AVIAN GERMINAL DISC REGION SECRETES FACTORS THAT STIMULATE PROLIFERATION AND INHIBIT PROGESTERONE PRODUCTION BY GRANULOSA-CELLS

Microscopic analysis of ovarian follicles in the domestic hen has reve aled differences in the cellular structure of granulosa cells that are dependent upon the location of granulosa cells relative to the germin al disc, which contains the female gamete. These differences appear as a morphological gradient, which implies variations in granulosa cell function. This observation prompted us to hypothesize that the germina l disc region (GDR) of the avian preovulatory follicle participates in the process of follicular growth by producing factors that act in a p aracrine manner to stimulate proliferation of and inhibit steroidogene sis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we aske d two questions: 1) Are physiological gradients of proliferation and s teroidogenesis present within the granulosa layer of a preovulatory fo llicle? 2) Does the GDR secrete factors that affect granulosa cell pro liferation and/or steroidogenesis? Incorporation of H-3-thymidine was used as a measure of proliferation, and production of progesterone was used as a measure of steroidogenesis. In the first experiment, 8-mm-d iameter sections were obtained from three morphologically distinct reg ions of the granulosa monolayer: 1) the GDR, 2) granulosa cells distal to the GDR (distal granulosa), and 3) granulosa cells midway between the GDR and distal granulosa cells (proximal granulosa cells). The GDR incorporated the most H-3-thymidine and produced the least progestero ne. Distal granulosa cells incorporated the least H-3-thymidine and pr oduced the most progesterone. Proximal granulosa cells incorporated an intermediate amount of H-3-thymidine and produced an intermediate amo unt of progesterone. To answer the second question, conditioned medium was prepared from GDRs and distal granulosa cells (control) obtained from the F1 (largest preovulatory follicle) and F3 (the third-largest preovulatory follicle) follicles. Sections (8-mm in diameter) of the d istal granulosa layer (F3 for H-3-thymidine incorporation, F1 for prog esterone production) were incubated in GDR-conditioned medium or granu losa cell-conditioned medium to determine whether factors secreted int o the medium by the GDR and distal granulosa cells affect granulosa ce ll proliferation and/or steroidogenesis. Certain samples of GDR-condit ioned medium and granulosa cell-conditioned medium were boiled, protea se-treated or charcoal-stripped. F3 and F1 GDRs produced heat- and pro tease-sensitive factors that promoted proliferation and inhibited prog esterone production by granulosa cells. These data indicate that diame trically opposed gradients of proliferation and steroidogenesis are pr esent within the granulosa layer of an individual preovulatory follicl e. Furthermore, the GDR produces proliferation-stimulating and steroid ogenesis-inhibiting factors that may act in an autocrine or paracrine manner to influence proliferation and steroidogenesis in granulosa cel ls.