FADD磷酸化對(duì)雄性小鼠精子發(fā)生影響的研究
發(fā)布時(shí)間：2018-12-21 07:22
【摘要】：FADD(Fas-associated death domain),最早是作為細(xì)胞程序性死亡(凋亡)過(guò)程中的接頭蛋白被人們熟識(shí)。而如今FADD的非凋亡功能已越來(lái)越受到人們的關(guān)注。目前,已發(fā)現(xiàn)FADD在細(xì)胞周期、細(xì)胞增殖等眾多過(guò)程中發(fā)揮重要功能。尤其是FADD-191Ser的磷酸化修飾,在各種生理、病理?xiàng)l件下,都發(fā)揮重要的調(diào)控功能。為了研究FADD-191Ser磷酸化修飾在生殖系統(tǒng)可能發(fā)揮的作用,本實(shí)驗(yàn)室與南京大學(xué)醫(yī)藥生物技術(shù)國(guó)家重點(diǎn)實(shí)驗(yàn)室合作,選用FADD~(-/-) tgD (Ser191Asp)小鼠(內(nèi)源性FADD敲除小鼠、同時(shí)轉(zhuǎn)入tgD(mFADD-S191D)模擬FADD的永久磷酸化,后文簡(jiǎn)稱(chēng)FADD(S191D)小鼠)作為研究對(duì)象,同窩的FADD~(+/-)小鼠作為對(duì)照,開(kāi)展實(shí)驗(yàn)。 在前期小鼠的繁殖過(guò)程中,我們發(fā)現(xiàn)FADD(S191D)小鼠是不育的,只能用FADD~(+/-)tgD和FADD~(+/-)小鼠交配的方式拿到。FADD(S191D)這一小鼠模型最明顯的表型特征就是瘦小。從FADD(S191D)雄性成年小鼠解剖特征上看,睪丸很小,睪丸重與小鼠體重的比值(臟器指數(shù))較對(duì)照組顯著降低。睪丸HE切片顯示,FADD(S191D)小鼠睪丸曲精小管管徑明顯減小,管腔中細(xì)胞層數(shù)減少,幾乎沒(méi)有或很少有減數(shù)分裂后的細(xì)胞,并且管腔中存在大量強(qiáng)嗜酸性染色、疑似凋亡的生精細(xì)胞。對(duì)睪丸各級(jí)生精細(xì)胞進(jìn)行計(jì)數(shù),發(fā)現(xiàn)其睪丸中精原細(xì)胞、精母細(xì)胞及圓形精子細(xì)胞均有不同程度的減少,其中以圓形精子細(xì)胞的下降最為明顯。而對(duì)能到達(dá)附睪尾的精子進(jìn)行形態(tài)學(xué)觀察和計(jì)數(shù),也發(fā)現(xiàn)附睪尾的精子明顯減少,精子的畸形率顯著上升。 為了尋找FADD(S191D)小鼠精子發(fā)生障礙的原因,我們?nèi)〕赡闒ADD(S191D)小鼠及同窩對(duì)照小鼠睪丸,分別作TUNEL染色,發(fā)現(xiàn)FADD(S191D)小鼠睪丸管腔中細(xì)胞的凋亡率顯著上升,凋亡存在于各級(jí)生精細(xì)胞,其中以精母細(xì)胞的凋亡為主;結(jié)合以單倍體圓形精子細(xì)胞數(shù)目下降最明顯的各級(jí)生精細(xì)胞的減少,這些結(jié)果表明FADD永久磷酸化對(duì)精子發(fā)生的各個(gè)階段,尤其是精母細(xì)胞的減數(shù)分裂產(chǎn)生了嚴(yán)重的影響。 為研究FADD(S191D)導(dǎo)致生精細(xì)胞減數(shù)分裂異常的機(jī)制,我們選用減數(shù)分裂不同時(shí)期幾個(gè)特定的Marker,進(jìn)行了睪丸精母細(xì)胞聯(lián)會(huì)復(fù)合體鋪展免疫熒光,來(lái)觀察FADD(S191D)小鼠睪丸減數(shù)分裂的進(jìn)程。結(jié)果發(fā)現(xiàn),與對(duì)照相比,FADD(S191D)小鼠粗線期精母細(xì)胞常染色體上部分DNA雙鏈斷裂(DSBs)尚未被修復(fù),且聯(lián)會(huì)復(fù)合體上重組位點(diǎn)Marker,錯(cuò)配修復(fù)蛋白MLH1位點(diǎn)數(shù)減少。這一結(jié)果顯示,FADD(S191D)小鼠睪丸減數(shù)分裂過(guò)程中,染色體DNA重組修復(fù)發(fā)生異常,從而使得精母細(xì)胞凋亡率增加,精子發(fā)生障礙。 那么為什么FADD的永久磷酸化會(huì)導(dǎo)致重組修復(fù)位點(diǎn)異常呢?有學(xué)者研究發(fā)現(xiàn)在體細(xì)胞中FADD在細(xì)胞核內(nèi)可以和MLH1相互作用,同時(shí)FADD還能與甲基島結(jié)合結(jié)構(gòu)域蛋白4(MBD4)相互作用,參與維持基因組的穩(wěn)定[1]。因此我們推測(cè)在本研究中FADD的磷酸化也是通過(guò)這一相互作用來(lái)影響精母細(xì)胞DNA修復(fù)的。為了驗(yàn)證這一假設(shè),因MLH1和MBD4均在細(xì)胞核內(nèi)發(fā)揮功能,我們分別提取FADD(S191D)及FADD~(+/-)小鼠睪丸細(xì)胞質(zhì)、細(xì)胞核蛋白,Western Blot驗(yàn)證這幾種蛋白在細(xì)胞核與細(xì)胞質(zhì)中的表達(dá)情況,結(jié)果顯示:無(wú)論是實(shí)驗(yàn)組還是對(duì)照組,FADD在小鼠睪丸中均主要在細(xì)胞核表達(dá);在FADD(S191D)小鼠睪丸中,MLH1及MBD4在細(xì)胞質(zhì)中的表達(dá)量增加,而在細(xì)胞核中的表達(dá)下降,提示FADD的磷酸化可能影響了MLH1及MBD4從細(xì)胞質(zhì)到細(xì)胞核的轉(zhuǎn)運(yùn),使其在細(xì)胞核中的表達(dá)水平下降,進(jìn)而引起DNA修復(fù)障礙。 綜上,我們通過(guò)對(duì)FADD在雄性生殖系統(tǒng)的表達(dá)和定位的研究及FADD(S191D)小鼠生殖系統(tǒng)表型的觀察,發(fā)現(xiàn)FADD永久磷酸化能導(dǎo)致精子發(fā)生障礙,對(duì)其發(fā)生機(jī)制的初步探討顯示FADD磷酸化能通過(guò)降低細(xì)胞核中MLH1及MBD4的水平而導(dǎo)致精母細(xì)胞DNA斷裂修復(fù)障礙,進(jìn)而引起精母細(xì)胞凋亡率增加,精子發(fā)生異常。以上有關(guān)FADD(S191D)小鼠的研究,為進(jìn)一步了解FADD蛋白通過(guò)Ser-191磷酸化修飾發(fā)揮的非凋亡功能提供了新的思路和理論依據(jù)。
[Abstract]:FADD (Fas-associated dead domain) is the first to be recognized as a linker protein in the process of programmed cell death (apoptosis). Now the non-apoptotic function of FADD is more and more concerned. At present, FADD has been found to play an important role in many processes such as cell cycle and cell proliferation. in particular, the phosphorylation modification of FADD-191Ser plays an important regulatory function under various physiological and pathological conditions. In order to study the potential role of FADD-191Ser phosphorylation in the reproductive system, the laboratory, in cooperation with the National Key Laboratory of the National Key Laboratory of the Medical Biotechnology of Nanjing University, selected FADD-(-/-) tgD (Ser191Asp) mouse (endogenous FADD knockout mice, while transferring to tgD (mFADD-S191D) to simulate the permanent phosphorylation of FADD, The FADD-(+/-) mice in the same fossa were used as the control, and the experiment was carried out. In the early-stage mouse reproduction, we found that FADD (S191D) mice were sterile and only can be mated with FADD-(+/-) tgD and FADD-(+/-) mice. The most obvious phenotypic characteristic of the mouse model obtained by the. FADD (S191D) is that The testis is small, the ratio of the testis weight to the weight of the mouse (organ index) is significantly higher than that of the control group, from the anatomical features of the FADD (S191D) male adult mouse. The results showed that the diameter of the small tube in the testis of the FADD (S191D) mouse was significantly reduced, the number of cell layers in the lumen of the tube was reduced, and there were little or no meiosis cells. The cells were counted, and the spermatogonial cells, spermatocytes and round sperm cells of the testis were reduced to a certain extent, in which the decrease of the round sperm cells was the most It is obvious that the sperm of the tail of the epididymis can be observed and counted, and the sperm of the tail of the epididymis is obviously reduced, and the abnormal rate of the sperm is remarkable. In order to find the cause of the spermatogenesis in FADD (S191D) mice, we take adult FADD (S191D) mouse and the testis of the same-pit control mouse to make TUNEL staining respectively. It is found that the apoptosis rate of the cells in the testis of FADD (S191D) mice is significantly increased, and the apoptosis is present at all levels of spermatogenic cells, in which spermatocytes are used. The results show that FADD permanent phosphorylation is responsible for the various stages of spermatogenesis, especially in the meiosis of spermatocytes. The effect of FADD (S191D) on the abnormal meiosis of spermatogenic cells was studied. In order to study the mechanism of FADD (S191D) to cause the abnormal meiosis of spermatogenic cells, we selected several specific Markers in different periods of meiosis, and then, the FADD (S191D) mice were observed. It was found that the partial DNA double-strand break (DSBs) on the normal chromosome of the FADD (S191D) mice had not been repaired, and the recombination site Marker and the mismatch repair protein M on the FADD (S191D) mice had not been repaired. The number of LH1 sites was decreased. The results showed that during the meiosis of the testis of the FADD (S191D) mice, the chromosomal DNA reconstructive repair was abnormal, so that the apoptosis rate of the spermatocytes increased. Plus, the spermatogenesis is an obstacle. So why FADD's permanent phosphorylation It is found that FADD can interact with MLH1 in the nucleus and the FADD can interact with the methyl island binding domain protein 4 (MBD4). The stability of the genome[1] is maintained. Therefore, we assume that the phosphorylation of FADD in this study is also by this interaction In order to verify this hypothesis, we extracted FADD (S191D) and FADD ~ (+/-) mouse testis cytoplasm, nuclear protein and Western Blot to verify the nuclear and cytoplasm. The expression of MLH1 and MBD4 in the cytoplasm of the testis of FADD (S191D) mice was increased in the testis of FADD (S191D) mice. The expression of FADD decreased in the nucleus, suggesting that the phosphorylation of FADD could affect the transfer of MLH1 and MBD4 from the cytoplasm to the nucleus, and the level of expression of MLH1 and MBD4 in the nucleus decreased. In order to induce DNA repair, we found FADD in the study of the expression and localization of FADD in the male reproductive system and the phenotype of the reproductive system of FADD (S191D) mice. Permanent phosphorylation can lead to a disorder of spermatogenesis, and a preliminary study of the mechanism is shown to show that FADD phosphorylation can cause the DNA of the spermatocyte to break and repair by reducing the level of MLH1 and MBD4 in the nucleus, which in turn causes the spermatogenesis. The apoptosis rate of the cells increased and the spermatogenesis was abnormal. The above studies on FADD (S191D) mice provide a further understanding of the non-apoptosis of the FADD protein through the Ser-191 phosphorylation modification.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363
本文編號(hào)：2388564
[Abstract]:FADD (Fas-associated dead domain) is the first to be recognized as a linker protein in the process of programmed cell death (apoptosis). Now the non-apoptotic function of FADD is more and more concerned. At present, FADD has been found to play an important role in many processes such as cell cycle and cell proliferation. in particular, the phosphorylation modification of FADD-191Ser plays an important regulatory function under various physiological and pathological conditions. In order to study the potential role of FADD-191Ser phosphorylation in the reproductive system, the laboratory, in cooperation with the National Key Laboratory of the National Key Laboratory of the Medical Biotechnology of Nanjing University, selected FADD-(-/-) tgD (Ser191Asp) mouse (endogenous FADD knockout mice, while transferring to tgD (mFADD-S191D) to simulate the permanent phosphorylation of FADD, The FADD-(+/-) mice in the same fossa were used as the control, and the experiment was carried out. In the early-stage mouse reproduction, we found that FADD (S191D) mice were sterile and only can be mated with FADD-(+/-) tgD and FADD-(+/-) mice. The most obvious phenotypic characteristic of the mouse model obtained by the. FADD (S191D) is that The testis is small, the ratio of the testis weight to the weight of the mouse (organ index) is significantly higher than that of the control group, from the anatomical features of the FADD (S191D) male adult mouse. The results showed that the diameter of the small tube in the testis of the FADD (S191D) mouse was significantly reduced, the number of cell layers in the lumen of the tube was reduced, and there were little or no meiosis cells. The cells were counted, and the spermatogonial cells, spermatocytes and round sperm cells of the testis were reduced to a certain extent, in which the decrease of the round sperm cells was the most It is obvious that the sperm of the tail of the epididymis can be observed and counted, and the sperm of the tail of the epididymis is obviously reduced, and the abnormal rate of the sperm is remarkable. In order to find the cause of the spermatogenesis in FADD (S191D) mice, we take adult FADD (S191D) mouse and the testis of the same-pit control mouse to make TUNEL staining respectively. It is found that the apoptosis rate of the cells in the testis of FADD (S191D) mice is significantly increased, and the apoptosis is present at all levels of spermatogenic cells, in which spermatocytes are used. The results show that FADD permanent phosphorylation is responsible for the various stages of spermatogenesis, especially in the meiosis of spermatocytes. The effect of FADD (S191D) on the abnormal meiosis of spermatogenic cells was studied. In order to study the mechanism of FADD (S191D) to cause the abnormal meiosis of spermatogenic cells, we selected several specific Markers in different periods of meiosis, and then, the FADD (S191D) mice were observed. It was found that the partial DNA double-strand break (DSBs) on the normal chromosome of the FADD (S191D) mice had not been repaired, and the recombination site Marker and the mismatch repair protein M on the FADD (S191D) mice had not been repaired. The number of LH1 sites was decreased. The results showed that during the meiosis of the testis of the FADD (S191D) mice, the chromosomal DNA reconstructive repair was abnormal, so that the apoptosis rate of the spermatocytes increased. Plus, the spermatogenesis is an obstacle. So why FADD's permanent phosphorylation It is found that FADD can interact with MLH1 in the nucleus and the FADD can interact with the methyl island binding domain protein 4 (MBD4). The stability of the genome[1] is maintained. Therefore, we assume that the phosphorylation of FADD in this study is also by this interaction In order to verify this hypothesis, we extracted FADD (S191D) and FADD ~ (+/-) mouse testis cytoplasm, nuclear protein and Western Blot to verify the nuclear and cytoplasm. The expression of MLH1 and MBD4 in the cytoplasm of the testis of FADD (S191D) mice was increased in the testis of FADD (S191D) mice. The expression of FADD decreased in the nucleus, suggesting that the phosphorylation of FADD could affect the transfer of MLH1 and MBD4 from the cytoplasm to the nucleus, and the level of expression of MLH1 and MBD4 in the nucleus decreased. In order to induce DNA repair, we found FADD in the study of the expression and localization of FADD in the male reproductive system and the phenotype of the reproductive system of FADD (S191D) mice. Permanent phosphorylation can lead to a disorder of spermatogenesis, and a preliminary study of the mechanism is shown to show that FADD phosphorylation can cause the DNA of the spermatocyte to break and repair by reducing the level of MLH1 and MBD4 in the nucleus, which in turn causes the spermatogenesis. The apoptosis rate of the cells increased and the spermatogenesis was abnormal. The above studies on FADD (S191D) mice provide a further understanding of the non-apoptosis of the FADD protein through the Ser-191 phosphorylation modification.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R363
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 顧鳴敏,王鑄鋼,黃薇,范麗安;六種與HLA相關(guān)聯(lián)疾病的易感基因研究進(jìn)展[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2003年04期
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