Desiccation and supra‐zero temperature storage of cat germinal vesicles lead to less structural damage and similar epigenetic alterations compared to cryopreservation

2020

BMC Genomics

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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.

Section: Vitrification Vs Dehydration Stresssupporting

confidence: 50%

Section: Introductionmentioning

confidence: 99%

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

Amelkina

2020

BMC Genomics

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“…Global DNA methylation (5‐methylcytosine) level appears not to be affected by lyophilization in rabbit sperm (Mercati et al, 2020). The proportion of cat germinal vesicles with tri‐methylation of histone H3K4me3 (detected by immunostaining) after microwave drying is lower as observed during cryopreservation (P. C. Lee & Comizzoli, 2019). While partial microwave drying of cat testicular tissues showed that histomorphometry (>80%) and viability (>50%) of seminiferous tubules can be preserved (Silva et al, 2020), no epigenetic or transcriptional data are reported yet.…”

Section: Damages and Stresses On Biological Structures And Functions ...mentioning

confidence: 94%

“…Similarly, percentages of cat oocytes' nuclei (germinal vesicles) with intact DNA (comet assay) after air drying are lower (Graves‐Herring et al, 2013), but not as much as what cryopreservation triggers. Regarding nuclear envelopes and chromatin configuration, structural damages are not extensive in microwave‐dried germinal vesicles compared to cryopreservation in the domestic cat (P. C. Lee & Comizzoli, 2019). DNA integrity (via TUNEL assay) after partial microwave‐assisted dehydration has also been reported in ovarian and testicular tissues, in which about 50% of cells had intact DNA (P.‐C.…”

Section: Damages and Stresses On Biological Structures And Functions ...mentioning

confidence: 99%

Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation

Molecular Reproduction Devel

Amelkina

Lee

2022

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Long‐term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long‐term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long‐term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.

“…Our aim in the current study was to validate and further characterize candidate proteins that can serve as markers of competent oocytes. Identification of these markers could help expand our understanding of oocyte development and be applied to improve ART by using them to assess the efficacy of female fertility preservation methods, especially ones focusing on preservation of the GV alone ( Graves-Herring et al, 2013 ; Elliott et al, 2015 ; Lee and Comizzoli, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Oocyte Meiotic Competence in the Domestic Cat Model: Novel Roles for Nuclear Proteins BRD2 and NPM1

Chavez

Lee

2021

Front. Cell Dev. Biol.

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To participate in fertilization and embryo development, oocytes stored within the mammalian female ovary must resume meiosis as they are arrested in meiotic prophase I. This ability to resume meiosis, known as meiotic competence, requires the tight regulation of cellular metabolism and chromatin configuration. Previously, we identified nuclear proteins associated with the transition from the pre-antral to the antral follicular stage, the time at which oocytes gain meiotic competence. In this study, the objective was to specifically investigate three candidate nuclear factors: bromodomain containing protein 2 (BRD2), nucleophosmin 1 (NPM1), and asparaginase-like 1 (ASRGL1). Although these three factors have been implicated with folliculogenesis or reproductive pathologies, their requirement during oocyte maturation is unproven in any system. Experiments were conducted using different stages of oocytes isolated from adult cat ovaries. The presence of candidate factors in developing oocytes was confirmed by immunostaining. While BRD2 and ASRGL1 protein increased between pre-antral and the antral stages, changes in NPM1 protein levels between stages were not observed. Using protein inhibition experiments, we found that most BRD2 or NPM1-inhibited oocytes were incapable of participating in fertilization or embryo development. Further exploration revealed that inhibition of BRD2 and NPM-1 in cumulus-oocyte-complexes prevented oocytes from maturing to the metaphase II stage. Rather, they remained at the germinal vesicle stage or arrested shortly after meiotic resumption. We therefore have identified novel factors playing critical roles in domestic cat oocyte meiotic competence. The identification of these factors will contribute to improvement of domestic cat assisted reproduction and could serve as biomarkers of meiotically competent oocytes in other species.