Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Bonn, Germany.
Theriogenology. 2011 Oct 15;76(7):1215-26. doi: 10.1016/j.theriogenology.2011.05.025. Epub 2011 Aug 5.
Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) maturational competence; (3) gene expression of certain molecular markers; and (4) in vitro embryo development after intracytoplasmic sperm injection. Equine oocytes were exposed to BCB stain and were classified as BCB+ or BCB- according to their ability to convert the dye from blue to colorless. Additionally, BCB+ and BCB- oocytes were subclassified as having a compact (Cp) or expanded (Ex) cumulus complex. As a result, BCB+ oocytes had a greater proportion of expanded cumulus oocyte complexes compared with BCB- oocytes (71.2% vs. 49.5%). Moreover, we observed a significant difference in oocyte diameter and volume between BCB+ and BCB- oocytes irrespective of cumulus morphology. BCB+ oocytes reached a higher maturation rate compared with BCB- oocytes (59.0% vs. 28.7%). Regarding the analyzed candidate genes, relative transcript abundance was significantly different for nine genes. The expression of eight genes was significantly higher (P < 0.05) for BCB+ oocytes, including ATPV6E, IF-3, TFAM, DNMT1, STAT3, Aurora-A, ODC1, and CKS2 whereas BCB- oocytes showed higher in expression of COX1. These results are in line with the observed developmental competence. Cleavage rate (45.9% vs. 29.0%) and percentage of embryos that reached the blastocyst stage (9.2% vs. 1.4%) were significantly higher for embryos derived from BCB+ oocytes compared with BCB- oocytes. In conclusion, the present study provides evidence that G6PDH-activity in immature equine oocytes is a useful predictor for subsequent in vitro developmental competence.
由于马属动物不成熟卵母细胞的发育能力差异很大，因此马属动物胚胎的体外生产效率仍然很低。与马属动物不同，在许多不同物种中，通过用灿烂甲酚蓝（BCB）染料对葡萄糖-6-磷酸脱氢酶（G6PDH）的活性进行染色，可以成功预测不成熟卵母细胞的体外发育能力。因此，本研究的目的是检测马属动物卵母细胞中 G6PDH 活性与以下各项之间的关联：（1）卵丘形态和卵母细胞的直径和体积等特性；（2）成熟能力；（3）某些分子标记物的基因表达；以及（4）胞质内精子注射后的体外胚胎发育。将马属动物卵母细胞暴露于 BCB 染色剂中，并根据其将染料从蓝色变为无色的能力将其分为 BCB+或 BCB-。此外，将 BCB+和 BCB-卵母细胞进一步细分为具有致密（Cp）或扩展（Ex）卵丘复合体的卵母细胞。结果显示，BCB+卵母细胞的扩展卵丘卵母细胞复合体比例高于 BCB-卵母细胞（71.2%比 49.5%）。此外，我们观察到 BCB+和 BCB-卵母细胞之间的卵母细胞直径和体积存在显著差异，而与卵丘形态无关。BCB+卵母细胞的成熟率高于 BCB-卵母细胞（59.0%比 28.7%）。关于分析的候选基因，九个基因的相对转录丰度存在显著差异。BCB+卵母细胞中 8 个基因的表达显著更高（P < 0.05），包括 ATPV6E、IF-3、TFAM、DNMT1、STAT3、Aurora-A、ODC1 和 CKS2，而 BCB-卵母细胞中 COX1 的表达更高。这些结果与观察到的发育能力一致。与 BCB-卵母细胞相比，BCB+卵母细胞的卵裂率（45.9%比 29.0%）和达到囊胚阶段的胚胎百分比（9.2%比 1.4%）显著更高。总之，本研究提供的证据表明，马属动物不成熟卵母细胞中的 G6PDH 活性是预测后续体外发育能力的有用指标。
