建立Spanx基因条件敲除小鼠模型及其对精子生育能力影响的研究

The mouse Spanxn gene is located on the X chromosome and specifically expressed in testes. The homologous Spanxn gene in human is a SPANX gene family, which contains two subfamilies SPANX-A/D and SPANX-N. Human SPANX gene family is post-meiotic expressed and is thought to be involved in the formation of acrosome and sperm nucleus. In our previous study on Globozoospermia patient's sperm with homozygous mutation of PICK1 gene, we found that compared with normal sperm, the expression of several SPANX proteins were significantly reduced in patient' s sperm. Moreover, we have found the expression of SPANX proteins was also significantly reduced in the donors'sperm with low fertility(no pregnancy in 12 AID treantment cycles). The Western Blotting result proved that the SPANX proteins is correlated to the fertility. However, the precise biological function of the SPANX gene family remains unclear. Besides, the SPANX gene was also found in many tumor cells and such as testescular germ cell tumor , melanoma, and probably involved in ovary development. In this project, we intend to use mice as a model since mice have a single form of SPANX, Spanxn，which will simplify the study of SPANX function. We will study the time and spacial expression pattern of Spanxn during mouse development. We will also establish a Spanxn knockout mice model and implement whole genome expression profiling and proteome analysis to uncover Spanxn function. Combining all the data above and with the use of bioinforatics, we intend to elucidate the molecular mechanisim and regulatory network of Spanxn. This project will establish Spanxn gene knockout model mice. It is meaningful to reveal the mechanism of Spanxn gene in spermatogenesis, especially in spermiogenesis stage, but also provide new model for ovary development and oncology research. Thus it will provide a theoretical basis and guidance for the clinical treatment of male infertility and may help us to find candidate targets for male contraceptive drugs and new contraceptive approach, and understand the mechanic basis of ovary development and the tumorigenesis

课题组前期研究发现,在生育力低下的精子和圆头精子中有SPANX蛋白家族多种蛋白表达下调;并证实SPANX蛋白与精子的生育能力密切相关,但具体作用机理未知。人类SPANX基因在减数分裂之后表达,推测该基因与精子核和顶体的形成相关，影响精子生育能力。此外,该基因在多种肿瘤和早衰卵巢中表达异常,提示其可能涉及肿瘤发生和卵巢发育。课题拟采用CRPSPR/Cas9技术，构建小鼠同源基因的条件敲除模型，结合临床上该基因与妊娠结局的相关性，探讨其与生育能力的关系，明确SPANX基因的功能；通过分析基因敲除后细胞和分子水平的改变，结合蛋白质相互作用研究，探索其调控的分子作用网络和机理。本课题拟建立SPANX对男性生育力评估体系，为男性不育的临床诊断和治疗提供指导，也为男性避孕药提供新的候选靶标,将有助于预防男性不育、改善男性生殖健康;动物模型的建立也为研究卵巢发育和肿瘤发生提供新的技术平台。

本项目进行了如下研究：(1)探索不同生育能力供精者精子在蛋白质组学方面的差异；(2)以睾丸生殖肿瘤细胞为模型，初步探究SPANX基因家族成员在体外对肿瘤细胞的影响；(3)构建Spanxn基因敲除小鼠模型，研究其对精子发生和雄鼠生育力的影响。.取得以下结果：(1)通过回顾性分析本中心人类精子库十年间的供精数据，发现共有87例供精者精液在历经12个AID治疗周期后无临床妊娠，而138例供精者精液在历经10个AID周期以内，达到我国规定的供精妊娠上限（5例妊娠），证明生育力存在差异；(2)通过非标记蛋白质组学技术，在低生育力精子中472个蛋白质表达下调和55个蛋白质表达上调，蛋白质免疫印迹证实SPANX蛋白家族成员在低生育力精子中表达下调；(3)利用免疫荧光染色检测SPANX蛋白家族成员在睾丸中的表达，结果显示SPANXA/D从次级精母细胞开始到精子发生完成均有表达，SPANXN主要表达于晚期精子细胞和精子中；在人类精子中，SPANXA/D主要定位在赤道板、尾部颈段和中段附近，SPANXN主要定位在顶体、头部和尾部中段；(4)利用蛋白质免疫印迹和RT-PCR检测小鼠中Spanxn基因的表达情况，结果表明小鼠Spanxn基因在睾丸中特异性表达；(5)通过合笼实验检测Spanxn敲除小鼠的生育能力，结果表明Spanxn基因敲除雄鼠是可育的，但是其生育力水平下降，具体表现为见栓率降低；(6)通过显微结构对比，发现Spanxn基因敲除后，雄鼠的睾丸组织学水平无明显变化，不影响精子发生；同时Spanxn基因敲除雄鼠的精子总数和活力、精子的自发和诱发顶体反应率与野生小鼠相比无统计学差异；(7)在Ntera-2和Tcam-2两种细胞系中过表达SPANXN3，结果显示这两种细胞系的增殖、迁移和侵袭能力均受到影响；.结论：(1)供精者的精子存在着生育力的差异，且这类差异与精液常规参数无关；(2)人类SPANX蛋白与精子的生育能力有关；(3)小鼠Spanxn基因在睾丸中特异性表达；(4) Spanxn敲除后，雄鼠小鼠生育力下降；(5) Spanxn基因敲除雄鼠的睾丸和精子发生无明显变化。(6)人类SPANXN3过表达，可以抑制Ntera-2和Tcam-2细胞在体外的增殖、迁移和侵袭。