Sharpe R M, Maddocks S, Millar M, Kerr J B, Saunders P T, McKinnell C
MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, Scotland, United Kingdom.
J Androl. 1992 Mar-Apr;13(2):172-84.
The aim of this study was to identify potential androgen-regulated proteins (ARP) that might mediate the supportive effects of testosterone on spermatogenesis. Adult rats were injected with ethane dimethane sulphonate (EDS) to destroy Leydig cells and thus induce complete testosterone withdrawal. Other EDS-treated rats were injected with 25 mg testosterone esters (TE) every 3 days to maintain quantitatively normal spermatogenesis. A timeframe for the study of androgen action on spermatogenesis was deduced from enumeration of degenerating germ cells at stage VII of the spermatogenic cycle in perfusion-fixed testes from rats in the early stages (4 to 8 days) after EDS treatment. Based on this data and changes in testicular interstitial fluid volume, long seminiferous tubule segments were isolated from control rats and from EDS-treated rats (+/- TE-supplementation) at stages II-V, VI-VIII, or IX-XII, 2 days to 6 days after EDS treatment. Seminiferous tubule segments were incubated for 22 hours with 60 microCi 35S-labelled methionine. Incorporation into newly synthesized proteins in the seminiferous tubule culture medium (= secreted proteins) or in seminiferous tubule lysates (= intracellular proteins) was determined by trichloroacetic acid-precipitation followed by analysis using two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis. In control rats, incorporation of 35S-methionine into proteins secreted by isolated seminiferous tubules was more than twice as great at stages VI-VIII than at stages II-V or IX-XII. This doubling in methionine incorporation into stages VI-VIII secreted proteins was abolished, however, 4 days after EDS treatment (when germ cell degeneration at stage VII was only just evident). A similar change occurred 4 days after testosterone withdrawal induced by immunoneutralization of luteinizing hormone. In the latter case and after EDS treatment, TE-supplementation of rats from day 0 maintained the normal control pattern of methionine incorporation into seminiferous tubule secreted proteins, although 6 days after EDS and TE treatment, incorporation into stages VI-VIII secreted proteins was 19% lower (P less than 0.05) than in the control group. In contrast, incorporation of methionine into proteins secreted by seminiferous tubules at stages II-V and IX-XII was unaffected by EDS and TE pretreatment, as was incorporation into intracellular proteins at all stages.(ABSTRACT TRUNCATED AT 400 WORDS)
本研究的目的是鉴定可能介导睾酮对精子发生支持作用的潜在雄激素调节蛋白（ARP）。成年大鼠注射乙烷二甲磺酸盐（EDS）以破坏睾丸间质细胞，从而诱导睾酮完全撤除。其他经EDS处理的大鼠每3天注射25mg睾酮酯（TE）以维持精子发生数量正常。通过对EDS处理后早期（4至8天）大鼠灌注固定睾丸生精周期VII期退化生殖细胞的计数，推导出雄激素对精子发生作用的研究时间框架。基于这些数据以及睾丸间质液体积的变化，在EDS处理后2天至6天的II-V、VI-VIII或IX-XII期，从对照大鼠和经EDS处理的大鼠（±TE补充）中分离出长的曲细精管段。将曲细精管段与60μCi 35S标记的蛋氨酸孵育22小时。通过三氯乙酸沉淀，然后使用二维十二烷基硫酸钠聚丙烯酰胺凝胶电泳进行分析来测定35S标记的蛋氨酸掺入曲细精管培养基（=分泌蛋白）或曲细精管裂解物（=细胞内蛋白）中新合成蛋白的情况。在对照大鼠中，在VI-VIII期，35S-蛋氨酸掺入分离的曲细精管分泌蛋白中的量比在II-V期或IX-XII期多两倍以上。然而，在EDS处理后4天（当VII期生殖细胞退化刚刚明显时），VI-VIII期分泌蛋白中蛋氨酸掺入量的这种加倍被消除。在通过免疫中和促黄体生成素诱导睾酮撤除后4天也发生了类似变化。在后一种情况下以及在EDS处理后，从第0天开始对大鼠补充TE可维持蛋氨酸掺入曲细精管分泌蛋白的正常对照模式，尽管在EDS和TE处理后6天，VI-VIII期分泌蛋白中的掺入量比对照组低19%（P<0.05）。相比之下，II-V期和IX-XII期曲细精管分泌蛋白中蛋氨酸的掺入不受EDS和TE预处理的影响，所有阶段细胞内蛋白的掺入情况也是如此。（摘要截短至400字）
