S型氯代甘油醇对精子获能相关蛋白磷酸化的影响及机制研究

发布时间：2018-06-09 05:21

本文选题：S型氯代甘油醇 + 精子特异的3-磷酸甘油脱氢酶(GAPDS)　；参考：《复旦大学》2012年博士论文

【摘要】：氯代甘油醇(a-chlorohydrin, ACH),化学名3-氯-1,2-丙二醇(3-mono-chloro-propane-1,2-diol,3-MCPD),是一种重要化工原料,普通人群可通过食用受污染的食物及以表氯醇树脂过滤的饮用水暴露ACH。ACH有R型((R)-α-chlorohydrin, RACH)和S型((S)-α-chlorohydrin, SACH)两种异构体。S型氯代甘油醇(SACH)被认为是经典的睾丸后毒物,短期低剂量经口染毒SACH可致多种雄性动物可逆性的不育。为研究SACH影响精子受精过程中可能的作用机制,本研究围绕SACH与大、小鼠/人精子获能相关蛋白磷酸化关系,深入分析SACH对精子获能调控信号通路的影响。第一部分S型氯代甘油醇对大鼠精子功能的影响本研究以改良的Biggers, Whitten, Whittingham培养液(BWW),37℃,5%CO2为大鼠精子获能孵育条件,观察6h获能过程中自发性顶体反应和蛋白酪氨酸磷酸化变化及体外精卵融合状况,以验证该孵育条件是否支持大鼠精子获能。结果显示,大鼠附睾尾精子在获能孵育之初几乎不发生顶体反应,蛋白酪氨酸磷酸化也处于低水平。随着孵育时间延长,精子自发性顶体反应率逐渐增加,在获能孵育6h顶体反应发生率接近60%；精子的蛋白酪氨酸磷酸化水平在4.5h之后开始增强,在6h磷酸化较为充分。在该孵育条件下,大鼠精卵融合率平均达到59.1%。以上结果表明,该孵育条件可充分支持大鼠精子获能。为观察SACH对大鼠精子功能影响,本研究在获能条件下以10μM、25μM、50μM和100μμMSACH染毒大鼠附睾尾精子6h,结果显示：25μμMSACH就可抑制精子运动速率,包括VAP,VCL和VSL以及精子头摆动幅度；精子超活化也受到抑制,25μM、50μM和100μM剂量组精子VCL≥400μm/s的比例与对照相比均大幅下降(P0.05)；自发性顶体反应可被10μM和25μμMSACH明显抑制,而浓度继续增加(≥50μμM)则与对照没有显著差别。SACH抑制大鼠精子蛋白酪氨酸磷酸化的作用非常显著,100μμMSACH可使85kDa条带蛋白酪氨酸磷酸化水平下降近90%,52kDa条带下降70%。但100μMSACH对大鼠精卵融合无显著影响,1.0mM或10mMSACH才可显著降低二细胞发生率。故SACH可显著抑制SD大鼠精子的运动速率、超活化以及蛋白酪氨酸磷酸化。第二部分s型氯代甘油醇对大鼠精子获能相关蛋白磷酸化的影响以cAMP/蛋白激酶A(PKA)介导的蛋白酪氨酸磷酸化是动物精子获能调控的关键信号途径。PKA通过磷酸化目标蛋白丝氨酸/苏氨酸(Ser/Thr)残基间接调控下游蛋白酪氨酸磷酸化。本研究结果显示,大鼠附睾尾精子在获能过程出现PKA底物磷酸化逐渐增强;在获能末期,磷脂酰肌醇-3激酶(PI3K)85kDa/55kDa亚基均被酪氨酸磷酸化。以SACH在获能条件下染毒大鼠精子,结果显示PKA底物磷酸化和PI3K85kDa/55kDa酪氨酸磷酸化可被50μM和100μMSACH显著抑制,提示PKA和PI3K活性可能受抑制。PKA活性受cAMP调控,本研究发现50μM和100μMSACH可减低大鼠精子cAMP水平,而加入cAMP类似物dbcAMP和磷酸二酯酶抑制剂IBMX可拮抗SACH对获能相关蛋白磷酸化的抑制作用,提示SACH通过抑制cAMP而影响下游获能相关磷酸化。SACH可抑制大鼠精子特异的3-磷酸甘油脱氢酶(GAPDS)(?)舌性,降低其ATP水平,而加入甘油可保护精子GAPDS、ATP、蛋白酪氨酸磷酸化、PKA底物磷酸化和PI3K酪氨酸磷酸化不受SACH抑制,表明糖酵解抑制是SACH影响精子蛋白磷酸化的原因。然而,研究未发现大鼠精子蛋白酪氨酸磷酸化被SACH抑制与其功能受损存在直接联系。第三部分s型氯代甘油醇对人和小鼠精子获能相关蛋白酪氨酸磷酸化的影响为了解其他动物精子在获能过程中蛋白磷酸化变化规律,本研究将人和小鼠精子在相应获能获能下孵育5h或2h,结果发现,人和小鼠精子在获能孵育条件下发生蛋白酪氨酸磷酸化,人精子的110kDa和85kDa条带磷酸化最为显著,而小鼠在83kDa条带最为显著,且均可被SACH抑制。本研究证实,甘油可以保护人精子蛋白酪氨酸磷酸化不受SACH抑制,且人精子更倾向于利用糖酵解途径产生的ATP磷酸化相关蛋白,据此推测,SACH可能通过与在大鼠相似途径抑制人精子蛋白磷酸化。与大鼠不同,在本研究中,小鼠精子PKA底物磷酸化在获能过程逐渐下降,但仍可被SACH抑制。SACH可抑制小鼠GAPDS活性,同时可影响小鼠精子与卵子黏附能力。为探讨精子体外获能受精过程的生理生化改变是否能作为评价雄性生殖毒物的方法,本研究以3-溴丙酮酸(BrPA)和奥硝唑,氟化物和新型污染物碘乙酸(IAA)等糖酵解抑制剂在获能条件下染毒人精子,结果表明这些化学物对人精子蛋白酪氨酸磷酸化有不同程度的抑制。提示某些环境化学物可影响精子功能的作用,外源化学物对体外精子的蛋白酪氨酸磷酸化影响有望作为雄性生殖毒物筛选测试的体外评价指标之
[Abstract]:Chloroglycerol (a-Chlorohydrin, ACH), chemical name 3- chlorine -1,2- propanediol (3-mono-chloro-propane-1,2-diol, 3-MCPD), is an important chemical raw material. Ordinary people can expose ACH.ACH has R type ((R) - alpha -chlorohydrin, RACH) and S type by eating contaminated food and drinking water filtered by epichlorohydrin resin. Two isomers,.S type chloroglycerol (SACH), are considered as the classic testicular posterior poison. Short term low dose of SACH can induce reversible sterility in many male animals. In order to study the possible mechanism of effect of SACH on sperm fertilization, this study focuses on the phosphorylation of SACH with large, rat / human sperm capacitated proteins. The effect of SACH on the regulation of signal transduction pathway in sperm capacitation.
Part 1 Effect of S chloroglycerol on sperm function in rats
In this study, a modified Biggers, Whitten, Whittingham culture solution (BWW), 37 C and 5%CO2 were used to incubate sperm in rats. The changes of spontaneous acrosome reaction and protein tyrosine phosphorylation and in vitro sperm fusion in the process of 6h capture were observed to verify whether the incubation conditions support the sperm capacitation in rats. The results showed that the epididymal tail of rats was found. When the sperm was incubated at the beginning of the incubation, there was almost no acrosome reaction, and the protein tyrosine phosphorylation was also low. As the incubation time extended, the spontaneous acrosome reaction rate of the sperm increased gradually. The rate of the acrosome reaction in the incubated 6h was close to 60%; the level of the protein tyrosine phosphorylation of the sperm began to increase after 4.5H, and in the phosphorylation of 6h, the rate of phosphorylation of the sperm protein tyrosine was increased. Under the hatching condition, the average rate of sperm egg fusion was above 59.1%.. The results showed that the incubation condition could fully support rat sperm capacitation.
To observe the effect of SACH on spermatozoa function in rats, the sperm 6h in epididymis of rats was exposed to 10 mu M, 25 mu M, 50 mu M and 100 mu MSACH. The results showed that the sperm motility was inhibited by 25 mu MSACH, including VAP, VCL and VSL, and the swing amplitude of sperm head; the activation of sperm Zi Chao was also suppressed, 25 mu M, 50, M and 100 US dose groups The proportion of sperm VCL > 400 m/s decreased significantly (P0.05), and the spontaneous acrosome reaction could be significantly inhibited by 10 M and 25 mu MSACH, while the concentration continued to increase (> 50 mu M), but there was no significant difference between the sperm protein tyrosine phosphorylation of the.SACH inhibition rats and the 100 u MSACH could make the 85kDa strip cheese. The phosphorylation level of ammonia acid decreased by nearly 90%, 52kDa strip decreased 70%. but 100 MSACH had no significant effect on sperm fusion in rats. 1.0mM or 10mMSACH could significantly reduce the two cell rate. Therefore, SACH could significantly inhibit the rate of sperm movement, super activation and protein tyrosine phosphorylation in SD rats.
The second part is the effect of s chloroglycerol on the phosphorylation of sperm capacitation related protein in rats.
Protein tyrosine phosphorylation mediated by cAMP/ protein kinase A (PKA) is the key signal pathway for the regulation of animal sperm capacitation,.PKA indirectly regulates downstream protein tyrosine phosphorylation through the phosphorylated target protein serine / threonine (Ser/Thr) residues. The results show that the phosphorylation of PKA substrate in the capacitation process of rat epididymal spermatozoa Gradually, at the end of the acquisition, phosphatidylinositol -3 kinase (PI3K) 85kDa/55kDa subunits were phosphorylated by tyrosine. SACH was poisoned by SACH under the acquired condition. The results showed that the phosphorylation of PKA substrate and PI3K85kDa/55kDa tyrosine phosphorylation could be significantly suppressed by 50 mu M and 100 mu MSACH, suggesting that the activity of PKA and PI3K may be inhibited by cA. MP regulation, the study found that 50 mu M and 100 MSACH can reduce the cAMP level of rat sperm, while dbcAMP and phosphodiesterase inhibitor IBMX, cAMP analogues, can antagonize the inhibitory effect of SACH on the phosphorylation of energy related proteins, suggesting that SACH can inhibit the spermatozoon specific 3- phosphoric acid in rats by inhibiting the downstream acquired phosphorylation.SACH by inhibiting cAMP. Glycerol dehydrogenase (GAPDS) is a tongue and reduces its ATP level, while glycerol can protect sperm GAPDS, ATP, protein tyrosine phosphorylation, PKA substrate phosphorylation and PI3K tyrosine phosphorylation are not inhibited by SACH, indicating that glycolytic inhibition is the cause of SACH affecting the phosphorylation of sperm protein. However, the study of sperm protein tyrosine phosphorylation was not found in rats. There is a direct link between SACH inhibition and impairment of its function.
The third part is the effect of s chloroglycerol on tyrosine phosphorylation of human sperm and mouse sperm capacitation related protein.
In order to understand the changes of protein phosphorylation of other animal sperm during the process of capacitation, the human and mouse sperm were incubated with 5h or 2H under the capacitated energy acquisition. The results showed that the human and mouse spermatozoa were phosphorylated with protein tyrosine, and the 110kDa and 85kDa bands of human sperm were most significant, and the mice were in 83kDa. The stripe is most significant and can be inhibited by SACH. This study confirms that glycerol can protect human sperm protein tyrosine phosphorylation from SACH inhibition, and human sperm is more inclined to use glycolytic pathway to produce ATP phosphorylation related proteins. Accordingly, it is presumed that SACH may inhibit the phosphorylation of human sperm protein by similar pathway in rats. In this study, the phosphorylation of PKA substrate in mouse spermatozoa decreased gradually in the capacitation process, but it could still be inhibited by SACH to inhibit the GAPDS activity of mice and to influence the adhesion ability of sperm and egg in mice. The effects of 3- bromide pyruvic acid (BrPA) and ornidazole, fluoride and new type of contaminant iodide acetate (IAA) and other glycolytic inhibitors on human spermatozoa under capacitated conditions showed that these chemicals inhibited the human sperm protein tyrosine phosphorylation in varying degrees. In vitro sperm protein tyrosine phosphorylation is expected to be an in vitro evaluation index for screening male reproductive toxicants.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

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