Establishment and Analysis of Cylicin1 and Cylicin2 deficient mice – a tool to study male factor infertility

发现Cylicin基因家族，Cylicin 1（Cylc 1）和Cylicin 2（Cylc 2）仅在睾丸中表达。 CYLC 1在人和牛精子的萼区被检测到，并呈现为近赤道的带状和漏斗状结构。Cylicin蛋白质的特征在于重复的、富含赖氨酸的三肽，其导致蛋白质的总正电荷，等电点为10.55。最常见的三肽由赖氨酸二肽和天冬氨酸或谷氨酸（KKD/KKE）组成。此外，CYLC 1含有富含脯氨酸的C-末端结构域。蛋白质的中心部分包含几个重复单元，预计这些重复单元形成短的α-螺旋。Cylicins的功能在很大程度上仍然未知。然而，精子形成过程中精子头部成形的作用进行了讨论，因为核周膜形成缺陷已被确定在结构异常，圆头精子在男性。猪CYLC 2进一步被证明是一种肌动蛋白结合蛋白，可能在圆形精子细胞的顶体区和成熟精子的顶体后区起细胞骨架调节剂的作用。近年来，一些不育男性被诊断为CYLC 1基因功能缺失突变，表明Cylcins可能是精子发生和男性生育所必需的，本项目旨在进一步全面了解Cylicin 1和/或Cylicin 2比例缺失的后果。我们设想开发一种男性因素不育的模型。建立并验证Cylicin 1和Cylicin 2缺陷小鼠品系。·分析Cylicin 1和Cylicin 2缺失的影响。·将Cyclicin 1突变与Cylicin 2突变杂交，分析对精子和生育力的冗余和补偿效应。·使用蛋白质组学分析任一蛋白质缺失的分子效应分析将有助于理解Cylicins的基本生物学，并将作为关于人类数据的原理证明。我们将能够产生一个Cylicin缺乏男性因素不育的小鼠模型，并将能够分析源于缺陷。最终，本文提出的研究将为更好地了解男性不育症奠定基础，并有助于制定治疗方案。

The family of Cylicin-genes, Cylicin 1 (Cylc1) and Cylicin 2 (Cylc2) are found exclusively expressed in the testis. CYLC1 is detected in the calyx region of human and bovine sperm and presents as a near-equatorial belt-like and funnel shaped structure. Cylicin proteins are characterized by repetitive, lysine-rich tripeptides, which result in an overall positive charge of the protein with an isoelectric point of 10.55. Most frequent, tripeptides consist of lysine dipeptides followed by aspartic or glutamic acid (KKD/KKE). Further, CYLC1 contains a proline-rich C-terminal domain. The central part of the protein comprises several repeating units, which are predicted to form short α-helices. The function of Cylicins remains largely unknown. However, a role in sperm head shaping during spermiogenesis is discussed, since defects in perinuclear theca formation have been identified in structurally abnormal, round-headed sperm in men. Boar CYLC2 was further shown to function as an actin-binding protein, which might serve as a cytoskeletal regulator in the acrosomal region of round spermatids and the postacrosomal region of mature sperm. Recently, several infertile men were diagnosed to harbor loss of function mutations in CYLC1 indicating that Cylcins could be essential for spermatogenesis and male fertility.The project aims at further understanding the consequences of loss of Cylicin 1 and/or Cylicin 2 ratio in a comprehensive matter. We envision to develop a model of male factor infertility.• To establish and validate Cylicin 1 and Cylicin 2 deficient mouse lines.• To analyze the effects of loss of Cylicin 1 and Cylicin 2.• To breed the Cyclicin 1 mutation with the Cylicin 2 mutation to analyze redundant and compensatory effects on sperm and fertility.• To analyze the molecular effects of loss of either protein using proteomicsAnalyses will help to understand the basic biology of Cylicins and will serve as a proof of principle with regard to the human data. We will be able to generate a murine model for Cylicin-deficient male factor infertility and will be able to analyze the defects originating from the deficiency. Ultimately, the research proposed here will lay the basis to better understand infertility in man and help in development of treatment regimen.