Bhardwaj Rahul, Ansari Mohd Matin, Pandey Sriti, Parmar Mehtab S, Chandra Vikash, Kumar G Sai, Sharma G Taru
Reproductive Physiology Lab, Division of Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Izatnagar, UP, India.
Reproductive Physiology Lab, Division of Physiology and Climatology, ICAR-Indian Veterinary Research Institute, Izatnagar, UP, India.
Theriogenology. 2016 Nov;86(8):2004-11. doi: 10.1016/j.theriogenology.2016.06.019. Epub 2016 Jun 27.
Selection of competent oocytes is crucial for successful in vitro embryo production and correlating expression profile of oocyte competence markers in cumulus cells with oocyte quality is an important preamble. In the present study, expression profile of oocyte competence markers (GREM1, EGFR, HAS2, and TNFAIP6) was correlated through brilliant cresyl blue (BCB) staining and subsequently with in vitro embryo production. Excellent to good quality buffalo, cumulus oocyte complexes (COCs) were stained with BCB (26 μM) and based on the blue coloration of cytoplasm COCs were divided in two groups as BCB(+ve) and BCB(-ve). Mean percentage of BCB(+ve) oocytes was significantly higher (P < 0.05) than BCB(-ve) oocytes (56.79 ± 1.22 vs. 43.20 ± 1.22), the mean oocyte diameter was also significantly larger (P < 0.05) for the BCB(+ve) group than that of BCB(-ve) group (145.7 ± 1.8 μm vs. 132.7 ± 1.9 μm). The cleavage rate (%), blastocyst rate (%), and total cell number was significantly (P < 0.05) higher in BCB(+ve) than BCB(-ve) group (71.15 ± 2.17 vs. 52.89 ± 2.65; 31.58 ± 1.11 vs. 7.73 ± 0.97, and 93.14 ± 2.42 vs. 71.42 ± 2.09, respectively). Relative mRNA expression of marker genes in cumulus cells increased significantly (P < 0.05) after maturation viz. GREM1 (10.13 folds), EGFR (9.04 folds), HAS2 (27.91 folds), and TNFAIP6 (64.81 folds). Brilliant cresyl blue positive oocytes showed significantly higher (P < 0.05) expression of GREM1, EGFR, and HAS2 transcripts than BCB(-ve) oocytes, however, no significant change in the expression of TNFAIP6 gene was observed. It is concluded from the study that GREM1, EGFR, and HAS2 could be used as molecular markers of oocyte competence for an improved buffalo embryo production in vitro.
选择有发育能力的卵母细胞对于体外胚胎生产的成功至关重要，并且将卵丘细胞中卵母细胞发育能力标志物的相关表达谱与卵母细胞质量相关联是重要的前提。在本研究中，通过亮甲酚蓝（BCB）染色将卵母细胞发育能力标志物（GREM1、EGFR、HAS2和TNFAIP6）的表达谱进行关联，随后与体外胚胎生产相关联。将优质到良好质量的水牛卵丘卵母细胞复合体（COCs）用BCB（26μM）染色，并根据细胞质的蓝色染色将COCs分为两组，即BCB（+）和BCB（-）。BCB（+）卵母细胞的平均百分比显著高于BCB（-）卵母细胞（P<0.05）（56.79±1.22对43.20±1.22），BCB（+）组的平均卵母细胞直径也显著大于BCB（-）组（P<0.05）（145.7±1.8μm对132.7±1.9μm）。BCB（+）组的卵裂率（%）、囊胚率（%）和总细胞数显著高于BCB（-）组（P<0.05）（71.15±2.17对52.89±2.65；31.58±1.11对7.73±0.97，以及93.14±2.42对71.42±2.09）。成熟后卵丘细胞中标志物基因的相对mRNA表达显著增加（P<0.05），即GREM1（10.13倍）、EGFR（9.04倍）、HAS2（27.91倍）和TNFAIP6（64.81倍）。亮甲酚蓝阳性卵母细胞显示GREM1、EGFR和HAS2转录本的表达显著高于BCB（-）卵母细胞（P<0.05），然而，未观察到TNFAIP6基因表达的显著变化。从该研究得出结论，GREM1、EGFR和HAS2可作为卵母细胞发育能力的分子标志物，以改善水牛体外胚胎生产。
