Cataldo L, Mastrangelo M A, Kleene K C
Department of Biology, University of Massachusetts, Boston, 100 Morrissey Boulevard, Boston, Massachusetts, 02125-3393, USA.
Exp Cell Res. 1997 Feb 25;231(1):206-13. doi: 10.1006/excr.1996.3447.
Male germ cells in mice develop normally at 32 degrees C and spermatogenesis is severely inhibited by higher temperatures, including abdominal temperature, 37 degrees C. To examine the effects of heat stress on protein synthesis in various testicular cell types, seminiferous tubules were cultured at 32 degrees or 37 degrees C for 70 min or 42.5 degrees or 44 degrees C for 10 min followed by incubation for 60 min at 32 degrees C. Cultures were labeled with [35S]methionine, and the proteins that are soluble in 4% trichloroacetic acid were analyzed by acid-urea polyacrylamide gel electrophoresis. This culture system preserves the cytoarchitecture of the seminiferous epithelium and avoids breaking late haploid cells (elongated spermatids) during tissue dissociation. Incorporation of [35S]methionine into histone H1t, the testis-specific subtype of histone H1, in pachytene primary spermatocytes (meiotic cells) was reduced by about 33-50% following incubation at 37 degrees and 42.5 degrees C and by >/=90% after incubation at 44 degrees C. In contrast, exposure to 37 degrees, 42.5 degrees, and 44 degrees C had minimal effects on incorporation into transition proteins 1 and 2 in elongated spermatids. To determine whether heat stress inhibits translational initiation, the distribution of several mRNAs in cytoplasmic extracts of cultured tubules was analyzed by sucrose gradients and Northern blots. Exposure to 37 degrees and 44 degrees C produces incremental reductions in the size of polysomes translating H1t mRNA in pachytene spermatocytes and the sulfated glycoprotein 2 mRNA in Sertoli cells, the somatic cell type in the germinal epithelium. Neither 37 degrees nor 44 degrees C reduces the size or proportion of polysomal protamine 2 mRNA in elongated spermatids. These results demonstrate that the initiation of translation in pachytene spermatocytes and Sertoli cells is inhibited by exposure to abdominal temperature and that elongated spermatids are much more resistant to thermal stress.
小鼠雄性生殖细胞在32℃时正常发育，而包括腹腔温度37℃在内的较高温度会严重抑制精子发生。为了研究热应激对各种睾丸细胞类型中蛋白质合成的影响，将生精小管在32℃或37℃培养70分钟，或在42.5℃或44℃培养10分钟，然后在32℃孵育60分钟。培养物用[35S]甲硫氨酸标记，用酸脲聚丙烯酰胺凝胶电泳分析可溶于4%三氯乙酸的蛋白质。该培养系统保留了生精上皮的细胞结构，避免了组织解离过程中晚期单倍体细胞（伸长的精子细胞）的破裂。在粗线期初级精母细胞（减数分裂细胞）中，将[35S]甲硫氨酸掺入组蛋白H1t（组蛋白H1的睾丸特异性亚型）在37℃和42.5℃孵育后减少约33 - 50%，在44℃孵育后减少≥90%。相比之下，暴露于37℃、42.5℃和44℃对伸长精子细胞中过渡蛋白1和2的掺入影响最小。为了确定热应激是否抑制翻译起始，通过蔗糖梯度和Northern印迹分析了培养小管细胞质提取物中几种mRNA的分布。暴露于37℃和44℃会使粗线期精母细胞中翻译H1t mRNA的多核糖体大小以及生精上皮中的体细胞类型支持细胞中硫酸化糖蛋白2 mRNA的多核糖体大小逐渐减小。37℃和44℃均未降低伸长精子细胞中多核糖体鱼精蛋白2 mRNA的大小或比例。这些结果表明，暴露于腹腔温度会抑制粗线期精母细胞和支持细胞中的翻译起始，而伸长的精子细胞对热应激的耐受性要强得多。
