Studies on the mechanism of granulosa cell luteinization will be pursued with an emphasis on local control processes. The role of a follicular fluid luteinization stimulator (LS) isolated from large (6-12mm) porcine follicles will be examined. The LS, which is a protein about 60,000 daltons, will be isolated from large porcine follicular fluid, purified and characterized chemically and physiologically. This LS will be assayed on the basis of its ability to enhance FSH induction of LH/hCG receptors and progesterone secretion in cultured porcine granulosa cells. The LS will be purified by an appropriate combination of alcohol precipitation, Sephacryl-S-300 column chromatography, ion exchange chromatography on DEAE trisacryl and HPLC ion exchange on a TSK 5 PW column. HPLC on a C3 reverse phase column and a tiyo soda 3000 SW gel exclusion column will be carried out as required. Fractions will be characterized by SDS gel electrophoresis and by protease assay. The final product will be subject to end group analysis and Manual Edman degradation. The cellular site of action of the purified LS will be examined in cultured porcine granulosa cells, i.e. its ability to alter de novo synthesis of LH/hCG receptors using incorporation of heavy amino acids, ability to alter adenyl cyclase, & ability to alter general protein synthesis. Rat granulosa cells will also be used as an experimental system. An antiserum, monoclonal and polyclonal, will be generated against porcine LS. The cross reactivity against LS activity in human follicular fluid will be examined. The ability of porcine granulosa and thecal cells to secrete LS will be examined under different hormonal conditions. The effect of LS in vivo will be examined initially in the immature rat and if positive results are obtained the observations will be carried out in the immature rhesus monkey. Alternately the effects of antiserum to LS in vivo to ovarian function will be examined in the immature rat or monkey. The role of a follicular fluid luteinization inhibitor (LI) present in small porcine follicles (1-3mm) and medium (3-5mm) sized follicles will be determined. The LI will be isolated and purified using FSH treated porcine granulosa cell cultures as a bioassay. The ability to inhibit FSH induction of LH/hCG receptors, progesterone secretion and morphological luteinization will be followed. An antiserum against LI will be prepared and evaluated for actions against LI in vitro and in vivo. The hormonal control of granulosa cell synthesis of LI activity will be examined. Interaction of LI and LS in control of follicular maturation will be considered.
