Department of Physiology, Institute of Biomedicine, University of Turku, Finland.
Biol Reprod. 2012 Jan 16;86(1):1-8. doi: 10.1095/biolreprod.111.092403. Print 2012 Jan.
Mammalian sperm gain their ability to fertilize the egg during transit through the epididymis and by interacting with proteins secreted by the epididymal epithelial cells. Certain members of the CRISP (cysteine-rich secretory protein) family form the major protein constituent of the luminal fluid in the mammalian epididymis. CRISP4 is the newest member of the CRISP family expressed predominantly in the epididymis. Its structure and expression pattern suggest a role in sperm maturation and/or sperm-egg interaction. To study the relevance of CRISP4 in reproduction, we have generated a Crisp4 iCre knock-in mouse model through insertion of the iCre recombinase coding cDNA into the Crisp4 locus. This allows using the mouse line both as a Crisp4 deficient model and as an epididymis-specific iCre-expressing mouse line applicable for the generation of conditional, epididymis-specific knockout mice. We show that the loss of CRISP4 leads to a deficiency of the spermatozoa to undergo progesterone-induced acrosome reaction and to a decreased fertilizing ability of the sperm in the in vitro fertilization conditions, although the mice remain fully fertile in normal mating. However, removal of the egg zona pellucida returned the fertilization potential of the CRISP4-deficient spermatozoa, and accordingly we detected a reduced number of Crisp4-deficient spermatozoa bound to oocytes as compared with the wild-type spermatozoa. We also demonstrate that iCre recombinase is expressed in a pattern similar to endogenous Crisp4 and is able to initiate the recombination event with its target sequences in vivo.
哺乳动物精子在通过附睾时获得受精卵子的能力，并通过与附睾上皮细胞分泌的蛋白质相互作用而获得。CRISP（富含半胱氨酸的分泌蛋白）家族的某些成员构成了哺乳动物附睾管腔液的主要蛋白质成分。CRISP4 是 CRISP 家族的最新成员，主要在附睾中表达。其结构和表达模式表明其在精子成熟和/或精子-卵子相互作用中发挥作用。为了研究 CRISP4 在生殖中的相关性，我们通过将 iCre 重组酶编码 cDNA 插入 Crisp4 基因座，生成了 Crisp4 iCre 敲入小鼠模型。这使得该小鼠系既可以作为 Crisp4 缺失模型，也可以作为附睾特异性 iCre 表达的小鼠系，适用于生成条件性、附睾特异性敲除小鼠。我们发现 CRISP4 的缺失导致精子无法进行孕酮诱导的顶体反应，并且在体外受精条件下精子的受精能力降低，尽管这些小鼠在正常交配中仍保持完全生育能力。然而，去除卵透明带恢复了 CRISP4 缺陷精子的受精能力，因此我们检测到与野生型精子相比，CRISP4 缺陷精子与卵母细胞的结合数量减少。我们还证明 iCre 重组酶的表达模式与内源性 Crisp4 相似，并且能够在体内启动与靶序列的重组事件。
