BMP4对隐睾生精功能的影响及其机制研究

Cryptorchidism results from the abnormal descending from abdominal cavity to scrotum by mesoderm-originated testis. The mechanism of male infertility of cryptorchidism needs to be further investigated. Through literature searching, we found that several investigators have reported their observation that bone morphogenetic protein-4 ( BMP4) may influence the development and spermatogenic function of adult testis in patients with a history of cryptorchidism in childhood. BMP4 can also influence the germ cell differention process from pluripotent stem cells in vitro. Both of these observations imply the important function of BMP4 , but the specific mechanisms remain unknown due to the lack of a proper model system. We speculate that BMP4 may play an important role in the the mesoderm decision and further spermatogenic cells commitment and function, which need to be investigated in a proper disease model system. Recently, transcription activator-like effectors (TALEs) emerges as a powerful and simple gene sequence-specific manipulation technology with high efficiency. We have established human cryptorchidism-specific induced pluripotent stem (iPS) cells in our previous research which could be used to model cryptorchidism in vitro . In this study, we will establish inducible BMP4 overexpression and inhibition system with cryptorchidism-specific induced pluripotent stem (iPS) cells . These indusible system will be used to investigate the germ cell differention process under different BMP4 expression pattern in vitro. Then the differentiation samples will be further analysed through realtime PCR, immunostaining, western blot, mRNA chip array, etc. The findings will then be verified again in a second-round differentiation experiment. Through these approaches, we are intending to explore the important function of BMP4 during mesoderm decision and further spermatogenic cells commitment of cryptorchidism-specific iPS cells. All these results will facilitate the understanding of the function and molecular mechanism of BMP4 on the spermatogenic potential of testis in cryptorchidism. In addition, these results will also provide a new theoretical basis for the understanding of cryptorchidism and new prevention measures proposed.

隐睾的发生源于个体在胚胎发育阶段起源于中胚层的睾丸的发育和下降发生了异常停顿，其导致成年后不育的发生机制有待研究；我们发现，BMP4在体内可影响睾丸的发育和生精功能，在体外也影响多能干细胞向中胚层生精细胞的诱导分化，我们推测其在中胚层生精细胞的发育过程中可能发挥重要作用，但机制不明。基于以上原因，本课题拟以前期已经建立的隐睾特异性iPS细胞作为隐睾的体外细胞模型，通过TALEs靶向基因操作技术，分别建立可调控的BMP4过表达和转录抑制的隐睾特异性iPS细胞系，以此为模型，研究其在不同的BMP4作用条件下向中胚层的生精细胞定向分化过程中的差异，通过对不同的细胞分化样本进行功能分析和进一步验证，研究BMP4影响隐睾特异性iPS细胞体外分化时向中胚层生精细胞发育过程中的关键作用及其机制，有助于我们理解睾丸和生精细胞发育中的关键因素，为隐睾等疾病所致生精功能异常的新的防治措施的提出提供依据。

生殖健康是生命科学研究的重点关注领域。隐睾是小儿泌尿外科常见的疾病，是指男婴出生后单侧或双侧睾丸末降至阴囊而停留在其正常下降通路之任何一处，其成年后不育是常见的并发症，发生机制未明。本课题组在前一个国家自然科学基金的资助下成功建立了隐睾特异性iPS细胞系，可用于隐睾生精机制研究的细胞模型。在此基础上，本课题首先成功建立了可诱导表达BMP4的隐睾特异性iPS细胞和对照组iPS细胞；研究建立了基于序贯添加BMP4、DHT和RA的三步法生精细胞体外诱导方案，BMP4使细胞从多能状态诱导分化到胚层决定阶段；DHT的目的是使细胞进入PGC并随后进入后续的分化成熟状态；RA的目的是使细胞进入减数分裂阶段。通过三步法诱导得到了比较稳定的生精细胞诱导效率；通过向各组培养系统中加入Doxycycline，激发BMP4 的表达，采集不同组别、不同时间点的分化样本，通过各种分析对诱导细胞的特性进行鉴定，发现BMP4在诱导iPS细胞体外分化时的胚层决定和向生精细胞发育过程中的关键作用，内源性表达BMP4比外源性添加BMP4因子将提高诱导分化效率和增加实验的稳定性，和正常对照相比，隐睾特异性iPS细胞向生精细胞的诱导分化效率在早期未见明显差异，在诱导分化后期、进入减数分裂阶段后有显著差异；与外源性补充BMP4因子相比，诱导表达的BMP4诱导体系的诱导结果更为稳定、诱导效率更高，其原因可能为外源补充的BMP4因子可能存在批次差异、培养环境中易于降解导致诱导环境中因子浓度不稳定等因素。本课题的研究初步显示BMP4可能与隐睾的睾丸生精功能有关，其是开启生精过程的关键因子。但BMP4对生精的影响主要体现在早期阶段，如能及早纠正其表达，辅之以生精后期的关键因素（如睾酮的适时分泌、RA信号分子的适时开启），将有可能改善精子的生成及其质量。但因本课题仅仅得到了进入减数分裂后的细胞，仍未得到具有精子形状的细胞，且进入减数分裂的人的生殖细胞因伦理限制无法进行在体功能实验，限制了本课题的进一步展开。