Sadeesh Em1, Meena Kataria, Balhara S, Ps Yadav. 1. Division of Animal Physiology and Reproduction, Central Institute for Research on Buffaloes, Hisar, 125001, India, sadeeshcirb@gmail.com.
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.
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.
Authors: N L Selokar; R A Shah; A P Saha; M Muzaffar; M Saini; M S Chauhan; R S Manik; P Palta; S K Singla Journal: Theriogenology Date: 2012-04-26 Impact factor: 2.740