Krummen L A, Baldwin D M
Department of Physiology and Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0576.
Endocrinology. 1988 Oct;123(4):1868-78. doi: 10.1210/endo-123-4-1868.
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.
本研究的目的是评估睾酮（T）对雄性垂体促黄体生成素（LH）亚基载脂蛋白合成、糖基化及释放的直接影响。将来自1周去势大鼠的细胞在无类固醇培养基中培养48小时，然后在含或不含10 nM T的培养基中培养48小时。随后，将细胞在含有[35S]甲硫氨酸（35S-Met）或[3H]葡糖胺（3H-Gln）的培养基中孵育2、4、6、8或12小时，培养基中含或不含1 nM促性腺激素释放激素（GnRH）（实验1），或者在含有前体物质且含或不含10 nM T和/或1 nM GnRH的培养基中孵育（实验2）。通过免疫沉淀法，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，测定放射性标记前体掺入LH亚基的情况。在实验1中，至少8小时内，前体掺入总蛋白（TP）和LH亚基的量随时间呈线性增加。GnRH不影响前体掺入总蛋白或LH亚基的35S-Met标记，但刺激3H-Gln呈线性时间依赖性地积累到总（细胞加培养基）LH亚基中，并刺激放射免疫法可检测的LH释放到培养基中。基于这些结果，在8小时孵育期间研究了T对LH亚基生物合成（含或不含GnRH）的影响。在实验2中，GnRH增强了两种LH亚基中3H-Gln（但不是35S-Met）的掺入。GnRH刺激了35S-Met LHα和3H-Gln LH亚基的释放，并增加了分泌型LH亚基的相对糖基化，而不改变细胞内LH亚基的相对糖基化。T抑制放射免疫法可检测的LH释放以及两种前体掺入总LH亚基和分泌型LH亚基（含或不含GnRH）。然而，只有分泌型LHα的相对糖基化被T（含或不含GnRH）降低。这些数据表明，T直接作用于垂体，抑制LH亚基载脂蛋白合成，并选择性抑制LHα糖基化。此外，这些数据支持以下假设，即LH糖基化的变化可能是GnRH和T调节LH释放的方式之一。
