Although the effects of gonadotropin releasing hormone (GnRH) and the sex steroids on LH release are well recognized, our knowledge of the physiological events associated with the regulation of LH biosynthesis is limited. Since GnRH is known to regulate both the release and synthesis of LH, it seems imperative that more knowledge be gained about the interrelationship between LH biosynthesis, storage and release. Obtaining such information is a critical prerequisite to developing a more rational approach to regulating fertility in health and disease. The overall objective of this proposal is to gain a better understanding of the physiological mechanisms regulating LH biosynthesis by GnRH and the sex steroids. The specific objectives of this proposal are: investigate the effects of the gonadal steroids and GnRH ON lh subunit apoprotein synthesis and glycosylation and determine whether responses obtained with continuous GnRH stimulation are different than those obtained using a more physiological pulsatile GnRH stimulation, 2) determine the effects of the gonadal steroids on cAMP stimulation of LH subunit biosynthesis, 3) determine whether a functional heterogeneity exist for gonadotrophs synthesizing LH in response to GnRH and whether it may be steroid hormone dependent, and 4) investigate the hormonal regulation of glycosylation and sulfation of LH subunits by GnRH and estradiol by comparing the incorporation of sulfate and various carbohydrates. Hormonal regulation of LH subunit biosynthesis will be investigated using 3-4 day cultured anterior pituitary cells from male and female rats. LH subunit biosynthesis will be determined by the incorporation of radiolabeled methionine and glucosamine (or other sugars and sulfate) into LH by immunoprecipitation of samples using an anti-LH antiserum highly specific for the LH alpha/beta dimer, and the immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radioactivity associated with the LH alpha and LH beta subunits. To determine if a functional heterogeneity of gonadotrophs exist and if it is influenced by sex steroids, pituitary cells will be separated by unit gravity sedimentation into large, medium, and small gonadotrophs to determine their capacity to synthesize LH. Together, these studies should contribute significantly to our understanding of the physiological regulation of LH biosynthesis and its relationship to GnRH stimulated release which in turn should potentially enhance the clinical utility of GnRH and its analogs for use as therapeutic agents.
