Wrenzycki C, Herrmann D, Carnwath J W, Niemann H
Institut für Tierzucht und Tierverhalten (FAL) Mariensee, Neustadt, Germany.
J Reprod Fertil. 1996 Sep;108(1):17-24. doi: 10.1530/jrf.0.1080017.
In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity with the published bovine genomic DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcript was stable through early development, or embryonic gene expression that had been active was then terminated prematurely. The differences in transcription between bovine embryos derived in vivo or in vitro indicate that culture conditions affect gene expression. This affords a tool for the further optimization of in vitro production systems for bovine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in vitro.
在本研究中，我们检测了体内和体外获得的牛胚胎中编码连接蛋白43（Cx43）的mRNA的存在情况。使用添加了激素和10%发情母牛血清用于成熟培养的TCM 199，从屠宰场获取的卵巢中体外培养出卵丘-卵母细胞复合体、从卵丘细胞中分离出的未成熟和成熟卵母细胞、受精卵、2-4细胞和8-16细胞胚胎、桑椹胚、囊胚和孵化囊胚。将成熟24小时的卵丘-卵母细胞复合体与公牛精子接触19小时，然后在添加了10%发情母牛血清的TCM 199中培养至所需发育阶段。从超排供体母牛收集体内获得的桑椹胚和囊胚。使用改良的酚-氯仿提取法从60-200个牛卵母细胞或胚胎的样本中提取总RNA，并通过逆转录聚合酶链反应进行分析。在逆转录之前，使用牛特异性引物通过聚合酶链反应检测经脱氧核糖核酸酶消化的胚胎RNA的等分试样，以控制残留基因组DNA污染。无DNA的总RNA在与Cx43特异性3'引物预孵育后进行逆转录。使用定义Cx43 516 bp片段的Cx43特异性引物通过聚合酶链反应扩增所得的cDNA。通过用Alu I进行限制性酶切分析和测序验证逆转录聚合酶链反应产物。对每个发育阶段的测定至少重复两次，各重复之间结果相同。在体内生长的牛桑椹胚和囊胚中检测到Cx43转录本。相比之下，虽然从卵丘-卵母细胞复合体到桑椹胚的体外早期阶段表达Cx43，但囊胚和孵化囊胚中未检测到该基因的可检测浓度的mRNA。限制性酶切显示出三个预测大小（139、177、200 bp）的片段。扩增产物与已发表的牛基因组DNA序列显示100%的同一性。在我们的体外条件下，Cx43基因要么从未被激活，这需要母体转录本在早期发育过程中保持稳定，要么已活跃的胚胎基因表达随后过早终止。体内或体外获得的牛胚胎之间转录的差异表明培养条件会影响基因表达。这为进一步优化牛胚胎的体外生产系统提供了一种工具，并通过定义体外生产的牛胚胎的转录表型有助于其生理特征的研究。
