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Nan Fang Yi Ke Da Xue Xue Bao = Journal... May 2022To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (HO)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the...
OBJECTIVE
To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (HO)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.
METHODS
GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to HO (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.
RESULTS
Exposure to HO significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells ( < 0.05 or 0.01). Treatment of the cells prior to HO exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate ( < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 ( < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells ( < 0.05).
CONCLUSION
Hyperoside protects GC-2 cells against HO- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.
Topics: Animals; Antioxidants; Heme Oxygenase-1; Hydrogen Peroxide; Kelch-Like ECH-Associated Protein 1; Male; Mice; NF-E2-Related Factor 2; Oxidative Stress; Quercetin; RNA, Messenger; Spermatocytes; Superoxide Dismutase
PubMed: 35673910
DOI: 10.12122/j.issn.1673-4254.2022.05.07 -
International Journal of Molecular... Dec 2021Long noncoding RNAs (lncRNAs) are composed of nucleotides located in the nucleus and cytoplasm; these are transcribed by RNA polymerase II and are greater than 200 nt in... (Review)
Review
Long noncoding RNAs (lncRNAs) are composed of nucleotides located in the nucleus and cytoplasm; these are transcribed by RNA polymerase II and are greater than 200 nt in length. LncRNAs fulfill important functions in a variety of biological processes, including genome imprinting, cell differentiation, apoptosis, stem cell pluripotency, X chromosome inactivation and nuclear transport. As high throughput sequencing technology develops, a substantial number of lncRNAs have been found to be related to a variety of biological processes, such as development of the testes, maintaining the self-renewal and differentiation of spermatogonial stem cells, and regulating spermatocyte meiosis. These indicate that lncRNAs can be used as biomarkers and potential therapeutic targets for male infertility. However, only a few comprehensive reviews have described the role of lncRNAs in male reproduction. In this paper, we summarize recent findings relating to the role of lncRNAs in spermatogenesis, their potential as biomarkers for male infertility and the relationship between reproductive arrest and transgenerational effects. Finally, we suggest specific targets for the treatment of male infertility from the perspective of lncRNAs.
Topics: Animals; Cell Proliferation; Humans; Infertility, Male; Male; Meiosis; RNA, Long Noncoding; Spermatocytes; Spermatogenesis
PubMed: 34948376
DOI: 10.3390/ijms222413579 -
Nature Communications Nov 2021The mammalian SWI/SNF nucleosome remodeler is essential for spermatogenesis. Here, we identify a role for ARID2, a PBAF (Polybromo - Brg1 Associated Factor)-specific...
The mammalian SWI/SNF nucleosome remodeler is essential for spermatogenesis. Here, we identify a role for ARID2, a PBAF (Polybromo - Brg1 Associated Factor)-specific subunit, in meiotic division. Arid2 spermatocytes arrest at metaphase-I and are deficient in spindle assembly, kinetochore-associated Polo-like kinase1 (PLK1), and centromeric targeting of Histone H3 threonine3 phosphorylation (H3T3P) and Histone H2A threonine120 phosphorylation (H2AT120P). By determining ARID2 and BRG1 genomic associations, we show that PBAF localizes to centromeres and promoters of genes known to govern spindle assembly and nuclear division in spermatocytes. Consistent with gene ontology of target genes, we also identify a role for ARID2 in centrosome stability. Additionally, misexpression of genes such as Aurkc and Ppp1cc (Pp1γ), known to govern chromosome segregation, potentially compromises the function of the chromosome passenger complex (CPC) and deposition of H3T3P, respectively. Our data support a model where-in PBAF activates genes essential for meiotic cell division.
Topics: Animals; Cell Cycle Proteins; Centromere; Chromatin; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Helicases; Gene Silencing; Histones; Male; Mammals; Meiosis; Metaphase; Mice; Mice, Knockout; Nuclear Proteins; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Spermatocytes; Spermatogenesis; Transcription Factors; Transcriptome; Polo-Like Kinase 1
PubMed: 34772938
DOI: 10.1038/s41467-021-26828-1 -
PLoS Genetics Sep 2021Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic...
Meiosis initiation and progression are regulated by both germ cells and gonadal somatic cells. However, little is known about what genes or proteins connecting somatic and germ cells are required for this regulation. Our results show that deficiency for adhesion molecule IGSF11, which is expressed in both Sertoli cells and germ cells, leads to male infertility in mice. Combining a new meiotic fluorescent reporter system with testicular cell transplantation, we demonstrated that IGSF11 is required in both somatic cells and spermatogenic cells for primary spermatocyte development. In the absence of IGSF11, spermatocytes proceed through pachytene, but the pericentric heterochromatin of nonhomologous chromosomes remains inappropriately clustered from late pachytene onward, resulting in undissolved interchromosomal interactions. Hi-C analysis reveals elevated levels of interchromosomal interactions occurring mostly at the chromosome ends. Collectively, our data elucidates that IGSF11 in somatic cells and germ cells is required for pericentric heterochromatin dissociation during diplotene in mouse primary spermatocytes.
Topics: Animals; Cell Adhesion Molecules; Chromosomes; Female; Genes, Reporter; Heterochromatin; Humans; Immunoglobulins; Male; Meiotic Prophase I; Mice; Sertoli Cells; Spermatocytes
PubMed: 34491997
DOI: 10.1371/journal.pgen.1009778 -
Oncotarget Jul 2017Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human...
Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human testicular function remains to be identified. GPR30 is a novel membrane estrogen receptor with high-affinity and low-capacity binding to estrogens. We demonstrated that estrogen receptor α (ERα), estrogen receptor β (ERβ) as well as GPR30 are expressed in mouse spermatocyte-derived GC-2 cells using Real-time PCR. We treated the cells with different doses of BPA and found that even low doses of BPA can inhibit GC-2 cell growth using MTT assay. To make sure which receptor is responsible for the biological function of BPA, we used ER down-regulator ICI and indicated that BPA could bind to GPR30. We also observed that BPA was able to induce Erk1/2 phosphorylation in GC-2 cells and proved that this process was mediated by GPR30-related EGFR-MAPK pathway using western blot. By Real-time PCR, we found that the expression of c-Fos was up-regulated and Cyclin D1 gene was down-regulated, in the presence of BPA and ICI. The results of MTT assay, comet assay and flow cytometry indicated that the activation of GPR30 induced by BPA inhibited the cell growth and induced cell apoptosis and ICI, GPR30 siRNA, EGFR inhibitor (AG), and MAPK (PD) inhibitor could partially reverse this effect. Immunohistochemistry on the testis of BPA -damaged mice showed that BPA induced spermatocyte apoptosis without affecting the seminiferous tubules and spermatocyte. In conclusion, BPA triggered spermatocyte apoptosis via GPR30.
Topics: Animals; Apoptosis; Benzhydryl Compounds; Biomarkers; Cell Line; Cell Proliferation; Dose-Response Relationship, Drug; ErbB Receptors; Gene Expression; Gene Expression Regulation; Genes, fos; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phenols; Phosphorylation; Receptors, Estrogen; Signal Transduction; Spermatocytes
PubMed: 28446726
DOI: 10.18632/oncotarget.16923 -
Cell Reports Jul 2017The meiotic functions of Emi2, an inhibitor of the APC/C complex, have been best characterized in oocytes where it mediates metaphase II arrest as a component of the...
The meiotic functions of Emi2, an inhibitor of the APC/C complex, have been best characterized in oocytes where it mediates metaphase II arrest as a component of the cytostatic factor. We generated knockout mice to determine the in vivo functions of Emi2-in particular, its functions in the testis, where Emi2 is expressed at high levels. Male and female Emi2 knockout mice are viable but sterile, indicating that Emi2 is essential for meiosis but dispensable for embryonic development and mitotic cell divisions. We found that, besides regulating cell-cycle arrest in mouse eggs, Emi2 is essential for meiosis I progression in spermatocytes. In the absence of Emi2, spermatocytes arrest in early diplotene of prophase I. This arrest is associated with decreased Cdk1 activity and was partially rescued by a knockin mouse model of elevated Cdk1 activity. Additionally, we detected expression of Emi2 in spermatids and sperm, suggesting potential post-meiotic functions for Emi2.
Topics: Animals; F-Box Proteins; Female; Gene Expression Regulation; Male; Meiotic Prophase I; Mice; Mice, Knockout; Spermatids; Spermatocytes; Spermatogenesis
PubMed: 28723571
DOI: 10.1016/j.celrep.2017.06.033 -
Molecular Biology of the Cell Dec 2020Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly...
Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly regulate specific meiotic stages is not known. Here, we combine synchronization of spermatogenesis, cytological analyses and single-cell RNAseq (scRNAseq) in the ertoli-ell ndrogen eceptor nockut (SCARKO) mutant and control mice, and demonstrate that SCARKO mutant spermatocytes exhibited normal expression and localization of key protein markers of meiotic prophase events, indicating that initiation of meiotic prophase is not androgen dependent. However, spermatocytes from SCARKO testes failed to acquire competence for the meiotic division phase. ScRNAseq analysis of wild-type and SCARKO mutant testes revealed a molecular transcriptomic block in an early meiotic prophase state (leptotene/zygotene) in mutant germ cells, and identified several misregulated genes in SCARKO Sertoli cells, many of which have been previously implicated in male infertility. Together, our coordinated cytological and scRNAseq analyses identified germ-cell intrinsic and extrinsic genes responsive to Sertoli-cell androgen signaling that promotes cellular states permissive for the meiotic division phase.
Topics: Androgens; Animals; Male; Meiosis; Meiotic Prophase I; Mice; Mice, Inbred C57BL; Mice, Knockout; Prophase; Receptors, Androgen; Sequence Analysis, RNA; Sertoli Cells; Signal Transduction; Single-Cell Analysis; Spermatocytes; Spermatogenesis; Testis
PubMed: 33026960
DOI: 10.1091/mbc.E20-05-0334 -
Cell Reports Jan 2024Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male...
Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. Maps male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.
Topics: Humans; Male; Mice; Animals; Chromatin; Meiosis; Pachytene Stage; Phase Separation; Meiotic Prophase I; Spermatocytes; Mammals
PubMed: 38175751
DOI: 10.1016/j.celrep.2023.113651 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Jun 2023To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells)...
OBJECTIVE
To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells.
METHODS
Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level.
RESULTS
Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 ( < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration ( < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) ( < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells ( < 0.05).
CONCLUSIONS
TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Topics: Animals; Male; Mice; GTP-Binding Proteins; Mitogen-Activated Protein Kinases; NF-kappa B; RNA, Messenger; Serine-Type D-Ala-D-Ala Carboxypeptidase; Signal Transduction; Spermatocytes; Tubulin
PubMed: 37439173
DOI: 10.12122/j.issn.1673-4254.2023.06.16 -
Biology of Reproduction Apr 2020In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male...
In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility.
Topics: Animals; Azoospermia; COS Cells; Chlorocebus aethiops; Fertility; Male; Meiosis; Metallothionein; Mice; Mice, Knockout; Spermatocytes; Spermatogenesis; Spermatogonia; Spermatozoa; Testis
PubMed: 31916570
DOI: 10.1093/biolre/ioaa002