Cyclic AMP has been recognized for almost 3 decades as an important chemical second messenger that mediates hormone action. In contrast, calcium (Ca2+) second-messenger signaling pathways are considerably less well understood. Our specific theme of study focuses on the mechanisms by which Ca2+ pathways mediate gonadotropic hormone action in the ovary. To this end, we have utilized single-cell digital fluorescence videomicroscopy of ovarian (granulosa) cells to investigate FSH and LH's regulation of intra-cellular free Ca2+ concentrations, [Ca2+]i. Notably, in gonadal target cells bearing native gonadotropin receptors (e.g. swine granulosa and rat Sertoli cells), FSH and LH both stimulate adenyl cyclase, but each gonadotropin also activates a distinctive [Ca2+]i signaling response. [Ca2+]i signaling by FSH versus LH can be distinguished by multiple criteria including: dependence on extracellular Ca2+ influx, mimicry by putative agonists of phospholipase C, time course, presumptive G-protein involvement, susceptibility to desensitization, etc. Accordingly, we hypothesize that FSH and LH receptors activate unique transducing mechanisms that stipulate their respective specificities of [Ca2+]i signal generation. Existence of multiple signaling pathways within target gonadal cells can allow for two or more signaling mechanisms to be activated simultaneously by a single ligand, sequentially as a function of effector concentrations, or by multiple stimuli occurring concurrently. This intracellular signaling strategy probably contributes to the essential pleiotropic cellular effects of FSH and LH on gonadal tissues. Based on this background, in this renewal proposal we will use primary cultures of untransformed swine granulosa cells to investigate: (i) the regulated biochemical mechanisms that govern the distinctive FSH versus LH- promoted [Ca2+]i signals in single ovarian cells; (ii) the mechanistic basis of FSH-versus LH-driven [Ca2+]i signal desensitization in individual target cells; and (iii) the specific temporal features of the FSH versus LH- stimulated second-messenger [Ca2+]i signals that direct expression of an important steroidogenic gene (cytochrome P450 cholesterol side-chain cleavage enzyme) within single ovarian (granulosa) cells. Such studies should provide significant new insights into the idiotypic mechanisms of second-messenger [Ca2+]i signaling by each of two primary gonadotropic hormones in native FSH and LH-receptor- bearing ovarian target cells, and should clarify the nature of the specific functional dependence of gene expression on [Ca2+]i changes in individual gonadal cells as activated by FSH versus LH. A greater comprehension of basic mechanisms of second-messenger-directed hormone action in reproductive cells is necessary to foster the development of novel rational strategies for fertility regulation in the human, domestic animal, and endangered wild species.
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