FADD磷酸化对雄性小鼠精子发生影响的研究
发布时间：2018-12-21 07:22
【摘要】：FADD(Fas-associated death domain),最早是作为细胞程序性死亡(凋亡)过程中的接头蛋白被人们熟识。而如今FADD的非凋亡功能已越来越受到人们的关注。目前,已发现FADD在细胞周期、细胞增殖等众多过程中发挥重要功能。尤其是FADD-191Ser的磷酸化修饰,在各种生理、病理条件下,都发挥重要的调控功能。为了研究FADD-191Ser磷酸化修饰在生殖系统可能发挥的作用,本实验室与南京大学医药生物技术国家重点实验室合作,选用FADD~(-/-) tgD (Ser191Asp)小鼠(内源性FADD敲除小鼠、同时转入tgD(mFADD-S191D)模拟FADD的永久磷酸化,后文简称FADD(S191D)小鼠)作为研究对象,同窝的FADD~(+/-)小鼠作为对照,开展实验。 在前期小鼠的繁殖过程中,我们发现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)这一小鼠模型最明显的表型特征就是瘦小。从FADD(S191D)雄性成年小鼠解剖特征上看,睾丸很小,睾丸重与小鼠体重的比值(脏器指数)较对照组显著降低。睾丸HE切片显示,FADD(S191D)小鼠睾丸曲精小管管径明显减小,管腔中细胞层数减少,几乎没有或很少有减数分裂后的细胞,并且管腔中存在大量强嗜酸性染色、疑似凋亡的生精细胞。对睾丸各级生精细胞进行计数,发现其睾丸中精原细胞、精母细胞及圆形精子细胞均有不同程度的减少,其中以圆形精子细胞的下降最为明显。而对能到达附睾尾的精子进行形态学观察和计数,也发现附睾尾的精子明显减少,精子的畸形率显著上升。 为了寻找FADD(S191D)小鼠精子发生障碍的原因,我们取成年FADD(S191D)小鼠及同窝对照小鼠睾丸,分别作TUNEL染色,发现FADD(S191D)小鼠睾丸管腔中细胞的凋亡率显著上升,凋亡存在于各级生精细胞,其中以精母细胞的凋亡为主;结合以单倍体圆形精子细胞数目下降最明显的各级生精细胞的减少,这些结果表明FADD永久磷酸化对精子发生的各个阶段,尤其是精母细胞的减数分裂产生了严重的影响。 为研究FADD(S191D)导致生精细胞减数分裂异常的机制,我们选用减数分裂不同时期几个特定的Marker,进行了睾丸精母细胞联会复合体铺展免疫荧光,来观察FADD(S191D)小鼠睾丸减数分裂的进程。结果发现,与对照相比,FADD(S191D)小鼠粗线期精母细胞常染色体上部分DNA双链断裂(DSBs)尚未被修复,且联会复合体上重组位点Marker,错配修复蛋白MLH1位点数减少。这一结果显示,FADD(S191D)小鼠睾丸减数分裂过程中,染色体DNA重组修复发生异常,从而使得精母细胞凋亡率增加,精子发生障碍。 那么为什么FADD的永久磷酸化会导致重组修复位点异常呢?有学者研究发现在体细胞中FADD在细胞核内可以和MLH1相互作用,同时FADD还能与甲基岛结合结构域蛋白4(MBD4)相互作用,参与维持基因组的稳定[1]。因此我们推测在本研究中FADD的磷酸化也是通过这一相互作用来影响精母细胞DNA修复的。为了验证这一假设,因MLH1和MBD4均在细胞核内发挥功能,我们分别提取FADD(S191D)及FADD~(+/-)小鼠睾丸细胞质、细胞核蛋白,Western Blot验证这几种蛋白在细胞核与细胞质中的表达情况,结果显示:无论是实验组还是对照组,FADD在小鼠睾丸中均主要在细胞核表达;在FADD(S191D)小鼠睾丸中,MLH1及MBD4在细胞质中的表达量增加,而在细胞核中的表达下降,提示FADD的磷酸化可能影响了MLH1及MBD4从细胞质到细胞核的转运,使其在细胞核中的表达水平下降,进而引起DNA修复障碍。 综上,我们通过对FADD在雄性生殖系统的表达和定位的研究及FADD(S191D)小鼠生殖系统表型的观察,发现FADD永久磷酸化能导致精子发生障碍,对其发生机制的初步探讨显示FADD磷酸化能通过降低细胞核中MLH1及MBD4的水平而导致精母细胞DNA断裂修复障碍,进而引起精母细胞凋亡率增加,精子发生异常。以上有关FADD(S191D)小鼠的研究,为进一步了解FADD蛋白通过Ser-191磷酸化修饰发挥的非凋亡功能提供了新的思路和理论依据。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【共引文献】
相关期刊论文 前1条
1 顾鸣敏,王铸钢,黄薇,范丽安;六种与HLA相关联疾病的易感基因研究进展[J];中华医学遗传学杂志;2003年04期
，本文编号：2388564
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