双酚A引起父源性生殖缺陷的小鼠模型建立与机制研究

发布时间：2018-06-23 02:44

  本文选题：双酚A + 生精功能　； 参考：《吉林大学》2015年博士论文

【摘要】：研究背景： 目前普遍认为，约有70%的出生缺陷是由环境因素与遗传因素共同作用的结果。由于胚胎发育对外界环境特别敏感，因而母源性因素对胚胎发育的影响一直是研究出生缺陷的主要角度。而对于父源性出生缺陷的研究却没有给予应有的重视。随着社会经济的发展，环境污染、不良生活习惯等各种环境因素使人类精液质量不断下降，而精液质量的持续下降很有可能提高父系出生缺陷的发生率。 双酚A（BPA）是一种人类普遍暴露的环境内分泌干扰物。据报道能对动物体和人体的神经系统、免疫系统、内分泌系统、生殖系统等产生不良影响。双酚A可通过多种机制作用于多种靶器官发挥生物学效应。BPA除了能够结合经典的雌激素受体ERα和ERβ，还能通过G蛋白偶联的膜雌激素受体GPR30的非基因组途径迅速发挥作用；此外，BPA的作用还涉及表观遗传学机制。 高剂量的双酚A对雄性生殖系统有害，特别是在组织器官形成和发育阶段的暴露能对实验动物造成生殖功能的危害，并且可以持续到成年期；低剂量BPA对雄性生殖系统的危害还存在着争议。BPA对子代生殖功能的影响目前也没有明确的结论，双酚A对精子的表观遗传修饰作用如DNA甲基化，组蛋白甲基化等的影响及其在传代中的意义都是有待研究的科学问题。 研究目的： 建立BPA引起精子异常而导致F1子代雄性生殖出生缺陷的小鼠模型，揭示BPA暴露小鼠精子的表观遗传学特征，初步揭示BPA引起精子异常与子代生殖缺陷的表观遗传学基础。 方法： 首先建立BPA雄性小鼠的暴露和传代模型。高剂量BPA以50mg/kg/day的剂量腹腔注射3周龄雄性C57BL/6J小鼠7天，5周后取样及繁殖，与6周龄正常雌性小鼠繁殖产生F1代，F1代雄性小鼠再与正常雌性小鼠繁殖产生F2代。检测F0、F1、F2代雄鼠精子数量、精子形态和睾丸组织形态学。抽提F0代小鼠精子DNA，MeDIP富集甲基化DNA后进行高通量测序及生物信息学分析，建立BPA暴露的小鼠精子DNA甲基化谱，并挑选差异甲基化基因和印记基因进行验证。分离F1代小鼠的精原细胞进行mRNA表达谱芯片检测和生物信息学分析，建立BPA暴露的F1代小鼠精原细胞表达谱。进行甲基化谱与表达谱的负相关分析。通过定量PCR技术、Westernblot、免疫组化等技术，检测BPA对DNA甲基化转移酶mRNA水平、组蛋白甲基化水平的影响。 低剂量BPA暴露分别以5μg/kg/day、50μg/kg/day的剂量灌胃给药3周龄C57BL/6J雄性小鼠5周，与6周龄正常雌性小鼠繁殖产生F1代。利用DNA甲基化结合蛋白MBD方法富集小鼠精子甲基化DNA，结合荧光定量PCR方法检测Tnnt2、Tectb等基因甲基化水平的相对变化；Western blot检测F1代小鼠精子中组蛋白甲基化水平变化。 利用小鼠精原干细胞C18-4细胞，体外研究BPA对小鼠精原干细胞增殖及表观遗传机制的影响。 结果： 1、高剂量BPA暴露对雄性小鼠及其子代的生精功能有不良影响。高剂量BPA引起F0代小鼠精子数量下降20.6%（P0.01），F1代小鼠精子数量下降12.1%（P0.05），F2代小鼠精子数下降9.2%（P0.01）。高剂量BPA暴露引起F0代小鼠精子畸形率增加了9.65%（P 0.05），对F1、F2代小鼠精子畸形率没有明显影响（P0.05）。高剂量BPA暴露能引起F1代出生性别比增加（P 0.05）。 2、F0代精子甲基化DNA高通量测序共筛选出1597个差异基因，其中BPA引起的低甲基化基因有1112个，占69.6%；高甲基化基因有379个，，占23.7%；106个基因部分低甲基化，部分高甲基化。 经进一步验证，F0代BPA组小鼠精子中印记基因Gtl2、H19、Igf2r、Kcnq1、Gnas、Impact、Lit1、Nespas、Mest、Peg3、Peg10、Snrpn甲基化水平与对照组没有统计学差异（P0.05）；BPA组小鼠精子中Magef1基因启动子区甲基化水平显著增加（P0.01）；BPA暴露组F0、F1两代小鼠精子中Tnnt2基因分别与对照组相比，甲基化水平都发生下降，而Magef1基因的甲基化水平相对增加。 高剂量BPA暴露导致F0代小鼠睾丸和精子整体DNA甲基化水平下降，睾丸中组蛋白H3K9Me3表达水平增加。 3、F1代小鼠精原细胞表达谱检测共得到2091个差异基因，BPA引起上调的基因有1546个，下调的基因545个。共有101个基因在精子DNA甲基化与精原细胞mRNA表达之间具有负相关性。 4、50μg/kg低剂量BPA暴露引起小鼠精子数量显著下降20.1%（P0.01），而5μg/kg低剂量BPA暴露对精子数量没有影响（P0.05）。两组低剂量BPA暴露对小鼠生殖器官脏器指数不产生明显影响（P0.05），但50μg/kg BPA能引起每胎产仔数减少（P0.05）。50μg/kg BPA能引起睾丸中的Tnnt2、Tectb基因甲基化水平发生变化。50μg/kg BPA能引起F1代小鼠精子中组蛋白H3K9Me3水平下降，H3K27Me3、H3K36Me3水平显著增加。 5、10-9M-10-6M BPA能促进精原干细胞C18-4细胞增殖（P0.05）；10-5M BPA能显著抑制细胞生长（P0.01）；10-5M BPA引起C18-4细胞凋亡率明显增加（P0.01），基因组整体DNA甲基化水平显著下降50%（P0.01），H3K27Me3，H3K36Me3的表达水平显著下降（P0.01）。 结论： （1）青春期雄性小鼠短暂地暴露于高剂量BPA，能对成年后及其F1子代的生精功能产生不良影响。高剂量BPA能引起F0代小鼠睾丸和精子总体DNA甲基化水平下降；F0代小鼠睾丸组蛋白H3K9Me3水平增加。BPA暴露引起F0、F1两代小鼠精子中Tnnt2基因甲基化水平发生相对下降、而Magef1基因甲基化水平相对增加。（2）50μg/kg/day低剂量BPA对小鼠生精功能产生不良影响，引起F1代小鼠精子组蛋白甲基化水平变化。（3）BPA对小鼠精原干细胞C18-4细胞增殖具有非单调剂量效应，10-5M高剂量BPA能影响整体DNA甲基化和组蛋白甲基化水平。
[Abstract]:Background of Study :

It is widely believed that about 70 % of the birth defects are the result of the common action of environmental factors and genetic factors . Because the embryonic development is especially sensitive to the external environment , the influence of maternal factor on embryo development has been the main angle for studying birth defects .

Biphenol A ( BPA ) is a universally exposed environment endocrine disturbance . It is reported that it can exert a bad influence on the nervous system , immune system , endocrine system and reproductive system of animal and human body . BPA can play a biological effect on various target organs through various mechanisms . BPA can play a role in the non - genomic pathway of membrane estrogen receptor GPRS 30 coupled with G protein in addition to classical estrogen receptor ER伪 and ER尾 .
In addition , BPA also involves an epigenetics mechanism .

High - dose bisphenol A is detrimental to the male reproductive system , especially in the formation and development stages of the tissue organ , which can harm the reproductive function of the experimental animals , and can last to adulthood ;
The influence of BPA on the reproductive function of the offspring is not clear . The influence of BPA on sperm ' s apparent genetic modification , such as DNA methylation , histone methylation and so on , is a scientific problem to be studied .

Purpose of study :

A mouse model of BPA - induced sperm abnormality leading to male reproductive birth defects in F1 offspring was established . The epigenetics characteristics of sperm in mice exposed to BPA were revealed , and the epigenetics basis of BPA - induced sperm abnormality and reproductive failure was preliminarily revealed .

Method :

The effects of BPA on DNA methylation level and histone methylation level in F1 generation mice were determined by quantitative PCR , Western blot and immunohistochemistry .

Low dose BPA exposure was 5 渭g / kg / day , 50 渭g / kg / day for 5 weeks , and F1 generation was produced in normal female mice at 6 weeks . The DNA methylation binding protein MBD method was used to enrich mouse sperm methylated DNA , and the relative changes of methylation level of Tnnt2 and Tectb were detected by fluorescence quantitative PCR .
Western blot was used to detect the level of histone methylation in the sperm of F1 mice .

The effects of BPA on the proliferation and apparent genetic mechanism of spermatogonial stem cells in mice were studied in vitro by using mouse spermatogonial stem cells C18 - 4 cells .

Results :

1 . High - dose BPA exposure had an adverse effect on the spermatogenic function of male mice and its progeny . High - dose BPA caused a decrease of sperm count by 20.6 % ( P0.01 ) , and the number of sperm in F1 generation decreased by 12.1 % ( P0.05 ) . The sperm deformity rate of F1 and F2 mice was increased by 9.65 % ( P0.05 ) .

2 . 1597 differentially expressed genes were screened by high - throughput sequencing of the methylated DNA of F0 . Among them , there were 1112 low - methylation genes induced by BPA , accounting for 69.6 % ;
There were 379 high methylated genes , accounting for 23 . 7 % ;
The 106 genes were partially methylated and partially methylated .

After further verification , the methylation level of imprinting genes Gtl2 , H19 , Igf2r , Kcnq1 , Gnas , Impact , Lit1 , Nespas , Mest , Peg3 , Peg10 and Snrpn in the sperm of F0 generation BPA group was not significantly different from that of the control group ( P0.05 ) .
The methylation level of Magef1 gene promoter in the sperm of BPA group increased significantly ( P0.01 ) .
Compared with the control group , the level of methylation of the Tnnt2 gene of F0 and F1 mice in BPA exposure group decreased , while the methylation level of Magef1 gene was relatively increased .

High - dose BPA exposure resulted in a decrease in the DNA methylation level in the testes and sperm of F0 mice and an increase in the level of histone H3K9Me3 expression in the testis .

3 . There were 2091 differentially expressed genes in F1 generation mouse spermatogenic cells , and there were 1546 genes which were up - regulated by BPA , and 545 downregulated genes . There were 101 genes which had negative correlation between DNA methylation of sperm and mRNA expression of spermatogenic cells .

4 銆

本文编号：2055398