本文選題：S型氯代甘油醇 + 精子特異的3-磷酸甘油脫氫酶(GAPDS)��； 參考：《復旦大學》2012年博士論文

【摘要】：氯代甘油醇(a-chlorohydrin, ACH),化學名3-氯-1,2-丙二醇(3-mono-chloro-propane-1,2-diol,3-MCPD),是一種重要化工原料,普通人群可通過食用受污染的食物及以表氯醇樹脂過濾的飲用水暴露ACH。ACH有R型((R)-α-chlorohydrin, RACH)和S型((S)-α-chlorohydrin, SACH)兩種異構體。S型氯代甘油醇(SACH)被認為是經典的睪丸后毒物,短期低劑量經口染毒SACH可致多種雄性動物可逆性的不育。為研究SACH影響精子受精過程中可能的作用機制,本研究圍繞SACH與大、小鼠/人精子獲能相關蛋白磷酸化關系,深入分析SACH對精子獲能調控信號通路的影響。 第一部分S型氯代甘油醇對大鼠精子功能的影響 本研究以改良的Biggers, Whitten, Whittingham培養(yǎng)液(BWW),37℃,5%CO2為大鼠精子獲能孵育條件,觀察6h獲能過程中自發(fā)性頂體反應和蛋白酪氨酸磷酸化變化及體外精卵融合狀況,以驗證該孵育條件是否支持大鼠精子獲能。結果顯示,大鼠附睪尾精子在獲能孵育之初幾乎不發(fā)生頂體反應,蛋白酪氨酸磷酸化也處于低水平。隨著孵育時間延長,精子自發(fā)性頂體反應率逐漸增加,在獲能孵育6h頂體反應發(fā)生率接近60%；精子的蛋白酪氨酸磷酸化水平在4.5h之后開始增強,在6h磷酸化較為充分。在該孵育條件下,大鼠精卵融合率平均達到59.1%。以上結果表明,該孵育條件可充分支持大鼠精子獲能。 為觀察SACH對大鼠精子功能影響,本研究在獲能條件下以10μM、25μM、50μM和100μμMSACH染毒大鼠附睪尾精子6h,結果顯示：25μμMSACH就可抑制精子運動速率,包括VAP,VCL和VSL以及精子頭擺動幅度；精子超活化也受到抑制,25μM、50μM和100μM劑量組精子VCL≥400μm/s的比例與對照相比均大幅下降(P0.05)；自發(fā)性頂體反應可被10μM和25μμMSACH明顯抑制,而濃度繼續(xù)增加(≥50μμM)則與對照沒有顯著差別。SACH抑制大鼠精子蛋白酪氨酸磷酸化的作用非常顯著,100μμMSACH可使85kDa條帶蛋白酪氨酸磷酸化水平下降近90%,52kDa條帶下降70%。但100μMSACH對大鼠精卵融合無顯著影響,1.0mM或10mMSACH才可顯著降低二細胞發(fā)生率。故SACH可顯著抑制SD大鼠精子的運動速率、超活化以及蛋白酪氨酸磷酸化。 第二部分s型氯代甘油醇對大鼠精子獲能相關蛋白磷酸化的影響 以cAMP/蛋白激酶A(PKA)介導的蛋白酪氨酸磷酸化是動物精子獲能調控的關鍵信號途徑。PKA通過磷酸化目標蛋白絲氨酸/蘇氨酸(Ser/Thr)殘基間接調控下游蛋白酪氨酸磷酸化。本研究結果顯示,大鼠附睪尾精子在獲能過程出現PKA底物磷酸化逐漸增強;在獲能末期,磷脂酰肌醇-3激酶(PI3K)85kDa/55kDa亞基均被酪氨酸磷酸化。以SACH在獲能條件下染毒大鼠精子,結果顯示PKA底物磷酸化和PI3K85kDa/55kDa酪氨酸磷酸化可被50μM和100μMSACH顯著抑制,提示PKA和PI3K活性可能受抑制。PKA活性受cAMP調控,本研究發(fā)現50μM和100μMSACH可減低大鼠精子cAMP水平,而加入cAMP類似物dbcAMP和磷酸二酯酶抑制劑IBMX可拮抗SACH對獲能相關蛋白磷酸化的抑制作用,提示SACH通過抑制cAMP而影響下游獲能相關磷酸化。SACH可抑制大鼠精子特異的3-磷酸甘油脫氫酶(GAPDS)(?)舌性,降低其ATP水平,而加入甘油可保護精子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影響精子蛋白磷酸化的原因。然而,研究未發(fā)現大鼠精子蛋白酪氨酸磷酸化被SACH抑制與其功能受損存在直接聯(lián)系。 第三部分s型氯代甘油醇對人和小鼠精子獲能相關蛋白酪氨酸磷酸化的影響 為了解其他動物精子在獲能過程中蛋白磷酸化變化規(guī)律,本研究將人和小鼠精子在相應獲能獲能下孵育5h或2h,結果發(fā)現,人和小鼠精子在獲能孵育條件下發(fā)生蛋白酪氨酸磷酸化,人精子的110kDa和85kDa條帶磷酸化最為顯著,而小鼠在83kDa條帶最為顯著,且均可被SACH抑制。本研究證實,甘油可以保護人精子蛋白酪氨酸磷酸化不受SACH抑制,且人精子更傾向于利用糖酵解途徑產生的ATP磷酸化相關蛋白,據此推測,SACH可能通過與在大鼠相似途徑抑制人精子蛋白磷酸化。與大鼠不同,在本研究中,小鼠精子PKA底物磷酸化在獲能過程逐漸下降,但仍可被SACH抑制。SACH可抑制小鼠GAPDS活性,同時可影響小鼠精子與卵子黏附能力。為探討精子體外獲能受精過程的生理生化改變是否能作為評價雄性生殖毒物的方法,本研究以3-溴丙酮酸(BrPA)和奧硝唑,氟化物和新型污染物碘乙酸(IAA)等糖酵解抑制劑在獲能條件下染毒人精子,結果表明這些化學物對人精子蛋白酪氨酸磷酸化有不同程度的抑制。提示某些環(huán)境化學物可影響精子功能的作用,外源化學物對體外精子的蛋白酪氨酸磷酸化影響有望作為雄性生殖毒物篩選測試的體外評價指標之
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R114

【參考文獻】

相關期刊論文 前1條

1 張皓;錢國慶;凌霄;趙建偉;鄭唯椺;陳麗;蔣頌輝;屈衛(wèi)東;;S型氯代甘油醇對雄性ICR小鼠生育力的影響[J];衛(wèi)生研究;2008年02期

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本文編號：1999124