Identification of germ cell-intrinsic players (gdnf and long non-coding rnas) involved in the spermatogonial stem cell fate of adult zebrafish
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2023-06-23
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Universidade Estadual Paulista (Unesp)
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A espermatogênese é um processo controlado por células-tronco no qual uma única célula-tronco espermatogonial (SSC) se diferencia em muitos espermatozoides haploides. A divisão de uma SSC pode resultar em duas células tronco (divisão simétrica), ou em uma célula tronco e outra diferenciada (divisão assimétrica). Independentemente do tipo de divisão celular, o processo de auto-renovação e diferenciação é controlado por numerosos sinais provenientes do tecido somático circundante, assim como também de fatores intrínsecos da própria SSC. Nesse sentido, estudos recentes mostraram que RNAs não codificantes (microRNAs, cirRNAs, lncRNAs) podem desempenhar um papel fundamental na reprodução, em particular na regulação da determinação sexual, diferenciação sexual e gametogênese. Entre os RNAs não codificantes, os lncRNAs se destacam por desempenhar papel regulatório na transcrição de "genes mestres" envolvidos em vários processos biológicos, incluindo a diferenciação e manutenção da pluripotência de células-tronco. Considerando esse contexto, este estudo teve como objetivo entender a sinalização Gdnf-Gfrα1 em testículos de zebrafish combinando abordagens in vivo, in silico e ex vivo, além dos efeitos biológicos do Fsh em lncRNAs testiculares. Nossos dados revelaram que o Gdnf, fator derivado de célula germinativa, está envolvido na manutenção da pluripotência das células germinativas por meio da criação de nichos espermatogoniais, dando suporte ao desenvolvimento de cistos espermatogônicos e inibindo a diferenciação tardia de espermatogônias de maneira autócrina e parácrina. Além disso, neste estudo, identificamos 5161 novos lncRNAs dos quais 76 foram diferencialmente regulados pelo rzfFsh. Além disso, análises de enriquecimento das DEGs demonstrou que esses transcritos estão amplamente relacionados à sinalização e sistemas orgânicos, como o sistema endócrino. Quando focamos nos mRNAs, encontramos 270 DEGs, sendo 174 “up-regulated” 96 “down-regulated”. Interessantemente, as vias mais enriquecidas estavam relacionadas à sinalização do hormônio da tireoide e esteroidogênese. Por fim, predições de interação entre lncRNAs-mRNA-proteínas mostraram que alguns lncRNAs podem interagir, e consequentemente, modular a expressão de genes de pluripotência de SSCs, como o pou5f3, nanos3 e nanog. Em conjunto, nossos dados indicam uma provável atividade do Fsh em criar um ambiente mais favorável para a diferenciação de espermatogônias tronco.
Spermatogenesis is a process controlled by stem cells in which a single spermatogonial 46 stem cell (SSC) differentiates into many haploid spermatozoa. The division of SSC follows either 47 the asymmetric model, where one stem cell produces a differentiated cell and a stem cell (self-48 renewal), or the symmetric model, where one stem cell produces two differentiated cells or two 49 stem cells. Regardless of the model, the process of self-renewal and differentiation is regulated by 50 numerous signals from the surrounding somatic tissue (the stem cell niche) and the germline stem 51 cells themselves. Additionally, recent studies have shown that non-coding RNAs (microRNAs, 52 circRNAs, lncRNAs) from the germline niche itself can play a crucial role in reproduction, 53 particularly in the regulation of sexual determination, sexual differentiation, and gametogenesis. 54 Among the non-coding RNAs, lncRNAs stand out for their regulatory role in the transcription of 55 "master genes" involved in various biological processes, including the differentiation and 56 maintenance of stem cell pluripotency. In this context, this study aimed to understand the Gdnf-57 Gfrα1 signaling in zebrafish testes by combining in vivo, in silico, and ex vivo approaches, as well 58 as the biological effects of Fsh on testicular lncRNAs. Our data revealed that Gdnf, now a germ 59 cell-derived factor, is involved in maintaining germline stem cell pluripotency through niche 60 creation, supporting the development of spermatogonial cysts and inhibiting late spermatogonial 61 differentiation in an autocrine and paracrine manner. Furthermore, in this study, we identified 5161 62 novel lncRNAs and 76 DEGs under the influence of rzfFsh, of which 46 were upregulated and 30 63 were downregulated. Additionally, enrichment analyses of the DEGs demonstrated that these 64 transcripts are broadly related to signaling and organ systems, such as the endocrine system. When 65 focusing on mRNAs, we found 270 DEGs, with 174 upregulated and 96 downregulated. 66 Interestingly, the most enriched pathways were related to thyroid hormone signaling and 67 steroidogenesis. Finally, predictions of interaction between lncRNAs-mRNAs-proteins showed 68 that some lncRNAs can interact, and consequently, modulate the expression of pluripotency genes 69 in SSCs, such as pou5f3, nanos3, and nanog. Collectively, our data indicate a likely activity of Fsh 70 in creating a more favorable environment for spermatogonial stem cell differentiation.
Spermatogenesis is a process controlled by stem cells in which a single spermatogonial 46 stem cell (SSC) differentiates into many haploid spermatozoa. The division of SSC follows either 47 the asymmetric model, where one stem cell produces a differentiated cell and a stem cell (self-48 renewal), or the symmetric model, where one stem cell produces two differentiated cells or two 49 stem cells. Regardless of the model, the process of self-renewal and differentiation is regulated by 50 numerous signals from the surrounding somatic tissue (the stem cell niche) and the germline stem 51 cells themselves. Additionally, recent studies have shown that non-coding RNAs (microRNAs, 52 circRNAs, lncRNAs) from the germline niche itself can play a crucial role in reproduction, 53 particularly in the regulation of sexual determination, sexual differentiation, and gametogenesis. 54 Among the non-coding RNAs, lncRNAs stand out for their regulatory role in the transcription of 55 "master genes" involved in various biological processes, including the differentiation and 56 maintenance of stem cell pluripotency. In this context, this study aimed to understand the Gdnf-57 Gfrα1 signaling in zebrafish testes by combining in vivo, in silico, and ex vivo approaches, as well 58 as the biological effects of Fsh on testicular lncRNAs. Our data revealed that Gdnf, now a germ 59 cell-derived factor, is involved in maintaining germline stem cell pluripotency through niche 60 creation, supporting the development of spermatogonial cysts and inhibiting late spermatogonial 61 differentiation in an autocrine and paracrine manner. Furthermore, in this study, we identified 5161 62 novel lncRNAs and 76 DEGs under the influence of rzfFsh, of which 46 were upregulated and 30 63 were downregulated. Additionally, enrichment analyses of the DEGs demonstrated that these 64 transcripts are broadly related to signaling and organ systems, such as the endocrine system. When 65 focusing on mRNAs, we found 270 DEGs, with 174 upregulated and 96 downregulated. 66 Interestingly, the most enriched pathways were related to thyroid hormone signaling and 67 steroidogenesis. Finally, predictions of interaction between lncRNAs-mRNAs-proteins showed 68 that some lncRNAs can interact, and consequently, modulate the expression of pluripotency genes 69 in SSCs, such as pou5f3, nanos3, and nanog. Collectively, our data indicate a likely activity of Fsh 70 in creating a more favorable environment for spermatogonial stem cell differentiation.
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Fsh, gfra1, gdnf, Spermatogenesis, Spermatogonia stem cell, Zebrafish