Burch Tanya, Yu Liang, Nyalwidhe Julius, Horcajadas Jose A, Bocca Silvina, Swanson R James, Oehninger Sergio
J Assist Reprod Genet. 2014 Jun;31(6):757-65. doi: 10.1007/s10815-014-0207-2.
To identify the secreted proteins of murine embryos grown in vitro.
Two-cell mouse embryos (n=432) were randomly allocated to culture to the blastocyst stage in protein-free and in protein-supplemented (3 % BSA) media. Proteins were separated by SDS-PAGE; bands were visualized by coomassie staining, followed by in-gel trypsin digestion and liquid chromatography-tandem mass spectrometry. RT-PCR and confocal microscopy were used to confirm gene/protein expression in blastocysts.
Of all individually identified proteins, 34 and 23 were found in embryos cultured without and with BSA, respectively, and 20 were common. Identified proteins having an N-terminal secretory sequence or transmembrane domains located on the extracellular backbone were postulated as secreted proteins. Gene and protein expression for two selected molecules were confirmed. Functional analysis revealed over-represented processes related to lipid metabolism, cyclase activity, and cell adhesion/membrane functions.
This study provided evidence to further characterize secreted proteins by mouse embryos grown from the 2-cell to the blastocyst stage in vitro. Because of homology between murine and human, these results may provide information to be translated to the clinical setting.
鉴定体外培养的小鼠胚胎分泌的蛋白质。
将432个二细胞期小鼠胚胎随机分配，分别在无蛋白培养基和添加蛋白（3%牛血清白蛋白）的培养基中培养至囊胚期。蛋白质通过SDS-PAGE分离；条带经考马斯亮蓝染色后可视化，然后进行胶内胰蛋白酶消化和液相色谱-串联质谱分析。采用RT-PCR和共聚焦显微镜确认囊胚中的基因/蛋白质表达。
在所有单独鉴定出的蛋白质中，分别在无牛血清白蛋白和有牛血清白蛋白培养的胚胎中发现了34种和23种，其中20种是共同的。具有N端分泌序列或位于细胞外骨架上的跨膜结构域的已鉴定蛋白质被假定为分泌蛋白。对两个选定分子的基因和蛋白质表达进行了确认。功能分析显示，与脂质代谢、环化酶活性以及细胞黏附/膜功能相关的过程出现频率过高。
本研究为进一步表征体外从二细胞期生长至囊胚期的小鼠胚胎分泌的蛋白质提供了证据。由于小鼠和人类之间存在同源性，这些结果可能为临床应用提供可转化的信息。
