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Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

ABSTRACT: Cryopreservation has a negative effect on the quality of oocytes and may be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. The purpose of the present study was to evaluate the detrimental effects on the developmental competence of somatic cell nuclear transferred (SCNT) mouse embryos using vitrified (cryopreserved) oocytes and to evaluate the recovery effects of melatonin on cryo-damage in cloned embryos. Development of SCNT embryos using cryopreserved oocyte cytoplasm (SCNT-CROC) was inferior to those using fresh cytoplasm (SCNT-FOC). Using RNA-sequencing analysis, we found upregulation of eight pro-apoptotic-related genes (Cyct, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, and Pmaip) in the SCNT-CROC group. Furthermore, the addition of melatonin, an agent that reduces apoptosis and ROS production, enhanced blastocyst formation rates in the SCNT-CROP group when compared with the melatonin-untreated group. Additionally, melatonin treatment increased the derivation efficiency of pluripotent stem cells from cloned embryos using cryopreserved oocyte.

SUBMITTER: Lee AR

PROVIDER: S-EPMC6411484 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

Lee Ah Reum AR Hong Kwonho K Choi Seo Hye SH Park Chanhyeok C Park Jae Kyun JK Lee Jin Il JI Bang Jae Il JI Seol Dong-Won DW Lee Jeoung Eun JE Lee Dong Ryul DR

Stem cell reports 20190221 3

Cryopreservation has a negative effect on the quality of oocytes and may be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. The purpose of the present study was to evaluate the detrimental effects on the developmental competence of somatic cell nuclear transferred (SCNT) mouse embryos using vitrified (cryopreserved) oocytes and to evaluate the recovery effects of melatonin on cryo-damage in cloned embryos. Development of SCNT embryos using cryopres ...[more]

PMID: 30799275

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Project description:The mammalian oocyte has the unique feature of supporting fertilization and normal development while being able of reprogramming the nuclei of somatic cells towards pluripotency and occasionally even totipotency. Whilst oocyte quality is known to decay with somatic aging it is not a given that different biological functions decay concurrently. In this study we tested whether oocyte reprogramming ability decreases with somatic aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT) at levels similar to ooplasts of young donors. Despite differences in transcriptome between ooplasts of old and young mice gene expression profiles of the resultant SCNT blastocysts were very similar. Furthermore albeit results showing global down-regulation of apoptosis-related genes in climacteric ooplasts and the reported beneficial effect of inhibiting p53 on transcription factor-induced (iPS) pluripotency reprogramming capacity of young ooplasts was not affected by specifically blocking p53. Our observations strongly suggest that the outcome of oocyte-induced reprogramming is determined by the availability of intrinsic reprogramming factors tightly regulated throughout aging as well as intracellular and environmental checkpoints during pre-implantation development towards selection of the successfully reprogrammed embryos. While oocytes are not regenerated but rather last for life we further propose that these cells can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction. Total mRNA was isolated for samples from young and climacteric mice (pools of 20 ooplasts and 20 SCNT blastocysts in duplicate) using a RNeasy Mini Kit (Qiagen Valencia CA USA) as described by the manufacturer (no DNase treatment). RNA concentrations as well as purity and integrity check were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit (Agilent Technologies Palo Alto CA USA). RiboAmp HS Plus Amplification Kit (MDS Analytical Technologies Ismaning Germany) was used to amplify the total mRNA with two rounds of amplification according to the manufacturerâ??s instructions. Amplified RNA was eluted with 15 ÂµL RE Buffer included in the RiboAmp HS Plus Amplification Kit. RNA concentrations were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit. Turbo Labeling CY3 Kit (MDS Analytical Technologies) was used to label 3 Âµg of amplified RNA with Cy3. Concentration and frequency of incorporation were measured with NanoPhotometer (Implen). Microarray wash and detection of the labeled RNA on GeneChips were carried out according to the manufacturerâ??s instructions (Agilent Technologies). Gene expression profiling was performed using Agilent Whole Mouse Genome Oligo Microarrays (4 x 44k each array containing 41174 features). Array image acquisition and feature extraction was performed using Agilent G2505B Microarray Scanner and Feature Extraction software v.9.5 (Agilent Technologies). 6 samples were analyzed. YoungOoplasts: young ooplasts 1 biological rep ClimactericOoplasts: climacteric ooplasts 1 biological rep YoungBlastocysts: young blastocysts 2 biological rep ClimactericBlastocysts: climacteric blastocysts 2 biological rep

Dataset Information

Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

Publications

Anti-apoptotic Regulation Contributes to the Successful Nuclear Reprogramming Using Cryopreserved Oocytes.

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