Throughout in vitro first spermatogenic wave: next-generation sequencing gene expression patterns of fresh and cryopreserved prepubertal mice testicular tissue explants

The in vitro spermatogenesis procedure, once optimized, should be proposed to survival prepubertal patients at high risk for malignant contamination in their testis, for example in case of acute leukaemia. Differentially expressed genes between the in vitro cultured testes and the aged-matched in vivo controls will be explored by a transcriptomic analysis using the RNA-Seq technology (single-read 50 bp, PolyA-enriched) at D0, D4, D16 and D30 of culture using our reference culture medium (one-step culture), and at the same time points after an initial culture step with growth factors (two-step culture). This approach will allow us to identify the signalling pathways or molecular mechanisms that are deregulated in vitro. Moreover, a comparison of transcriptomic analyses to cryopreserved testicular fragments will allow us to identify the potential impact of cryopreservation procedures for fragments after thawing (D0) and explants at the end of culture with our reference culture medium (D30). Overall design: Testicular fragments from 6.5 days postpartum (dpp) mice were cultured from fresh or controlled slow frozen (CSF) testicular tissue. Cultures were stopped at key time points of the first spermatogenic wave: day 4, D16, and D30 for fresh tissue in order to evaluate the impact of in vitro culture and stopped at D30 for frozen-thawed tissue to evaluate the impact of cryopreservation. Testes from mice of 6.5, 10.5, 22.5, and 36.5 dpp correspond to respective age-matched in vivo controls. RNAs from testicular explants (n=3×6 fragments per condition) as well as their respective in vivo controls (n=3×6 fragments per condition) were extracted and analysed by RNA-seq. Biological replicates samples: in vivo 6.5 (fresh: n=3; CSF: n=2), 10.5 (n=3), 22.5 (n=3), 36.5 (n=2) dpp; in vitro D4 (n=3), D16 (n=2), and D30 (fresh tissue: n=3; CSF: n=1).