Regulation of luteinizing hormone subunit biosynthesis in cultured male anterior pituitary cells: effects of gonadotropin-releasing hormone and testosterone.
The purpose of this study was to evaluate the direct effects of testosterone (T) on LH subunit apoprotein synthesis, glycosylation, and release by the male pituitary. Cells from 1-week castrate rats were cultured for 48 h in steroid-free medium, followed by 48 h in medium with or without 10 nM T. The cells were then incubated for 2, 4, 6, 8, or 12 h in medium containing [35S]methionine (35S-Met) or [3H]glucosamine (3H-Gln), with or without 1 nM GnRH (Exp 1) or in medium containing precursors with or without 10 nM T and/or 1 nM GnRH (Exp 2). Radiolabeled precursor incorporation into LH subunits was determined by immunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Exp 1, precursor incorporation into total protein (TP) and LH subunits increased linearly over time for at least 8 h. GnRH did not affect precursor incorporation into total protein or 35S-Met labeling of LH subunits, but stimulated a linear time-dependent accumulation of 3H-Gln into total (cells plus media) LH subunits and release of radioimmunoassayable LH into the medium. Based on these results, the effects of T on LH subunit biosynthesis (with or without GnRH) were studied during an 8-h incubation. In Exp 2, GnRH enhanced total 3H-Gln (but not 35S-Met) incorporation into both LH subunits. GnRH stimulated the release of 35S-Met LH alpha and 3H-Gln LH subunits and increased the relative glycosylation of secreted LH subunits without altering the relative glycosylation of intracellular LH subunits. T inhibited radioimmunoassayable LH release and incorporation of both precursors into total and secreted LH subunits (with or without GnRH). However, only the relative glycosylation of secreted LH alpha was reduced by T (with or without GnRH). These data indicate that T acts directly at the pituitary to inhibit LH subunit apoprotein synthesis and selectively inhibit LH alpha glycosylation. Further, these data support the hypothesis that changes in LH glycosylation may be one of the ways by which GnRH and T regulate LH release.
D000818:Animals; D002478:Cells, Cultured; D005944:Glucosamine / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007700:Kinetics; D007986:Luteinizing Hormone / Q000096:biosynthesis*; D046911:Macromolecular Substances; D008297:Male; D008715:Methionine / Q000378:metabolism; D009919:Orchiectomy; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012016:Reference Values; D013739:Testosterone / Q000494:pharmacology*
D046911:Macromolecular Substances; D007987:Gonadotropin-Releasing Hormone; D013739:Testosterone; D007986:Luteinizing Hormone; D008715:Methionine; D005944:Glucosamine