RESUMO
Centromere pairing is crucial for synapsis in meiosis. This study delves into the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, specifically focusing on F-box protein 47 (FBXO47), in mouse meiosis. Here, we revealed that FBXO47 is localized at the centromere and it regulates centromere pairing cooperatively with SKP1 to ensure proper synapsis in pachynema. The absence of FBXO47 causes defective centromeres, resulting in incomplete centromere pairing, which leads to corruption of SC at centromeric ends and along chromosome axes, triggering premature dissociation of chromosomes and pachytene arrest. FBXO47 deficient pachytene spermatocytes exhibited drastically reduced SKP1 expression at centromeres and chromosomes. Additionally, FBXO47 stabilizes SKP1 by down-regulating its ubiquitination in HEK293T cells. In essence, we propose that FBXO47 collaborates with SKP1 to facilitate centromeric SCF formation in spermatocytes. In summary, we posit that the centromeric SCF E3 ligase complex regulates centromere pairing for pachynema progression in mice.
Assuntos
Centrômero , Pareamento Cromossômico , Proteínas F-Box , Espermatócitos , Animais , Masculino , Centrômero/metabolismo , Centrômero/genética , Camundongos , Espermatócitos/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Humanos , Células HEK293 , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Meiose , Camundongos Knockout , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Camundongos Endogâmicos C57BLRESUMO
Male mouse meiosis has been traditionally studied using descriptive methods like histological sections and spreading or squashing techniques, which allow the observation of fixed meiocytes in either wildtype or genetically modified mice. For these studies, the sacrifice of the males and the extraction of the testicles are required to obtain the material of study. Other functional in vivo studies include the administration of intravenous or intraperitoneal drugs, or the exposure to mutagenic agents or generators of DNA damage, in order to study their impact on meiosis progression. However, in these studies, the exposure times or drug concentration are important limitations to consider when acknowledging animal welfare. Recently, several approaches have been proposed to offer alternative methodologies that allow the in vitro study of spermatocytes with a considerable reduction in the use of animals. Here we revisit and validate an optimal technique of organotypic culture of fragments of seminiferous tubules for meiotic studies. This technique is a trustable methodology to develop functional studies that preserve the histological configuration of the seminiferous tubule, aim homogeneity of the procedures (the use of the same animal for different study conditions), and allow procedures that would compromise the animal welfare. Therefore, this methodology is highly recommendable for the study of meiosis and spermatogenesis, while it supports the principle of 3R's for animal research.
Assuntos
Meiose , Sistemas Microfisiológicos , Túbulos Seminíferos , Animais , Masculino , Camundongos , Técnicas de Cultura de Órgãos/métodos , Túbulos Seminíferos/citologia , Espermatócitos/citologia , EspermatogêneseRESUMO
During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.
Assuntos
Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Meiose , Camundongos Knockout , Rad51 Recombinase , Espermatócitos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Animais , Masculino , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Camundongos , Espermatócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Dano ao DNA , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Cromatina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genéticaRESUMO
Chromatin undergoes extensive remodeling during meiosis, leading to specific patterns of gene expression and chromosome organization, which ultimately controls fundamental meiotic processes such as recombination and homologous chromosome associations. Recent game-changing advances have been made by analysis of chromatin binding sites of meiotic specific proteins genome-wide in mouse spermatocytes. However, further progress is still highly dependent on the reliable isolation of sufficient quantities of spermatocytes at specific stages of prophase I. Here, we describe a combination of methodologies we adapted for rapid and reliable isolation of synchronized fixed mouse spermatocytes. We show that chromatin isolated from these cells can be used to study chromatin-binding sites by ChIP-seq. High-quality data we obtained from INO80 ChIP-seq in zygotene cells was used for functional analysis of chromatin-binding sites.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Espermatócitos , Animais , Espermatócitos/metabolismo , Espermatócitos/citologia , Camundongos , Masculino , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Cromatina/genética , Cromatina/metabolismo , Meiose/genética , Imunoprecipitação da Cromatina/métodos , Sítios de LigaçãoRESUMO
Mammalian meiosis is a highly specialized cell division process, resulting in the production of genetically unique haploid cells. However, the molecular mechanisms governing meiosis remain largely unknown, primarily due to the difficulty in isolating pure sub-populations of spermatocytes. Definitive molecular, biochemical, and functional investigations of the meiosis process require the isolation of these individual homogeneous sub-populations of spermatocytes. Here, we present an approach that enables the purification of homogeneous spermatocytes from mouse testis at desired sub-stages. This approach consists of two strategic steps. The first is to synchronize spermatogenesis, aiming to minimize the diversity and complexity of testicular germ cells. The second involves utilizing mouse models with germ cell-specific fluorescent markers to differentiate the desired subtype from other cells in the testis. By employing fluorescence-activated cell sorting (FACS), this approach yields highly pure populations of spermatocytes at each sub-stage. When combined with other massively parallel sequencing techniques and in vitro cell culture methods, this approach will enhance our comprehension of the molecular mechanisms underlying mammalian meiosis and promote in vitro gametogenesis.
Assuntos
Separação Celular , Citometria de Fluxo , Meiose , Espermatócitos , Espermatogênese , Testículo , Animais , Masculino , Espermatócitos/citologia , Espermatócitos/metabolismo , Camundongos , Testículo/citologia , Testículo/metabolismo , Citometria de Fluxo/métodos , Separação Celular/métodosRESUMO
Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. Mouse TRIP13 and its ortholog Pch2 are instrumental in remodeling HORMA domain proteins. HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed homologs. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. Trip13-null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These major phenotypes are consistent with reported phenotypes of Trip13 hypomorph alleles. Trip13 heterozygous mice exhibit meiotic defects that are less severe than the Trip13-null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. Terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon synapsis in diverse organisms.
Assuntos
Meiose , Espermatócitos , Complexo Sinaptonêmico , Animais , Camundongos , Masculino , Complexo Sinaptonêmico/metabolismo , Complexo Sinaptonêmico/genética , Espermatócitos/metabolismo , Pareamento Cromossômico , Telômero/metabolismo , Telômero/genética , Feminino , Camundongos Knockout , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , ATPases Associadas a Diversas Atividades CelularesRESUMO
BACKGROUND: Non-obstructive azoospermia (NOA) is the most severe form of male infertility and affects approximately 1% of men worldwide. Fanconi anemia (FA) genes were known for their essential role in DNA repair and growing evidence showed the crucial role of FA pathway in NOA. However, the underlying mechanisms for Fance deficiency lead to a serious deficit and delayed maturation of male germ cells remain unclear. METHODS: We used Fance deficiency mouse model for experiments, and collected testes or epididymides from mice at 8 weeks (8W), 17.5 days post coitum (dpc), and postnatal 11 (P11) to P23. The mice referred to three genotypes: wildtype (Fance +/+), heterozygous (Fance +/-), and homozygous (Fance -/-). Hematoxylin and eosin staining, immunofluorescence staining, and surface spread of spermatocytes were performed to explore the mechanisms for NOA of Fance -/- mice. Each experiment was conducted with a minimum of three biological replicates and Kruskal-Wallis with Dunn's correction was used for statistical analysis. RESULTS: In the present study, we found that the adult male Fance -/- mice exhibited massive germ cell loss in seminiferous tubules and dramatically decreased sperms in epididymides. During the embryonic period, the number of Fance -/- prospermatogonia decreased significantly, without impacts on the proliferation (Ki-67, PCNA) and apoptosis (cleaved PARP, cleaved Caspase 3) status. The DNA double-strand breaks (γH2AX) increased at the cellular level of Fance -/- prospermatogonia, potentially associated with the increased nonhomologous end joining (53BP1) and decreased homologous recombination (RAD51) activity. Besides, Fance deficiency impeded the progression of meiotic prophase I of spermatocytes. The mechanisms entailed the reduced recruitment of the DNA end resection protein RPA2 at leptotene and recombinases RAD51 and DMC1 at zygotene. It also involved impaired removal of RPA2 at zygotene and FANCD2 foci at pachytene. And the accelerated initial formation of crossover at early pachytene, which is indicated by MLH1. CONCLUSIONS: Fance deficiency caused massive male germ cell loss involved in the imbalance of DNA damage repair in prospermatogonia and altered dynamics of proteins in homologous recombination, DNA end resection, and crossover, providing new insights into the etiology and molecular basis of NOA.
Assuntos
Azoospermia , Dano ao DNA , Reparo do DNA , Camundongos Knockout , Espermatócitos , Espermatogênese , Masculino , Animais , Espermatócitos/metabolismo , Reparo do DNA/genética , Camundongos , Azoospermia/genética , Azoospermia/patologia , Azoospermia/metabolismo , Dano ao DNA/genética , Espermatogênese/genética , Testículo/metabolismo , Testículo/patologia , Camundongos Endogâmicos C57BLRESUMO
Alternative cleavage and polyadenylation (APA) often results in production of mRNA isoforms with either longer or shorter 3' UTRs from the same genetic locus, potentially impacting mRNA translation, localization, and stability. Developmentally regulated APA can thus make major contributions to cell type-specific gene expression programs as cells differentiate. During Drosophila spermatogenesis, â¼500 genes undergo APA when proliferating spermatogonia differentiate into spermatocytes, producing transcripts with shortened 3' UTRs, leading to profound stage-specific changes in the proteins expressed. The molecular mechanisms that specify usage of upstream polyadenylation sites in spermatocytes are thus key to understanding the changes in cell state. Here, we show that upregulation of PCF11 and Cbc, the two components of cleavage factor II (CFII), orchestrates APA during Drosophila spermatogenesis. Knockdown of PCF11 or cbc in spermatocytes caused dysregulation of APA, with many transcripts normally cleaved at a proximal site in spermatocytes now cleaved at their distal site, as in spermatogonia. Forced overexpression of CFII components in spermatogonia switched cleavage of some transcripts to the proximal site normally used in spermatocytes. Our findings reveal a developmental mechanism where changes in expression of specific cleavage factors can direct cell type-specific APA at selected genes.
Assuntos
Linhagem da Célula , Poliadenilação , Espermatócitos , Espermatogênese , Animais , Poliadenilação/genética , Masculino , Espermatogênese/genética , Espermatócitos/metabolismo , Espermatócitos/citologia , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/citologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Espermatogônias/citologia , Espermatogônias/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
BACKGROUND: Zinc oxide nanoparticle (ZnO NP) is one of the metal nanomaterials with extensive use in many fields such as feed additive and textile, which is an emerging threat to human health due to widely distributed in the environment. Thus, there is an urgent need to understand the toxic effects associated with ZnO NPs. Although previous studies have found accumulation of ZnO NPs in testis, the molecular mechanism of ZnO NPs dominated a decline in male fertility have not been elucidated. RESULTS: We reported that ZnO NPs exposure caused testicular dysfunction and identified spermatocytes as the primary damaged site induced by ZnO NPs. ZnO NPs led to the dysfunction of spermatocytes, including impaired cell proliferation and mitochondrial damage. In addition, we found that ZnO NPs induced ferroptosis of spermatocytes through the increase of intracellular chelatable iron content and lipid peroxidation level. Moreover, the transcriptome analysis of testis indicated that ZnO NPs weakened the expression of miR-342-5p, which can target Erc1 to block the NF-κB pathway. Eventually, ferroptosis of spermatocytes was ameliorated by suppressing the expression of Erc1. CONCLUSIONS: The present study reveals a novel mechanism in that miR-342-5p targeted Erc1 to activate NF-κB signaling pathway is required for ZnO NPs-induced ferroptosis, and provide potential targets for further research on the prevention and treatment of male reproductive disorders related to ZnO NPs.
Assuntos
Ferroptose , MicroRNAs , NF-kappa B , Transdução de Sinais , Espermatócitos , Testículo , Óxido de Zinco , Animais , Masculino , Camundongos , Proliferação de Células/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Nanopartículas Metálicas/química , MicroRNAs/metabolismo , MicroRNAs/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatócitos/efeitos dos fármacos , Testículo/metabolismo , Testículo/efeitos dos fármacos , Óxido de Zinco/farmacologia , Óxido de Zinco/químicaRESUMO
Drosophila spermatogenesis involves the renewal of germline stem cells, meiosis of spermatocytes, and morphological transformation of spermatids into mature sperm. We previously demonstrated that Ocnus (ocn) plays an essential role in spermatogenesis. The ValRS-m (Valyl-tRNA synthetase, mitochondrial) gene was down-regulated in ocn RNAi testes. Here, we found that ValRS-m-knockdown induced complete sterility in male flies. The depletion of ValRS-m blocked mitochondrial behavior and ATP synthesis, thus inhibiting the transition from spermatogonia to spermatocytes, and eventually, inducing the accumulation of spermatogonia during spermatogenesis. To understand the intrinsic reason for this, we further conducted transcriptome-sequencing analysis for control and ValRS-m-knockdown testes. The differentially expressed genes (DEGs) between these two groups were selected with a fold change of ≥2 or ≤1/2. Compared with the control group, 4725 genes were down-regulated (dDEGs) and 2985 genes were up-regulated (uDEGs) in the ValRS-m RNAi group. The dDEGs were mainly concentrated in the glycolytic pathway and pyruvate metabolic pathway, and the uDEGs were primarily related to ribosomal biogenesis. A total of 28 DEGs associated with mitochondria and 6 meiosis-related genes were verified to be suppressed when ValRS-m was deficient. Overall, these results suggest that ValRS-m plays a wide and vital role in mitochondrial behavior and spermatogonia differentiation in Drosophila.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Infertilidade Masculina , Espermatogênese , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/deficiência , Espermatogênese/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Testículo/metabolismo , Meiose/genética , Espermatogônias/metabolismo , Perfilação da Expressão Gênica , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Espermatócitos/metabolismo , TranscriptomaRESUMO
Male infertility is a serious ongoing problem, whose causes have not yet been clearly identified. However, since human exposure to silver nanoparticles (AgNPs) has recently increased due to their beneficial properties, the present study aimed to determine the impact of small-size AgNPs on mouse spermatogonia (GC-1 spg) and spermatocytes [GC-2 spd(ts)] in vitro models as well as the ability of these nanostructures to induce inflammation. The results showed a significant dose- and time-dependent decrease in the metabolic activity in both cell models, which was correlated with an increase in the intracellular ROS level. Moreover, increased activity of caspase-9 and -3, together with enhanced expression of CASP3 and p(S15)-p53 proteins, was detected. Further studies indicated a decrease in ΔΨm after the AgNP-treatment, which proves induction of apoptosis with engagement of an intrinsic pathway. The PARP1 protein expression, the activity and protein expression of antioxidant enzymes, the GSH level, and the increased level of p-ERK1/2 indicate not only the engagement of DNA damage but also the occurrence of oxidative stress. The small-size AgNPs were able to induce inflammation, proved by increased protein expression of NF-κB, p-IκBα, and NLRP3, which indicate damage to spermatogonia and spermatocyte cells. Moreover, the PGC-1α/PPARγ and NRF2/Keap1 pathways were engaged in the observed effect. The spermatogonial cells were characterized by a stronger inflammation-based response to AgNPs, which may be correlated with the TNFα/TRAF2-based pathway. Summarizing, the obtained results prove that AgNPs impair the function of testis-derived cells by inducing the redox imbalance and inflammation process; therefore, these NPs should be carefully implemented in the human environment.
Assuntos
Inflamação , Nanopartículas Metálicas , Prata , Espermatócitos , Espermatogônias , Masculino , Prata/toxicidade , Prata/química , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/química , Animais , Camundongos , Inflamação/induzido quimicamente , Espermatogônias/efeitos dos fármacos , Espermatócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular , Infertilidade Masculina/induzido quimicamenteRESUMO
Triptolide (TP) is a major bioactive compound derived from Tripterygium wilfordii Hook. F. (TwHF) known for its medicinal properties, but it also exhibits potential toxic effects. It has been demonstrated to induce severe male reproductive toxicity, yet the precise mechanism behind this remains unclear, which limits its broad clinical application. This study aimed to investigate the mechanisms underlying testicular damage and spermatogenesis dysfunction induced by TP in mice, using both mouse models and the spermatocyte-derived cell line GC-2spd. In the present study, it was found that TP displayed significant testicular microstructure damaged and spermatogenesis defects including lower concentration and abnormal morphology by promoting ROS formation, MDA production and restraining GSH level, glutathione peroxidase 4 (GPX4) expression in vivo. Furthermore, Ferrostatin-1 (FER-1), a ferroptosis inhibitor, was found to significantly reduce the accumulation of lipid peroxidation, alleviate testicular microstructural damage, and enhance spermatogenic function in mice. Besides, notably decreased cell viability, collapsed mitochondrial membrane potential, and elevated DNA damage were observed in vitro. The above-mentioned phenomenon could be reversed by pre-treatment of FER-1, indicating that ferroptosis participated in the TP-mediated spermatogenesis dysfunction. Mechanistically, TP could enhance GPX4 ubiquitin degradation via triggering K63-linked polyubiquitination of GPX4, thereby stimulating ferroptosis in spermatocytes. Functionally, GPX4 deletion intensified ferroptosis and exacerbated DNA damage in GC-2 cells, while GPX4 overexpression mitigated ferroptosis induced by TP. Overall, these findings for the first time indicated a vital role of ferroptosis in TP induced-testicular injury and spermatogenic dysfunction through promoting GPX4 K63-linked polyubiquitination, which hopefully offers a potential therapeutic avenue for TP-related male reproductive damage. In addition, this study also provides a theoretical foundation for the improved clinical application of TP or TwHF in the future.
Assuntos
Diterpenos , Compostos de Epóxi , Ferroptose , Fenantrenos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Espermatócitos , Espermatogênese , Ubiquitinação , Masculino , Animais , Fenantrenos/farmacologia , Espermatogênese/efeitos dos fármacos , Diterpenos/farmacologia , Compostos de Epóxi/toxicidade , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Camundongos , Ferroptose/efeitos dos fármacos , Espermatócitos/efeitos dos fármacos , Espermatócitos/metabolismo , Ubiquitinação/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo , Testículo/patologia , Linhagem Celular , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Lisina/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacosRESUMO
BACKGROUND: Polystyrene nanoplastics (PS-NPs), are ubiquitous pollution sources in human environments, posing significant biosafety and health risks. While recent studies, including our own, have illustrated that PS-NPs can breach the blood-testis barrier and impact germ cells, there remains a gap in understanding their effects on specific spermatogenic cells such as spermatocytes. METHODS AND RESULTS: Herein, we employed an integrated approach encompassing phenotype, metabolomics, and transcriptomics analyses to assess the molecular impact of PS-NPs on mouse spermatocyte-derived GC-2spd(ts) cells. Optimal exposure conditions were determined as 24 h with 50 nm PS-NPs at 12.5 µg/mL and 90 nm PS-NPs at 50 µg/mL for subsequent multi-omics analysis. Our findings revealed that PS-NPs significantly influenced proliferation and viability, causing alterations in transcriptome and metabolome profiles. Transcriptomics analysis of GC-2spd(ts) cells exposed to PS-NPs indicated the pivotal involvement of cell proliferation and cycle, autophagy, ferroptosis, and redox reaction pathways in PS-NP-induced effects on the proliferation and viability of GC-2spd(ts) cells. Furthermore, metabolomics analysis identified major changes in amino acid metabolism, cyanoamino acid metabolism, and purine and pyrimidine metabolism following PS-NP exposure. CONCLUSION: Our integrated approach, combining metabolomics and transcriptomics profiles with phenotype data, enhances our understanding of the adverse effects of PS-NPs on germ cells.
Assuntos
Metabolômica , Poliestirenos , Espermatócitos , Transcriptoma , Animais , Masculino , Camundongos , Espermatócitos/efeitos dos fármacos , Poliestirenos/toxicidade , Transcriptoma/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nanopartículas/toxicidade , Metaboloma/efeitos dos fármacos , Microplásticos/toxicidadeRESUMO
BACKGROUND: Triclosan (TCS), as an endocrine disrupter, has been found to affect male fertility. However, the potential molecular mechanism is still unknown. We aimed to investigate whether the toxic effects of TCS on spermatocyte cells was mediated by the regulation of microRNA-20a-5â¯P on PTEN. METHODS: GC-2 and TM4 cells were treated with TCS (0.5-80⯵M) for 24 or 48â¯hours. Effect of TCS on proliferation of GC-2 and TM4 cells was detected using a cell counting kit-8 (CCK8) assay. Expression of miR-17 family and autophagy genes were detected. The interaction between miR-20a-5â¯P and PTEN was determined by a dual-luciferase reporter assay. RESULTS: TCS decreased cell proliferation of GC-2 and TM4 cells. Expression of autophagy-related genes and miR-17 family was altered by TCS. PTEN expression was significantly increased, whereas the expression of miR-20a-5â¯P was significantly decreased in GC-2 and TM4 cells. As predicted in relevant databases, there is a binding site of miR-20a-5â¯P in PTEN. The expression of PTEN was significantly down-regulated by the miR-20a-5â¯P mimic. CONCLUSION: As a downstream target of miR-20a-5â¯P, PTEN functioned in the autophagy process of which TCS inhibited the proliferation of spermatocyte cells. Our results provided new ideas for revealing the molecular mechanism and protective strategy on male infertility.
Assuntos
Autofagia , Proliferação de Células , MicroRNAs , PTEN Fosfo-Hidrolase , Espermatócitos , Triclosan , MicroRNAs/genética , MicroRNAs/metabolismo , Masculino , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular , Triclosan/toxicidade , Humanos , Espermatócitos/efeitos dos fármacos , Disruptores Endócrinos/toxicidade , AnimaisRESUMO
BACKGROUND: Spermatogenesis is a temperature-sensitive process, and elevation in temperature hampers this process quickly and significantly. We studied the molecular effects of testicular heating on piRNAs and gene expression in rat testicular germ cells. METHODS: We generated a cryptorchid rat model by displacing the testis from the scrotal sac (34 °C) to the abdominal area (37 °C) and sacrificed animals after 1 day, 3 days, and 5 days. Pachytene spermatocytes and round spermatids were purified using elutriation centrifugation and percoll gradient methods. We performed transcriptome sequencing in pachytene spermatocytes and round spermatids to identify differentially expressed piRNAs and their probable targets, i.e., TE transcripts and mRNAs. RESULTS: As a result of heat stress, we observed significant upregulation of piRNAs and TE transcripts in testicular germ cells. In addition to this, piRNA biogenesis machinery and heat shock proteins (Hsp70 and Hsp90 family members) were upregulated. mRNAs have also been proposed as targets for piRNAs; therefore, we shortlisted certain piRNA-mRNA pairs with an inverse relationship of expression. We observed that in testicular heat stress, the heat shock proteins go hand-in-hand with the upregulation of piRNA biogenesis machinery. The dysregulation of piRNAs in heat-stressed germ cells, increased ping-pong activity, and disturbed expression of piRNA target transcripts suggest a connection between piRNAs, mRNAs, and TE transcripts. CONCLUSIONS: In heat stress, piRNAs, piRNA machinery, and heat shock proteins are activated to deal with low levels of stress, which is followed by a rescue approach in prolonged stressaccompained by high TE activity to allow genetic mutations, perhaps for survival and adaptability.
Assuntos
Resposta ao Choque Térmico , RNA Interferente Pequeno , Espermátides , Espermatócitos , Testículo , Animais , Masculino , Espermátides/metabolismo , Espermatócitos/metabolismo , RNA Interferente Pequeno/genética , Ratos , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Testículo/metabolismo , Espermatogênese/genética , Espermatogênese/fisiologia , Estágio Paquíteno/genética , Ratos Sprague-Dawley , RNA de Interação com PiwiRESUMO
Spermatogenesis requires precise posttranslational control in the endoplasmic reticulum (ER), but the mechanism remains largely unknown. The protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases responsible for catalyzing the disulfide bond formation of nascent proteins. In this study, we generated 14 strains of KO mice lacking the PDI family enzymes and found that only PDI deficiency caused spermatogenesis defects. Both inducible whole-body PDI-KO (UBC-Cre/Pdifl/fl) mice and premeiotic PDI-KO (Stra8-Cre/Pdifl/fl) mice experienced a significant decrease in germ cells, testicular atrophy, oligospermia, and complete male infertility. Stra8-Cre/Pdifl/fl spermatocytes had significantly upregulated ER stress-related proteins (GRP78 and XBP1) and apoptosis-related proteins (Cleaved caspase-3 and BAX), together with cell apoptosis. PDI deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene spermatocytes. Quantitative mass spectrometry indicated that PDI deficiency downregulated vital proteins in spermatogenesis such as HSPA4L, SHCBP1L, and DDX4, consistent with the proteins' physical association with PDI in normal testes tissue. Furthermore, PDI served as a thiol oxidase for disulfide bond formation of SHCBP1L. Thus, PDI plays an essential role in protein quality control for spermatogenesis in mice.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas , Espermatogênese , Testículo , Animais , Masculino , Espermatogênese/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Camundongos , Testículo/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Apoptose , Espermatócitos/metabolismo , Estresse do Retículo Endoplasmático , Oligospermia/genética , Oligospermia/metabolismo , Oligospermia/patologiaRESUMO
Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.
Assuntos
Centríolos , Segregação de Cromossomos , Proteínas Associadas aos Microtúbulos , Centro Organizador dos Microtúbulos , Espermatócitos , Espermatogênese , Centríolos/metabolismo , Centríolos/genética , Animais , Masculino , Camundongos , Espermatogênese/genética , Espermatócitos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Mouse HORMAD1 is a phospho-protein involved in multiple functions during meiotic prophase I. To obtain insight into the significance of its phosphorylation, we generated phospho-specific antibodies against two serine residues, Ser307 and Ser378, representing each of two serine clusters in mouse HORMAD1. The Ser307 phosphorylation is detectable from early leptotene substage in both wild-type and Spo11-/- spermatocytes, indicating that Ser307 is a primary and SPO11-independent phosphorylation site. In contrast, the Ser378 phosphorylation is negligible at earlier substages in wild-type and Spo11-/- spermatocytes. After mid-zygotene substage, the Ser378 phosphorylation is abundant on unsynapsed chromosome axes in wild-type spermatocytes and is detected only in a part of unsynapsed chromosome axes in Spo11-/- spermatocytes. We also generated a non-phosphorylated Ser307-specific antibody and found that Ser307 is phosphorylated on sex chromosome axes but is almost entirely unphosphorylated on desynapsed chromosome axes in diplotene spermatocytes. These results demonstrated a substage-specific phosphorylation status of mouse HORMAD1, which might be associated with multiple substage-specific functions.
Assuntos
Prófase Meiótica I , Serina , Espermatócitos , Animais , Fosforilação , Masculino , Camundongos , Serina/metabolismo , Espermatócitos/metabolismo , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Camundongos Endogâmicos C57BL , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Camundongos Knockout , Cromossomos Sexuais/genética , Cromossomos Sexuais/metabolismoRESUMO
Male cases diagnosed COVID-19 with more complications and higher mortality compared with females, and the overall consequences of male sex hormones and semen parameters deterioration were observed in COVID-19 patients, whereas the involvement and mechanism for spermatogenic cell remains unclear. The study was aimed to investigate the infection mode of S protein (D614G) pseudovirus (pseu-S-D614G) to spermatogenic cells, as well as the influence on cell growth. Both mouse spermatogonia (GC-1 cell, immortalized spermatogonia) and spermatocyte (GC-2 cell, immortalized spermatocytes) were used to detect the infection of pseu-S-D614G of SARS-CoV-2, and further explored the effect of SARS-CoV-2-spike protein (S-protein) and SARS-CoV-2-spike protein (omicron) (O-protein) on GC-1 cell apoptosis and proliferation. The data showed that the pseu-S-D614G invaded into GC-1 cells through either human ACE2 (hACE2) or human CD147 (hCD147), whereas GC-2 cells were insensitive to viral infection. In addition, the apoptosis and proliferation suppression inflicted by S-protein and O-protein on GC-1 cells was through Bax-Caspase3 signaling rather than arresting cell cycle progression. These findings suggest that CD147, apart from ACE2, may be a potential receptor for SARS-CoV-2 infection in testicular tissues, and that the apoptotic effect was induced in spermatogonia cells by S-protein or O-protein, eventually resulted in the damage to male fertility.
Assuntos
Enzima de Conversão de Angiotensina 2 , Apoptose , Basigina , COVID-19 , SARS-CoV-2 , Espermatogônias , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Masculino , Camundongos , Enzima de Conversão de Angiotensina 2/metabolismo , Apoptose/fisiologia , Basigina/metabolismo , Linhagem Celular , Proliferação de Células , COVID-19/metabolismo , Espermatócitos/metabolismo , Espermatócitos/virologia , Espermatogônias/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
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.