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Frontiers in Endocrinology 2021Fine particulate matter (PM)-induced male reproductive toxicity arouses global public health concerns. However, the mechanisms of toxicity remain unclear. This study...
Analysis by Metabolomics and Transcriptomics for the Energy Metabolism Disorder and the Aryl Hydrocarbon Receptor Activation in Male Reproduction of Mice and GC-2spd Cells Exposed to PM.
Fine particulate matter (PM)-induced male reproductive toxicity arouses global public health concerns. However, the mechanisms of toxicity remain unclear. This study aimed to further investigate toxicity pathways by exposure to PM and through the application of metabolomics and transcriptomics. , spermatocyte-derived GC-2spd cells were treated with 0, 25, 50, 100 μg/mL PM for 48 h. , the real-world exposure of PM for mouse was established. Forty-five male C57BL/6 mice were exposed to filtered air, unfiltered air, and concentrated ambient PM in Tangshan of China for 8 weeks, respectively. The results and showed that PM exposure inhibited GC-2spd cell proliferation and reduced sperm motility. Mitochondrial damage was observed after PM treatment. Increased Humanin and MOTS-c levels and decreased mitochondrial respiratory indicated that mitochondrial function was disturbed. Furthermore, nontargeted metabolomics analysis revealed that PM exposure could disturb the citrate cycle (TCA cycle) and reduce amino acids and nucleotide synthesis. Mechanically, the aryl hydrocarbon receptor (AhR) pathway was activated after exposure to PM, with a significant increase in CYP1A1 expression. Further studies showed that PM exposure significantly increased both intracellular and mitochondrial reactive oxygen species (ROS) and activated NRF2 antioxidative pathway. With the RNA-sequencing technique, the differentially expressed genes induced by PM exposure were mainly enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, of which was the most significantly changed gene. Our findings demonstrated that PM exposure could induce spermatocyte damage and energy metabolism disorder. The activation of the aryl hydrocarbon receptor might be involved in the mechanism of male reproductive toxicity.
Topics: Air Pollutants; Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Citric Acid Cycle; Cytochrome P-450 Enzyme System; Energy Metabolism; Gene Expression Profiling; Male; Metabolome; Metabolomics; Mice; Mice, Inbred C57BL; Mitochondria; Particulate Matter; Reactive Oxygen Species; Receptors, Aryl Hydrocarbon; Reproduction; Sperm Motility; Spermatocytes; Transcriptome; Xenobiotics
PubMed: 35046903
DOI: 10.3389/fendo.2021.807374 -
Cells Feb 2020The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open...
The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.
Topics: Animals; Cell Nucleus; Genome, Human; Heterochromatin; Histones; Humans; Male; Mice; Mice, Knockout; NIH 3T3 Cells; RNA Recognition Motif Proteins; RNA, Nuclear; RNA-Binding Motifs; Spermatocytes; Spermatogenesis; Transfection
PubMed: 32085641
DOI: 10.3390/cells9020474 -
Frontiers in Endocrinology 2021To achieve spermatogenesis , one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or...
To achieve spermatogenesis , one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the -generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional , meiotic crossover formation should be closely monitored when mimicking gametogenesis to prevent generation of aneuploid gametes.
Topics: Aneuploidy; Animals; Azoospermia; Cell Differentiation; Cell Line; Cell Proliferation; Chromosome Pairing; Chromosomes; Male; Meiosis; Mice; Mice, Inbred DBA; Sertoli Cells; Spermatocytes; Spermatogenesis; Spermatozoa; Testis
PubMed: 34721307
DOI: 10.3389/fendo.2021.761249 -
American Journal of Human Genetics Jun 2024Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes...
Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.
Topics: Humans; Spermatogenesis; Male; DNA Methylation; Genome, Human; Spermatids; Spermatocytes; DNA Transposable Elements; Spermatozoa; Meiosis; Transcription Factors
PubMed: 38759652
DOI: 10.1016/j.ajhg.2024.04.017 -
Chromosoma Dec 2022In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recombination and segregation. However, when the pairing...
In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recombination and segregation. However, when the pairing process begins, it is still under investigation. Contrasting data exists in Mus musculus, since both leptotene DSB-dependent and preleptotene DSB-independent mechanisms have been described. To unravel this contention, we examined homologous pairing in pre-meiotic and meiotic Mus musculus cells using a three-dimensional fluorescence in situ hybridization-based protocol, which enables the analysis of the entire karyotype using DNA painting probes. Our data establishes in an unambiguously manner that 73.83% of homologous chromosomes are already paired at premeiotic stages (spermatogonia-early preleptotene spermatocytes). The percentage of paired homologous chromosomes increases to 84.60% at mid-preleptotene-zygotene stage, reaching 100% at pachytene stage. Importantly, our results demonstrate a high percentage of homologous pairing observed before the onset of meiosis; this pairing does not occur randomly, as the percentage was higher than that observed in somatic cells (19.47%) and between nonhomologous chromosomes (41.1%). Finally, we have also observed that premeiotic homologous pairing is asynchronous and independent of the chromosome size, GC content, or presence of NOR regions.
Topics: Animals; Mice; Male; In Situ Hybridization, Fluorescence; Meiosis; Meiotic Prophase I; Karyotype; Spermatocytes
PubMed: 35960388
DOI: 10.1007/s00412-022-00777-0 -
Reproductive Biology and Endocrinology... Jun 2018Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen....
BACKGROUND
Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment.
METHODS
Testes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs.
RESULTS
Heat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types.
CONCLUSION
The study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.
Topics: Animals; Gene Expression; Gene Expression Profiling; Hot Temperature; Male; Rats; Rats, Sprague-Dawley; Spermatocytes; Spermatogenesis
PubMed: 29859541
DOI: 10.1186/s12958-018-0372-8 -
Molecular Cell Dec 2021In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal...
In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.
Topics: Animals; Argonaute Proteins; Cell Separation; Drosophila melanogaster; Female; Flow Cytometry; Gene Expression; Gene Silencing; Genetic Techniques; Male; Methylation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Processing, Post-Translational; RNA, Double-Stranded; RNA, Small Interfering; Spermatocytes; Spermatogonia; Testis
PubMed: 34626567
DOI: 10.1016/j.molcel.2021.09.012 -
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 -
Molecular Biology Reports Feb 2023The treatment with chemotherapy may develop secondary tumors as a result of chemo genotoxicity. Sperm defects is another complication associated with chemo treatment. In...
BACKGROUND
The treatment with chemotherapy may develop secondary tumors as a result of chemo genotoxicity. Sperm defects is another complication associated with chemo treatment. In this study the genotoxicity of vinblastine (VB) was estimated in both somatic and germ cells.
MATERIALS
85 mice were taken. Four single doses of VB at 3, 4.5, 6 and 10 mg/kg and three successive doses at 3, 4.5 and 6 mg/kg were taken for estimation of chromosomal aberrations (CAs). Four single doses of VB were involved in estimating the DNA fragmentation, and comet assay. For sperm abnormalities mice were injected with three successive doses of VB at 3, 4.5, and 6 mg/kg.
RESULTS
The results demonstrated a significant frequency of DNA fragmentation in spleen cells and in the percentage of CAs in bone marrow. Numerical and structural aberrations were recorded with a pronounced number of polyploidy metaphases which reached (11.60%) after treatment with 6 mg/kg for three successive days vs zero for control. VB also induced a significant percentage of CAs in spermatocytes in the form of univalent. Sperm defects in the form of coiled tail, absence of acrosome and shapeless head and a significant DNA damage in the testes were recorded. The frequency of sperm abnormalities reached 11.06 ± 0.14 after treatment with highest tested dose (6 mg/kg) vs 3.04 ± 0.19 for control.
CONCLUSION
VB is genotoxic in somatic and germ cells. Sperm defects induced by VB are of serious concern to future generations and may affect the fertility of cancer survivors.
Topics: Male; Animals; Mice; Vinblastine; Semen; Spermatozoa; DNA Damage; Spermatocytes; Chromosome Aberrations
PubMed: 36394708
DOI: 10.1007/s11033-022-08061-1 -
International Journal of Molecular... Dec 2021MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic...
MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic development. Despite the unique mitochondrial organization in sperm flagella, the biological requirement in sperm development and functions remain undefined. Here, using sperm-specific Cre drivers, we show that either or knockout in haploid germ cells does not affect male fertility. The and double knockout mice were further analyzed. We found no differences in testis morphology and weight between -deficient mice and their wild-type littermate controls. Spermatogenesis was normal in double knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules, indicating that sperm formation was not disrupted upon MFN deficiency. Collectively, our findings reveal that both MFN1 and MFN2 are dispensable for sperm development and functions in mice.
Topics: Animals; Female; GTP Phosphohydrolases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Dynamics; Sperm Maturation; Spermatocytes; Spermatogenesis; Spermatozoa; Testis
PubMed: 34948301
DOI: 10.3390/ijms222413507