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Testicular organoids and novel in vitro approaches to study spermatogonial stem cells and spermatogenesis in fish

Grant number: 20/03569-8
Support type:Regular Research Grants
Duration: June 01, 2021 - May 31, 2023
Field of knowledge:Agronomical Sciences - Fishery Resources and Fishery Engineering - Inland Water Fishery Resources
Principal researcher:Rafael Henrique Nóbrega
Grantee:Rafael Henrique Nóbrega
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Assoc. researchers: Hamid Reza Habibi ; Lázaro Wender Oliveira de Jesus ; Luiz Renato de França

Abstract

Testicular organoids are three dimensional clusters derived from testicular cells that exhibit in vitro same cytoarchitecture and functionality of a testis in vivo. To achieve this, the organoids are originated from stem cells (spermatogonial stem cells) and somatic cells that recapitulate the testis morphogenesis, and mimic the complex cellular and molecular interactions present in the organ. Therefore, organoids are powerful tools to study testis development, spermatogenesis per se, cellular interactions, and regulation of the spermatogonial stem cells in their niche. Moreover, testicular organoids can be excellent models for toxicological studies. They can be used for high-throughput screening for drug toxicity, as well as platforms to understand how environmental toxicants affect spermatogenesis. To our knowledge, there are no testicular organoids developed for fish until now. Thus, if developed, this technology will allow a more detailed investigation on fish spermatogenesis, and the effects of environmental contaminants on spermatogonial stem cells. Another interesting tool is the LNA" GapmeRs system to perform in vitro knockdown studies. This technology has been shown to be very efficient to inhibit the expression of specific transcripts. In comparison with knockout technologies (TALENT or CRISPR/CAS9), this method is faster, and can be applied for any target gene without lethality. Thus, LNA" GapmeRs can be a powerful tool to investigate the role of selected target genes on fish spermatogenesis and spermatogonial stem cell regulation. This research proposal, in a first moment, aims to generate 3D testicular organoids using zebrafish and native fish species, such as pirarucu (Arapaima gigas), tambaqui (Colossoma macropomum) and lambari (Astyanax altiparanae). Subsequently, to evaluate the efficiency of the employed method, testicular organoids will be characterized by morphological and functional assays. Once characterized, testicular organoids from zebrafish will be exposed to environmental contaminants to evaluate the effects of theses contaminants on the spermatogonial stem cells. Finally, we will use LNA" GapmeRs system to examine the roles of selected target genes and long non-coding RNAs in the zebrafish spermatogenesis. In summary, this research proposal, to be developed in collaboration with University of Calgary (Canada), Vrije Universiteit Brussel (Belgium) and Federal University of Minas Gerais (UFMG) and Alagoas (UFAL), will allow the development of modern technologies for the study of fish spermatogenesis/spermatogonial stem cells, from functional, toxicological and zootechnical aspects. (AU)