Expression of histone 1 (H1) and testis-specific histone 1 (H1t) genes during stallion spermatogenesis

In eukaryotic cells, the major protein constituents of the chromatin are histones, which can be divided into five classes, identified as H1, H2A, H2B, H3 and H4. During normal spermatogenesis, a testis-specific H1t is expressed in primary spermatocytes and believed to facilitate histone to protamine exchanges during spermiogenesis. In equine testes we detected the H1 protein at 22kDa by western blot analysis while H1t was detected at 29kDa. H1 protein was found to be expressed in all germ cells up to elongating spermatids (Sc) at stage IV. In peripubertal animals, there was a prolonged expression up to elongating spermatids (Sd1) at stage V. A fragment of the equine H1t gene was cloned (GenBank Accession No. AJ865320). The mRNA expression of H1t was found at the level in spermatogonia and in primary spermatocytes up to mid-pachytene at stage VIII/I, whereas H1t protein was found to be expressed up to round spermatides (Sa/Sb1) at stage VIII/I. In peripubertal animals, the H1t protein expression was detected up to elongating spermatids (Sb2) at stage II. Analysis of testes of different ages (< or =2 years) and (> or =3 years) by real-time RT-PCR revealed an increase of H1t mRNA expression, with a wide range of individual variety between 2 and 4 years old animals indicating a stable expression in animals older than 4 years old. This is the first study to show the testis-specific H1t in the stallion and gives evidence that the well-known peripubertal infertility in the stallion may be related to an insufficient histone to protamine exchange. The pattern of protamine gene expression, however, has still to be elucidated.     

cDNA cloning and expression of Xenopus sperm-specific basic nuclear protein 5 (SP5) gene

As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1–6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)⁺ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1–6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0. © 1994 Wiley-Liss, Inc.     

Mouse transition protein 1 is translationally regulated during the postmeiotic stages of spermatogenesis

Transition protein 1 (TP1) is a small basic nuclear protein that functions in chromatin condensation during spermatogenesis in mammals. Here, recently identified cDNA clones encoding mouse transition protein 1(mTP1) were used to characterize the expression of the mTP1 mRNA during spermatogenesis. Southern blot analysis demonstrates that there is a single copy of the gene for transition protein 1 in the mouse genome. Northern blot analysis demonstrates that mTP1 mRNA is a polyadenylated mRNA approximately 600 bases long, which is first detected at the round spermatid stage of spermatogenesis. mTP1 mRNA is not detectable in poly(A)+ RNAs isolated from mouse brain, kidney, liver, or thigh muscle. mTP1 mRNA is translationally regulated in that it is first detected in round spermatids, but no protein product is detectable until approximately 3 days later in elongating spermatids. In total cellular RNA isolated from stages in which mTP1 is synthesized, the mTP1 mRNA is present as a heterogeneous class of mRNAs that vary in size from about 480 to 600 bases. The shortened, heterogeneous mTP1 mRNAs are found in the polysome region of sucrose gradients, while the longer, more homogeneous mTP1 mRNAs are present in the postmonosomal fractions.     

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.     

Expression of the mouse testicular histone gene H1t during spermatogenesis

 The testicular H1 histone variant, H1t, is synthesized during spermatogenesis in mammalian male germ cells. In situ hybridization and immunohistochemical techniques were used to assign the expression of either the H1t mRNA or the H1t protein to specific cell stages of spermatogenesis. Our results show the presence of the H1t mRNA only in the late and mid-pachytene stages, whereas the protein occurs first in pachytene spermatocytes, and persists until later stages from round up to elongated spermatids. Accepted: 1 March 1996     

Stage- and cell-specific expression of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat seminiferous epithelium.

Expression of mRNAs in the rat testis encoding cyclic AMP (cAMP)-dependent protein kinases (PKAs) was studied. A microdissection method was used to isolate 10 pools of seminiferous tubules representing various stages of the cycle of the seminiferous epithelium in combination with Northern blots and in situ hybridization. The results showed a differential expression of the four isoforms of the regulatory subunits (PKA-R) at various stages of the cycle. RI alpha mRNA was detected at approximately the same levels at all stages while expression of RI beta mRNA was low at stages XIII-III, started to increase at stages IV-V, and reached a maximum at stages VIII-XI. The level of RII alpha mRNA was low at stages II-VI, increased markedly at stage VIIa,b, and reached maximal levels at stages VIIc,d and VIII, followed by a reduced expression at later stages, RII beta mRNA levels increased significantly at stage VI with maximal levels at stages VII and VIII. In situ hybridization of sections from the adult rat testis revealed RI alpha mRNA in the layers of pachytene spermatocytes and round spermatids of all stages. RI beta mRNA was detected over late pachytene spermatocytes and round spermatids of stages VII-XIII. RII alpha mRNA was seen in the layers of round spermatids of stages VII-VIII and elongating spermatids of later stages while RII beta mRNA was detected only in the round spermatid region of stages VII-VIII and in some tubules of stages I-VI. These data show that mRNAs encoding PKA-R are expressed in a stage-specific manner in differentiating male germ cells with different patterns of expression for each subunit; this suggests specific roles for these protein kinases at different times of spermatogenesis.     

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.     

Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells

The induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) were measured in the spermatogenic cells of mice using the alkaline elution technique. The animals were injected with [3H]thymidine and sacrificed on subsequent days to examine selectively cohorts of radiolabeled cells in the successive stages of maturation. A significantly increased frequency of SSB was observed in the unirradiated early spermatocytes and late spermatids, associated with genetic recombination and chromatin compaction, respectively. The frequency of SSBs induced by irradiation of animals in vivo remained constant from the early spermatocyte through mid-spermatid stages and decreased significantly only after the cells matured to the late spermatid stage. The frequency of SSBs after in vitro irradiation of testicular cell suspensions also decreased as round spermatids matured to late spermatids. Such decreases for both modes of irradiation may result from maturation-dependent alterations in chromatin in late spermatids, such as condensation and replacement of histones with protamines, rather than from changes in oxygen tension. Rejoining of SSBs in vivo was efficient in the spermatocytes and early spermatids but declined in late spermatids. Possible reasons for the discrepancy between the greater number of unrepaired lesions and lower susceptibility to mutation induction in late spermatids than in round spermatids are discussed.     

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma. Olufunmilade A. Omisanjo is a scholarship holder of the German Academic Exchange Service (DAAD). Sonja Hartmann is a member of the German Research Foundation (DFG) Research Training Group 533 Cell–cell-Interaction in Reproduction.     

Changes in the structural organization of chromatin during spermatogenesis in the rat

In this study, histone H4 was shown to be extensively hyperacetylated in mid-spermatids of the rat during the time period when the entire complement of histones is replaced by basic spermatidal transition proteins. The degree of hyperacetylation of histone H4 was minimal in pachytene spermatocytes. Therefore, the hyperacetylation appears to be directly involved in the histone replacement process late in spermatogenesis in mid-spermatids. In order to investigate further the possible effects of histone H4 hyperacetylation and the other dramatic changes in the nuclear proteins on the structure of chromatin in germinal cells, we examined the thermal denaturation profiles of chromatin from various purified germinal cell types. Our analyses revealed that chromatins from pachytene spermatocytes and early spermatids have similar thermal denaturation profiles, with their major thermal transitions slightly lower than those for rat liver. However, the major thermal transitions for chromatin from mid-spermatids are much lower than those from pachytene spermatocytes and early-spermatids. We propose that the greatly lowered thermal stability of mid-spermatid chromatin represents a dramatic relaxation or decondensation of the chromatin in this cell type in preparation for the replacement of histone by the basic spermatidal transition proteins and that the decondensation is due in large part to the extensive histones hyperacetylation which occurs in these cells.     

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.     

Changes in mRNA length accompany translational regulation of the somatic and testis-specific cytochrome c genes during spermatogenesis in the mouse

The mouse testis contains two isotypes of cytochrome c, which differ in 14 of 104 amino acids: cytochrome cs is present in all somatic tissues and cytochrome cT is testis specific. The regulation of cytochrome cS and cytochrome cT gene expression during spermatogenesis was examined by Northern blot analysis using specific cDNA probes. Total RNA was isolated from adult tissues, enriched germinal cell populations and polysomal gradients of total testis and isolated germinal cells. Three cytochrome cS mRNAs were detected averaging 1.3 kb, 1.1 kb and 0.7 kb in all tissues examined; an additional 1.7 kb mRNA was observed in testis. Isolated germinal cells through prepuberal pachytene spermatocytes contained only the three smaller mRNAs; the 1.7 kb mRNA was enriched in round spermatids. All three smaller cytochrome cS mRNAs were present on polysomes; the 1.7 kb mRNA was non-polysomal. Cytochrome cT mRNA of 0.6-0.9 kb was detected in testis; mRNA levels were low in early spermatogonia and peaked in prepuberal pachytene spermatocytes. In adult pachytene spermatocytes, a subset of the cytochrome cT mRNAs, 0.7-0.9 kb, was present on polysomes; a shortened size class, 0.6-0.75 kb, was non-polysomal. A distinct, primarily non-polysomal, cytochrome cT 0.7 kb mRNA was present in round spermatids. These results indicate that (1) both cytochrome cS and cytochrome cT mRNAs are present in early meiotic cells, (2) a 1.7 kb cytochrome cS mRNA is post-meiotically expressed and non-polysomal and (3) cytochrome cS and cytochrome cT mRNAs are each developmentally and translationally regulated during spermatogenesis.     

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。     

A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.