192 EFFECT OF DIFFERENT CULTURE MEDIUM ON THE PRODUCTION OF GRANULOSA SCNT EMBRYOS IN WATER BUFFALO (BUBALUS BUBALIS) AND THEIR mRNA TRANSCRIPT ANALYSIS AT DEVELOPMENTAL STAGES IN COMPARISON WITH IN VITRO FERTILIZATION-PRODUCED EMBRYOS

Abstract

Success of NT is as low as 0.2 to 3.4% in cloned offspring, and many cloned embryos arrest development before implantation. Expression patterns of mRNA in NT embryos at critical time points play an important role in epigenetic reprogramming and are affected by external factors (Dean et al. 2001 PNAS 98, 13 734–13 738). Embryo culture system can affect the silencing or activations of a particular gene at fetal development stages (Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901). The study was aimed to analyze the expression of genes responsible for growth and embryogenesis (IGF1, IGF2, IGF1R & IGF2R), cellular metabolism (Glut1), gap junction (Cx43) with internal standard (GAPDH) in SCNT embryos in TCM199+FBS, TCM199+PVA and CR1aa+BSA culture media. Passage-5 granulosa cells were used as donor cells after analyzing their proliferation, senescence and ploidy levels. IVM oocytes after nuclear transfer were activated electrically (1.5 kV cm^–2, 15 μs) and chemically (ionomycin, 6DMAP, CHX & CytoB) and were cultured up to blastocyst stage. For control IVM oocytes were fertilized with BO medium capacitated frozen semen in TCM199+10%FBS. The cDNA was prepared from single 2, 4, 8, 16-cell, morula and blastocyst embryos using cell to cDNA kit (Ambion). Relative expression of candidate genes was quantified using real-time PCR with ΔΔCT method. Data was analyzed for one-way ANOVA and Post-Hoc Duncan multiple range tests at P ≤ 0.05 level of significance. Blastocyst production was significantly different among groups with the greatest rate (22.36%) in TCM199+FBS than in TCM199+PVA (15.6%) & CR1aa+BSA (19.21%). Cx43 expression was normal in TCM199+FBS as compared with other media. This could be related to modifications in intercellular communication between other connexin proteins in serum. Glut1 level was abnormal in all NT embryo culture groups. Glucose transport efficiency increased in response to glucose starvation in mediums and can be related to greater Glut1 expression (Gardner and Kaye 1995 Reprod. Fertil. Dev. 7, 41–50). IGF1 & IGF2 were up regulated and IGF1R & IGF2R were down regulated in all NT embryo culture groups than in IVF. The temporal and spatial expression of these transcripts determined by real-time software (Stratagene) was significantly affected by the presence of exogenous protein in medium. IGF1R & IGF2R transcripts showed aberrant reprogramming of donor cells, which affect chromatin remodeling, and is highly correlated to imprinted genes (Giraldo et al. 2008 Biol. Reprod. 78, 832–840). In TCM199+PVA & CR1aa+BSA regulation of all transcripts was more aberrant than TCM199+BSA as compared with IVF embryos. Altered mRNA levels in NT embryos at certain stages are indicative of their vulnerability to grow probably due to suboptimal culture conditions. To improve the efficiency and production of healthy clones, expression analysis of important gene in preimplantation embryos can be correlated with developmental competence of SCNT embryos before transfer in surrogates.