SLO3 KO MOUSE: A TOOL TO REVEAL VOLTAGE-DEPENDENT PROCESSES IN SPERM FERTILITY

描述(申请人提供)：精子获能是一系列鲜为人知的分子过程，发生在女性生殖道体内，对精子使卵子受精的能力是绝对必要的。获能的一个关键成分是精子质膜的超极化，但超极化的分子机制尚不清楚。我们之前通过使用SLO_3基因敲除(SLO_3-/-)小鼠表明，SLO_3钾通道是导致精子获能过程中发生超极化的主要离子通道，并且SLO_3-/-是男性不育。我们还证明了来自SL03-/-小鼠的精子在精子活力和顶体反应(AR)方面存在严重缺陷。由于SLO_3通道是获能期间膜电位(Em)变化的主要决定因素，现在应该可以揭示获能介质的成分如何触发SLO_3通道的激活，以及SLO_3通道的激活如何影响在获能和AR期间活跃的其他信号通路。由于SLO_3-/-精子损害了精子的活力和AR，而且这两种情况都需要[Ca~(2+)]i的升高，我们将研究SLO_3通道的激活和Em随后的变化如何影响获能和AR过程中发生的[Ca~(2+)]_i变化。这项建议的总体目标是揭示精子膜电位(Em)变化在获能和AR中的作用。SLO_3-/-小鼠现在是一个无价的工具，它使我们能够获得对精子Em的更大的实验控制，并将揭示Em在所检查的精子生理的每个元素中的重要性。SLO_3通道对受精是必不可少的这一事实也表明，该基因内的遗传变异可能解释了男性个体之间生育能力的差异。SLO_3通道也是设计和测试可用作避孕药的这一基本通道专用阻滞剂的理想靶点。最后，了解精子的电学性质和外部离子环境的基本方面可能对提高临床试管受精程序的效率具有重要意义。 与公共卫生相关：slo3基因编码一种只在哺乳动物中发现的高电导K+通道，并仅在男性生殖细胞中表达。利用SLO_3基因敲除小鼠品系和各种分子、生理和转基因技术，我们将揭示对精子获能和顶体反应至关重要的电压敏感过程，这些信息可能会影响体外受精和避孕领域。

DESCRIPTION (provided by applicant): Sperm capacitation is a poorly understood series of molecular processes that occur in vivo in the female genital tract, and is absolutely essential to a sperm's ability to fertilize an egg. A key component of capacitation is the hyperpolarization of the sperm plasma membrane, but the molecular mechanism underlying hyperpolarization was unknown. We previously showed by using a SLO3 knock-out (SLO3 -/-) mouse that the SLO3 potassium channel is the main ion channel responsible for the hyperpolarization that occurs during sperm capacitation and that the SLO3 -/- is male infertile. We also demonstrated that sperm from SLO3 -/- mice have major deficits in sperm motility as well as in the Acrosome Reaction (AR). Since SLO3 channels are the major determinant of changes in membrane potential (Em) that occur during capacitation, it should now be possible to reveal how the components of the capacitating media trigger the activation of SLO3 channels and how SLO3 channel activation affects other signaling pathways that are active during capacitation and the AR. Since SLO3 -/- sperm have impaired motility and AR, and both events require increases in [Ca2+]i, we will investigate how SLO3 channel activation and subsequent changes in Em affect the [Ca2+]i changes that occur during capacitation and the AR. The overall objective of this proposal is to reveal the role of sperm membrane potential (Em) changes in capacitation and the AR. The SLO3 -/- mouse now represents an invaluable tool which permits us to gain greater experimental control over sperm Em and will reveal the importance of Em in each element of sperm physiology examined. The fact that SLO3 channels are essential to fertilization also suggests the possibility that genetic variation within this gene accounts for differences in fertility among male individuals. SLO3 channels also represent an ideal target for designing and testing blockers specific for this essential channel that can be used as contraceptive drugs. Finally, knowledge of the electrical properties of sperm and the essential aspects of the external ionic environment may have important implications for improving the efficiency of clinical IVF procedures. PUBLIC HEALTH RELEVANCE: The slo3 gene encodes a high conductance K+ channel found only in mammals and expressed exclusively in male germ cells. Using the SLO3 knock out mouse line and a variety of molecular, physiological, and transgenic techniques we will reveal voltage-sensitive processes that are essential for sperm capacitation and the acrosome reaction, information that may impact the field of in-vitro fertilization and contraception.