Cryopreservation Causes Genetic and Epigenetic Changes in Zebrafish Genital Ridges

Cryopreservation is an important tool routinely employed in Assisted Reproduction Technologies (ARTs) and germplasm banking. For several years, the assessment of global DNA fragmentation seemed to be enough to ensure the integrity of genetic material. However, cryopreservation can produce molecular alterations in key genes and transcripts undetectable by traditional assays, such modifications could interfere with normal embryo development. We used zebrafish as a model to study the effect of cryopreservation on key transcripts and genes. We employed an optimized cryopreservation protocol for genital ridges (GRs) containing primordial germ cells (PGCs) considered one of the best cell sources for gene banking. Our results indicated that cryopreservation produced a decrease in most of the zebrafish studied transcripts (cxcr4b, pou5f1, vasa and sox2) and upregulation of heat shock proteins (hsp70, hsp90). The observed downregulation could not always be explained by promoter hypermethylation (only the vasa promoter underwent clear hypermethylation). To corroborate this, we used human spermatozoa (transcriptionally inactive cells) obtaining a reduction in some transcripts (eIF2S1, and LHCGR). Our results also demonstrated that this effect was caused by freezing/thawing rather than exposure to cryoprotectants (CPAs). Finally, we employed real-time PCR (qPCR) technology to quantify the number of lesions produced by cryopreservation in the studied zebrafish genes, observing very different vulnerability to damage among them. All these data suggest that molecular alterations caused by cryopreservation should be studied in detail in order to ensure the total safety of the technique.