易位染色体在减数分裂前期I的行为与生精功能关系的研究

About 1% patients are translocation carriers in infertile male. Different carriers usually have different spermatogenetic abilities. However, little was known about the mechanisms which affect the sperm number in translocation carriers. The translocated chromosomes and their homologous align themselves in a cross shape known as a quadrivalent,previous studies and our previous results in this project indicate that if the quadrivalent associated with sex body, the patient was almostly no sperm in semen. On the basis of these findings, we propose the following hypothesis: The MSCI function was disrupted when the quadricalent associated with sex body, which caused spermatogenesis failure in these carriers. In order to uncover the mehamisms involed, we will analyse the translocated chromosome behaviour in detail by using chromosome spread combined with immunofluorescence and FISH (Fluorescence In Situ Hybridization). Furthermore,In order to find the resons account for determination of the translocated assosiate with sex body in pachytene stage, we will analyse the DSB repair progress on the translocated chromosomes during meiosis I. In addition,we will detect if the MSCI function were disrupted when the quadrivalent associated with the sex body by using kinds of technologies,such as deep sequencing, mRNA FISH, immunofluorescence and so on. Overall,our research in this project will uncove the molecular mechanisms which afect the sperm number in translocation carriers from a new angle,hoping to provide new theoretical support for clinical treatment.

染色体易位患者约占男性不育病人的1%，不同染色体易位患者生精能力（正常、少精、无精）差异较大，而决定患者生精数量的分子机制尚不明确，本课题前期研究及既往的研究均提示减数分裂过程中易位染色体配对、联会形成的“四射体”是否与性泡（sex body）相连会直接影响患者生精数量，基于以上发现，本课题首次提出假说即“四射体与性泡结合可能影响性泡的功能，从而导致生精障碍”。为验证这一假说，本研究采用染色体微铺展结合免疫荧光以及FISH等技术分析易位染色体在减数分裂前期I的行为。利用易位染色体DSB（double-strand break）标记物分析其修复进程来探讨决定“四射体”是否与性泡结合的机制。进一步利用二代测序、mRNA原位杂交、免疫荧光等技术分析“四射体”与性泡结合后减数分裂性染色体基因沉默机制是否正常。本课题将从全新的角度阐述不同易位染色体患者生精能力差异的原因，为临床治疗提供新的理论支持。

本课收集不育病人的睾丸活检组织标本，结果显示易位染色体患者生精障碍与否与其是否在减数分裂过程中与性泡结合有关，如果与性泡结合就表现为严重的生精障碍，此时性泡的功能可能会受到影响，而易位染色体上DSB 不能得到修复是导致其与性泡相连的真正原因。另外，利用本项目建立起来的平台，检测非梗阻性无精子症患者精母细胞减数分裂进程，发现一例非常特殊的病例，并进入深入研究其导致生精障碍的原因。同时课题组积极拓宽研究思路，从端粒，减数分裂，肥胖等多角度深入研究男性生育力低下的相关机制。首次证明了精子端粒长度与生精功能和男性患者出生时父亲的年龄显著相关，精子端粒长度 与个体年龄、精子数目、出生时父母亲年龄呈显著正相关，精子端粒长度与优胚率、可移植胚胎率成正相关，但是临床妊娠率并无显著影响；精液密度梯度离心法可以富集具有较长端粒、活力高且DNA损伤较低的精子；精液中端粒长度与精子密度正相关；男性超重/肥胖引起氧化应激反应增加，造成精子端粒长度变短、线粒体活性降低和DNA碎片增加，进而导致IVF周期不良妊娠结局；外周血端粒长度缩短表明生精功能下降，且与原发性无精症发病有显著的相关性。