Mota Gustavo Bruno, Oliveira e Silva Ingrid, de Souza Danielle Kaiser, Tuany Flavia, Pereira Michele Munk, Camargo Luiz Sergio de Almeida, Rosa e Silva Alzira Amélia Martins
Laboratory for the Study of Reproduction,Biological Institute,University of Brasília,DF,70910-900,Brazil.
Embrapa Dairy Cattle,Juiz de Fora,MG,36038-330,Brazil.
Zygote. 2015 Aug;23(4):563-72. doi: 10.1017/S0967199414000239. Epub 2014 Jun 10.
The aim of this study was to evaluate the dose-response effect of insulin, plus follicle-simulating hormone (FSH) at a fixed concentration, in a serum-free defined culture medium (DCM) on the in vitro maturation of bovine cumulus-oocyte complexes (COCs). For oocyte nuclear maturation, the expression levels of GDF9, GLUT1, PRDX1 and HSP70.1 transcripts related to oocyte and embryo developmental competence were analysed. For in vitro maturation (IVM), cumulus-oocyte complexes from slaughterhouse ovaries were distributed into four groups based on insulin concentration added to serum-free DCM, which was composed of alpha minimum essential medium (α-MEM), as basal medium: (1) DCM control: 0 ng/ml; (2) DCM1: 1 ng/ml; (3) DCM10: 10 ng/ml; and (4) DCM100: 100 ng/ml. After IVM, the nuclear status of a sample of oocytes was analysed and the other oocytes were submitted for in vitro fertilization (IVF) and in vitro culture (IVC). Different concentrations of insulin did not affect significantly the nuclear maturation and cleavage rate (72 h post-insemination) across all groups. Blastocyst rate (192 h post-insemination) did not differ in DCM control (24.3%), DCM1 (27.0%) and DCM10 (26.3%) groups, but the DCM100 (36.1%) group showed a greater blastocyst rate (P 0.05) was observed at the different insulin concentrations. The results indicated that insulin added to DCM influenced levels of transcripts related to cellular stress (HSP70-1 and PRDX1) and oocyte competence (GDF9) in bovine oocytes and at higher concentrations enhanced blastocyst production.
本研究的目的是评估在无血清限定培养基(DCM)中,胰岛素与固定浓度的促卵泡激素(FSH)联合作用对牛卵丘-卵母细胞复合体(COC)体外成熟的剂量反应效应。对于卵母细胞核成熟,分析了与卵母细胞和胚胎发育能力相关的GDF9、GLUT1、PRDX1和HSP70.1转录本的表达水平。对于体外成熟(IVM),将来自屠宰场卵巢的卵丘-卵母细胞复合体根据添加到无血清DCM中的胰岛素浓度分为四组,该无血清DCM由α-最低必需培养基(α-MEM)作为基础培养基组成:(1)DCM对照组:0 ng/ml;(2)DCM1组:1 ng/ml;(3)DCM10组:10 ng/ml;(4)DCM100组:100 ng/ml。IVM后,分析一部分卵母细胞的核状态,另一部分卵母细胞进行体外受精(IVF)和体外培养(IVC)。不同浓度的胰岛素对所有组的核成熟和受精后72小时的分裂率均无显著影响。受精后192小时的囊胚率在DCM对照组(24.3%)、DCM1组(27.0%)和DCM10组(26.3%)中无差异,但DCM100组(36.1%)的囊胚率更高(P<0.05)。在不同胰岛素浓度下观察到,添加到DCM中的胰岛素会影响牛卵母细胞中与细胞应激(HSP70-1和PRDX1)和卵母细胞能力(GDF9)相关的转录本水平,且较高浓度时会提高囊胚产量。