Isolation and proteomic analysis of the sperm annulus

描述（由申请人提供）： 随着世界人口以对数速度增长，在大约40年内，地球上的人口将达到大约100亿。因此，对更多避孕选择的需求，特别是针对男性的需求，预计也会增加。为了确定男性避孕的新目标，必须进行研究以了解精子成为功能性配子所必须经历的关键事件。考虑到这一目标，我们建议研究环，一个大分子分隔蛋白结构位于精子尾部。没有环带的精子在结构上是正常的，但它们不能游泳，也不能进行获能。这种情况已经在实验室中用Septin 4敲除小鼠以及临床上在不育男性群体中显示。总之，这些数据确定了环在精子功能中起着至关重要的作用，但其作用机制尚不清楚。我们建议分离环，并确定其蛋白质组使用质谱。将使用低和高严格条件来分离环以鉴定尽可能多的环相关蛋白。我们将在精子功能的四个不同阶段（不活动、活动、活动过度和受精能力）分离环。将通过免疫荧光法确认已识别的瓣环和瓣环相关蛋白。还将鉴定改变其蛋白酪氨酸磷酸化的蛋白。从收集到的信息中，我们将开发关于环如何发挥作用的假设，并通过抑制拟议的功能和观察效果在野生型精子中进行测试，以及在sept 4-/-精子中进行拯救实验，看看功能是否可以恢复。了解环在精子功能中的重要作用将促进我们对这些机制如何共同作用使精子细胞成为功能性配子的理解。这一信息可能会导致新的治疗不育男性以及新的睾丸后男性避孕药。 公共卫生相关性：该项目的目标是分离精子环并确定其蛋白质组。这些数据将推动septin，避孕和生殖生物学领域的发展。这项工作可能会产生不孕症的新疗法以及新的男性避孕目标。

DESCRIPTION (provided by applicant): With world population expanding at a logarithmic rate, the planet will reach approximately 10 billion people in about 40 years. The demand for more contraceptive options, especially targeting the male, thus is also expected to rise. To identify new targets for male contraception, studies must be undertaken to understand critical events that sperm must undergo to become functional gametes. With this goal in mind, we propose to study the annulus, a macromolecular septin structure located on the sperm tail. Sperm that are produced without an annulus are otherwise normal structurally, but they fail to swim and cannot undergo capacitation. This condition has been shown in the laboratory with septin 4 knockout mice as well as clinically in a population of infertile men. Together, these data establish that the annulus plays a vital role in sperm function, but its mechanisms of action are unknown. We propose to isolate the annulus and determine its proteome using mass spectrometry. Both low and high stringency conditions will be used to isolate the annulus to identify as many annulus-associating proteins as possible. We will isolate the annulus at four different stages of sperm function (immotile, motile, hypermotile, and fertilization competent). Identified annulus and annulus-associated proteins will be confirmed by immunofluorescence. The identification of proteins that alter their protein tyrosine phosphorylation will also be done. From the information gathered, we will develop hypotheses about how the annulus is functioning and test them in wild-type sperm by inhibiting the proposed function and observing the effect, as well as rescue experiments in sept4-/- sperm to see if function can be restored. Understanding the vital role the annulus plays in sperm function will advance our comprehension of how these mechanisms work together to make a sperm cell become a functional gamete. This information may lead to new therapeutics for infertile men as well as novel post- testicular male contraceptives. PUBLIC HEALTH RELEVANCE: The goal of this project is to isolate the sperm annulus and determine its proteome. This data will advance the fields of septin, contraceptive, and reproductive biology. Putative new therapies for infertility as well as novel male contraceptive targets may arise from this work.