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Taking A Molecular Approach To A Clinical Problem: Sperm Capacitation As The Missing Link For Successful In Vitro Fertilization In The Horse

机译：采取分子方法解决临床问题：精子获能是马成功体外受精的缺失环节

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摘要

Ejaculated mammalian sperm must reside within the female reproductive tract for a finite period of time before acquiring the ability to fertilize an oocyte. The complex biochemical and physiological changes that occur during this process are collectively termed „capacitation‟. Capacitation comprises the molecular events that prepare the sperm for acrosomal exocytosis and a change in the pattern of flagellar motility called hyperactivation. Both of these processes are required for fertilization. In several domestic and laboratory species, as well as in humans, these events can be recapitulated in the laboratory by incubating sperm in a defined medium. Two in vitro markers of capacitation include time dependent increases in protein tyrosine phosphorylation and high rates of agonistinduced acrosomal exocytosis. In contrast to other species, capacitation of stallion sperm has been extremely difficult to achieve with inconsistent results reported between and within laboratories. Moreover, despite numerous attempts, only two foals have ever been produced via in vitro fertilization (IVF) technology. Evidence suggests that this is ultimately due to the inability to appropriately capacitate stallion sperm in the laboratory. Therefore, the objectives of this research were to define and characterize: 1) the incubation conditions that supported capacitation of stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and agonist-induced acrosomal exocytosis; 2) the molecular pathways involved in both of these capacitation-dependent events; 3) the motion parameters associated with hyperactivated motility; and 4) the incubation conditions that supported successful in vitro fertilization of mare oocytes. We determined that incubation of stallion sperm in modified Whitten‟s containing 25 mM sodium bicarbonate and 7 mg/ml BSA supported significant time-dependent increases in protein tyrosine phosphorylation and significant percentages of progesterone-induced acrosomal exocytosis. We also demonstrated that the serine/threonine kinase protein kinase A (PKA) plays a critical role in the protein tyrosine phosphorylation pathway and that the guanine nucleotide exchange factor EPAC plays a role in acrosomal exocytosis. Moreover, by characterizing the motion parameters associated with procaine-induced hyperactivation in stallion sperm in juxtaposition with our capacitating conditions, we were able to achieve 60.7% in vitro fertilization rates of mare oocytes (0-30% previously reported).
机译：在获得使卵母细胞受精的能力之前，已射精的哺乳动物精子必须在女性生殖道中停留一段有限的时间。在此过程中发生的复杂的生化和生理变化统称为“获能”。获能包括使精子为顶体胞吐作用做好准备的分子事件，以及鞭毛运动模式的改变（称为过度激活）。这两个过程都是施肥所必需的。在一些家庭和实验室物种中，以及在人类中，可以通过在定义的培养基中孵育精子在实验室中概括这些事件。两种获能的体外标记包括蛋白质酪氨酸磷酸化的时间依赖性增加和激动剂诱导的顶体胞吐作用的高发生率。与其他物种相反，由于实验室之间和实验室之间报告的结果不一致，很难实现种马精子的获能。此外，尽管进行了许多尝试，但通过体外受精（IVF）技术仅生产了两只小马驹。有证据表明，这最终归因于实验室中无法适当地给种马精子注入能力。因此，本研究的目的是确定和表征：1）支持种马精子获能的孵化条件，如蛋白酪氨酸磷酸化和激动剂诱导的顶体胞吐作用增加所证明； 2）这两个与获能有关的事件均涉及的分子途径； 3）与超活化运动相关的运动参数； 4）支持母体卵母细胞体外受精成功的培养条件。我们确定在包含25 mM碳酸氢钠和7 mg / ml BSA的改良Whitten's中孵育种马精子可支持蛋白质酪氨酸磷酸化的显着时间依赖性增加以及孕酮诱导的顶体胞吐作用的显着百分比。我们还证明，丝氨酸/苏氨酸激酶蛋白激酶A（PKA）在蛋白酪氨酸磷酸化途径中起关键作用，而鸟嘌呤核苷酸交换因子EPAC在顶体胞吐作用中起作用。此外，通过在电容条件下表征与普鲁卡因诱导的种马精子并列放置相关的运动参数，我们能够实现母体卵母细胞体外受精率达到60.7％（先前报道为0-30％）。

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    McPartlin Lori;

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  • 年度 2010
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