We have studied the de novo biosynthesis and secretion of LH subunits in pituitary quarters from orchiectomized and intact control adult male rats and their regulation by GftRH. After labeling with [35S]cystine ([35S]Cys), [35S]methionine, or [3H]glucosamine ([3H]GlcN) in the presence or absence of 10−8 M GnRH, tissue lysates arid media were immunoprecipitated with antisera to LHβ, then LHα (after removal of TSH by immunoprecipitatiori with ariti-TSHβ), and the products were analyzed by sodium dodecyl sulfate gradient gel electrophoresis. During a 12-min pulse labeling with [35S]rriethionine, three forms of immunoreactive a were labeled at 21,000, 18,000, and 12,000 mol wt. After a 30-min chase with excess unlabeled methionine, the 12,000 form decreased from 10% to 3% of total radioactivity, while the 21,000 form increased from 57% to 69%, implying a precursor-product relationship. Neither orchiectomy nor GnRH had any effect on [35S]Cys or [3H]GlcN incorporation into intracellular or secreted total proteins. After a 6-h continuous labeling, incorporation of [35S] Cys into intracellular combined LHa in castrates was 158% of the control value, combined LHβ was 304%, and free a was 466%. The [3H]GlcN to [35S]Cys ratio, reflecting relative glycosylation, was unchanged in castrates for total proteins or LHα and somewhat decreased for LHβ and free α. Orchiectomy increased [35S]Cys-labeled secreted LHβ and free α to 183% and 231% of control values, respectively. Relative glycosylation of secreted LHα, LHβ, and free α was unchanged in castrates. These data indicate that orchiectomy stimulates LH and free α apoprotein synthesis and secretion without increasing relative glycosylation, while acute in vitro GnRH exposure increases relative glycosylation with little or no effect on apoprotein synthesis. Sex steroids and GnRH appear to regulate synthesis, glycosylation, and secretion in distinct patterns for the different subunits.
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