Loi P, Ledda S, Filia F, Gallus M, Cappai P, Naitana S
Istituto di Fisiologia Veterinaria, Università di Sassari.
Boll Soc Ital Biol Sper. 1990 Dec;66(12):1173-9.
Four cell embryos collected by laparatomy from Sardinian breed ewes superovulated with FSH-p (16 mg Sigma), were divested of their zonae pellucidae (ZP) by micromanipulation or chemical methods (pronase 0.5%, tyrode pH 2.2). The blastomeres were separated by pipetting using a flame polished pasteur pipette in a Ca free medium (PBS. Sigma) and were inserted into previously evacuated Z.P. using a Leitz micromanipulator. The Z.P. were removed either mechanically or with acid tyrode; pronase was unable to digest them after incubation at 30 degrees C for 120 minutes. The single blastomeres were cocultured on a monolayer of ovine oviductal epithelial cells in TCM 199 + 10 FCS at 38 degrees C in 5% CO2 for 60 hours. No developments were observed in blastomeres obtained by acid digestion of the ZP while 50% of the other blastomeres continued their development until the 16 cell stages. Our results suggest that coculture with oviductal epithelial cell monolayers can support in vitro development of single ovine blastomeres.
通过剖腹术从用FSH-p（16毫克西格玛）进行超数排卵的撒丁岛品种母羊收集的4细胞胚胎，通过显微操作或化学方法（0.5%的链霉蛋白酶，pH值为2.2的台氏液）去除其透明带（ZP）。在无钙培养基（PBS，西格玛）中使用火焰抛光的巴氏吸管通过吹打分离卵裂球，并使用徕卡显微操作器将其插入预先抽空的透明带中。透明带通过机械方法或用酸性台氏液去除；在30摄氏度孵育120分钟后，链霉蛋白酶无法消化它们。将单个卵裂球在含有10%胎牛血清的TCM 199中，于38摄氏度、5%二氧化碳条件下，在绵羊输卵管上皮细胞单层上共培养60小时。通过酸性消化透明带获得的卵裂球未观察到发育，而其他卵裂球中有50%继续发育直至16细胞阶段。我们的结果表明，与输卵管上皮细胞单层共培养可以支持单个绵羊卵裂球的体外发育。
