messenger ribonucleic acid expression in ovarian theca-interstitial cells stimulated to differentiate in vitro

Currently available evidence supports the hypothesis that insulin-like growth factor-I (IGF-I) may play a role in stimulating ovarian theca-interstitial cell (TIC) differentiation in preantral follicles. The purpose of the present studies was to examine the potential role of IGF-I in TIC differentiation by determining the effects of IGF-I on cholesterol side-chain cleavage cytochrome P450 (P450SCC) mRNA expression in TIC stimulated to differentiate in vitro. TIC were isolated from the ovaries of hypophysectomized immature rats by Percoll gradient centrifugation and cultured in the presence and absence of LH and IGF-I up to 6 days. At various times cytoplasmic RNA was extracted from the TIC and P450SCC mRNA was measured by specific assay using reverse transcription followed by the polymerase chain reaction. Increasing concentrations of LH (0-1 microgram/ml) stimulated a dose-related increase in P450SCC mRNA (ED50 = 36.2 +/- 5.5 ng/ml) which reached maximal levels at 100 ng/ml of LH. Addition of IGF-I (30 ng/ml) caused a small increase in P450SCC mRNA over TIC treated with LH alone but did not alter the ED50 for LH stimulation. IGF-I alone also stimulated an increase in P450SCC mRNA which reached approximately 3-fold over unstimulated levels at 100 ng/ml. In the presence of LH, IGF-I stimulated a dose-related increase in P450SCC mRNA (ED50 = 1.2 +/- 0.05 ng/ml). Time-course studies revealed that expression of P450SCC mRNA was greatest at 2 days in TIC treated with IGF-I alone, LH alone or LH plus IGF-I and then declined at 4 and 6 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Preantral follicles stimulate luteinizing hormone independent differentiation of ovarian theca-interstitial cells by an intrafollicular paracrine mechanism.

Ovarian thecal cells are thought to differentiate from fibroblast-like precursor cells in the stroma adjacent to developing follicles. Since the precursor cells do not contain LH receptors, a regulator other than LH must initiate thecal differentiation. These studies were designed to test the hypothesis that preantral follicles secrete substances that can stimulate thecal differentiation. Preantral follicles devoid of theca were obtained by limited enzymatic dispersal of 26-day old rat ovaries. Follicles were cultured (5 follicles/well) in 96-well plates containing serum-free medium to generate follicle-conditioned medium (FCM). Isolated theca-interstitial cells (TIC) were cultured (2 days) in 50% FCM, to bioassay for androgen-stimulating activity. Androsterone production was measured by RIA. FCM from follicles of increasing size with 1, 2, 3, 4 or 5 layers of granulosa cells (GC) stimulated increasing amounts of androsterone suggesting that secretion of androgen-stimulating activity is developmentally regulated in preantral follicles. The androgen-stimulating activity of 7.5-fold concentrated FCM was markedly increased above control levels or the levels stimulated by insulin-like growth factor-I (100 ng/ ml), transforming growth factor-α (100 ng/ml), transforming growth-factor-β1 (10 ng/ml), inhibin A (300 ng/ml), activin A (100 ng/ml), or Müllerian inhibiting substance (MIS; 300 ng/ml) suggesting that the bioactive substances were distinct from these intrafollicular growth factors. rFSH stimulated a > 10-fold increase in androgenstimulating activity demonstrating that the bioactivity is hormonally regulated. The bioactivity was sensitive to trypsin digestion but was not inhibited by indomethacin (10 μm) suggesting that it is peptide not prostaglandin in nature. Gel filtration chromatography indicated that the M of the bioactive peptides in FCM ranged from 19 500 to 23 600. Taken together our results demonstrate that preantral follicles secrete thecal differentiating factors (TDFs) that are developmentally and hormonally regulated by FSH. The properties of the TDFs are markedly different from known intrafollicular growth factors and may represent a new paracrine regulator in the ovary that can stimulate LH-independent thecal differentiation.