BACKGROUND: Recent studies have demonstrated that prolonged sperm storage adversely affects offspring through epigenetics, yet its broader effects on other molecular levels such as transcription and proteomics in progeny have been rarely explored. RESULTS: We employed comprehensive multi-omics approaches to uncover storage-induced epigenetic changes in sperm and their effects on embryonic development and offspring health. Sperm from common carp (Cyprinus carpio) was stored in vitro in artificial seminal plasma for 14 days, and the impacts of storage on functional properties of sperm and progeny development were investigated. We combined DNA methylome, transcriptomic and proteomic data to elucidate the potential mechanisms by which sperm storage influences progeny development. Prolonged in vitro storage significantly reduced sperm motility and fertilising ability which coincided with changes in the DNA methylation pattern. Integrated analyses of the offspring DNA methylome, comparative transcriptomics and cardiac performance measurements revealed storage-induced alterations of genes associated with nervous system development, myocardial morphogenesis and cellular responses to stimuli. Proteomic analyses showed that in addition to visual perception and nervous system function, pathways of the immunity system were also enriched. Results provide strong evidence of the epigenetic inheritance of the offspring's performances when short-term stored sperm was used for fertilisation. CONCLUSIONS: Short-term sperm storage induces heritable molecular and phenotypic changes in offspring, raising concerns over the potential intergenerational consequences of assisted reproductive practices in aquaculture and possibly other vertebrates.

• Keywords
• Epigenetic inheritance, Epigenetics, Fish sperm, Offspring development, Sperm ageing,
• MeSH
• Embryonic Development * MeSH
• Epigenesis, Genetic MeSH
• Carps * genetics   physiology   embryology   MeSH
• DNA Methylation MeSH
• Multiomics MeSH
• Proteomics MeSH
• Spermatozoa * physiology   metabolism   MeSH
• Transcriptome MeSH
• Semen Preservation * adverse effects   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

• MeSH
• Chromatin drug effects   genetics   MeSH
• Dimethyl Sulfoxide pharmacology   MeSH
• DNA Breaks, Double-Stranded drug effects   MeSH
• Fibroblasts MeSH
• Cryopreservation methods   MeSH
• Cryoprotective Agents pharmacology   MeSH
• Skin cytology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• S Phase drug effects   MeSH
• Cell Survival drug effects   genetics   MeSH
• Freezing adverse effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Chromatin MeSH
• Dimethyl Sulfoxide MeSH
• Cryoprotective Agents MeSH