Isolation and characterization of TH3, a germ cell-specific variant of histone 3 in rat testis

We have identified and purified TH3, a germ cell-specific histone. It has been characterized by amino acid analysis, tryptic peptide mapping, labeling with cystine, and by electrophoretic mobility as a variant of H3. On fully reduced Triton/acid/urea gels its mobility is retarded more than that of the somatic variants H3.2 and H3.3, but less than that of H3.1; it migrates between the H2As and H1s. Germinal cells from adult and sexually immature testes were purified by centrifugal elutriation followed by Percoll density gradient separation in order to study the distribution and synthesis of TH3. TH3 is found in significant levels in spermatogonia and in similar or slightly higher amounts in spermatocytes and round spermatids. The synthesis of TH3 takes place in the spermatogonia but not in spermatocytes, in contrast to the other testis-specific histones, TH2A, H1t, and TH2B. Therefore, TH3 may have a different role in spermatogenesis than do the other testis-specific histone variants.     

Histone variants in rat spermatogonia and primary spermatocytes

The levels and synthesis of histone variants have been directly measured in spermatogonia and in various stages of primary spermatocytes purified from the rat testis. These measurements were made possible by the development of a procedure, employing centrifugal elutriation and density gradient centrifugation, to separate highly enriched populations of such cells from immature rat testes at the early stages of spermatogenesis. The results show a difference in regulation of the synthesis and accumulation of testis-specific histones H1t, TH2A, TH2B, and TH3. TH3 is present and actively synthesized in A and B spermatogonia. The testis-enriched variants, H2A.X and H1a, are also present at their maximal levels in A spermatogonia. No detectable amounts of H1t, and at most, low levels of TH2A and TH2B could be found in spermatogonia. While TH2A and TH2B are already present and actively synthesized in early primary spermatocytes (around the preleptotene stage), H1t does not accumulate until the pachytene stage.     

Histone gene expression during sea urchin spermatogenesis: an in situ hybridization study

The expression of testis-specific and adult somatic histone genes in sea urchin testis was investigated by in situ hybridization. The testis-specific histone genes (Sp H2B-1 of Strongylocentrotus purpuratus and Sp H2B-2 of Lytechinus pictus) were expressed exclusively in a subset of male germ line cells. These cells are morphologically identical to replicating cells pulse-labelled with 3H-thymidine. Genes coding for histones expressed in adult somatic and late embryo cells (H2A-beta for S. purpuratus and H3-1 for L. pictus) were expressed in the same germ line cells, as well as in the supportive cells (nutritive phagocytes) of the gonad. All histone mRNAs detected in the male germ lineage declined precipitously by the early spermatid stage, before cytoplasmic reduction. The data suggest that both testis-specific and adult somatic histone genes are expressed in proliferating male germ line cells. Testis-specific gene expression is restricted to spermatogonia and premeiotic spermatids, but somatic histone expression is not. The decline of histone mRNA in nondividing spermatids is not merely a consequence of cytoplasmic shedding, but probably reflects mRNA turnover.     

Nuclear proteins in spermatogenesis

Mammalian somatic type histone variants are replaced or supplemented in early primary spermatocytes and possibly spermatogonia by testis specific and testis enriched histone variants. The testis complement of histones is replaced entirely by transition basic proteins in mid-spermatids. This transition is accompanied by a dramatic reduction of thermal stability of mid-spermatid chromatin which may be due in part to hyperacetylation of histone H4. The transition basic proteins are replaced by protamines which are arginine-rich and contain cysteine.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

质谱法分析大鼠精子发生过程中组蛋白H1变异体的多样性

目的为探究连接组蛋白H1在精子发生过程染色体重构中的功能,了解一共有多少种连接组蛋白H1参与各期生精细胞的染色体的构建。方法分离高纯度的SD大鼠的各期生精细胞,提取组蛋白,应用SDS-PAGE分离组蛋白的各组分,组蛋白（H1）经过蛋白酶（Glu-c和Arg-c）酶切,应用质谱进行检测。结果鉴定了组蛋白H1的体细胞亚型（H1.1-H1.5）和睾丸特异的连接组蛋白亚型（H1t）。组蛋白H1t分别表达在精原细胞,精母细胞和圆形精子细胞中。结论大鼠精子发生过程中,其主要连接组蛋白H1的种类是：H1.1-H1.5和H1t。     

Characterization of histone H2A.X expression in testis and specific labeling of germ cells at the commitment stage of meiosis with histone H2A.X promoter-enhanced green fluorescent protein transgene

To study the complex molecular mechanisms of mammalian spermatogenesis, it would be useful to be able to isolate cells at each stage of differentiation, especially at the stage in which the cells switch from mitosis to meiosis. Currently, no useful marker proteins or gene promoters specific to this important stage are known. We report here a transgenic mouse line that under the control of the promoter for a histone variant, H2A.X, expressed an enhanced green fluorescent protein (EGFP) in cells at the stage of the mitosis-meiosis switch. Endogenous H2A.X is expressed in type A spermatogonia through meiotic prophase spermatocytes in testis and in some somatic cells. However, despite the fact that its expression was driven by the H2A.X promoter, the EGFP expressed in the transgenic mice specifically labeled only the intermediate spermatogonia stage through the meiotic prophase spermatocyte stage in transgenic mice containing the -600-base pair H2A.X promoter/EGFP construct. Type A spermatogonia and somatic cells of other organs were not labeled. This expression pattern made it possible to isolate living cells from the testis of the transgenic mice at the stage of the mitosis-meiosis switch in spermatogenesis using EGFP fluorescence.     

Increased accessibility of the N-terminus of testis-specific histone TH2B to antibodies in elongating spermatids

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N-terminus of the testis-specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross-reacted with TH2B because of sequence homology at the N-termini of TH and TH2B. The epitope was localized to the N-terminus of TH2B as trypsin-digested chromatin which lacked the N-terminal tail did not react with anti-TH and preincubating anti-TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10–12 immunoreacted intensely with anti-TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N-terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones. © 1995 wiley-Liss, Inc.