Initial response of ovarian tissue transcriptome to vitrification or microwave-assisted dehydration in the domestic cat model

Abstract

Background 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. 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. Results The 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. Conclusions Our 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.

Transcriptome dynamics in developing testes of domestic cats and impact of age on tissue resilience to cryopreservation

“…Thus, getting information on the whole transcriptome changes in the tissue during preservation is essential for understanding the mechanisms of stress response and recovery of cells as a system. For instance, our group recently demonstrated how RNA-seq and resulting functional networks can help to understand the response of ovarian tissue to various stresses caused by preservation [ 34 ].…”

Feline microRNAome in ovary and testis: Exploration of in-silico miRNA-mRNA networks involved in gonadal function and cellular stress response

“…Interestingly, NOTCH2 has been shown to be a direct target of miR-34c with its expression greatly decreased in miR34c transfected cells (Bouhallier et al, 2010). In our study, NOTCH2 paralog NOTCH1 has been predicted as a DE target of several miRNAs from the major cluster, including miR-34b-3p, miR-34c-5p, miR-146a-5p, miR-200c-3p, miR-449-5p, and miR-92b-3p. Tissue preservation protocols, such as vitrification, slowfreezing and recently developed microwave-assisted dehydration, expose cells to non-physiological conditions which may disturb cellular homeostasis and lead to cellular Frontiers in Genetics frontiersin.org stress (Lee et al, 2019;Amelkina and Comizzoli, 2020). Protocols are constantly being optimized to reduce the severity of cellular stress and mitigate any potential damage to proteins, DNA, RNA, and lipids.…”

“…It is also an important biomedical model, carrying a similarity with human genome with high degree of conserved synteny and serving as a model for ∼250 genetic diseases that are analogous to human disorders ( O’Brien et al, 2002 ; Pontius et al, 2007 ; Tamazian et al, 2014 ). Recently, our group has been focusing on elucidating transcriptome dynamics in the ovarian and testicular tissue of domestic cat to better understand molecular mechanisms behind gonadal development and response of tissues to stress caused by preservation protocols ( Amelkina and Comizzoli, 2020 ; Amelkina et al, 2021 ). We have reported major changes in mRNA expression in the ovarian tissue in response to vitrification and microwave-assisted preservation ( Amelkina and Comizzoli, 2020 ), as well as gradual shifts in transcriptome dynamics throughout testicular development ( Amelkina et al, 2021 ).…”

“…Recently, our group has been focusing on elucidating transcriptome dynamics in the ovarian and testicular tissue of domestic cat to better understand molecular mechanisms behind gonadal development and response of tissues to stress caused by preservation protocols ( Amelkina and Comizzoli, 2020 ; Amelkina et al, 2021 ). We have reported major changes in mRNA expression in the ovarian tissue in response to vitrification and microwave-assisted preservation ( Amelkina and Comizzoli, 2020 ), as well as gradual shifts in transcriptome dynamics throughout testicular development ( Amelkina et al, 2021 ). To better understand the processes observed in these gonadal tissues, we need to go further and unravel the mechanisms of gene expression regulation.…”

Long-term preservation of germ cells and gonadal tissues at ambient temperatures

“…The feasibility of applying this drying technique to gonadal tissue has also been explored. Drying of cat ovarian tissue showed limited impact on morphology but altered transcriptional activity and gene expression , Amelkina & Comizzoli 2020. Recently, drying of cat testicular tissues showed that structural integrity and cell viability could be maintained at an acceptable level (Silva et al 2020).…”