The proposed research is designed to test the hypothesis that the nonhuman primate (monkey) corpus luteum is a heterogeneous gland consisting of dynamic subpopulations of luteal cells which differ on the basis of structure, function and regulation by hormonal substances. The long-term objectives include elucidation of the origin(s), paracrine interactions and regulatory mechanisms for luteal cell subtypes which combine to produce the normal activity and lifespan of the primate corpus luteum. The initial aims are: (1) to distinguish and separate luteal cells into highly-enriched subpopulations based on differences in physical properties (cell size and organelles) and in binding of fluorescent-tagged hormones, (2) to characterize the ability of purified cell populations to produce steroid hormones and prostaglandins and to respond to luteotropic and luteolytic agents, and (3) to begin in vitro studies on possible cell-to-cell interactions influencing the steroidogenic activity of luteal cells. Collagenase-dispersed cells will be prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. Subpopulations of cells will be distinguished and separated by multiparameter flow cytometry, based on differences in forward and 90 degrees light scatter (a function of cell size/shape and density of organelles) and the binding of fluorescent-tagged hCG and prostaglandins. Sorted groups of cells will be incubated in vitro as acute suspensions to examine (a) their capacity to produce progesterone, estrogens and prostaglandins (PGs) E2, F2 alpha, D2 and I2, and (b) their sensitivity to gonadotropic hormones (LH and CG), various PGs and estrogens. Finally, different groups of cells will be cultured individually or together to elucidate cell-to-cell interactions which promote or inhibit steroidogenesis. Distinguishing the activities and regulatory mechanisms for luteal cell subtypes within the monkey corpus luteum could revise dramatically theories on the control of luteal function in primates. The project would provide the framework for further studies elucidating the origins, functions and changes in specific cell types during the lifespan of the corpus luteum in the normal menstrual cycle, throughout pregnancy and in situations associated with luteal dysfunction.
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