Differential Effects of Heat Shock on Translation of Normal mRNAs in Primary Spermatocytes, Elongated Spermatids, and Sertoli Cells in Seminiferous Tubule Culture

Abstract

Male germ cells in mice develop normally at 32°C and spermatogenesis is severely inhibited by higher temperatures, including abdominal temperature, 37°C. To examine the effects of heat stress on protein synthesis in various testicular cell types, seminiferous tubules were cultured at 32° or 37°C for 70 min or 42.5° or 44°C for 10 min followed by incubation for 60 min at 32°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° and 42.5°C and by ≥90% after incubation at 44°C. In contrast, exposure to 37°, 42.5°, and 44°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° and 44°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° nor 44°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.