RESUMEN

DNA meiotic recombinase 1 (disrupted meiotic cDNA, Dmc1) protein is homologous to the Escherichia coli RecA protein, was first identified in Saccharomyces cerevisiae. This gene has been well studied as an essential role in meiosis in many species. However, studies on the dmc1 gene in reptiles are limited. In this study, a cDNA fragment of 1,111 bp was obtained from the gonadal tissues of the Chinese soft-shell turtle via RT-PCR, containing a 60 bp 3' UTR, a 22 bp 5' UTR, and an ORF of 1,029 bp encoding 342 amino acids, named Psdmc1. Multiple sequence alignments showed that the deduced protein has high similarity (>95 %) to tetrapod Dmc1 proteins, while being slightly lower (86-88 %) to fish species.Phylogenetic tree analysis showed that PsDmc1 was clustered with the other turtles' Dmc1 and close to the reptiles', but far away from the teleost's. RT-PCR and RT-qPCR analyses showed that the Psdmc1 gene was specifically expressed in the gonads, and much higher in testis than the ovary, especially highest in one year-old testis. In situ hybridization results showed that the Psdmc1 was mainly expressed in the perinuclear cytoplasm of primary and secondary spermatocytes, weakly in spermatogonia of the testes. These results indicated that dmc1 would be majorly involved in the developing testis, and play an essential role in the germ cells' meiosis. The findings of this study will provide a basis for further investigations on the mechanisms behind the germ cells' development and differentiation in Chinese soft-shell turtles, even in the reptiles.

Asunto(s)

Gametogénesis , Filogenia , Tortugas , Animales , Tortugas/genética , Tortugas/metabolismo , Masculino , Gametogénesis/genética , Femenino , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Testículo/metabolismo , Clonación Molecular , Secuencia de Aminoácidos , Meiosis/genética , Ovario/metabolismo , Espermatocitos/metabolismo , Pueblos del Este de Asia

RESUMEN

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.

Asunto(s)

Centrómero , Emparejamiento Cromosómico , Proteínas F-Box , Espermatocitos , Animales , Masculino , Centrómero/metabolismo , Centrómero/genética , Ratones , Espermatocitos/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Humanos , Células HEK293 , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Meiosis , Ratones Noqueados , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Ratones Endogámicos C57BL

RESUMEN

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, ∼500 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.

Asunto(s)

Linaje de la Célula , Poliadenilación , Espermatocitos , Espermatogénesis , Animales , Poliadenilación/genética , Masculino , Espermatogénesis/genética , Espermatocitos/metabolismo , Espermatocitos/citología , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/genética , Células Madre Adultas/metabolismo , Células Madre Adultas/citología , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Espermatogonias/citología , Espermatogonias/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética

RESUMEN

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.

Asunto(s)

Secuenciación de Inmunoprecipitación de Cromatina , Cromatina , Espermatocitos , Animales , Espermatocitos/metabolismo , Espermatocitos/citología , Ratones , Masculino , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Meiosis/genética , Inmunoprecipitación de Cromatina/métodos , Sitios de Unión

RESUMEN

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.

Asunto(s)

Separación Celular , Citometría de Flujo , Meiosis , Espermatocitos , Espermatogénesis , Testículo , Animales , Masculino , Espermatocitos/citología , Espermatocitos/metabolismo , Ratones , Testículo/citología , Testículo/metabolismo , Citometría de Flujo/métodos , Separación Celular/métodos

RESUMEN

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.

Asunto(s)

Meiosis , Espermatocitos , Complejo Sinaptonémico , Animales , Ratones , Masculino , Complejo Sinaptonémico/metabolismo , Complejo Sinaptonémico/genética , Espermatocitos/metabolismo , Emparejamiento Cromosómico , Telómero/metabolismo , Telómero/genética , Femenino , Ratones Noqueados , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , ATPasas Asociadas con Actividades Celulares Diversas

RESUMEN

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.

Asunto(s)

Azoospermia , Daño del ADN , Reparación del ADN , Ratones Noqueados , Espermatocitos , Espermatogénesis , Masculino , Animales , Espermatocitos/metabolismo , Reparación del ADN/genética , Ratones , Azoospermia/genética , Azoospermia/patología , Azoospermia/metabolismo , Daño del ADN/genética , Espermatogénesis/genética , Testículo/metabolismo , Testículo/patología , Ratones Endogámicos C57BL

RESUMEN

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.

Asunto(s)

Proteínas de Ciclo Celular , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Meiosis , Ratones Noqueados , Recombinasa Rad51 , Espermatocitos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Animales , Masculino , Meiosis/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Ratones , Espermatocitos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Recombinación Homóloga , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Cromatina/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Proteínas de Unión a Fosfato/genética

RESUMEN

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.

Asunto(s)

Diterpenos , Compuestos Epoxi , Ferroptosis , Fenantrenos , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Espermatocitos , Espermatogénesis , Ubiquitinación , Masculino , Animales , Fenantrenos/farmacología , Espermatogénesis/efectos de los fármacos , Diterpenos/farmacología , Compuestos Epoxi/toxicidad , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Ratones , Ferroptosis/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Espermatocitos/metabolismo , Ubiquitinación/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismo , Testículo/patología , Línea Celular , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Lisina/metabolismo , Peroxidación de Lípido/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Ferroptosis , MicroARNs , FN-kappa B , Transducción de Señal , Espermatocitos , Testículo , Óxido de Zinc , Animales , Masculino , Ratones , Proliferación Celular/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Nanopartículas del Metal/química , MicroARNs/metabolismo , MicroARNs/genética , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Espermatocitos/metabolismo , Espermatocitos/efectos de los fármacos , Testículo/metabolismo , Testículo/efectos de los fármacos , Óxido de Zinc/farmacología , Óxido de Zinc/química

RESUMEN

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.

Asunto(s)

Proteínas de Drosophila , Drosophila melanogaster , Infertilidad Masculina , Espermatogénesis , Animales , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/deficiencia , Espermatogénesis/genética , Mitocondrias/metabolismo , Mitocondrias/genética , Testículo/metabolismo , Meiosis/genética , Espermatogonias/metabolismo , Perfilación de la Expresión Génica , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Espermatocitos/metabolismo , Transcriptoma

RESUMEN

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.

Asunto(s)

Respuesta al Choque Térmico , ARN Interferente Pequeño , Espermátides , Espermatocitos , Testículo , Animales , Masculino , Espermátides/metabolismo , Espermatocitos/metabolismo , ARN Interferente Pequeño/genética , Ratas , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Testículo/metabolismo , Espermatogénesis/genética , Espermatogénesis/fisiología , Fase Paquiteno/genética , Ratas Sprague-Dawley , ARN de Interacción con Piwi

RESUMEN

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.

Asunto(s)

Chaperón BiP del Retículo Endoplásmico , Ratones Noqueados , Proteína Disulfuro Isomerasas , Espermatogénesis , Testículo , Animales , Masculino , Espermatogénesis/genética , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/genética , Ratones , Testículo/metabolismo , Chaperón BiP del Retículo Endoplásmico/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Apoptosis , Espermatocitos/metabolismo , Estrés del Retículo Endoplásmico , Oligospermia/genética , Oligospermia/metabolismo , Oligospermia/patología

RESUMEN

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.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Apoptosis , Basigina , COVID-19 , SARS-CoV-2 , Espermatogonias , Glicoproteína de la Espiga del Coronavirus , Animales , Humanos , Masculino , Ratones , Enzima Convertidora de Angiotensina 2/metabolismo , Apoptosis/fisiología , Basigina/metabolismo , Línea Celular , Proliferación Celular , COVID-19/metabolismo , Espermatocitos/metabolismo , Espermatocitos/virología , Espermatogonias/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo

RESUMEN

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.

Asunto(s)

Centriolos , Segregación Cromosómica , Proteínas Asociadas a Microtúbulos , Centro Organizador de los Microtúbulos , Espermatocitos , Espermatogénesis , Centriolos/metabolismo , Centriolos/genética , Animales , Masculino , Ratones , Espermatogénesis/genética , Espermatocitos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Meiosis/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética

RESUMEN

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.

Asunto(s)

Profase Meiótica I , Serina , Espermatocitos , Animales , Fosforilación , Masculino , Ratones , Serina/metabolismo , Espermatocitos/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Ratones Endogámicos C57BL , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Ratones Noqueados , Cromosomas Sexuales/genética , Cromosomas Sexuales/metabolismo

RESUMEN

As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole's conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4-null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication.

Asunto(s)

Centriolos , Proteínas de Drosophila , Microtúbulos , Espermatocitos , Centriolos/metabolismo , Centriolos/ultraestructura , Centriolos/genética , Animales , Masculino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Espermatocitos/metabolismo , Microtúbulos/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Espermátides/metabolismo , Espermátides/citología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Mutación , Drosophila

RESUMEN

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.

Asunto(s)

Metilación de ADN , Genoma Humano , Espermatogénesis , Humanos , Espermatogénesis/genética , Masculino , Espermátides/metabolismo , Espermatocitos/metabolismo , Elementos Transponibles de ADN/genética , Espermatozoides/metabolismo , Meiosis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Coatomer Protein Complex-II (COPII) mediates anterograde vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Here, we report that the COPII coatomer complex is constructed dependent on a small GTPase, Sar1, in spermatocytes before and during Drosophila male meiosis. COPII-containing foci co-localized with transitional endoplasmic reticulum (tER)-Golgi units. They showed dynamic distribution along astral microtubules and accumulated around the spindle pole, but they were not localized on the cleavage furrow (CF) sites. The depletion of the four COPII coatomer subunits, Sec16, or Sar1 that regulate COPII assembly resulted in multinucleated cell production after meiosis, suggesting that cytokinesis failed in both or either of the meiotic divisions. Although contractile actomyosin and anilloseptin rings were formed once plasma membrane ingression was initiated, they were frequently removed from the plasma membrane during furrowing. We explored the factors conveyed toward the CF sites in the membrane via COPII-mediated vesicles. DE-cadherin-containing vesicles were formed depending on Sar1 and were accumulated in the cleavage sites. Furthermore, COPII depletion inhibited de novo plasma membrane insertion. These findings suggest that COPII vesicles supply the factors essential for the anchoring and/or constriction of the contractile rings at cleavage sites during male meiosis in Drosophila.

Asunto(s)

Vesículas Cubiertas por Proteínas de Revestimiento , Citocinesis , Proteínas de Drosophila , Meiosis , Proteínas de Transporte Vesicular , Animales , Masculino , Cadherinas/metabolismo , Membrana Celular/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Citocinesis/fisiología , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Meiosis/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Espermatocitos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

Asunto(s)

Roturas del ADN de Doble Cadena , Animales , Masculino , Ratones , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Mutación con Pérdida de Función , Mamíferos/metabolismo , Meiosis/genética , Mutación , Espermatocitos/metabolismo , Células Germinativas/metabolismo , Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo