【作者】 董宏山；

【导师】 杨万喜； 陈小章；

【作者基本信息】 浙江大学 ， 动物学， 2008， 硕士

【摘要】 精子发生过程中,哺乳动物通过细胞程序性死亡,或称为凋亡调节生精细胞的生成,以达到与支持细胞比例协调和组织内动态平衡,特别是在第一个生精波到来时,生精细胞出现大量的凋亡。同时,凋亡在成熟的精子中也会发生。细胞凋亡是精子发生过程中的重要事件,但是凋亡超出正常范围,会导致成熟精子数量过少或无精症。因此凋亡和与之相对的存活机制之间的动态平衡至关重要。以往的研究表明细胞凋亡主要通过线粒体途径和死亡受体途径内外两条通路进行,受Bcl-2家族凋亡因子(Bax,Bak,Bcl-xs,Bad)和抗凋亡因子(Bcl-2,Bcl-xL,Mcl,A1)的表达水平调节,同时多个半胱氨酸蛋白酶家族caspase成员参与凋亡的激活过程。选择何种凋亡途径依赖于外来刺激的种类。与凋亡相对的,动物体内存在许多可以抑制凋亡的存活因子,诸如视黄酸受体A,促卵泡成熟激素,雄性激素,促黄体生成激素等。存活因子抑制或阻断凋亡信号,虽然已经发现许多抑制凋亡的存活因子,但其详细的调控机制还不明确。我们发现一种激素M(?)llerian inhibitingsubstance(MIS)可以与精子细胞表面的膜蛋白受体相结合,以往的研究表明YWK-Ⅱ膜蛋白在胞浆一侧可以与Go蛋白相结合,因此,MIS与YWK-Ⅱ膜蛋白、Go蛋白很可能形成重要的信号通路。MIS的基本功能是在性别分化中促进穆勒氏管的退行性变化,而最新的研究表明MIS参与细胞抗凋亡的过程中。结合起来看,我们提出假设:MIS可以通过精子膜蛋白YWK-Ⅱ受体与Go蛋白一起共同起抑制凋亡,提升精子存活能力。为了检测和验证我们提出的假设,我们利用Western Blot检测了不同发育阶段的小鼠睾丸中YWK-Ⅱ蛋白的表达和MIS的表达情况,利用RT-PCR技术检测了YWK-Ⅱ在基因水平的表达情况。为了获得直接的实验证掘,我们以小鼠为动物模型进行显微操作,将MIS与YWK-Ⅱ的抗体注射到小鼠曲细精管中。48小时后利用计算机辅助检测技术检测显微操作后小鼠精子的各项参数,如运动能力,精子记数。在Western Blot实验中,我们发现YWK-Ⅱ蛋白的表达随小鼠发育而不断增强,与我们的推测YWK-Ⅱ在成年小鼠体内对成熟精子抗凋亡起作用相吻合,而MIS的表达则在小鼠发育早期逐渐降低,小鼠成年以后则稳定在较高的水平不变。说明MIS在小鼠发育早期起性别分化的重要作用。小鼠发育成熟后,这种分化作用不再需要,则其表达削弱,但仍维持较高水平。MIS则与YWK-Ⅱ一起提升精子的存活能力。从动物实验模型的显微注射实验中,我们得到了直接的证据。注射MIS则与YWK-Ⅱ抗体的处理组小鼠精子不仅在精子记数上显著降低,而且精子的运动能力也剧烈下降,这一结果直接证明了MIS与YWK-Ⅱ蛋白受体形成通路共同起抗凋亡,提升精子存活能力的假设。更多还原

【Abstract】 Apoptosis, or can be called cell programed death is believed the primary model to regulate the ratio of germ cell to sertoli cell and homeostasis in mammals during spermatogenesis. As we know, a massive wave of apoptosis will occur during the first wave of spermatogenesis. Physiological apoptosis also can be observed in mature sperm. Apoptosis is vital for the process of spermatogenesis, but excessive apoptosis will cause many reproductive dieases, such as, azoospermia, Oligozoospermia. It’s very important to keep the balance of sperm apoptosis and survival.Previous studies indicated that two major pathways ,the mitochondrial pathway and the cell death receptor pathway are involved in the process of apoptosis in mammals. Sperm apoptosis is regulated by the expression of apoptosis factors (Bax, Bak, Bcl-xs, Bad) and anti-apoptosis factors (Bcl-2, Bcl-xL, Mcl, A1), at the same time, many caspase proteins involve in the process. Which passway will be induced depend on the external signals. Contrast to apoptosis, there are many anti-apoptosis factors, such as, Retinoic Acid (RA), Follicle-stimulating Hormone (FSH), testoterone, Luteinizing hormone (LH). The anti-apoptosis factors can block the apoptosis signals, though the detailed mechanism is unclear. We recently found that Müllerian inhibiting substance(MIS)can bind to YWK-II receptor, which is a membrane protein expressing on sperm. Previous research demonstrated that YWK-II receptor can bind to Go protein. It’s possible that MIS can form a key passway together with YWK-II and Go protein. The primary role of MIS is sex determination and induce regression of the Müllerian ducts. New reports indicate that MIS involve in the process of anti-apoptosis. Taken together, we hypothese that MIS can inhibit apoptosis and promote sperm survival together with YWK-II and Go protein.In order to verify our hypothese, we check the expression of YWK-II and MIS in different ages’ mouse testis by Western Blot. We also check the expression of YWK-II on gene level by RT-PCR. With the purpose of getting the direct proofs, we perform the experiments of injecting the antibodies of YWK-II and MIS into the seminiferous tuble of mouse testis. 48 hours later, we check the parameters of sperm, such as sperm motility and sperm count, with computer assisted sperm analysis (CASA). In the Western Blot experiment, we found that the expression of YWK-II increase gradually during development. It’s coincident with the hypothesis about anti-apoptosis roles of YWK-II in adult mouse. On the contrary, the expression of MIS decrease gradually in the newborn mouse during development. The expression of MIS is stable and maintain at a relative high level in adult mouse. It suggest that the key role of MIS is sex determination in the newborn mouse and it become unimportant in adult mouse. The expression of MIS reduce gradually, but it’s considerable in the adult mouse. Maybe MIS and promote sperm survival by YWK-II receptor. We get the direct evidences from the in vivo experiments in mouse model. The sperm motility and sperm count decrease obviously in the antibody treament groups. It demonstrates that MIS can inhibit apoptosis and promote sperm survival though YWK-II receptor.更多还原