In recent years, a large number
of studies implicated cryopreservation to increased occurrence
of various forms
of mutations, increased DNA fragmentation and the event
of apoptosis. However, the evidence emerged from such studies is somewhat inconclusive; and the current study argued that analysing the DNA damage response (DDR) from the cryopreserved cells would help characterise the nature
of the putative DNA damage resulting from cryopreservation. To that end, the current study set out to investigate the effects
of different cryopreservation parameters on the genome in the context
of the event
of double strand breaks (DSBs), single strand breaks (SSBs), and various forms
of sequence alteration using 51/4 h post fertilisation (hpf) zebrafish (Danio rerio) embryos as the model organism. The cryopreservation parameters under which the investigation was carried out were chilling
of the embryos at 0 掳C for up to 120 min, treatment
of the embryos with two cryoprotective additives (CPA), MeOH and Me
2SO, for 30 min, and cooling
of the embryos to 鈭?5 掳C and subsequent warming. Assays for DSB-activated DDR proteins and SSB-activated DDR proteins in 51/4 hpf zebrafish (Danio rerio) were developed under the current study. The assays were then utilised to investigate the event
of DSBs and SSBs in the genome
of the embryos treated with different cryopreservation parameters. The study also analysed the expression pr
ofiles
of a set
of genes unique to the base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (MMR) as indicators
of the occurrence
of various forms
of sequence alteration in the genome
of the embryos treated with the cryopreservation parameters. The study found that chilling and CPA treatment did not induce DSBs or SSBs but up-regulated the MMR and BER mechanisms, respectively. CPA treatment also down-regulated NER and MMR mechanisms. Cooling and warming, on the contrary, did not induce DSBs but induced SSBs in the genome, which were repaired when the embryos were provided with a recovery time. Cooling and warming also up-regulated the NER and BER mechanisms in the embryos. The findings
of the study provided important insights into how eukaryotic cells respond to different cryopreservation parameters, which will significantly enhance the knowledge regarding the effects
of cryopreservation on the genome
of biological objects.
Source of funding: Overseas Research Students Awards Scheme (ORSAS) and University of Bedfordshire
Conflict of interest: None declared.