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Environmental Toxicology and... Dec 2016As a widespread environmental contaminant, bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) has been implicated in male reproductive function injury. Previous studies...
As a widespread environmental contaminant, bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) has been implicated in male reproductive function injury. Previous studies have investigated the mechanisms of DNA damage and oxidative stress caused by BPA; however, little is known regarding its impact on DNA methylation. In this paper, we assessed the adverse effects of BPA on mouse spermatocytes and investigated a potential role of DNA methylation. We demonstrated that BPA exposure inhibited cell proliferation, reduced the DNA replication capacity, and triggered apoptosis in GC-2 cells. In addition, the global DNA methylation levels increased, and the relative expression levels of DNA methyltransferases (DNMTs) varied following BPA exposure. Thousands of distinct methylated sites were screened using microarray analysis. The expressions of myosin-binding protein H (mybph) and protein kinase C δ (prkcd) were verified to be regulated by DNA methylation. These findings indicate that BPA had toxicity in spermatocytes, and DNA methylation may play a vital role in the regulation of BPA-triggered spermatocyte toxicity.
Topics: Animals; Apoptosis; Benzhydryl Compounds; Cell Culture Techniques; Cell Line; Cell Proliferation; CpG Islands; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Replication; Environmental Pollutants; Male; Mice; Phenols; Spermatocytes
PubMed: 27855348
DOI: 10.1016/j.etap.2016.11.003 -
Molecular & General Genetics : MGG Nov 1996We have identified a novel Drosophila gene, degenerative spermatocyte (des), that may be required for the initiation of meiosis in spermatogenesis. In des mutant testes,...
We have identified a novel Drosophila gene, degenerative spermatocyte (des), that may be required for the initiation of meiosis in spermatogenesis. In des mutant testes, the primary spermatocytes become mature in size but degenerate without initiating meiotic chromosome condensation in their nuclei. We have mapped the locus of the des gene at 26A on the left arm of the second chromosome, and identified two related, differentially spliced transcripts (des-1, des-2) from the locus. The genomic DNA fragment encoding both transcripts is able to rescue the des phenotype when introduced into mutant flies. The expression of the des-1 transcript is significantly suppressed in the des mutant testes, whereas expression of the des-2 transcript is undetectable in both wild-type and mutant testes, indicating that the des-1 transcript encodes the des function in spermatogenesis. The des-1 transcript is selectively expressed in primary spermatocytes during normal spermatogenesis. The nucleotide sequence of the des-1 transcript predicts that it encodes a novel transmembrane protein. These results raise the possibility that the des gene product may be required for interactions between primary spermatocytes and surrounding somatic cells.
Topics: Amino Acid Sequence; Animals; Base Sequence; Chromosome Mapping; DNA, Complementary; Drosophila; Drosophila Proteins; Female; Gene Expression; Genes, Insect; Heterozygote; Male; Meiosis; Membrane Proteins; Molecular Sequence Data; Mutation; Phenotype; RNA, Messenger; Sequence Analysis, DNA; Spermatocytes; Spermatogenesis; Testis
PubMed: 9003299
DOI: 10.1007/s004380050308 -
Molecular Medicine Reports Jan 2019The aim of the present study was to investigate the underlying mechanisms of hypoxia‑induced microRNA (miR)‑210 effects on mouse GC‑2spd (GC‑2) cells. GC‑2...
The aim of the present study was to investigate the underlying mechanisms of hypoxia‑induced microRNA (miR)‑210 effects on mouse GC‑2spd (GC‑2) cells. GC‑2 cells were subjected to hypoxia or normoxia for 12, 24, 48 and 72 h. Apoptosis of GC‑2 cells was detected using terminal deoxynucleotidyl‑transferase‑meditated dUTP nick end labeling and flow cytometry. Reverse transcription‑quantitative polymerase chain reaction was performed to analyze the expression of miR‑210. Hypoxia‑inducible factor‑1α (HIF‑1α), caspase‑3, B‑cell lymphoma 2, apoptosis regulator BAX and Kruppel‑like factor 7 (KLF7) protein expression levels were detected by western blotting. Luciferase reporter gene assays were used to assess the targeting effects of miR‑210 on KLF7. Hypoxia induced GC‑2 cell apoptosis and increased the expression of HIF‑1α and pro‑apoptotic proteins; however, decreased anti‑apoptotic protein expression levels. Furthermore, hypoxia resulted in the upregulation of miR‑210 in GC‑2 cells. HIF‑1α and miR‑210 were involved in the apoptosis of GC‑2 cells by mediating the expression of apoptosis‑associated proteins. Furthermore, KLF7 was directly targeted by miR‑210 to influence the apoptosis of GC‑2 cells subjected to hypoxia. The results suggested that hypoxia‑induced miR‑210 stimulated the activation of the apoptosis signaling pathway and contributed to the apoptosis of GC‑2 cells by targeting KLF7.
Topics: Animals; Apoptosis; Cells, Cultured; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kruppel-Like Transcription Factors; Male; Mice; MicroRNAs; Signal Transduction; Spermatocytes
PubMed: 30431115
DOI: 10.3892/mmr.2018.9644 -
Basic & Clinical Pharmacology &... Feb 2020Methotrexate (MTX) is widely used to treat cancers and systemic autoimmune diseases. However, it is severely toxic to healthy cells, especially those of the reproductive...
Methotrexate (MTX) is widely used to treat cancers and systemic autoimmune diseases. However, it is severely toxic to healthy cells, especially those of the reproductive system, and therefore poses a great risk to patient fertility. In addition, the underlying mechanism of MTX-induced reproductive toxicity has not yet been fully elucidated. Here, a spermatocyte cell line (GC2) was used as an in vitro model system to determine whether MTX induces autophagy and apoptosis, and to elucidate the role of reactive oxygen species (ROS) and Ca in these two processes. Treatment with MTX resulted in a dramatic decrease in cell viability, inhibition of cell proliferation, collapse of the mitochondrial membrane potential and activation of caspase 3, suggesting that MTX induced apoptosis. Moreover, MTX activated autophagy, as indicated by conversion of LC3-I to LC3-II (microtubule-associated protein 1 light chain 3) and an increase in the number of LC3 puncta. Furthermore, MTX triggered ROS overproduction, rather than a Ca burst. Intriguingly, eliminating excess ROS significantly alleviated MTX-induced apoptosis and autophagy. In addition, inhibiting autophagy significantly reversed apoptosis and promoted cell survival, indicating that autophagy aggravated MTX-induced apoptosis in GC2 cells. Taken together, these results suggest that ROS signalling, not Ca , is critical in mediating MTX-induced autophagy and apoptosis and autophagy serves as a promoted partner of apoptosis to deteriorate MTX-induced cytotoxicity in GC2 cells. The findings from this study provide new perspectives for evaluating the reproductive toxicity of MTX.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Autophagy; Calcium; Cell Line; Cell Proliferation; Cell Survival; Male; Membrane Potential, Mitochondrial; Methotrexate; Mice; Reactive Oxygen Species; Signal Transduction; Spermatocytes
PubMed: 31420979
DOI: 10.1111/bcpt.13306 -
Advances in Anatomy, Embryology, and... 2017Heat shock transcription factors (HSFs), as regulators of heat shock proteins (HSPs) expression, are well known for their cytoprotective functions during cellular... (Review)
Review
Heat shock transcription factors (HSFs), as regulators of heat shock proteins (HSPs) expression, are well known for their cytoprotective functions during cellular stress. They also play important yet less recognized roles in gametogenesis. All HSF family members are expressed during mammalian spermatogenesis, mainly in spermatocytes and round spermatids which are characterized by extensive chromatin remodeling. Different HSFs could cooperate to maintain proper spermatogenesis. Cooperation of HSF1 and HSF2 is especially well established since their double knockout results in meiosis arrest, spermatocyte apoptosis, and male infertility. Both factors are also involved in the repackaging of the DNA during spermatid differentiation. They can form heterotrimers regulating the basal level of transcription of target genes. Moreover, HSF1/HSF2 interactions are lost in elevated temperatures which can impair the transcription of genes essential for spermatogenesis. In most mammals, spermatogenesis occurs a few degrees below the body temperature and spermatogenic cells are extremely heat-sensitive. Pro-survival pathways are not induced by heat stress (e.g., cryptorchidism) in meiotic and postmeiotic cells. Instead, male germ cells are actively eliminated by apoptosis, which prevents transition of the potentially damaged genetic material to the next generation. Such a response depends on the transcriptional activity of HSF1 which in contrary to most somatic cells, acts as a proapoptotic factor in spermatogenic cells. HSF1 activation could be the main trigger of impaired spermatogenesis related not only to elevated temperature but also to other stress conditions; therefore, HSF1 has been proposed to be the quality control factor in male germ cells.
Topics: Animals; Heat-Shock Proteins; Heat-Shock Response; Humans; Infertility, Male; Male; Mammals; Spermatocytes; Spermatogenesis
PubMed: 28389750
DOI: 10.1007/978-3-319-51409-3_3 -
Genetics Aug 2017A key aspect of germ cell development is to establish germline sexual identity and initiate a sex-specific developmental program to promote spermatogenesis or oogenesis....
A key aspect of germ cell development is to establish germline sexual identity and initiate a sex-specific developmental program to promote spermatogenesis or oogenesis. Previously, we have identified the histone reader Plant Homeodomain Finger 7 (PHF7) as an important regulator of male germline identity. To understand how PHF7 directs sexual differentiation of the male germline, we investigated the downstream targets of PHF7 by combining transcriptome analyses, which reveal genes regulated by , with genomic profiling of histone H3K4me2, the chromatin mark that is bound by PHF7. Through these genomic experiments, we identify a novel spermatocyte factor Receptor Accessory Protein Like 1 (REEPL1) that can promote spermatogenesis and whose expression is kept off by PHF7 in the spermatogonial stage. Loss of significantly rescues the spermatogenesis defects in mutants, indicating that regulation of is an essential aspect of PHF7 function. Further, increasing REEPL1 expression facilitates spermatogenic differentiation. These results indicate that PHF7 controls spermatogenesis by regulating the expression patterns of important male germline genes.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Histones; Homeodomain Proteins; Male; Spermatocytes; Spermatogenesis
PubMed: 28588035
DOI: 10.1534/genetics.117.199935 -
Differentiation; Research in Biological... 2020Long noncoding RNAs (lncRNAs) have been shown to execute key roles in spermatogenesis. However, little is known about how lncRNAs gene expression is itself regulated in...
Long noncoding RNAs (lncRNAs) have been shown to execute key roles in spermatogenesis. However, little is known about how lncRNAs gene expression is itself regulated in the germ cells of testis. We previously demonstrated that high expression of lncRNA-Gm2044 exists in spermatocytes and can regulate male germ cell proliferation. Here, the transcriptional regulation of lnRNA-Gm2044 expression in spermatocytes and the downstream signaling were further explored. A bioinformatics assessment predicted two potential binding-sites for the spermatocyte-specific transcription factor A-MYB in the promoter region of lncRNA-Gm2044. Our results proved that the transcription factor A-MYB promotes the expression of lncRNA-Gm2044 in mouse spermatocyte-derived GC-2spd(ts) cells. ChIP and luciferase assays verified that A-MYB mainly binds to the distal promoter region (-819 bp relative to the transcription start site) of lncRNA-Gm2044 and regulates lncRNA-Gm2044 expression through the -819 bp binding-site. In addition, we confirmed that lncRNA-Gm2044 functions as a miR-335-3p sponge to enhance the levels of miR-335-3p's direct target protein, Sycp1. Furthermore, A-MYB can up-regulate Sycp1 expression and down-regulate GC-2spd(ts) cell proliferation by activating its target, lncRNA-Gm2044. Overexpression of lncRNA-Gm2044 or knockdown of miR-335-3p can, at least partially, rescue the effects of A-MYB on Sycp1 expression and GC-2spd(ts) cell proliferation.Taken together, our results provide new information on the mechanistic roles of lncRNA-miRNA in transcription factor A-MYB regulation of spermatocyte function.
Topics: Animals; Cell Differentiation; DNA-Binding Proteins; Gene Expression Regulation, Developmental; Male; Mice; MicroRNAs; Proto-Oncogene Proteins c-myb; RNA, Long Noncoding; Spermatocytes; Trans-Activators; Transcription, Genetic; Transcriptional Activation
PubMed: 32585553
DOI: 10.1016/j.diff.2020.05.004 -
Molecular Biology of the Cell May 2010The scaffolding protein anillin is required for completion of cytokinesis. Anillin binds filamentous (F) actin, nonmuscle myosin II, and septins and in cell culture...
The scaffolding protein anillin is required for completion of cytokinesis. Anillin binds filamentous (F) actin, nonmuscle myosin II, and septins and in cell culture models has been shown to restrict actomyosin contractility to the cleavage furrow. Whether anillin also serves this function during the incomplete cytokinesis that occurs in developing germ cells has remained unclear. Here, we show that anillin is required for cytokinesis in dividing Drosophila melanogaster spermatocytes and that anillin, septins, and myosin II stably associate with the cleavage furrow in wild-type cells. Anillin is necessary for recruitment of septins to the cleavage furrow and for maintenance of F-actin and myosin II at the equator in late stages of cytokinesis. Remarkably, expression of DE-cadherin suppresses the cytokinesis defect of anillin-depleted spermatocytes. DE-cadherin recruits beta-catenin (armadillo) and alpha-catenin to the cleavage furrow and stabilizes F-actin at the equator. Similarly, E-cadherin expression suppresses the cytokinesis defect caused by anillin knockdown in mouse L-fibroblast cells. Our results show that the anillin-septin and cadherin-catenin complexes can serve as alternative cassettes to promote tight physical coupling of F-actin and myosin II to the cleavage furrow and successful completion of cytokinesis.
Topics: Actins; Actomyosin; Animals; Animals, Genetically Modified; Cadherins; Catenins; Contractile Proteins; Cytokinesis; Drosophila Proteins; Drosophila melanogaster; Fluorescence Recovery After Photobleaching; GTP Phosphohydrolases; L Cells; Luminescent Proteins; Male; Mice; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Myosin Type II; Protein Binding; RNA Interference; Septins; Spermatocytes; Transfection
PubMed: 20237160
DOI: 10.1091/mbc.e09-08-0714 -
Cytoskeleton (Hoboken, N.J.) Nov 2012Cytokinesis separates the genomic material and organelles of a dividing cell equitably into two physically distinct daughter cells at the end of cell division. This... (Review)
Review
Cytokinesis separates the genomic material and organelles of a dividing cell equitably into two physically distinct daughter cells at the end of cell division. This highly choreographed process involves coordinated reorganization and regulated action of the actin and microtubule cytoskeletal systems, an assortment of motor proteins, and membrane trafficking components. Due to their large size, the ease with which exquisite cytological analysis may be performed on them, and the availability of numerous mutants and other genetic tools, Drosophila spermatocytes have provided an excellent system for exploring the mechanistic basis for the temporally programmed and precise spatially localized events of cytokinesis. Mutants defective in male meiotic cytokinesis can be easily identified in forward genetic screens by the production of multinucleate spermatids. In addition, the weak spindle assembly checkpoint in spermatocytes, which causes only a small delay of anaphase onset in the presence of unattached chromosomes, allows investigation of whether gene products required for spindle assembly and chromosome segregation are also involved in cytokinesis. Perhaps due to the large size of spermatocytes and the requirement for two rapid-fire rounds of division without intervening S or growth phases during meiosis, male meiotic mutants have also revealed much about molecular mechanisms underlying new membrane addition during cytokinesis. Finally, cell type-specific differences in the events that set up and complete cytokinesis are emerging from comparison of spermatocytes with cells undergoing mitosis either elsewhere in the organism or in tissue culture.
Topics: Animals; Cell Cycle Checkpoints; Cytokinesis; Drosophila melanogaster; Male; Meiosis; Mutation; Spermatocytes; Spindle Apparatus
PubMed: 22927345
DOI: 10.1002/cm.21063 -
Genes & Genetic Systems Jun 2022Meiosis is a crucial process for spermatogenesis and oogenesis. Initiation of meiosis coincides with spermatocyte differentiation and is followed by meiotic prophase, a... (Review)
Review
Meiosis is a crucial process for spermatogenesis and oogenesis. Initiation of meiosis coincides with spermatocyte differentiation and is followed by meiotic prophase, a prolonged G2 phase that ensures the completion of numerous meiosis-specific chromosome events. During meiotic prophase, chromosomes are organized into axis-loop structures, which underlie meiosis-specific events such as meiotic recombination and homolog synapsis. In spermatocytes, meiotic prophase is accompanied by robust alterations of gene expression programs and chromatin status for subsequent sperm production. The mechanisms regulating meiotic initiation and subsequent meiotic prophase programs are enigmatic. Recently, we discovered MEIOSIN (Meiosis initiator), a DNA-binding protein that directs the switch from mitosis to meiosis. This review mainly focuses on how MEIOSIN is involved in meiotic initiation and the meiotic prophase program during spermatogenesis. Further, we discuss the downstream genes activated by MEIOSIN, which are crucial for meiotic prophase-specific events, from the viewpoint of chromosome dynamics and the gene expression program.
Topics: Chromosome Pairing; Humans; Male; Meiosis; Mitosis; Spermatocytes; Spermatogenesis
PubMed: 34955498
DOI: 10.1266/ggs.21-00054