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Testicular organoids and knock-down in vitro via GAPMERS as tools to study the spermatogonial stem cells in zebrafish (Danio rerio)

Grant number: 19/26306-5
Support type:Scholarships abroad - Research
Effective date (Start): October 01, 2020
Effective date (End): January 31, 2021
Field of knowledge:Agronomical Sciences - Fishery Resources and Fishery Engineering - Inland Water Fishery Resources
Principal Investigator:Rafael Henrique Nóbrega
Grantee:Rafael Henrique Nóbrega
Host: Hamid Reza Habibi
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
Local de pesquisa : University of Calgary, Canada  

Abstract

Testicular organoids are in vitro three dimensional clusters derived from testicular cells. These clusters exhibit the 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 models 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 also about 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 in vitro expression of specific transcripts. In comparison to 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 as model. Subsequently, to evaluate the efficiency of the employed method, testicular organoids will be characterized by morphological and functional assays. Once characterized, testicular organoids will be exposed to environmental contaminants to evaluate the effects of theses contaminants on the zebrafish spermatogonial stem cells. Finally, we will use LNA" GapmeRs system to examine the roles of selected target genes in the zebrafish spermatogenesis. In summary, this research proposal, to be developed in the University of Calgary, will allow the development of modern technologies for the study of fish spermatogenesis/spermatogonial stem cells, from functional and toxicological aspects. Furthermore, this will be an excellent opportunity for learning, strengthen the existing collaborative research with Dr. Hamid Habibi, and for further applying these new technologies in Brazil.