OVARIAN THECAL INTERSTITIAL ANDROGEN SYNTHESIS IS ENHANCED BY A FOLLICLE-STIMULATING HORMONE-STIMULATED PARACRINE MECHANISM

To obtain direct evidence for FSH-stimulated paracrine signaling in th e ovary, 21-day-old intact or hypophysectomized female Wistar rats rec eived four sc injections of recombinant human FSH (rhFSH; total dose, 16-72 IU) at 12-h intervals. Ovaries were removed 48 h after the first injection to extract total RNA for Northern analysis of 17-hydroxylas e/C-17-20-lyase (cytochrome P450c17alpha) mRNA or to isolate thecal/in terstitial cells for assessment of basal and hLH-responsive androgen s ynthesis in vitro. In situ hybridization with a S-35-labeled cytochrom e P450c17alpha cRNA probe confirmed that expression of the cytochrome P450c17alpha gene was specific to thecal/interstitial cells. The appro ximately 2.0-kilobase P450c17alpha mRNA signal in ovarian total RNA fr om intact animals was increased approximately 5-fold by treatment with rhFSH (total dose, 72 IU) or PMSG (15 IU). This effect was shown to b e dose dependent, with a approximately 2-fold increase in response to 16 IU (total dose) rhFSH. P450c17alpha mRNA levels in isolated granulo sa and thecal/interstitial cell total RNA from intact animals were com pared to establish which was the principal cellular site of P450c17alp ha mRNA expression. The P450c17alpha mRNA signal was undetectable in c ontrol granulosa cells and only barely discernible after treatment wit h 72 IU (total dose) rhFSH. In contrast, P450c17alpha mRNA was abundan t in control thecal/interstitial mRNA, and its level was increased 4- to 6-fold by treatment with rhFSH. Treatment of hypopsysectomized anim als with rhFSH did not consistently alter ovarian P450c17alpha mRNA le vels. During culture for 48 h in serum-free medium, basal androgen (an drostenedione plus androsterone) production by thecal/interstitial cel ls from intact animals was unaffected by treatment with rhFSH in vivo, but hLH-stimulated androgen production by these cells was enhanced ap proximately 2-fold. Neither basal nor hLH-responsive androgen producti on by thecal/ interstitial cells from hypophysectomized animals was al tered by previous treatment with rhFSH in vivo. Treatment of thecal/in terstitial cell cultures from both intact and hypophysectomized animal s with inhibin (0.1-30 ng/ml), a putative granulosa-derived paracrine factor, did not measurably affect basal androgen synthesis, but potent ly enhanced LH-responsive androgen synthesis in vitro. Similarly, trea tment of thecal/interstitial cell cultures with conditioned medium fro m FSH-treated granulosa cell cultures significantly enhanced LH-respon sive, but not basal, androgen production. We conclude that treatment o f pituitary-intact rats with ''pure'' FSH modulates thecal/interstitia l cell androgen synthesis. Granulosa cells, but not thecal cells, poss ess FSH receptors, and thecal/interstitial cells are the principal ova rian sites of P450c17alpha expression. Thus, factors produced by FSH-s timulated granulosa cells must function as paracrine signals in vivo. In hypophysectomized animals, the absence of any effect of FSH on thes e parameters of thecal/interstitial cell function emphasizes the relev ance of paracrine signaling to modulation of LH action, rather than di rect regulation of thecal/interstitial function per se.