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 revealed differences in the cellular structure of granulosa cells that are dependent upon the location of granulosa cells relative to the germinal 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 germinal disc region (GDR) of the avian preovulatory follicle participates in the process of follicular growth by producing factors that act in a paracrine manner to stimulate proliferation of and inhibit steroidogenesis in the granulosa layer, establishing a gradient in the morphology and physiology of the granulosa layer. To test our hypothesis, we asked two questions: 1) Are physiological gradients of proliferation and steroidogenesis present within the granulosa layer of a preovulatory follicle? 2) Does the GDR secrete factors that affect granulosa cell proliferation and/or steroidogenesis? Incorporation of 3H-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-diameter sections were obtained from three morphologically distinct regions 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 3H-thymidine and produced the least progesterone. Distal granulosa cells incorporated the least 3H-thymidine and produced the most progesterone. Proximal granulosa cells incorporated an intermediate amount of 3H-thymidine and produced an intermediate amount 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 distal granulosa layer (F3 for 3H-thymidine incorporation, F1 for progesterone production) were incubated in GDR-conditioned medium or granulosa cell-conditioned medium to determine whether factors secreted into the medium by the GDR and distal granulosa cells affect granulosa cell proliferation and/or steroidogenesis. Certain samples of GDR-conditioned medium and granulosa cell-conditioned medium were boiled, protease-treated or charcoal-stripped. F3 and F1 GDRs produced heat- and protease-sensitive factors that promoted proliferation and inhibited progesterone production by granulosa cells. These data indicate that diametrically opposed gradients of proliferation and steroidogenesis are present within me granulosa layer of an individual preovulatory follicle. Furthermore, the GDR produces proliferation-stimulating and steroidogenesis-inhibiting factors that may act in an autocrine or paracrine manner to influence proliferation and steroidogenesis in granulosa cells.