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The patterns of transcription and translation of the ribosomal protein L32 (Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5' RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5' terminal oligopyrimidine tract (5' TOP) that is used is somatic cells. The 5' TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and approximately 62% of Rpl32 mRNAs lack a 5' TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5' TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5' TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.

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.

Round spermatids and elongating spermatids were purified from a suspension of mouse testicular cells by sedimentation at unit gravity coupled with density gradient centrifugation through Percoll. Following separation, the two cell types were fractionated into polysomal and non-polysomal compartments. By comparison with round spermatids, elongating spermatids contain about one-half as much cytoplasmic RNA per cell, one sixth as much poly(A)+ RNA per cell and one-half the concentration of poly (A)+ mRNA in their cytoplasm. About two-thirds of the poly(A)+ messenger RNA (mRNA) was in the non-polysomal fraction in both cell types. Polypeptides whose synthesis was directed by cell-free translation of purified mRNA from each cell fraction were analyzed by two-dimensional gel electrophoresis. At the level of detection provided by the electrophoretic methods used, the majority of peptides from the polysomal and non-polysomal compartments for each cell type were similar. However, between the two cell types, approx. 5-10% of the polypeptides in the polysomal and non-polysomal fractions differed markedly in abundance. When the polypeptides encoded by the polysomal and non-polysomal mRNA from round spermatids were compared to the polypeptides encoded in the equivalent fractions from elongating spermatids, a significant reduction in number of polypeptides from elongating spermatids was seen. The presence of specific mRNAs in the non-polysomal fraction of round spermatids and in the polysomal fraction of elongating spermatids suggests that storage of mRNA in the cytoplasm and subsequent utilization provides a source of mRNA for proteins expressed at a time during spermiogenesis when transcription has terminated.

Following intratesticular injection of [35S]methionine, the multiple isoforms of actin and tubulin from highly purified mouse testicular meiotic and post-meiotic cells have been analysed by high resolution two-dimensional gel electrophoresis. In pachytene spermatocytes both beta and gamma actin are synthesized, gamma actin being made in a significantly greater amount. The relative proportion of synthesis of beta and gamma actin changes during spermiogenesis, beta actin increasing and gamma actin decreasing in round spermatids, elongating spermatids, and residual bodies. Both alpha and beta tubulin are synthesized in approximately equal proportion in pachytene spermatocytes. In addition to the tubulin isoforms synthesized during meiosis, at least one new form of both alpha and beta tubulin first appears in post-meiotic (haploid) cells. In elongating spermatids and residual bodies, the synthesis of alpha tubulin is drastically reduced.