Inhibin A and B secretion in mouse preantral follicle culture

Conditioned media from single mouse ovarian follicles cultured from the early preantral stage up to complete maturity were analysed for different immunoreactive inhibin forms. The inhibin assays measured (i) alpha-specific inhibin, as represented by a mix of 32 and and 57 kDa inhibins, inhibin precursors, alpha-subunit and its precursors; (ii) dimeric inhibin A; and (iii) dimeric inhibin B. The validity of these assays for the measurement of mouse inhibin was established. All forms of inhibin were secreted in culture media from the preantral follicle stage onwards. Inhibin B was the most sensitive marker for proliferation of early stage follicles, while inhibin A secretion became predominant at later stages, when antral-like cavities were formed in granulosa cell masses. Supplementation of standard culture medium with recombinant follicle stimulating hormone appeared to be the predominant regulator of inhibin secretion; addition of recombinant luteinizing hormone throughout the culture period did not cause any major shifts in the expression of dimeric inhibin or alpha-specific inhibin forms. In the absence of theca cells during isolation and culture (as reflected by absence of oestrogen secretion), follicles grew at a reduced rate, and produced lower inhibin concentration in conditioned medium. These data suggest (i) that monitoring of dimeric inhibins can provide useful markers of the growth and differentiation of cultured follicles and (ii) that dimeric inhibins A and B are secreted at an earlier stage in vitro than in vivo.