共查询到20条相似文献,搜索用时 31 毫秒
1.
Kula N. Jha Lily Wong Patricia M. Zerfas Rukman S. De Silva Ying-Xin Fan Nikolay A. Spiridonov Gibbes R. Johnson 《The Journal of biological chemistry》2010,285(45):35180-35187
We previously reported the identification of small serine/threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (Kd ∼10 nm), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaperone that is critical to the HSP90-mediated activation of SSTK. 相似文献
2.
Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases 总被引:14,自引:0,他引:14
Hazzouri M Pivot-Pajot C Faure AK Usson Y Pelletier R Sèle B Khochbin S Rousseaux S 《European journal of cell biology》2000,79(12):950-960
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. 相似文献
3.
4.
Characterization of E3Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones 总被引:7,自引:0,他引:7
During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation. 相似文献
5.
Suphamungmee W Apisawetakan S Weerachatyanukul W Wanichanon C Sretarugsa P Poomtong T Sobhon P 《Molecular reproduction and development》2005,70(2):211-221
The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species. 相似文献
6.
Two basic proteins, protamines P1 and P2, are present in chromatin of mouse spermatozoa. Protamine P1, the less abundant protein in mouse, has a homolog in most mammals, and its synthesis follows a conventional route. In contrast, protamine P2 has been found only in certain other mammals, including humans, and it is synthesized as a precursor nearly twice as long as the mature protein. Processing of this precursor is not yet understood, although it necessarily takes place in elongating spermatids and is likely to play a role in the chromatin condensation occurring in these haploid cells. We have fractionated basic proteins from mouse testis chromatin and have identified six proteins on electrophoretic gels which, like protamines, are insoluble in SDS. All six were also soluble at the same trichloroacetic acid concentration as protamine P2 and were present in chromatin of elongating spermatids. Radioactive labelling patterns acquired by these SDS-insoluble proteins during translation in vitro of testis RNA indicate that the largest represents the precursor of protamine P2, and suggest that the others represent intermediates generated by proteolytic cleavage of the precursor. Results from pulse 3H labelling in vivo were also consistent with the conclusion that a precursor/product relationship exists between these proteins and protamine P2. Conclusions concerning the kinetics of processing have, in addition, been drawn from this data. Hypotheses concerning possible functional roles played by the precursor are presented. 相似文献
7.
Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Govin J Escoffier E Rousseaux S Kuhn L Ferro M Thévenon J Catena R Davidson I Garin J Khochbin S Caron C 《The Journal of cell biology》2007,176(3):283-294
During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants. 相似文献
8.
Nair M Nagamori I Sun P Mishra DP Rhéaume C Li B Sassone-Corsi P Dai X 《Developmental biology》2008,320(2):446-455
9.
Sophie Rousseaux Cécile Caron Christophe Pivot-Pajot Anne Karen Faure Mira Hazzouri Bernard Sele Saadi Khochbin 《Andrologie》2003,13(2):139-147
Round spermatids are post-meiotic cells with a haploid genome contained in a nucleus, with a structure initially similar to that of the somatic cell nucleus. During spermatogenesis, the spermatid nucleus undergoes drastic remodelling during which it first elongates and then condenses into the very specific and tightly packaged structure of the sperm nucleus. During this remodelling dthe histones are replaced by transition proteins, which, in turn, are replaced by protamines, the specific nuclear proteins of the spermatozoa. Immediately prior to their replacement, the histones are hyperacetylated. The first part of our work was to precisely characterise the changes in histone acetylation during murine spermatogenesis. We have shown that the core histones H2A, H2B, H3 and H4 are hyperacetylated in the elongating spermatids. We have also shown that these changes in acetylation are associated with degradation of the enzymes responsible for histone deacetylation, histone deacetylases or HDACs, while histone acetyl transferases are still present in these cells. The histone acetylation pattern was also investigated during human spermatogenesis, revealing that histone hyperacetylation in the nucleus of elongating spermatids, which appears to be conserved during the course of evolution, also occurs during human spermatogenesis. Moreover, our data obtained from the testes of men with severely altered spermatogenesis, including SCO syndromes (Sertoli Cells Only Syndromes), show that a global hyperacetylation of the Sertoli cell nuclei is associated with an absence of meiotic and post-meiotic cells. This suggests that the global histone acetylation variations observed during spermatogenesis are part of a signalling pathway involving germ cell — Sertoli cell communication. Altogether, these data provide a basis for a better understanding of the mechanisms and identification of the factors involved in post-meiotic remodelling of chromatin. 相似文献
10.
Meyer-Ficca ML Lonchar JD Ihara M Meistrich ML Austin CA Meyer RG 《Biology of reproduction》2011,84(5):900-909
To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling. 相似文献
11.
12.
Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice 总被引:8,自引:0,他引:8
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Zhao M Shirley CR Yu YE Mohapatra B Zhang Y Unni E Deng JM Arango NA Terry NH Weil MM Russell LD Behringer RR Meistrich ML 《Molecular and cellular biology》2001,21(21):7243-7255
During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation. 相似文献
13.
Transcriptional and translational control of the message for transition protein 1, a major chromosomal protein of mammalian spermatids 总被引:6,自引:0,他引:6
The spermatid transition proteins comprise a set of basic chromosomal proteins that appear during the period when spermatids are undergoing nuclear elongation and condensation, about midway between the end of meiosis and the release of spermatozoa from the seminiferous tubule. The transition proteins replace the histones but are themselves subsequently replaced by protamines, and they are not found in sperm nuclei. We have used a cDNA clone for the smallest transition protein (TP1, 54 amino acids) to show that its message first appears postmeiotically in late round spermatids. Thus production of TP1 is an example of haploid gene expression. The message remains translationally inactive for some 3-4 days before translation occurs in early elongating spermatids. While translationally repressed, TP1 message is nonpolysomal and has a discrete size of about 590 bases, including a 140 residue poly(A) tail. In contrast, polysome-associated message is of heterogeneous size due to variability of poly(A) lengths. 相似文献
14.
Worawittayawong P Leigh C Weerachatyanukul W Manochantr S Sobhon P Breed WG Sretarugsa P 《Cell and tissue research》2008,334(1):135-144
Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species. 相似文献
15.
Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity. 相似文献
16.
Emmanual Unni Artur Mayerhofer Yun Zhang Yogendra M. Bhatnagar Lonnie D. Russell Marvin L. Meistrich 《Molecular reproduction and development》1995,42(2):210-219
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. 相似文献
17.
Nayernia K Drabent B Adham IM Möschner M Wolf S Meinhardt A Engel W 《Biology of reproduction》2003,69(6):1973-1978
In recent years, much knowledge about the functions of defined genes in spermatogenesis has been gained by making use of mouse transgenic and gene knockout models. Single null mutations in mouse genes encoding four male germ cell proteins, transition protein 2 (Tnp-2), proacrosin (Acr), histone H1.1 (H1.1), and histone H1t (H1t), have been generated and analyzed. Tnp-2 is believed to participate in the removal of the nuclear histones and initial condensation of the spermatid nucleus. Proacrosin is an acrosomal protease synthesized as a proenzyme and activated into acrosin during the acrosome reaction. The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues and germ cells during the S-phase of the cell cycle. The histone gene H1t is expressed exclusively in spermatocytes and may have a function in establishing an open chromatin structure for the replacement of histones by transition proteins and protamines. Male mutant mice lacking any of these proteins show no apparent defects in spermatogenesis or fertility. To examine the synergistic effects of these proteins in spermatogenesis and during fertilization, two lines of triple null mice (Tnp-2-/-/Acr-/-/H1.1-/- and Tnp-2-/-/Acr-/-/H1t-/-) were established. Both lines are fertile and show normal sperm parameters, which clearly demonstrate the functional redundancy of these proteins in male mouse fertility. However, sperm only deficient for Acr (Acr-/-) are able to compete significantly with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1.1-/- (70.7% vs. 29.3%) but not with sperm from triple knockout mice Tnp-2-/-/Acr-/-/H1t-/- (53.6% vs. 46.4%). These results are consistent with a model that suggests that some sperm proteins play a role during sperm competition. 相似文献
18.
19.
20.
Lena Arvalo Gina Esther Merges Simon Schneider Franka Enow Oben Isabelle Sophie Neumann Hubert Schorle 《PLoS genetics》2022,18(6)
Protamines are unique sperm-specific proteins that package and protect paternal chromatin until fertilization. A subset of mammalian species expresses two protamines (PRM1 and PRM2), while in others PRM1 is sufficient for sperm chromatin packaging. Alterations of the species-specific ratio between PRM1 and PRM2 are associated with infertility. Unlike PRM1, PRM2 is generated as a precursor protein consisting of a highly conserved N-terminal domain, termed cleaved PRM2 (cP2), which is consecutively trimmed off during chromatin condensation. The carboxyterminal part, called mature PRM2 (mP2), interacts with DNA and together with PRM1, mediates chromatin-hypercondensation. The removal of the cP2 domain is believed to be imperative for proper chromatin condensation, yet, the role of cP2 is not yet understood. We generated mice lacking the cP2 domain while the mP2 is still expressed. We show that the cP2 domain is indispensable for complete sperm chromatin protamination and male mouse fertility. cP2 deficient sperm show incomplete protamine incorporation and a severely altered protamine ratio, retention of transition proteins and aberrant retention of the testis specific histone variant H2A.L.2. During epididymal transit, cP2 deficient sperm seem to undergo ROS mediated degradation leading to complete DNA fragmentation. The cP2 domain therefore seems to be a key aspect in the complex crosstalk between histones, transition proteins and protamines during sperm chromatin condensation. Overall, we present the first step towards understanding the role of the cP2 domain in paternal chromatin packaging and open up avenues for further research. 相似文献