RESUMO
Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.
Assuntos
Masculino , Camundongos , Animais , Reprogramação Celular/genética , Tetraploidia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Metilação de DNA , Espermatogônias/metabolismo , Células Germinativas/metabolismoRESUMO
With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
Assuntos
Animais , Masculino , Cabras , Células-Tronco , Espermatogônias/metabolismo , Proliferação de Células , Citometria de Fluxo , Testículo/metabolismoRESUMO
Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.
Assuntos
Camundongos , Masculino , Animais , Espermatogônias/metabolismo , Meiose/genética , Diferenciação Celular , Tretinoína/farmacologia , Mitose , Testículo/metabolismoRESUMO
Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.
Assuntos
Adulto , Humanos , Masculino , Diferenciação Celular , Proliferação de Células , Fatores de Transcrição Forkhead/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismoRESUMO
The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.
Assuntos
Animais , Masculino , Camundongos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Infertilidade Masculina , Meiose/genética , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Ligação a Fosfato/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Rad51 Recombinase/genética , Reação em Cadeia da Polimerase em Tempo Real , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismoRESUMO
Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility. However, the exact mechanisms underlying the behavior of spermatogonia, including spermatogonial stem cell (SSC) self-renewal and spermatogonial proliferation and differentiation, are not fully understood. Recent studies demonstrated that the mTOR complex 1 (mTORC1) signaling pathway plays a crucial role in spermatogonial development, but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined. In this study, we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia. The Mtor knockout (KO) mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age. These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre. Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice, suggesting that spermatogonial differentiation was inhibited. Spermatogonial proliferation was also impaired in Mtor KO mice, leading to a diminished spermatogonial pool and total germ cell population. Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.
Assuntos
Animais , Masculino , Camundongos , Proliferação de Células/genética , Fertilidade/genética , Camundongos Knockout , Espermatogênese/genética , Espermatogônias/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Testículo/metabolismoRESUMO
Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.
Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.
Assuntos
Animais , Masculino , Camundongos , Espermatogônias/citologia , Espermatogônias/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Poliésteres/química , Diferenciação Celular/genética , Sobrevivência Celular , Imunofluorescência , Proliferação de Células/genética , Alicerces Teciduais , Nanofibras/química , Reação em Cadeia da Polimerase em Tempo Real , Animais Recém-NascidosRESUMO
While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2-A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.
Assuntos
Animais , Masculino , Camundongos , Diferenciação Celular , Imunofluorescência , Regulação da Expressão Gênica/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteínas Proto-Oncogênicas c-kit/genética , Túbulos Seminíferos/citologia , Espermatogênese , Espermatogônias/metabolismo , Testículo/citologia , Fixação de Tecidos , Fatores de Transcrição/genéticaRESUMO
Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.
Assuntos
Animais , Masculino , Ratos , Proteínas de Fluorescência Verde , Lentivirus , Ratos Transgênicos , Espermatogônias/metabolismoRESUMO
To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.
Assuntos
Animais , Masculino , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Camundongos Knockout , Espermatogênese/genética , Espermatogônias/metabolismo , Testículo/metabolismoRESUMO
Earlier studies have shown that of the four genes (Hsp60A, Hsp60B, Hsp60C, Hsp60D genes) predicted to encode the conserved Hsp60 family chaperones in Drosophila melanogaster, the Hsp60A gene (at the 10A polytene region) is expressed in all cell types of the organism and is essential from early embryonic stages, while the Hsp60B gene (at 21D region) is expressed only in testis, being essential for sperm individualization. In the present study, we characterized the Hsp60C gene (at 25F region), which shows high sequence homology with the other three Hsp60 genes of D. melanogaster. In situ hybridization of Hsp60C-specific riboprobe shows that expression of this gene begins in late embryonic stages (stage 14 onwards), particularly in the developing tracheal system and salivary glands; during larval and adult stages, it is widely expressed in many cell types but much more strongly in tracheae and in developing and differentiating germ cells. A P-insertion mutant (Hsp60C(1)) allele with the P transposon inserted at -251 position of the Hsp60C gene promoter was generated. This early larval recessive lethal mutation significantly reduces levels of Hsp60C transcripts in developing tracheae and this is associated with a variety of defects in the tracheal system, including lack of liquid clearance. About 10% of the homozygotes survive as weak, shortlived and completely sterile adults. Testes of the surviving mutant males are significantly smaller, with fewer spermatocytes, most of which do not develop beyond the round spermatid stage. In situ and Northern hybridizations show significantly reduced levels of the Hsp60C transcripts in Hsp60C(1) homozygous adult males. The absence of early meiotic stages in the Hsp60C(1) homozygous testes contrasts with the effect of testis-specific Hsp60B (21D) gene, whose mutation affects individualization of sperm bundles later in spermiogenesis. In view of the specific effects in tracheal development and in early stages of spermatogenesis, it is likely that, besides its functions as a chaperone, Hsp60C may have signalling functions and may also be involved in cation transport across the developing tracheal epithelial cells.
Assuntos
Sequência de Aminoácidos , Animais , Chaperonina 60/genética , Proteínas de Drosophila/genética , Feminino , Fertilidade/genética , Genes Recessivos , Homozigoto , Hibridização In Situ , Larva/genética , Masculino , Dados de Sequência Molecular , Mutação , Homologia de Sequência , Espermatócitos/metabolismo , Espermatogônias/metabolismoRESUMO
La fisiología del envejecimiento implica cambios morfológicos que interfieren con la función testicular normal. El hombre senil mantiene la potencia fecundante, aunque su eficacia disminuye. Posiblemente, asociada a una presentación histopatológica testicular. Se utilizaron muestras de tejidos testicular de 3 individuos seniles (69 años), sometidos a orquiectomía terapéutica y de un joven adulto (25 años). Fallecido por causa desconocida. Las gónadas fueron procesadas por técnicas histológicas para Hematoxina-P.A.S. Al microscopio se evaluaron la morfometría, celularidad e histopatología del epitelio seminífero. En los resultados se obtuvo una reducción del diámetro tubular y altura del epitelio seminífero en los individuos seniles, al ser comparados con el individuo joven. Paralelamente, en los testículos de individuos seniles se describio una drástica disminución en el número de células de De Sertoli, y un poco menor para espermatogonias tipo A oscuras, A claras y B; con una relación gonia/Sertori alterada. Asociado a lo anterior, se encontró un porcentaje reducido de espermátidas redondas y alargadas y de espermatozoides, en lumen por túbulo. La evaluación histopatológica en los individuos seniles reveló un epitelio seminífero severamente dañado, con presencia de vacuolización, discontinuidad del epitelio y detención de la espermatogénesis. Por lo tanto, se concluye la existencia de un notorio efecto adverso del progreso de la edad hacia senil, en la estructura histológica testicular, involucrando las diferentes poblaciones celulares y la relación entre ellas, como también la integridad del epitelio seminífero