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Genes Feb 2019We studied and compared the nucleolar expression or nucleoli from specific bivalents in spermatocytes of the standard 2n=40, of Robertsonian (Rb) homozygotes 2n = 24...
We studied and compared the nucleolar expression or nucleoli from specific bivalents in spermatocytes of the standard 2n=40, of Robertsonian (Rb) homozygotes 2n = 24 and heterozygotes 2n = 32. We analyzed 200 nuclear microspreads of each specific nucleolar chromosome and spermatocyte karyotype, using FISH to identify specific nucleolar bivalents, immunofluorescence for both fibrillarin of the nucleolus and the synaptonemal complex of the bivalents, and DAPI for heterochromatin. There was nucleolar expression in all the chromosomal conditions studied. By specific nucleolar bivalent, the quantitative relative nucleolar expression was higher in the bivalent 12 than in its derivatives, lower in the bivalent 15 than in its derivatives and higher in the bivalent 16 than its Rb derivatives. In the interactions between non-homologous chromosomal domains, the nucleolar bivalents were preferentially associated through pericentromeric heterochromatin with other bivalents of similar morphology and sometimes with other nucleolar bivalents. We suggest that the nucleolar expression in Rb nucleolar chromosomes is modified as a consequence of different localization of ribosomal genes (NOR) in the Rb chromosomes, its proximity to heterochromatin and its associations with chromosomes of the same morphology.
Topics: Animals; Cell Nucleolus; Chromosomes; Homozygote; Male; Mice; Spermatocytes; Translocation, Genetic
PubMed: 30736350
DOI: 10.3390/genes10020120 -
Pathology Oncology Research : POR Jan 2017
Topics: Brain; Brain Neoplasms; Humans; Male; Neoplasm Metastasis; Neoplasms, Germ Cell and Embryonal; Seminoma; Spermatocytes; Testicular Neoplasms
PubMed: 27815811
DOI: 10.1007/s12253-016-0142-x -
Epigenetics Apr 2016H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the...
H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.
Topics: Amino Acid Sequence; Animals; Cell Nucleolus; Chromatin; DNA, Ribosomal; Gene Expression Regulation, Developmental; Germ Cells; Histones; Male; Mice; Mouse Embryonic Stem Cells; Sequence Homology, Amino Acid; Spermatids; Spermatocytes; Spermatogenesis; Testis; Transcription, Genetic
PubMed: 27018843
DOI: 10.1080/15592294.2016.1159369 -
Development (Cambridge, England) Jul 2016The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from...
The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. Here we describe the identification and initial characterization of dany, a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC and tTAFs, the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, we propose that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization.
Topics: Amino Acid Sequence; Animals; Cell Cycle Checkpoints; Cell Nucleus; Cell Nucleus Shape; Chromatin; Chromosomes; Drosophila Proteins; Drosophila melanogaster; Fertility; Gene Expression Regulation; Genes, Insect; Male; Meiosis; Mutation; Nuclear Envelope; Nuclear Proteins; Spermatocytes; Spermatogenesis; Subcellular Fractions; Testis; Transcription, Genetic
PubMed: 27436041
DOI: 10.1242/dev.134759 -
Asian Journal of Andrology 2017Male germ cell development is a well-defined process occurring in numerous seminiferous tubules of the testis. Uncovering testicular novel genes related to intrinsic...
Male germ cell development is a well-defined process occurring in numerous seminiferous tubules of the testis. Uncovering testicular novel genes related to intrinsic regulation of spermatogenesis is essential for the understanding of spermatogenesis. In the present study, we investigated mouse Mageg2, which belongs to a group of melanoma-associated antigens (MAGEs). Mageg2 is transcribed in the testis specifically, and its expression level is increased at the pachytene spermatocyte stage, indicating that Mageg2 is expressed predominantly in germ cells. We generated an antibody against mouse MAGEG2 for further characterization at the protein level. Immunoblot analysis suggested that MAGEG2 has specific testicular expression and the expression primarily occurred in pachytene spermatocytes. Proteomic analyses demonstrated that mouse MAGEG2 binded to testicular germ cell-specific serine/threonine-protein kinase 31 (STK31) and heat shock protein 9 (HSPA9). Direct binding with both interaction partners was confirmed by co-immunoprecipitation. We found that STK31 and HSPA9 bind MAGEG2 directly but not with each other. Interestingly, MAGEG2 reduced the kinase activity of STK31. Our study suggests that mouse MAGEG2 has at least two functions, including chaperone activity related to HSPA9 and regulation of pachytene spermatocyte-specific kinase, STK31. Altogether, our results provide the first information about MAGEG2 at the transcript and protein levels and suggest its potential molecular functions.
Topics: Animals; Heat-Shock Proteins; Male; Mice; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Proteins; Spermatocytes; Spermatogenesis; Testis
PubMed: 27852984
DOI: 10.4103/1008-682X.192033 -
BMC Bioinformatics Feb 2013Mammalian germ cells undergo meiosis to produce sperm or eggs, haploid cells that are primed to meet and propagate life. Meiosis is initiated by retinoic acid and...
BACKGROUND
Mammalian germ cells undergo meiosis to produce sperm or eggs, haploid cells that are primed to meet and propagate life. Meiosis is initiated by retinoic acid and meiotic prophase is the first and most complex stage of meiosis when homologous chromosomes pair to exchange genetic information. Errors in meiosis can lead to infertility and birth defects. However, despite the importance of this process, germ cell-specific gene expression patterns during meiosis remain undefined due to difficulty in obtaining pure germ cell samples, especially in females, where prophase occurs in the embryonic ovary. Indeed, mixed signals from both germ cells and somatic cells complicate gonadal transcriptome studies.
RESULTS
We developed a machine-learning method for identifying germ cell-specific patterns of gene expression in microarray data from mammalian gonads, specifically during meiotic initiation and prophase. At 10% recall, the method detected spermatocyte genes and oocyte genes with 90% and 94% precision, respectively. Our method outperformed gonadal expression levels and gonadal expression correlations in predicting germ cell-specific expression. Top-predicted spermatocyte and oocyte genes were both preferentially localized to the X chromosome and significantly enriched for essential genes. Also identified were transcription factors and microRNAs that might regulate germ cell-specific expression. Finally, we experimentally validated Rps6ka3, a top-predicted X-linked spermatocyte gene. Protein localization studies in the mouse testis revealed germ cell-specific expression of RPS6KA3, mainly detected in the cytoplasm of spermatogonia and prophase spermatocytes.
CONCLUSIONS
We have demonstrated that, through the use of machine-learning methods, it is possible to detect germ cell-specific expression from gonadal microarray data. Results from this study improve our understanding of the transition from germ cells to meiocytes in the mammalian gonad. Further, this approach is applicable to other tissues for which isolating cell populations remains difficult.
Topics: Animals; Artificial Intelligence; Female; Gene Expression Profiling; Gene Expression Regulation; Germ Cells; Male; Meiotic Prophase I; Mice; MicroRNAs; Oocytes; Spermatocytes; Support Vector Machine; Transcription Factors
PubMed: 23445120
DOI: 10.1186/1471-2105-14-72 -
Oncotarget Jan 2017CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis...
CD147 null mutant male mice are infertile with arrested spermatogenesis and increased apoptotic germ cells. Our previous studies have shown that CD147 prevents apoptosis in mouse spermatocytes but not spermatogonia. However, the underlying mechanism remains elusive. In the present study, we aim to determine the CD147-regulated apoptotic pathway in mouse spermatocytes. Our results showed that immunodepletion of CD147 triggered apoptosis through extrinsic apoptotic pathway in mouse testis and spermatocyte cell line (GC-2 cells), accompanied by activation of non-canonical NFκB signaling and suppression of canonical NFκB signaling. Furthermore, CD147 was found to interact with TRAF2, a factor known to regulate NFκB and extrinsic apoptotic signaling, and interfering CD147 led to the decrease of TRAF2. Consistently, depletion of CD147 by CRISPR/Cas9 technique in GC-2 cells down-regulated TRAF2 and resulted in cell death with suppressed canonical NFκB and activated non-canonical NFκB signaling. On the contrary, interfering of CD147 had no effect on NFκB signaling pathways as well as TRAF2 protein level in mouse spermatogonia cell line (GC-1 cells). Taken together, these results suggested that CD147 plays a key role in reducing extrinsic apoptosis in spermatocytes, but not spermatogonia, through modulating NFκB signaling pathway.
Topics: Animals; Apoptosis; Basigin; Cell Line; Cell Survival; Gene Knockout Techniques; Gene Targeting; Male; Mice; NF-kappa B; Signal Transduction; Spermatocytes; TNF Receptor-Associated Factor 2
PubMed: 27902973
DOI: 10.18632/oncotarget.13624 -
Journal of Cellular and Molecular... Oct 2018Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2...
Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30- and 60-day-old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double-strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co-immunoprecipitated with a testis-enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC-2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC-2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.
Topics: Adaptor Proteins, Signal Transducing; Animals; Carrier Proteins; DNA Breaks, Double-Stranded; DNA Repair; Gene Expression Regulation, Developmental; Male; Meiosis; Mice; Spermatocytes; Spermatogenesis; Spermatozoa; Testicular Hormones
PubMed: 30055035
DOI: 10.1111/jcmm.13751 -
Developmental Cell Jan 2020GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein...
GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.
Topics: Animals; Caenorhabditis elegans; DNA Damage; DNA Repair; DNA Replication; DNA Topoisomerases, Type II; DNA-Binding Proteins; Genome; Genomic Instability; Germ Cells; Male; Mice; Mice, Inbred C57BL; Mitosis; Mutation; Nuclear Proteins; Spermatocytes; Spermatogenesis
PubMed: 31839538
DOI: 10.1016/j.devcel.2019.11.006 -
Cellular and Molecular Life Sciences :... Aug 2022Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation,...
Meiosis, a highly conserved process in organisms from fungi to mammals, is subjected to protein phosphorylation regulation. Due to the low abundance of phosphorylation, there is a lack of systemic characterization of phosphorylation regulation of meiosis in mammals. Using the phosphoproteomic approach, we profiled large-scale phosphoproteome of purified primary spermatocytes undergoing meiosis I, and identified 14,660 phosphorylation sites in 4419 phosphoproteins. Kinase-substrate phosphorylation network analysis followed by in vitro meiosis study showed that CDK9 was essential for meiosis progression to metaphase I and had enriched substrate phosphorylation sites in proteins involved in meiotic cell cycle. In addition, histones and epigenetic factors were found to be widely phosphorylated. Among those, HASPIN was found to be essential for male fertility. Haspin knockout led to misalignment of chromosomes, apoptosis of metaphase spermatocytes and a decreased number of sperm by deregulation of H3T3ph, chromosomal passenger complex (CPC) and spindle assembly checkpoint (SAC). The complicated protein phosphorylation and its important regulatory functions in meiosis indicated that in-depth studies of phosphorylation-mediated signaling could help us elucidate the mechanisms of meiosis.
Topics: Animals; Histones; Male; Mammals; Meiosis; Metaphase; Mice; Phosphorylation; Semen; Spermatocytes
PubMed: 35930080
DOI: 10.1007/s00018-022-04507-8