McPartlin L A, Suarez S S, Czaya C A, Hinrichs K, Bedford-Guaus S J
Departments of Clinical Sciences and Biological and Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Biol Reprod. 2009 Jul;81(1):199-206. doi: 10.1095/biolreprod.108.074880. Epub 2009 Feb 4.
Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The objective of this study was to characterize stallion sperm hyperactivation and to test whether hyperactivation of capacitated sperm supported equine IVF. Treatment of sperm with procaine, an anesthetic shown to induce hyperactivation in other mammalian species, resulted in the decrease of three motility variables indicative of hyperactivation: straight line velocity (P = 0.029), straightness (P = 0.001), and linearity (P = 0.002). We demonstrated that procaine-induced hyperactivation was not regulated by changes in protein tyrosine phosphorylation and that it did not induce acrosomal exocytosis in capacitated sperm compared with calcium ionophore (P > 0.05), similar to findings in the bovine. Most notably, by coupling our capacitating conditions with the induction of hyperactivation using procaine, we have achieved the novel result of substantial and reproducible percentages of fertilized mare oocytes (60.7%) in our IVF experiments. Conversely, sperm incubated in capacitating conditions but not treated with procaine did not fertilize (0%). These results support the hypothesis that capacitation and hyperactivation are required for successful IVF in the equine.
获能是一个复杂且尚未被充分理解的过程，它涵盖了精子为成功使卵母细胞受精而必须经历的所有分子变化。马的体外受精一直难以实现，体外受精（IVF）率较低（0%-33%）就证明了这一点；此外，使用IVF仅产生过两匹小马驹。在添加了牛血清白蛋白和碳酸氢钠的改良惠滕氏培养液中孵育种公马精子，会产生显著的时间依赖性蛋白酪氨酸磷酸化，并诱导顶体胞吐，这与获能一致。本研究的目的是表征种公马精子的超活化，并测试获能精子的超活化是否支持马的体外受精。用普鲁卡因处理精子，普鲁卡因是一种已证明能在其他哺乳动物物种中诱导超活化的麻醉剂，导致三个指示超活化的运动变量降低：直线速度（P = 0.029）、直线性（P = 0.001）和线性度（P = 0.002）。我们证明，普鲁卡因诱导的超活化不受蛋白酪氨酸磷酸化变化的调节，并且与钙离子载体相比，它不会在获能精子中诱导顶体胞吐（P > 0.05），这与牛的研究结果相似。最值得注意的是，通过将我们的获能条件与使用普鲁卡因诱导超活化相结合，我们在体外受精实验中取得了新的成果，即有相当比例且可重复的母马卵母细胞受精（60.7%）。相反，在获能条件下孵育但未用普鲁卡因处理的精子未受精（0%）。这些结果支持了这样一种假设，即获能和超活化是马成功进行体外受精所必需的。
