Concerted metabolism of steroid hormones produced by cocultured ovarian cell types

Ovaries of immature, intact rats were dispersed by collagenase-DNase treatment and cultured in serum-free medium (ovarian cell culture). The hormonal responsiveness of the ovarian cell was compared to that exhibited by pure granulosa cells isolated via needle puncturing. Surprisingly, despite the fact that the majority of the cultured cells should have been comprised of granulosa type, no follicle-stimulating hormone-inducible progesterone or 20α-OH-progesterone (20α-OH-P) could be detected by radioimmunoassay, as typically occurs in cultures of pure granulosa cells. Therefore, in order to unravel the cause for the different responsiveness between the granulosa and the ovarian cell, we applied thin-layer chromatography analysis to follow the metabolic fate of added radioactive pregnenolone to intact monolayers in culture. Such TLC analysis revealed that, after priming with follicle-stimulating hormone, added [³H]pregnenolone was converted to progesterone which was rapidly reduced and finally accumulated as 5α-pregnane-3α,20α-diol (pregnanediol). In addition to pregnanediol, a second class of steroid hormones accumulated in the coculture medium, namely androsterone and 5α-pregnane-3α,17α,20α-triol (pregnanetriol). These latter two were specific products of the ovarian coculture, indicating the presence of theca-interstitial cells bearing 17α-hydroxylase and 17,20-lyase activities. Pregnanediol, rather than progesterne, was found to be the progestin precursor for androgen formation. We thus conclude that due to exchange of steroid metabolites between the cocultured cell types, the final steroid products are different by far from the expected contributions of each individually cultured cell type. Moreover, these findings reveal an additional aspect of the 'two-cell theory', suggesting a granulosa-thecal concerted metabolism of progestin steroids, rather than exchange of aromatizable androgens.