Lopes E F, Marques L S, Duranti R G, de Oliveira A T D, Lopes R F F, Rodrigues J L
Laboratório de Embriologia e Biotécnicas da Reprodução, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Laboratório de Biotecnologia Animal Aplicada, Departamento de Ciências Morfológicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Reprod Domest Anim. 2015 Oct;50(5):763-70. doi: 10.1111/rda.12585. Epub 2015 Aug 17.
Oocyte selection based on the brilliant cresyl blue (BCB) staining test has been successfully used to differentiate between competent and incompetent bovine oocytes. Here, the expression of genes involved in transport of monocarboxylates (Mct1-4) and oogenesis specific genes (Bmp15, Gdf9 and Has2) in BCB+ and BCB- selected immature and mature bovine cumulus-oocyte complexes (COC) was evaluated. In order to find specific molecular markers to characterize successful oocyte maturation, our study was also aimed at identifying the expression of Mcts and oogenesis specific genes in denuded oocytes and cumulus cells. Immature COCs morphological appropriate were (i) stained with 26 mm BCB for 90 min before IVM, (ii) exposed to same incubation conditions as stained COCs, but without BCB (holding group) or (iii) transferred into a maturation medium immediately after morphological selection (control group). mRNA expression was investigated by RT-PCR in COCs before and after IVM. No relationship was observed in the relative expression of Has2, Gdf9, Bmp15 or Mct1, 2 and 4 transcripts between BCB+ and BCB- COCs. Transcripts analysis showed that Gdf9 and Bmp15 in BCB+, BCB- and holding groups were up-regulated (p < 0.05) before IVM, while Has2 was up-regulated (p < 0.01) after IVM in the control group. Other genes remained stable during maturation (Mct1, 2 and 4). Our results showed, for the first time, Mct1, 2 and 4 expression in bovine COCs. Mct1 and Mct4 transcripts were present in denuded oocytes and cumulus cell, while Mct2 was detected only in cumulus cells. These differences between the three isoforms in localization suggest unique roles for each in monocarboxylate transport during maturation.
基于灿烂甲酚蓝（BCB）染色试验的卵母细胞选择已成功用于区分有能力和无能力的牛卵母细胞。在此，评估了参与单羧酸转运的基因（Mct1 - 4）和卵子发生特异性基因（Bmp15、Gdf9和Has2）在BCB +和BCB -选择的未成熟和成熟牛卵丘 - 卵母细胞复合体（COC）中的表达。为了找到表征成功卵母细胞成熟的特定分子标记，我们的研究还旨在鉴定裸卵和卵丘细胞中Mcts和卵子发生特异性基因的表达。形态合适的未成熟COC：（i）在体外成熟（IVM）前用26 mM BCB染色90分钟，（ii）暴露于与染色COC相同的孵育条件，但不使用BCB（对照组）或（iii）在形态选择后立即转移到成熟培养基中（对照组）。通过RT - PCR研究IVM前后COC中的mRNA表达。在BCB +和BCB - COC之间未观察到Has2、Gdf9、Bmp15或Mct1、2和4转录本的相对表达之间的关系。转录本分析表明，BCB +、BCB -和对照组中，IVM前Gdf9和Bmp15上调（p < 0.05），而对照组中IVM后Has2上调（p < 0.01）。其他基因在成熟过程中保持稳定（Mct1、2和4）。我们的结果首次显示了牛COC中Mct1、2和4的表达。Mct1和Mct4转录本存在于裸卵和卵丘细胞中，而Mct2仅在卵丘细胞中检测到。这三种同工型在定位上的这些差异表明它们在成熟过程中的单羧酸转运中各自具有独特作用。
