【作者】 何晨；

【导师】 刘强；

【作者基本信息】 上海交通大学 ， 人体解剖与组织胚胎学， 2020， 硕士

【摘要】 精子在睾丸中产生后并不具备与卵子融合的受精能力,需在附睾中获得前向运动力,随后在雌性生殖道内先后经历获能及顶体反应,穿过透明带,与卵子融合,完成受精过程。若精子获能或顶体反应发生异常,均会导致男性生育力下降。目前,获能和顶体反应已经有了比较广泛的研究,但其具体机制研究尚不完全明确。本研究在前期筛选影响精子获能和顶体反应的小分子化合物过程基础上,探索所发现的两种小分子化合物AC220和ZK 93426对精子获能及顶体反应的影响并进一步探索其作用机制。AC220是FMS样酪氨酸激酶3(FMS-like tyrosine kinase-3,FLT3)有效的选择性抑制剂,而ZK 93426属于β-咔啉衍生物,是一种选择性苯二氮卓类受体拮抗剂。我们首先检测了AC220对小鼠和人精子顶体反应的作用,结果显示,AC220显著抑制顶体反应。随后实验证明,AC220对小鼠精子存活及运动力无明显影响。进一步检测AC220对获能的作用发现,其明显抑制了获能过程中相关蛋白酪氨酸磷酸化,并且获能后的精子中加入AC220可以下调蛋白酪氨酸磷酸化,提示AC220抑制精子顶体反应是通过下调蛋白酪氨酸磷酸化实现的。PKA激动剂dbc AMP对AC220的作用显示,dbc AMP可以逆转AC220对小鼠精子获能的抑制作用,提示AC220作用于PKA通路上游。另外,对于AC220的靶蛋白FLT3分析发现,FLT3存在于小鼠睾丸生精细胞中,并且定位于小鼠精子的头部,但定位于人精子的颈部。另一方面,我们检测了ZK 93426对小鼠和人精子获能的影响,结果显示,ZK 93426明显促进了获能过程中相关蛋白酪氨酸磷酸化,而这种促进作用在缺少钙离子的获能培养液中更为明显,免疫荧光法也进一步证实了这个结论。随后发现ZK 93426对小鼠精子运动力无明显影响。我们分别选用精子钙离子通道Cat Sper抑制剂Mibefradil和NNC 55-0396及PKA通路抑制剂H89进一步探索其机制发现,对Cat Sper的抑制并不会影响ZK 93426对于获能相关蛋白酪氨酸磷酸化的显著促进作用,而H89可抑制其作用。综上所述,AC220可以抑制精子获能相关的蛋白酪氨酸磷酸化,并通过下调蛋白酪氨酸磷酸化抑制顶体反应;ZK 93426可促进获能相关的蛋白酪氨酸磷酸化,在没有外源钙离子存在时这种促进作用更显著;AC220和ZK 93426可能通过作用于PKA通路的上游发挥相应的作用,但ZK 93426不依赖于Cat Sper通道发挥作用。本文研究结果将有助于揭示精子获能中的信号通路和分子机制,为临床不明原因男性不育的诊断和治疗提供新的思路。更多还原

【Abstract】 Spermatozoa produced in the testis don’t have the ability to fertilize the egg.It needs to gain forward movement ability in the epididymis,undergoes capacitation and acrosome reaction in the female reproductive tract,passes through the zona pellucida,and integrates with the egg to complete the fertilization.If capacitation or acrosome reaction is abnormal,male fertility will decrease.At present,there have been extensive studies on capacitation and acrosome reaction,but the specific mechanism has not been completely resolved.Previously AC220 and ZK 93426 were found during the screening of small molecular compounds that affect capacitation and acrosome reaction.The aim of this study was to explore the effect of these two compounds on sperm capacitation and acrosome reaction and to further explore the underlying mechanism.AC220 is a potent,selective and efficacious FMS-like tyrosine kinase-3(FLT3)inhibitor while ZK93426 is a selective benzodiazepine receptor antagonist,it belongs to β-carboline derivatite.We first detected the effect of AC220 on the acrosome reaction of mouse and human sperm and the result showed that AC220 could inhibit the acrosome reaction significantly.Later experiment showed that AC220 had no significant effect on mouse sperm viability and motility.Further examination revealed that AC220 could significantly inhibit capacitation-related protein tyrosine phosphorylation.Furthermore,PKA pathway agonist dbc AMP can reverse the inhibitory effect of AC220 on capacitation.In addition,we analyzed FLT3,which is the target protein of AC220 and found FLT3 is present in mouse spermatogenic cells,and FLT3 is located in the head of mouse sperm and in the neck of human sperm.On the other hand,we tested the effect of ZK 93426 on mouse and human sperm capacitation.The results showed that ZK 93426 significantly promoted capacitation-related protein tyrosine phosphorylation.The promoting effect was more intensive in HMB in the absence of calcium and immunofluorescence confirmed this conclusion.ZK 93426 was subsequently found to have no significant effect on mouse sperm motility.We selected the Cat Sper channel inhibitors Mibefradil and NNC 55-0396 and the PKA pathway inhibitor H89 to further explore their mechanism and found that Mibefradil and NNC 55-0396 couldn’t inhibit the promoting effect of ZK 93426 on capacitation-related tyrosine phosphorylation while H89 could inhibit its effect.In summary,AC220 inhibits the acrosome reaction by down-regulating protein tyrosine phosphorylation.ZK 93426 promotes capacitation-related protein tyrosine phosphorylation.AC220 and ZK 93426 may play a corresponding role by acting upstream of the PKA pathway but the promoting effect of ZK 93426 is independent of the Cat Sper channel.This study could help reveal the signal pathways and molecular mechanism in sperm capacitation,and provide new ideas for the diagnosis and treatment of male infertility.更多还原