Mota Gustavo Bruno, Batista Ribrio Ivan Tavares Pereira, Serapião Raquel Varella, Boité Mariana Cortes, Viana João Henrique Moreira, Torres Ciro Alexandre Alves, de Almeida Camargo Luiz Sergio
Departamento de Zootecnia, Universidade Federal de Viçosa, Viçosa, MG, 36571-000, Brazil.
Zygote. 2010 Aug;18(3):209-16. doi: 10.1017/S0967199409990219. Epub 2009 Nov 26.
The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.
本研究的目的是评估用亮甲酚蓝染料（BCB）对未成熟牛卵母细胞进行筛选以及转录本MATER和ZAR1的表达情况。将来自屠宰场卵巢的卵丘 - 卵母细胞复合体（COCs）置于稀释于mDPBS中的BCB中，在38.5℃、饱和湿度的空气中孵育60分钟。孵育后，根据细胞质颜色将这些COCs分为两组：BCB +（细胞质染色）或BCB -（细胞质未染色）。对照组在形态学筛选后立即进行体外成熟（IVM），而持留对照组的COCs则暴露于不含BCB但与BCB +组和BCB -组相同孵育条件的mDPBS中。所有组的COCs均进行体外成熟、体外受精（IVF）和体外培养（IVC）。对照组（65.3%）和BCB +组（64.4%）之间的受精后72小时的卵裂率相似，但高于（p < 0.05）持留对照组（49.8%）和BCB -组（51.3%）。BCB +组（18.5%）和对照组（16.3%）之间的受精后192小时的囊胚率无差异，但高于（p < 0.05）BCB -组（8.4%）。持留对照组（14.2%）、对照组和BCB +组之间的囊胚率无差异。通过实时PCR评估从对照组、持留对照组、BCB +组和BCB -组收集的未成熟卵母细胞中MATER和ZAR1基因的相对表达。尽管持留对照组、BCB +组和BCB -组中MATER的相对表达与对照组相比下调，但各组间MATER和ZAR1转录本的相对表达无统计学差异（p > 0.05）。结果表明，BCB染料可检测出具有不同发育能力的未成熟卵母细胞群体，尽管未观察到使用暴露于或未暴露于BCB的卵母细胞在体外胚胎生产方面有改善。暴露于BCB的未成熟卵母细胞的发育能力似乎与MATER和ZAR1转录本表达的变化无关。
