Nickson D A, Boyd J S, Eckersall P D, Ferguson J M, Harvey M J, Renton J P
Department of Surgery/Reproduction, University of Glasgow Veterinary School, UK.
J Reprod Fertil Suppl. 1993;47:231-40.
Oocytes were collected from ovaries of bitches, at various stages of the oestrous cycle, after routine sterilization. Cumulus-enclosed oocytes were cultured for 0-72 h in a modified M-199 medium containing 10% oestrous bitch serum and 20 micrograms oestradiol ml-1. For oocytes surrounded by two or more layers of cumulus cells, an increase in the expression of mRNA transcripts for zona pellucida glycoprotein 3 (ZP3) was seen and reached peak levels after 48 h culture in vitro. Histological assessment showed that 39% of these oocytes had extruded their first polar body after 24 h culture in vitro. When these in vitro matured oocytes were transferred to oviduct cell monolayers and inseminated with fresh dog spermatozoa in Medium-199 supplemented with 10% fetal calf serum and 50 mg gentamicin sulfate ml-1, penetration of the zona pellucida started 1 h after insemination for oocytes that had been cultured for 48 and 72 h. At 12 h after insemination both male and female pronuclei were seen in 37.5% and 20% of the oocytes incubated for 48 and 72 h, respectively. No further development was seen.
在常规绝育后，从处于发情周期不同阶段的母犬卵巢中采集卵母细胞。将卵丘包裹的卵母细胞在含有10%发情母犬血清和20微克/毫升雌二醇的改良M-199培养基中培养0至72小时。对于被两层或更多层卵丘细胞包围的卵母细胞，可见透明带糖蛋白3（ZP3）的mRNA转录本表达增加，并在体外培养48小时后达到峰值水平。组织学评估显示，这些卵母细胞在体外培养24小时后，39%排出了第一极体。当将这些体外成熟的卵母细胞转移到输卵管细胞单层，并在添加有10%胎牛血清和50毫克/毫升硫酸庆大霉素的M-199培养基中用新鲜犬精子授精时，对于培养48小时和72小时的卵母细胞，授精1小时后开始穿透透明带。授精12小时后，在分别培养48小时和72小时的卵母细胞中，37.5%和20%可见雄原核和雌原核。未见进一步发育。
