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Reproductive Biology Mar 2022D-Aspartate (D-Asp) and its methylated form N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones. d-Asp also...
D-Aspartate (D-Asp) and its methylated form N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones. d-Asp also induces spermatogonia proliferation directly by activating the ERK/Aurora B pathway. In the present study, a mouse spermatocyte-derived cell line (GC-2) which represents a stage between preleptotene spermatocyte and round spermatids was exposed to 200 μM d-Asp or 50 μM NMDA for 30 min, 2 h, and 4 h to explore the influence of these amino acids on cell proliferation and mitochondrial activities occurring during this process. By Western blotting analyses, the expressions of AMPAR (GluA1-GluA2/3 subunits), cell proliferation as well as mitochondria functionality markers were determined at different incubation times. The results revealed that d-Asp or NMDA stimulate proliferation and meiosis in the GC-2 cells via the AMPAR/ERK/Akt pathway, which led to increased levels of the PCNA, p-H3, and SYCP3 proteins. The effects of d-Asp and NMDA on the mitochondrial functionality of the GC-2 cells strongly suggested an active role of these amino acids in germ cell maturation. In both d-Asp- and NMDA-treated GC-2 cells mitochondrial biogenesis as well as mitochondrial fusion are increased while mitochondria fission is inhibited. Finally, the findings showed that NMDA significantly increased the expressions of the CII, CIII, CIV, and CV complexes of oxidative phosphorylation system (OXPHOS), whereas d-Asp induced a significant increase in the expressions only of the CIV and CV complexes. The present study provides novel insights into the mechanisms underlying the role of d-Asp and NMDA in promoting spermatogenesis.
Topics: Animals; D-Aspartic Acid; Male; Mice; N-Methylaspartate; Spermatocytes; Spermatogenesis; Spermatogonia
PubMed: 35032869
DOI: 10.1016/j.repbio.2021.100601 -
Toxicology and Applied Pharmacology Sep 19952-Methoxyethanol (ME) produces testicular lesions characterized by pachytene spermatocyte degeneration in rats and guinea pigs which differ in onset, severity, and...
2-Methoxyethanol (ME) produces testicular lesions characterized by pachytene spermatocyte degeneration in rats and guinea pigs which differ in onset, severity, and morphological characteristics. In the rat, degenerating spermatocytes appear necrotic at 24 hr, while in the guinea pig they appear apoptotic 96 hr after the start of three daily doses. To further examine if the spermatocyte degeneration in both species represented necrosis or apoptosis, the extent and nature of nuclear DNA fragmentation after ME exposure were assessed both visually using an in situ nucleotide 3' end-labeling (ISEL) procedure and by DNA gel electrophoresis. Testes from rats given a single oral dose of ME (200 mg/kg) showed the expected pachytene spermatocyte degeneration 24 hr after dosing, with the nuclear chromatin degradation typical of necrosis. In contrast, testes from guinea pigs given daily oral doses of ME (200 mg/kg) showed spermatocyte degeneration at only 96 hr after the start of dosing, with marked peripheral nuclear chromatin condensation characteristic of apoptosis. Coincident with the appearance of morphologic changes, degenerating spermatocytes in both species contained fragmented DNA as revealed by the ISEL procedure. The pattern of DNA fragmentation on agarose gels in both species consisted of ordered multiples or "ladders" of approximately 200 base pairs, a hallmark of apoptosis, with their appearance coincident with the time course of morphologic spermatocyte degeneration and ISEL staining. Preliminary data reveal the appearance of divalent metal cation-dependent endonuclease activity at pH 7.0 in ME-treated immature (24-day-old) rat testis that produces a similar pattern of DNA fragmentation and which appears to be distinct from activity associated with the spontaneous germ cell degeneration observed in testes of this age. In summary, in vivo ME exposure induces spermatocyte apoptosis in both the rat and guinea pig despite differing morphological classifications and time of onset of cell death. Future studies will focus on further characterization of the testicular endonuclease in the rat and the potential role of increased intracellular Ca2+ as a "triggering" stimulus in ME-induced spermatocyte apoptosis.
Topics: Animals; Apoptosis; DNA; DNA Damage; Endonucleases; Ethylene Glycols; Guinea Pigs; Immunosuppressive Agents; Male; Rats; Rats, Inbred F344; Species Specificity; Spermatocytes; Testis
PubMed: 7676444
DOI: 10.1006/taap.1995.1173 -
Molecular Reproduction and Development Aug 2006Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by...
Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.
Topics: Animals; Apoptosis; Chromatin; DNA Breaks, Double-Stranded; DNA Damage; Germ Cells; Histones; Male; Meiosis; Mice; Mice, Inbred C57BL; Rad51 Recombinase; Spermatocytes
PubMed: 16700071
DOI: 10.1002/mrd.20508 -
Sheng Li Ke Xue Jin Zhan [Progress in... Apr 2008
Review
Topics: Animals; Cyclin-Dependent Kinase 2; Female; Humans; Male; Meiosis; Mice; Oocytes; Spermatocytes
PubMed: 18664186
DOI: No ID Found -
Transgenic Research Jun 2004We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene... (Comparative Study)
Comparative Study
We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V-VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.
Topics: Animals; Crosses, Genetic; DNA-Binding Proteins; Galactosides; Gene Expression; Histological Techniques; Indoles; Integrases; Male; Mice; Mice, Transgenic; Nuclear Proteins; Promoter Regions, Genetic; Spermatocytes; Testis
PubMed: 15359605
DOI: 10.1023/b:trag.0000034716.73957.f7 -
ELife May 2021The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by...
The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.
Topics: Animals; Cell Differentiation; Gene Expression Regulation, Developmental; Giant Cells; Male; Mice; Mice, Inbred C57BL; Spermatocytes; Spermatogenesis; Spermatogonia; Vesicular Transport Proteins
PubMed: 33973520
DOI: 10.7554/eLife.59759 -
Molecular Human Reproduction Nov 2019The purification of individual male germ cell populations is integral for the molecular and biochemical characterisation of specific spermatogenic phases. Although a...
The purification of individual male germ cell populations is integral for the molecular and biochemical characterisation of specific spermatogenic phases. Although a number of more contemporary techniques have been developed, velocity sedimentation using the STAPUT method remains as a gold standard for this purpose. The gentle nature of the technique, wherein germ cell subpopulations are separated by sedimentation at unit gravity, results in the isolation of viable and high-purity cells. We provide an updated and simplified step-by-step version of the STAPUT protocol for the purification of mouse male germ cells. As per the original method, the protocol described herein allows for the purification of mouse spermatocyte and round spermatids, however it also allows for successful purification of elongating, and elongated spermatid populations, and is optimised for the preservation of cellular ultrastructure. This method yields sufficient numbers of high-purity cells from one adult mouse for RNA or protein extraction or for immunolocalisation studies.
Topics: Animals; Centrifugation, Density Gradient; Fluorescent Antibody Technique; Male; Mice; Spermatids; Spermatocytes; Spermatogenesis
PubMed: 31642475
DOI: 10.1093/molehr/gaz056 -
Cell Biology International Sep 2018The aim of this study was to investigate the underlying mechanisms of hypoxia-induced apoptosis of GC-2spd (GC-2) cells. The GC-2 cells were treated with or without...
The aim of this study was to investigate the underlying mechanisms of hypoxia-induced apoptosis of GC-2spd (GC-2) cells. The GC-2 cells were treated with or without hypoxia for 12, 24, 48, and 72 h. Apoptosis of GC-2 cells was detected using TUNEL and flow cytometry. Fluorescence microscopic was used to observe the autophagy of GC-2 cells. Hypoxia-inducible factor-1alpha (HIF-1α), apoptosis-related protein and marker protein of autophagy levels were measured by Western blotting. Hypoxia induced apoptosis and autophagy of GC-2 cells, and increased expression of HIF-1α, LC3-II, Beclin-1, and pro-apoptotic protein, but reduced p62 and anti-apoptotic protein level. Meanwhile, hypoxia-induced HIF-1α mediated expression of apoptosis and autophagy-related protein in GC-2 cell. Furthermore, autophagy regulated hypoxia-induced apoptosis of GC-2 cell. Our data suggest that hypoxia induces apoptosis of GC-2 cell by activation of autophagy involving activation of the apoptosis signaling pathway under the hypoxic condition.
Topics: Animals; Apoptosis; Autophagy; Beclin-1; Blotting, Western; Cell Hypoxia; Cell Line; Cell Line, Tumor; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Microtubule-Associated Proteins; Signal Transduction; Spermatocytes
PubMed: 29660204
DOI: 10.1002/cbin.10971 -
Cytogenetics and Cell Genetics 1994
Review
Topics: Animals; Cell Survival; Humans; Male; Recombination, Genetic; Spermatocytes; Spermatogenesis; X Chromosome; Y Chromosome
PubMed: 8258305
DOI: 10.1159/000133648 -
Proceedings of the National Academy of... Feb 2021Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a...
Meiosis is a specialized cell division that creates haploid germ cells from diploid progenitors. Through differential RNA expression analyses, we previously identified a number of mouse genes that were dramatically elevated in spermatocytes, relative to their very low expression in spermatogonia and somatic organs. Here, we investigated in detail one of these genes, and independently conclude that it encodes a male germline-specific protein, in agreement with a recent report. We demonstrated that it is essential for pachynema progression in spermatocytes and named it male pachynema-specific (MAPS) protein. Mice lacking ( ) suffered from pachytene arrest and spermatocyte death, leading to male infertility, whereas female fertility was not affected. Interestingly, pubertal spermatocytes were arrested at early pachytene stage, accompanied by defects in DNA double-strand break (DSB) repair, crossover formation, and XY body formation. In contrast, adult spermatocytes only exhibited partially defective crossover but nonetheless were delayed or failed in progression from early to mid- and late pachytene stage, resulting in cell death. Furthermore, we report a significant transcriptional dysregulation in autosomes and XY chromosomes in both pubertal and adult pachytene spermatocytes, including failed meiotic sex chromosome inactivation (MSCI). Further experiments revealed that MAPS overexpression in vitro dramatically decreased the ubiquitination levels of cellular proteins. Conversely, in pachytene cells, protein ubiquitination was dramatically increased, likely contributing to the large-scale disruption in gene expression in pachytene cells. Thus, MAPS is a protein essential for pachynema progression in male mice, possibly in mammals in general.
Topics: Animals; Chromosome Pairing; DNA Repair; Female; Infertility, Male; Male; Meiosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Nuclear Proteins; Pachytene Stage; Sex Chromosomes; Spermatocytes; Spermatogenesis
PubMed: 33602822
DOI: 10.1073/pnas.2025421118