Division of Reproduction, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
Theriogenology. 2012 Feb;77(3):588-99. doi: 10.1016/j.theriogenology.2011.08.035. Epub 2011 Oct 5.
Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO(2). Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.
精子获能发生在输卵管中，蛋白质酪氨酸磷酸化是精子获能和获得受精能力的关键步骤。冷冻保存的精子在输卵管中显示出蛋白质酪氨酸磷酸化表达的改变。本研究比较了两种冷冻方法（常规-常规冷冻（CF）和简化-简化冷冻（SF）方法）对猪精子在输卵管液（ODF）刺激下发生蛋白质酪氨酸磷酸化能力的影响。将冷冻保存的猪精子与排卵前和排卵后的 ODF 在 38°C 和 5%CO2 下孵育 6 小时。每隔 1 小时取精子样本进行分析，以评估运动学和蛋白质酪氨酸磷酸化。使用流式细胞术测量精子中总蛋白质酪氨酸磷酸化，使用共聚焦显微镜评估不同的磷酸化模式。解冻后，两种冷冻方法对解冻后精子活力、速度和总酪氨酸磷酸化没有显著差异，尽管冷冻方法在孵育过程中对这些参数的 ODF 影响有显著差异（P<0.05）。CF 方法冷冻的精子在孵育过程中对排卵前 ODF 的磷酸化比例明显更高（P<0.001），而 SF 方法冷冻的精子则没有引起如此显著的反应，因为在孵育过程中的任何给定时间，处理之间酪氨酸磷酸化精子的比例都没有显著差异。如果使用 CF 方法，无论是尾部还是整个精子磷酸化的精子比例都会增加，以响应排卵前（EODF）和排卵后输卵管液。然而，如果使用 SF 方法，仅在 EODF 处理组中观察到这些模式的显著增加。本研究表明，与排卵后流体相比，排卵前输卵管的 ODF 诱导猪精子发生更高比例的酪氨酸磷酸化，并且冷冻方法显著影响解冻后精子对猪 ODF 的反应。
