Tulake Kuerban, Wang Xuguang, Chen Yong, Yu Chucai, Jing Binyu, Li Heping
College of Wildlife Resources, Northeast Forestry University, Harbin, China; Laboratory of Animal Reproduction Biology and Technology, College of Animal Science, Xinjiang Agricultural University, Urumqi, Xinjiang, China.
Laboratory of Animal Reproduction Biology and Technology, College of Animal Science, Xinjiang Agricultural University, Urumqi, Xinjiang, China.
Anim Reprod Sci. 2015 Mar;154:68-78. doi: 10.1016/j.anireprosci.2014.11.020. Epub 2015 Jan 14.
High efficiency of in vitro capacitation of deer sperm has not yet been achieved as low sperm penetration rates were reported in in vitro fertilization studies. Our main goal in this study was to identify the changes of frozen-thawed sperm of the rare red deer Tarim wapiti (Cervus elaphus yarkandensis) and detect the effect of bovine serum albumin (BSA), serum, and heparin on the protein tyrosine phosphorylation of frozen-thawed sperm. The frozen-thawed sperm of Tarim wapiti was suspended in improved modified tyrode-albumin-lactate-pyruvate medium and cultured in 5% CO2 at 38.5°C, and the status of protein tyrosine phosphorylation of sperm was detected by Western blotting. Although the results showed that the type number and expression of protein tyrosine phosphorylation of frozen-thawed wapiti sperm were decreased, the tyrosine-phosphorylated proteins such as 10, 14, 40, 47, and 55kDa were increased significantly during the process of capacitation culture (1-2h). In addition, tyrosine-phosphorylated proteins were promoted by BSA rather than serum, and estrus sheep serum (ESS) rather than estrus deer serum. When ESS and heparin were used together at 4h after capacitation, four main tyrosine phosphorylation proteins (10±2, 14±2, 25±3, and 47±3kDa) had a significantly higher expression than that at 2h after capacitation. We demonstrated that these proteins were involved in wapiti sperm in vitro capacitation, heparin in the incubation media was necessary for the capacitation and tyrosine phosphorylation protein was promoted by ESS.
由于体外受精研究中报道的精子穿透率较低，鹿精子的体外获能效率尚未实现。本研究的主要目标是确定珍稀马鹿塔里木亚种（Cervus elaphus yarkandensis）冻融精子的变化，并检测牛血清白蛋白（BSA）、血清和肝素对冻融精子蛋白酪氨酸磷酸化的影响。将塔里木马鹿的冻融精子悬浮于改良的台氏白蛋白乳酸丙酮酸培养基中，在38.5℃、5%二氧化碳条件下培养，通过蛋白质免疫印迹法检测精子蛋白酪氨酸磷酸化状态。虽然结果显示冻融马鹿精子蛋白酪氨酸磷酸化的类型数量和表达降低，但在获能培养过程（1-2小时）中，10、14、40、47和55kDa等酪氨酸磷酸化蛋白显著增加。此外，酪氨酸磷酸化蛋白受BSA而非血清、发情绵羊血清（ESS）而非发情马鹿血清的促进。当在获能后4小时同时使用ESS和肝素时，四种主要的酪氨酸磷酸化蛋白（10±2、14±2、25±3和47±3kDa）的表达显著高于获能后2小时。我们证明这些蛋白参与马鹿精子的体外获能，孵育培养基中的肝素是获能所必需的，并且酪氨酸磷酸化蛋白受ESS促进。
