FLOW CYTOMETRY AND MOLECULAR GENETICS OF FETAL CELLS

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The long term objective is to determine the hormonal and other factors regulating the growth and regression of ovarian follicles from primordial to antral states in various species of mammals. The specific objectives include: 1. A method has been worked out to isolate and purify in large numbers primordial follicles (PFS) from pig ovaries which makes this hitherto unexplored population available for descriptive and experimental analyses. It is proposed to improve the methodology to increase the yield of PFS; carry out morphological studies on in situ and freed PFS (SEM, TEM, thick sections (1 mu m) and H&E serial sections, histochemical and immunocytochemical investigations); compare in vitro protein synthesis of the PF complex with oocytes of mature pig oocytes and intermediate stages; to culture PFS for several days in the presence and absence or appropriate hormones and growth factors and analyze changes in protein synthesis, cam;p accumulation and morphology (transformation of squamous pregranulosa cells into a cuboidal "growing" granulosal compartment?). 2. Projects involving enzymatically isolated intact preantral follicles from hamster ovaries will be continued to analyze short and long term dependency of small growing follicles on gonadotropin and growth factor(s) support. With suitable hormonal treatments can cultured preantral follicles be converted to functionally normal mature antral follicles? In short term incubations, FSH and LH both stimulate DNA synthesis of large preantral and antral follicles but when given together DNA synthesis is reduced below baseline levels. What are the mechanisms involved in FSH-LH synergism and antagonism? Are inositol lipids involved, along with camp, as intermediaries of FSH/LH activity? 3. In the hamster, several lines of in vivo and in vitro evidence indicate that preantral follicles are responsive to gonadotropins which challenges the prevalent belief that their development is anhormonal. Is the hamster an exception or are preantral follicles in pig, rate, mouse and rabbit influenced by FSH and/or LH? 4. Is the immune system in the form of antibody - dependent cell mediated toxicity involved in vitro follicular atresia and in vivo spontaneous or induced degeneration? Are macrophages the key intermediary cell?