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.