睾丸热激促进CatSper1 DNA疫苗抗雄性生育作用及机制研究

At present, the male contraceptive options are limited to the condom and vasectomy, while many men wish to carry contraceptive responsibility. The lack of availability of effective and satisfactory contraceptive methods for many couples and adolescents caused millions of unintended pregnancies annually. Therefore, developing new approaches for male contraception is urgently needed. Immunocontraception may provide viable and valuable alternatives to the currently few options for male contraception. It has the features of long-acting, reversible, and easy to administer. Sperm specific antigen with key function should be target with high performance and low side effects for male immunocontraception. However, insufficient immunoreactive intensity and binding of the antibodies to sperm because of the existence of blood-testis-barrier (BTB) may be the main constrained bottleneck problems. Cation channel of sperm 1 (CatSper1) is an unique sperm cation channel which is required for sperm hyperactivation and male fertility. Our previous study provides evidence that immunization with B-cell epitopes in transmembrane domains and pore region of CatSper1 can cause significant antifertility effects in male mice, while no obvious side effect has been observed. Scrotal heat stress can reversibly open BTB. The present proposal is aimed to develop a mild scrotal heat stress which can open mouse BTB, and reveal its application and mechanism on enhancing the antifertility effect of CatSper DNA vaccine on male mice. Different adjuvants and poly(lactic-co-glycolic acid) will be applied and compared to enhance the immune effect of the DNA vaccine. Results of the project may provide a novel approach for enhancing the antifertility effect of DNA vaccine targeting sperm specific antigens.

男性避孕方法少，而男性有承担避孕的责任和参与避孕的热情，研究男性避孕方法，对计划生育及减少意外妊娠等有很好的价值和意义。免疫避孕有使用简单、有效期长、副作用少等特点，被认为是潜在的理想男性避孕方法。针对功能关键的精子特异性抗原的免疫避孕更是能增加避孕效果及减少副作用，但存在免疫反应强度不够，及因血睾屏障存在而导致抗体与精子结合少的瓶颈问题。CatSper1是精子特异性钙通道，是精子超活化﹑穿过透明带所必需，敲除该基因雄鼠完全不育；我们前期研究表明以CatSper1为靶点的表位疫苗有较好的抗雄性生育效果，且无明显毒副作用。睾丸热激是能使血睾屏障可逆性开放的安全方式，本课题拟摸索一种能使血睾屏障开放得温和的睾丸热激条件，研究其是否能增加抗体通过而促进CatSper1 DNA疫苗的抗雄性生育效果及机制；并从疫苗构建及包被等方面提高免疫反应强度。为提高精子特异性抗原用于男性免疫避孕的效果提供新思路

研究男性避孕方法对减少意外妊娠等有很好的价值和意义。针对功能关键的精子特异性抗原的免疫避孕能增加避孕效果、减少副作用，但存在免疫反应强度不够，及因血睾屏障存在而导致抗体与精子结合少的瓶颈问题。CatSper1是精子特异性钙通道，是精子超活化﹑穿过透明带所必需，敲除该基因雄鼠完全不育。我们前期研究表明以CatSper1为靶点的表位疫苗有较好的抗雄性生育效果，且无明显毒副作用；睾丸热激是能使血睾屏障可逆性开放的安全方式。本课题首先构建Catsper1 B细胞表位DNA融合疫苗（Catsper1 B细胞表位DNA联合免疫佐剂通用辅助T细胞表位PADRE以及C3d3和GMCSF，以增加免疫原性，以下简称融合疫苗）并体外验证了其重组质粒目的基因的表达。接下来研究两种物理方式，即阴囊热激和低频低强度超声波对小鼠血睾屏障物质通透性的影响，探索一种可能提高血睾屏障对抗体通透性的方法和实验条件。研究显示将小鼠单次置于43℃水浴进行阴囊热激30min，或2.5w/cm²超声持续5min均可一过性打开小鼠血睾屏障，观察到生物素进入生精小管，但只有热激组可以观察到IgG、IgM和附睾小体均进入到生精小管管腔中，两种方法均不引起小鼠睾丸组织病理性损伤。最后我们联合融合疫苗和热激对小鼠进行在体免疫避孕实验，结果提示，实验组的雄鼠交配能力不受影响，但是雌鼠受孕率却显著降低，且附睾精子的总活力和前向运动精子率都显著下降，而在免疫处理后28天雄鼠精子数量、活率均恢复正常水平。.综上，我们成功构建了以Catsper1 B细胞表位DNA为基础的融合疫苗，探索了打开小鼠血睾屏障的方法和条件，从而解决了先前实验中出现的免疫强度不够、大分子抗体很难通过血睾屏障的问题；并且在体对小鼠进行融合疫苗免疫注射并辅以热激处理，获得了预期的避孕效果以及可逆的抗生育力效果，为男性免疫的临床实施提供了可靠的理论基础。圆满完成本课题的工作计划。发表论文1篇，修回一篇，培养硕士研究生2名。