Direct effects of nerve growth factor on thecal cells from antral ovarian follicles

TrkA, the nerve growth factor (NGF) tyrosine kinase receptor, is expressed not only in the nervous system, but also in nonneural cells, including disc rete cellular subsets of the endocrine and immune system. In the rat ovary, trkA receptor abundance increases strikingly in thecal-interstitial cells during the hours preceding the first ovulation. Blockade of either trkA tra nsducing capacity or NGF biological activity inhibited ovulation, suggestin g a role for NGF in the ovulatory process of this species. To identify some of the processes that may be affected by trkA activation in the thecal com partment, we used purified thecal cells/thecal fibroblasts from bovine ovar ies (heretofore referred to as thecal cells). Ribonuclease protection assay s employing bovine-specific cRNA probes demonstrated the presence of the me ssenger RNAs (mRNAs) encoding NGF and its receptors, p75 NTR and trkA, in t he thecal compartment of small, medium, and large antral follicles and show ed that trkA mRNA is also expressed in granulosa cells. In situ hybridizati on and immunohistochemical examination of intact ovaries confirmed these ce llular sites of NGF and trkA synthesis. TrkA mRNA, but not NGF mRNA, was lo st within 48 h of placing thecal cells in culture. Thus, to study trkA-medi ated actions of NGF on these cells me transiently expressed the receptor by transfection with a vector containing a full-length rat trkA complementary DNA under transcriptional control of the cytamegalovirus promoter. Because ovulation is preceded by an LH-dependent increase in androgen and progeste rone production, the ability of NGF to modify the release of these steroids was determined in freshly plated cells still containing endogenous trkA re ceptors and in cells undergoing luteinization in culture that were transien tly transfected with the trkA-encoding plasmid. NGF stimulated bath androge n and progesterone release in freshly plated thecal cells, but not in lutei nizing cells provided with trkA receptors. As ovulation in rodents requires an increased formation of PGE, and has been shown to be antedated by proli feration of thecal fibroblasts, we determined the ability of NGF to affect these parameters in trkA-transfected thecal cells. The neurotrophin rapidly stimulated PGE, release and amplified the early steroidal response to hCG in trkA-expressing cells, but not in cells lacking the receptor. Likewise, NGF stimulated [H-3]thymidine incorporation into trkA-containing cells, but not into cells that had lost the receptor in culture. Induction of ovulati on in immature rats by gonadotropin treatment verified that an increased ce ll proliferation in the thecal compartment, determined by the incorporation of bromodeoxyuridine into cell nuclei, occurs 4-5 h before ovulation in th is species. These results suggest that the contribution of NGF to the ovula tory process includes a stimulatory effect of the neurotrophin on steroidog enesis, PGE(2) formation, and proliferative activity of thecal compartment cells.