Abstract
BackgroundLong term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Compared to single cell preservation, optimization of protocols for tissues is highly complex because of the diversity of cells responding differently to non-physiological conditions. Using the prepubertal domestic cat as a model, the objective was to study immediate effects of vitrification or microwave-assisted dehydration on the global transcriptome dynamics in the ovarian cortex. RNA sequencing was performed on ovarian tissues (n = 6 individuals) from different conditions: fresh tissue after dissection (F), vitrified/warmed tissue (V), tissue dehydrated for 5 min (D5) or 10 min (D10) followed by rehydration. Differential gene expression analysis was performed for comparison pairs V vs. F, D10 vs. F, D5 vs. F and D10 vs. D5, and networks were built based on results of functional enrichment and in silico protein-protein interactions.ResultsThe impact of the vitrification protocol was already measurable within 20 min after warming and involved upregulation of the expression of seven mitochondrial DNA genes related to mitochondrial respiration. The analysis of D10 vs. F revealed, 30 min after rehydration, major downregulation of gene expression with enrichment of in silico interacting genes in Ras, Rap1, PI3K-Akt and MAPK signaling pathways. However, comparison of D5 vs. F showed negligible effects of the shorter dehydration protocol with two genes enriched in Ras signaling. Comparison of D10 vs. D5 showed downregulation of only seven genes. Vitrification and dehydration protocols mainly changed the expression of different genes and functional terms, but some of the differentially expressed genes formed a major in silico protein-protein interaction cluster enriched for mitochondrial respiration and Ras/MAPK signaling pathways.ConclusionsOur results showed, for the first time, different effects of vitrification and microwave-assisted dehydration protocols on the global transcriptome of the ovarian cortex (using the domestic cat as a biomedical model). Acquired data and networks built on the basis of differentially expressed genes (1) can help to better understand stress responses to non-physiological stresses and (2) can be used as directions for future preservation protocol optimizations.
Highlights
Long term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes
RNA sequencing (RNA-seq) was performed on groups representing different state of ovarian tissue: fresh tissue after ovary dissection, tissue subjected to vitrification and warming, tissue subjected to microwave-assisted dehydration and rehydration
Summary of the acquired dataset Ovaries from six prepubertal domestic cats were each divided into four groups: fresh tissue after ovary dissection (F), tissue subjected to vitrification and warming (V), tissue subjected to microwave-assisted dehydration for 5 min (D5) or 10 min (D10) with subsequent rehydration (Fig. 1)
Summary
Long term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Ovarian cortical preservation can potentially be performed at any age or reproductive status of females, making it a valuable biomaterial in biomedical science [2] and in conservation of rare and endangered genotypes [3]. Two methods of ovarian tissue preservation have been extensively developed: slow-freezing and vitrification [4]. The procedure of vitrification has been optimized by increasing the viscosity of the solution, which prevents the crystal formation and avoids cellular damage [5]. Protocols have been optimized to avoid this issue, e.g., by adjusting the concentration of CPAs or by exposing samples to low temperature before vitrification to decrease the rate of potential energy and cell metabolism, making molecules less mobile and more stagnant but still disordered [7, 8]
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