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Journal of Assisted Reproduction and Genetics
Published in: Journal of Assisted Reproduction and Genetics 11/2014

01-11-2014 | Embryo Biology

Expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocytes extract and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized embryos

Authors: Sadeesh EM, Meena Kataria, Balhara S, PS Yadav

Published in: Journal of Assisted Reproduction and Genetics | Issue 11/2014

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Abstract

Purpose

To compare the expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocyte extracts and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized (IVF) embryos.

Methods

Hand-made cloned embryos were produced using oocyte extracts treated donor cells and brilliant cresyl blue (BCB) stained recipient cytoplasts. IVF embryos were produced by culturing 15–20 COCs in BO capacitated sperms from frozen thawed buffalo semen and the mRNA expression patterns of genes implicated in metabolism (GLUT1), pluripotency (OCT4), DNA methylation (DNMT1), pro- apoptosis (BAX) and anti-apoptosis (BCL2) were evaluated at 8- to16- cell stage embryos.

Results

A significantly (P < 0.05) higher number of 8- to16- cell and blastocyst stages (73.9 %, 32.8 %, respectively) were reported in hand-made cloning (HMC) as compared to in vitro fertilization (49.2 %, 24.2 %, respectively). The amount of RNA recovered from 8- to 16- cell embryos of HMC and in vitro fertilization did not appear to be influenced by the method of embryo generation (3.76 ± 0.61 and 3.82 ± 0.62 ng/μl for HMC and in vitro fertilization embryos, respectively). There were no differences in the expression of the mRNA transcripts of genes (GLUT1, OCT4, DNMT1, BAX and BCL2) were analysed by real-time PCR between hand-made cloned and IVF embryos.

Conclusions

Pre-treatment of donor cells with oocyte extracts and selection of developmentally competent oocytes through BCB staining for recipient cytoplast preparations may enhance expression of developmentally important genes GLUT1, OCT4, DNMT1, BAX, and BCL2 in hand-made cloned embryos at levels similar to IVF counterparts. These results also support the notion that if developmental differences observed in HMC and in vitro fertilization produced foetuses and neonates are the results of aberrant gene expression during the pre-implantation stage, those differences in expression are subtle or appear after the maternal to zygotic transition stage of development.
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Metadata
Title
Expression profile of developmentally important genes between hand-made cloned buffalo embryos produced from reprogramming of donor cell with oocytes extract and selection of recipient cytoplast through brilliant cresyl blue staining and in vitro fertilized embryos
Authors
Sadeesh EM
Meena Kataria
Balhara S
PS Yadav
Publication date
01-11-2014
Publisher
Springer US
Published in
Journal of Assisted Reproduction and Genetics / Issue 11/2014
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI
https://doi.org/10.1007/s10815-014-0316-y

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