Reproductive Physiology and ETT Laboratory, Veterinary Physiology and Climatology Division, Indian Veterinary Research Institute, Izatnagar, India.
Cell Biol Int. 2010 Mar 29;34(5):463-8. doi: 10.1042/CBI20090295.
The present study was designed to compare the expression profile of two developmentally important genes (HSP-70.1 and GLUT-1) and TCN (total cell number) count in fast (group A) and slow (group B) cleaved buffalo embryos to access their in vitro developmental competence. Buffalo COCs (cumulus oocyte complexes) were collected from local abattoir ovaries and subjected to in vitro maturation in: TCM-199 supplemented with 10% FBS (fetal bovine serum), BSA (3 mg/ml), sodium pyruvate (0.25 mM) and 20 ng/ml EGF (epidermal growth factor) at 38.5 degrees C under 5% CO2. In vitro derived embryos were collected at 4-8, 8-16 cell, morula and blastocyst stages at specific time points for gene expression analysis and total cell count. A semiquantitative RT-PCR (reverse transcriptase-PCR) assay was used to determine the HSP-70.1 and GLUT-1 transcripts. Results showed that developmental competence and TCN count in fast (group A)-cleaving embryos was significantly (P<0.05) higher than in the slow group (group B). The gene transcript of HSP-70.1 and GLUT-1 was expressed in oocytes (immature and mature) and throughout the embryonic developmental stages in the fast group (group A), while in the slow (group B) cleaving embryos, the expression of HSP-70.1 was absent in all the embryonic developmental stages, and expression of GLUT-1 was absent after 8-16 cell stage. In conclusion, TCN count and expression profile of HSP-70.1 and GLUT-1 genes in buffalo embryos are different taking into account the cleavage rate. Quality of such embryos for research purposes, TCN and expression profiling of developmentally important genes should be employed to optimize the in vitro culture system to produce superior quality of embryos.
本研究旨在比较两种发育重要基因（HSP-70.1 和 GLUT-1）和 TCN（总细胞数）在快速（A 组）和慢速（B 组）分裂水牛胚胎中的表达谱，以评估其体外发育能力。从当地屠宰场卵巢中收集水牛 COC（卵丘-卵母细胞复合物），并在 TCM-199 中进行体外成熟，其中含有 10% FBS（胎牛血清）、BSA（3mg/ml）、丙酮酸钠（0.25mM）和 20ng/ml EGF（表皮生长因子），在 38.5°C 下 5%CO2 中培养。在特定时间点收集 4-8、8-16 细胞、桑椹胚和囊胚阶段的体外胚胎，进行基因表达分析和总细胞计数。使用半定量 RT-PCR（逆转录酶-PCR）测定 HSP-70.1 和 GLUT-1 转录本。结果表明，快速（A 组）分裂胚胎的发育能力和 TCN 计数明显（P<0.05）高于慢速组（B 组）。HSP-70.1 和 GLUT-1 基因的转录本在快速组（A 组）的卵母细胞（不成熟和成熟）和整个胚胎发育阶段均有表达，而在慢速组（B 组）的分裂胚胎中，HSP-70.1 的表达在所有胚胎发育阶段均不存在，GLUT-1 的表达在 8-16 细胞阶段后不存在。综上所述，考虑到卵裂率，水牛胚胎的 TCN 计数和 HSP-70.1 和 GLUT-1 基因的表达谱存在差异。为了研究目的，应采用 TCN 和发育重要基因的表达谱来优化体外培养系统，以产生优质胚胎。
