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1.
Development ; 150(3)2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36718792

RESUMO

Spermatogenesis depends on the crosstalk of Sertoli cells (SCs) and germ cells. However, the gene regulatory network establishing the communications between SCs and germ cells remains unclear. Here, we report that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) in SCs is essential for the establishment of crosstalk between SCs and germ cells. Conditional knockout of hnRNPH1 in mouse SCs leads to compromised blood-testis barrier function, delayed meiotic progression, increased germ cell apoptosis, sloughing of germ cells and, eventually, infertility of mice. Mechanistically, we discovered that hnRNPH1 could interact with the splicing regulator PTBP1 in SCs to regulate the pre-mRNA alternative splicing of the target genes functionally related to cell adhesion. Interestingly, we also found hnRNPH1 could cooperate with the androgen receptor, one of the SC-specific transcription factors, to modulate the transcription level of a group of genes associated with the cell-cell junction and EGFR pathway by directly binding to the gene promoters. Collectively, our findings reveal a crucial role for hnRNPH1 in SCs during spermatogenesis and uncover a potential molecular regulatory network involving hnRNPH1 in establishing Sertoli-germ cell crosstalk.


Assuntos
Células de Sertoli , Espermatogênese , Animais , Masculino , Camundongos , Fertilidade/fisiologia , Células Germinativas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Camundongos Knockout , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/genética , Testículo/metabolismo , Fatores de Transcrição/metabolismo
2.
Int J Mol Sci ; 23(4)2022 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-35216057

RESUMO

Protein methyltransferases play various physiological and pathological roles through methylating histone and non-histone targets. Many histone methyltransferases have been reported to regulate the development of spermatogenic cells. However, the specific function of non-histone methyltransferases during spermatogenesis remains unclear. In this study, we found that METTL21A, a non-histone methyltransferase, is highly expressed in mouse testes. In order to elucidate the role of METTL21A in spermatogenesis, we generated a Mettl21a global knockout mouse model using CRISPR/Cas9 technology. Unexpectedly, our results showed that knockout males are fertile without apparent defects in the processes of male germ cell development, including spermatogonial differentiation, meiosis, and sperm maturation. Furthermore, the ablation of METTL21A does not affect the expression and localization of its known targeting proteins in testes. Together, our data demonstrated that METTL21A is not essential for mouse spermatogenesis and male fertility.


Assuntos
Fertilidade/fisiologia , Histona Metiltransferases/metabolismo , Espermatogênese/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Masculino , Meiose/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Espermatogônias/metabolismo , Testículo/metabolismo
3.
Epilepsia ; 59(8): 1621-1630, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30009426

RESUMO

OBJECTIVE: To identify the causative gene of autosomal dominant paroxysmal kinesigenic dyskinesia and benign familial infantile seizures (PKD/BFIS) in a large Chinese family and explore the potential pathogenic mechanism of a PRRT2 (proline-rich transmembrane protein 2) variant. METHODS: Genetic testing was performed via whole exome sequencing. Western blotting and immunofluorescence were used to analyze the protein expression level and subcellular localization of the PRRT2 mutant in HeLa cells and N2A cells. Coimmunoprecipitation was conducted to investigate the interaction of the PRRT2 mutant with syntaxin 1B (STX1B). RESULTS: In a large Chinese family with autosomal dominant PKD/BFIS showing wide phenotypic heterogeneity, including patients suffering from PKD, BFIS, or epilepsy and asymptomatic variant carriers, a c.621dupA variant in PRRT2 was identified in the proband and was shown to cosegregate with the phenotype in this family. This variant results in premature termination at codon 224, producing a truncated protein (p.Ser208Ilefs*17) in which the two conserved hydrophobic segments and the cytoplasmic loop are missing. Both the expression and subcellular localization of PRRT2 are strongly affected by the c.621dupA variant. In addition, we found that PRRT2 directly interacts with STX1B, a SNARE protein critical for neurotransmitter release, whereas the truncated variant p.Ser208Ilefs*17 lacking the helix-loop-helix domain fails to bind to STX1B. SIGNIFICANCE: Our findings identified a PRRT2 variant in a family with PKD/BFIS and confirmed STX1B as a new binding partner of PRRT2, which suggested that the loss of the interaction between PRRT2 and STX1B may contribute to the pathogenesis of PKD/BFIS.


Assuntos
Distonia/genética , Epilepsia Neonatal Benigna/genética , Saúde da Família , Proteínas de Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Sintaxina 1/genética , Adolescente , Adulto , Animais , Povo Asiático , Linhagem Celular Transformada , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Imunoprecipitação , Masculino , Pessoa de Meia-Idade , Transfecção
4.
Am J Hum Genet ; 93(5): 957-66, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24207120

RESUMO

Many ion channel genes have been associated with human genetic pain disorders. Here we report two large Chinese families with autosomal-dominant episodic pain. We performed a genome-wide linkage scan with microsatellite markers after excluding mutations in three known genes (SCN9A, SCN10A, and TRPA1) that cause similar pain syndrome to our findings, and we mapped the genetic locus to a 7.81 Mb region on chromosome 3p22.3-p21.32. By using whole-exome sequencing followed by conventional Sanger sequencing, we identified two missense mutations in the gene encoding voltage-gated sodium channel Nav1.9 (SCN11A): c.673C>T (p.Arg225Cys) and c.2423C>G (p.Ala808Gly) (one in each family). Each mutation showed a perfect cosegregation with the pain phenotype in the corresponding family, and neither of them was detected in 1,021 normal individuals. Both missense mutations were predicted to change a highly conserved amino acid residue of the human Nav1.9 channel. We expressed the two SCN11A mutants in mouse dorsal root ganglion (DRG) neurons and showed that both mutations enhanced the channel's electrical activities and induced hyperexcitablity of DRG neurons. Taken together, our results suggest that gain-of-function mutations in SCN11A can be causative of an autosomal-dominant episodic pain disorder.


Assuntos
Dor/genética , Animais , Povo Asiático/genética , Canais de Cálcio/genética , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Ligação Genética , Marcadores Genéticos , Humanos , Masculino , Camundongos , Repetições de Microssatélites , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/patologia , Dor/patologia , Linhagem , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/genética
5.
Gene ; 893: 147883, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-37839768

RESUMO

Genetic and epigenetic changes in sperm caused by male aging may be essential factors affecting semen parameters, but the effects and specific molecular mechanisms of aging on male reproduction have not been fully clarified. In this study, to explore the effect of aging on male fertility and seek the potential molecular etiology, we performed high-throughput RNA-sequencing in isolated spermatogenic cells, including pachytene spermatocytes (marked by the completion of chromosome synapsis) and round spermatids (produced by the separation of sister chromatids) from the elderly and the young men. Functional enrichment analysis of differentially expressed genes (DEGs) in round spermatids between the elderly and young showed that they were significantly enriched in gamete generation, spindle assembly, and cilium movement involved in cell motility. In addition, the expression levels of DEGs in round spermatids (post-meiotic cells) were found to be more susceptible to age. Furthermore, ten genes (AURKA, CCNB1, CDC20, CCNB2, KIF2C, KIAA0101, NR5A1, PLK1, PTTG1, RAD51AP1) were identified to be the hub genes involved in the regulation of sperm quality in the elderly through Protein-Protein Interaction (PPI) network construction and measuring semantic among GO terms and gene products. Our data provide aging-related molecular alterations in meiotic and post-meiotic spermatogenic cells, and the information gained from this study may explain the abnormal aging-related male fertility decline.


Assuntos
Sêmen , Espermátides , Masculino , Humanos , Idoso , Espermátides/metabolismo , Espermatozoides/metabolismo , Perfilação da Expressão Gênica , Fertilidade/genética , Espermatogênese/genética
6.
Cell Rep ; 43(4): 114113, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38625792

RESUMO

The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility, but the developmental origins of the SSC pool remain unclear. Here, we document that hnRNPU is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, hnRNPU deletion leads to the accumulation of ProSG and disrupts the process of T1-ProSG to T2-ProSG transition. Single-cell transcriptional analyses reveal that germ cells are in a mitotically quiescent state and lose their unique identity upon hnRNPU depletion. We further show that hnRNPU could bind to Vrk1, Slx4, and Dazl transcripts that have been identified to suffer aberrant alternative splicing in hnRNPU-deficient testes. These observations offer important insights into SSC pool establishment and may have translational implications for male fertility.


Assuntos
Espermatogênese , Espermatogônias , Animais , Masculino , Camundongos , Células-Tronco Germinativas Adultas/metabolismo , Processamento Alternativo/genética , Diferenciação Celular , Espermatogênese/genética , Espermatogônias/metabolismo , Espermatogônias/citologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Testículo/metabolismo , Testículo/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo
7.
Genome Biol ; 25(1): 193, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-39030605

RESUMO

BACKGROUND: The mitosis-to-meiosis switch during spermatogenesis requires dynamic changes in gene expression. However, the regulation of meiotic transcriptional and post-transcriptional machinery during this transition remains elusive. RESULTS: We report that methyltransferase-like protein 16 (METTL16), an N6-methyladenosine (m6A) writer, is required for mitosis-to-meiosis transition during spermatogenesis. Germline conditional knockout of Mettl16 in male mice impairs spermatogonial differentiation and meiosis initiation. Mechanistically, METTL16 interacts with splicing factors to regulate the alternative splicing of meiosis-related genes such as Stag3. Ribosome profiling reveals that the translation efficiency of many meiotic genes is dysregulated in METTL16-deficient testes. m6A-sequencing shows that ablation of METTL16 causes upregulation of the m6A-enriched transcripts and downregulation of the m6A-depleted transcripts, similar to Meioc and/or Ythdc2 mutants. Further in vivo and in vitro experiments demonstrate that the methyltransferase activity site (PP185-186AA) of METTL16 is necessary for spermatogenesis. CONCLUSIONS: Our findings support a molecular model wherein the m6A writer METTL16-mediated alternative splicing and translation efficiency regulation are required to control the mitosis-to-meiosis germ cell fate decision in mice, with implications for understanding meiosis-related male fertility disorders.


Assuntos
Adenosina , Processamento Alternativo , Meiose , Metiltransferases , Espermatogênese , Animais , Espermatogênese/genética , Masculino , Metiltransferases/metabolismo , Metiltransferases/genética , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Biossíntese de Proteínas , Camundongos Knockout , Mitose , Testículo/metabolismo , Espermatogônias/metabolismo
8.
J Cell Biol ; 222(5)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36930220

RESUMO

Pachytene piRNA biogenesis is a hallmark of the germline, distinct from another wave of pre-pachytene piRNA biogenesis with regard to the lack of a secondary amplification process known as the Ping-pong cycle. However, the underlying molecular mechanism and the venue for the suppression of the Ping-pong cycle remain elusive. Here, we showed that a testis-specific protein, ADAD2, interacts with a TDRD family member protein RNF17 and is associated with P-bodies. Importantly, ADAD2 directs RNF17 to repress Ping-pong activity in pachytene piRNA biogenesis. The P-body localization of RNF17 requires the intrinsically disordered domain of ADAD2. Deletion of Adad2 or Rnf17 causes the mislocalization of each other and subsequent Ping-pong activity derepression, secondary piRNAs overproduced, and disruption of P-body integrity at the meiotic stage, thereby leading to spermatogenesis arrested at the round spermatid stage. Collectively, by identifying the ADAD2-dependent mechanism, our study reveals a novel function of P-bodies in suppressing Ping-pong activity in pachytene piRNA biogenesis.


Assuntos
RNA de Interação com Piwi , Corpos de Processamento , Masculino , Prófase Meiótica I , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espermatogênese/genética
10.
Front Immunol ; 12: 631044, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33613576

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic has been raging around the world since January 2020. Pregnancy places the women in a unique immune scenario which may allow severe COVID-19 disease. In this regard, the potential unknown effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on mothers and fetuses have attracted considerable attention. There is no clear consistent evidence of the changes in the immune status of pregnant women after recovery from COVID-19. In this study, we use multiparameter flow cytometry and Luminex assay to determine the immune cell subsets and cytokines, respectively, in the peripheral blood and umbilical cord blood from pregnant women recovering from COVID-19 about 3 months (n=5). Our results showed decreased percentages of Tc2, Tfh17, memory B cells, virus-specific NK cells, and increased percentages of naive B cells in the peripheral blood. Serum levels of IL-1ra and MCP-1 showed a decreased tendency in late recovery stage (LRS) patients. Meanwhile, there was no significant difference in immune cell subsets in the umbilical cord blood. The placentas from LRS patients showed increased CD68+ macrophages infiltration and mild hypoxic features. The inflammatory damage of the placenta may be related to the antiviral response. Since the receptors, ACE2 and TMPRSS2, utilized by SARS-CoV-2 are not co-expressed in the placenta, so it is extremely rare for SARS-CoV-2 to cause infection through this route and the impact on the fetus is negligible.


Assuntos
Linfócitos B/imunologia , COVID-19/imunologia , Sangue Fetal/imunologia , Centro Germinativo/imunologia , Placenta/imunologia , SARS-CoV-2/fisiologia , Células Th17/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Autoantígenos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Memória Imunológica , Imunofenotipagem , Células Matadoras Naturais , Gravidez , Receptores de Interleucina-1/metabolismo , Serina Endopeptidases/metabolismo
11.
Theranostics ; 11(20): 10030-10046, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34815802

RESUMO

Background: Sertoli cells are essential regulators of testicular fate in the differentiating gonad; however, its role and underlying molecular mechanism of regulating testicular development in prepubertal testes are poorly understood. Although several critical regulatory factors of Sertoli cell development and function have been identified, identifying extrinsic factors that regulate gonocyte proliferation and migration processes during neonatal testis development remains largely unknown. Methods: We used the Sertoli cell-specific conditional knockout strategy (Cre/Loxp) in mice and molecular biological analyses (Luciferase assay, ChIP-qPCR, RNA-Seq, etc.) in vitro and in vivo to study the physiological roles of hnRNPU in Sertoli cells on regulating testicular development in prepubertal testes. Results: We identified a co-transcription factor, hnRNPU, which is highly expressed in mouse and human Sertoli cells and required for neonatal Sertoli cell and pre-pubertal testicular development. Conditional knockout of hnRNPU in murine Sertoli cells leads to severe testicular atrophy and male sterility, characterized by rapid depletion of both Sertoli cells and germ cells and failure of spermatogonia proliferation and migration during pre-pubertal testicular development. At molecular levels, we found that hnRNPU interacts with two Sertoli cell markers WT1 and SOX9, and enhances the expression of two transcriptional factors, Sox8 and Sox9, in Sertoli cells by directly binding to their promoter regions. Further RNA-Seq and bioinformatics analyses revealed the transcriptome-wide of key genes essential for Sertoli cell and germ cell fate control, such as biological adhesion, proliferation and migration, were deregulated in Sertoli cell-specific hnRNPU mutant testes. Conclusion: Our findings demonstrate an essential role of hnRNPU in Sertoli cells for prepubertal testicular development and testis microenvironment maintenance and define a new insight for our understanding of male infertility therapy.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Células de Sertoli/metabolismo , Proteínas WT1/metabolismo , Animais , Diferenciação Celular/genética , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOXE/metabolismo , Testículo/embriologia , Testículo/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genética , Proteínas WT1/genética
12.
Sci Rep ; 7(1): 17850, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29259219

RESUMO

PiT2 is a member of the inorganic phosphate transporter family, and is extensively expressed in the nervous system. It was found that loop7 domain of PiT2 is not required for retroviral recognition and transport function. The exact functions of loop7 remain poorly understood. Here we show that loop7 of PiT2 is necessary for the transport of PiT2 protein to the cell surface. Further, loop7 is also related to the outgrowth of neurite in Neuro2A cells interacts with the light chain 1 of microtubule-associated protein 1B (MAP1B). PiT2 with mutated MAP1B binding sites affect neurite outgrowth whereas Pi transport function deficient mutants of PiT2 do not. We also show that Drosophila dPiT interacts with microtubule-associated protein Futsch, and dPiT is crucial for the normal development of neuromuscular junctions (NMJs). These results indicate that PiT2 might participate in the regulation of neuronal outgrowth by interacting with MAP1B and independently of its Pi transport function in the nervous system.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Drosophila , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/metabolismo , Junção Neuromuscular/metabolismo , Fosfatos/metabolismo
14.
Nat Genet ; 44(3): 254-6, 2012 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-22327515

RESUMO

Familial idiopathic basal ganglia calcification (IBGC) is a genetic condition with a wide spectrum of neuropsychiatric symptoms, including parkinsonism and dementia. Here, we identified mutations in SLC20A2, encoding the type III sodium-dependent phosphate transporter 2 (PiT2), in IBGC-affected families of varied ancestry, and we observed significantly impaired phosphate transport activity for all assayed PiT2 mutants in Xenopus laevis oocytes. Our results implicate altered phosphate homeostasis in the etiology of IBGC.


Assuntos
Doenças dos Gânglios da Base/genética , Calcinose/genética , Cromossomos Humanos Par 8/genética , Homeostase/genética , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Animais , Povo Asiático , Doenças dos Gânglios da Base/metabolismo , Sequência de Bases , Calcinose/metabolismo , Ligação Genética , Marcadores Genéticos/genética , Homeostase/fisiologia , Humanos , Escore Lod , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Oócitos/metabolismo , Linhagem , Análise de Sequência de DNA , Xenopus laevis
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