A83-01 and DMH1 effects in the zebrafish spermatogonial niche: Unraveling the roles of TGF-β and BMP signaling in the Fsh-mediated spermatogonial fate

Author(s)

da Costa, Daniel Fernandes ; Ribeiro, Amanda de Oliveira ; Ricci, Juliana Morena Bonita ; Rodrigues, Maira da Silva ; de Oliveira, Marcos Antonio ; da Rosa, Ivana Felipe ; Doretto, Lucas Benites ; Nakajima, Rafael Takahiro ; Nobrega, Rafael Henrique

• Document type: Journal article
• Source: Gene; v. 897, p. 12-pg., 2023-12-21.

Abstract

Transforming growth factor-beta (TGF-beta) and bone morphogenetic protein (BMP) signaling has fundamental roles in the regulation of the stem cell niche for both embryonic and adult stem cells. In zebrafish, male germ stem cell niche is regulated by follicle-stimulating hormone (Fsh) through different members of the TGF-beta superfamily. On the other hand, the specific roles of TGF-beta and BMP signaling pathways are unknown in the zebrafish male germ stem cell niche. Considering this lack of information, the present study aimed to investigate the pharmacological inhibition of TGF-beta (A83-01) and BMP (DMH1) signaling pathways in the presence of recombinant zebrafish Fsh using testicular explants. We also reanalyzed single cell-RNA sequencing (sc-RNA-seq) dataset from adult zebrafish testes to identify the testicular cellular sites of smad expression, and to understand the physiological significance of the changes in smad transcript levels after inhibition of TGF-beta or BMP pathways. Our results showed that A83-01 potentiated the pro-stimulatory effects of Fsh on spermatogonial differentiation leading to an increase in the proportion area occupied by differentiated spermatogonia with concomitant reduction of type A undifferentiated (Aund) spermatogonia. In agreement, expression analysis showed lower mRNA levels for the pluripotency gene pou5f3, and increased expression of dazl (marker of type B spermatogonia and spermatocyte) and igf3 (pro-stimulatory growth factor) following the co-treatment with TGF-beta inhibitor and Fsh. Contrariwise, the inhibition of BMP signaling nullified the pro-stimulatory effects of Fsh, resulting in a reduction of differentiated spermatogonia and increased proportion area occupied by type Aund spermatogonia. Supporting this evidence, BMP signaling inhibition increased the mRNA levels of pluripotency genes nanog and pou5f3, and decreased dazl levels when compared to control. The sc-RNA-seq data unveiled a distinctive pattern of smad expression among testicular cells, primarily observed in spermatogonia (smad 2, 3a, 3b, 8), spermatocytes (smad 2, 3a, 8), Sertoli cells (smad 1, 3a, 3b), and Leydig cells (smad 1, 2). This finding supports the notion that inhibition of TGF-beta and BMP signaling pathways may predominantly impact cellular components within the spermatogonial niche, namely spermatogonia, Sertoli, and Leydig cells. In conclusion, our study demonstrated that TGF-beta and BMP signaling pathways exert antagonistic roles in the zebrafish germ stem cell niche. The members of the TGF-beta subfamily are mainly involved in maintaining the undifferentiated state of spermatogonia, while the BMP subfamily promotes spermatogonial differentiation. Therefore, in the complex regulation of the germ stem cell niche by Fsh, members of the BMP subfamily (pro-differentiation) should be more predominant in the niche than those belonging to the TGF-beta (anti-differentiation). Overall, these findings are not only relevant for understanding the regulation of germ stem cell niche but may also be useful for expanding in vitro the number of undifferentiated spermatogonia more efficiently than using recombinant hormones or growth factors. (AU)

• FAPESP's process: 18/16595-7 - Kisspeptins and its role in reproduction and gonadal development in teleost fish.
• Grantee: Juliana Morena Bonita Ricci
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 22/10421-2 - New insights on lncRNAS involved in gonadal maturation of teleosts: basic knowledge and development of biotechnological strategies to delay puberty
• Grantee: Rafael Takahiro Nakajima
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/15793-7 - Effects of hypothyroidism on testicular function of Danio rerio and relation between thyroid hormones and follicle stimulating hormone (Fsh)
• Grantee: Maira da Silva Rodrigues
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 20/15237-0 - Development of testicular organoids and their use for potential sperm maturation in Zebrafish (Danio rerio) and Tambaqui (Colossoma macropomum)
• Grantee: Ivana Felipe da Rosa
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 19/05643-3 - Identification of lncRNAs in germinative and somatic cells in testis of Zebrafish (Danio rerio) and its use as potential biomarkers of enviromental contamination
• Grantee: Lucas Benites Doretto
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 22/10389-1 - New insights on gonadal growth factors of Lambari, Astyanax altiparanae: basic knowledge and development of biotechnological strategies to delay puberty
• Grantee: Amanda de Oliveira Ribeiro
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 14/25313-4 - The role of AMH on sex differentiation and gametogenesis of teleost fish
• Grantee: Marcos Antônio de Oliveira
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 21/06742-5 - New insights on gonadal signals (growth factors and lncRNAs) that regulate gonadal maturation in fish: basic knowledge and development of biotechnological strategies to delay puberty
• Grantee: Rafael Henrique Nóbrega
• Support Opportunities: Research Grants - Young Investigators Grants - Phase 2