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Context: Spermatogonial stem cells (SSCs) in adult fish are valuable cells for surrogate production and genetic resources preservation and regeneration in fish. Indeed, SSCs transplanted into recipient fry generate functional spermatozoa or oocytes depending on the sex of the recipient fish, thereby bypassing the inability to cryopreserve fish oocytes or embryos. Amplification of SSCs in culture would help to increase the amount of material available for genetic resource cryopreservation. However, there is still a huge lack of knowledge in fish on the molecular mechanisms involved in the proliferative properties and stemness maintenance of adult SSCs. It has been shown that epigenetic actors maintain the stemness status of the germ stem cell in mammals.  The cryopreservation procedures can alter the epigenetic conformity of the cryopreserved cells. There is a need to evaluate the risk of epigenetic alterations that could occur in cryopreserved SSC.  

Objectives: The objective of MethylStemGerm project is to characterize the DNA methylation pattern of the SSC that is favorable to the maintenance of their stemness properties. The DNA methylation profiles will be determined in SSCs and in differentiated B spermatogonia using RRBS (reduced representation of bisulfite sequencing). In contrast to SSC, differentiated spermatogonia are committed towards germ cell differentiation and do not colonize the gonad of recipient. The localization and identification of differentially methylated CpG sites between SSCs and differentiated spermatogonia will provide information on genomic regions or potential genes involved in the stemness status of the SSC. Additionally, the stability of the DNA methylation pattern of the SSCs after cryopreservation will be assessed, in order to estimate potential alteration of the SSC stemness status or potential epigenetic alteration of the derived gametes.

Expectations: The project will help to characterize the DNA methylation pattern required for the maintenance of the stemness status of cryopreserved or in vitro amplified SSCs. This will give a better mastering of this cell type for cryobanking and for the preservation of the genetic resources in fish.