本文選題：雙酚A + 生精功能　； 參考：《吉林大學(xué)》2015年博士論文

【摘要】：研究背景： 目前普遍認為，約有70%的出生缺陷是由環(huán)境因素與遺傳因素共同作用的結(jié)果。由于胚胎發(fā)育對外界環(huán)境特別敏感，因而母源性因素對胚胎發(fā)育的影響一直是研究出生缺陷的主要角度。而對于父源性出生缺陷的研究卻沒有給予應(yīng)有的重視。隨著社會經(jīng)濟的發(fā)展，環(huán)境污染、不良生活習(xí)慣等各種環(huán)境因素使人類精液質(zhì)量不斷下降，而精液質(zhì)量的持續(xù)下降很有可能提高父系出生缺陷的發(fā)生率。 雙酚A（BPA）是一種人類普遍暴露的環(huán)境內(nèi)分泌干擾物。據(jù)報道能對動物體和人體的神經(jīng)系統(tǒng)、免疫系統(tǒng)、內(nèi)分泌系統(tǒng)、生殖系統(tǒng)等產(chǎn)生不良影響。雙酚A可通過多種機制作用于多種靶器官發(fā)揮生物學(xué)效應(yīng)。BPA除了能夠結(jié)合經(jīng)典的雌激素受體ERα和ERβ，還能通過G蛋白偶聯(lián)的膜雌激素受體GPR30的非基因組途徑迅速發(fā)揮作用；此外，BPA的作用還涉及表觀遺傳學(xué)機制。 高劑量的雙酚A對雄性生殖系統(tǒng)有害，特別是在組織器官形成和發(fā)育階段的暴露能對實驗動物造成生殖功能的危害，并且可以持續(xù)到成年期；低劑量BPA對雄性生殖系統(tǒng)的危害還存在著爭議。BPA對子代生殖功能的影響目前也沒有明確的結(jié)論，雙酚A對精子的表觀遺傳修飾作用如DNA甲基化，組蛋白甲基化等的影響及其在傳代中的意義都是有待研究的科學(xué)問題。 研究目的： 建立BPA引起精子異常而導(dǎo)致F1子代雄性生殖出生缺陷的小鼠模型，揭示BPA暴露小鼠精子的表觀遺傳學(xué)特征，初步揭示BPA引起精子異常與子代生殖缺陷的表觀遺傳學(xué)基礎(chǔ)。 方法： 首先建立BPA雄性小鼠的暴露和傳代模型。高劑量BPA以50mg/kg/day的劑量腹腔注射3周齡雄性C57BL/6J小鼠7天，5周后取樣及繁殖，與6周齡正常雌性小鼠繁殖產(chǎn)生F1代，F(xiàn)1代雄性小鼠再與正常雌性小鼠繁殖產(chǎn)生F2代。檢測F0、F1、F2代雄鼠精子數(shù)量、精子形態(tài)和睪丸組織形態(tài)學(xué)。抽提F0代小鼠精子DNA，MeDIP富集甲基化DNA后進行高通量測序及生物信息學(xué)分析，建立BPA暴露的小鼠精子DNA甲基化譜，并挑選差異甲基化基因和印記基因進行驗證。分離F1代小鼠的精原細胞進行mRNA表達譜芯片檢測和生物信息學(xué)分析，建立BPA暴露的F1代小鼠精原細胞表達譜。進行甲基化譜與表達譜的負相關(guān)分析。通過定量PCR技術(shù)、Westernblot、免疫組化等技術(shù)，檢測BPA對DNA甲基化轉(zhuǎn)移酶mRNA水平、組蛋白甲基化水平的影響。 低劑量BPA暴露分別以5μg/kg/day、50μg/kg/day的劑量灌胃給藥3周齡C57BL/6J雄性小鼠5周，與6周齡正常雌性小鼠繁殖產(chǎn)生F1代。利用DNA甲基化結(jié)合蛋白MBD方法富集小鼠精子甲基化DNA，結(jié)合熒光定量PCR方法檢測Tnnt2、Tectb等基因甲基化水平的相對變化；Western blot檢測F1代小鼠精子中組蛋白甲基化水平變化。 利用小鼠精原干細胞C18-4細胞，體外研究BPA對小鼠精原干細胞增殖及表觀遺傳機制的影響。 結(jié)果： 1、高劑量BPA暴露對雄性小鼠及其子代的生精功能有不良影響。高劑量BPA引起F0代小鼠精子數(shù)量下降20.6%（P0.01），F(xiàn)1代小鼠精子數(shù)量下降12.1%（P0.05），F(xiàn)2代小鼠精子數(shù)下降9.2%（P0.01）。高劑量BPA暴露引起F0代小鼠精子畸形率增加了9.65%（P 0.05），對F1、F2代小鼠精子畸形率沒有明顯影響（P0.05）。高劑量BPA暴露能引起F1代出生性別比增加（P 0.05）。 2、F0代精子甲基化DNA高通量測序共篩選出1597個差異基因，其中BPA引起的低甲基化基因有1112個，占69.6%；高甲基化基因有379個，，占23.7%；106個基因部分低甲基化，部分高甲基化。 經(jīng)進一步驗證，F(xiàn)0代BPA組小鼠精子中印記基因Gtl2、H19、Igf2r、Kcnq1、Gnas、Impact、Lit1、Nespas、Mest、Peg3、Peg10、Snrpn甲基化水平與對照組沒有統(tǒng)計學(xué)差異（P0.05）；BPA組小鼠精子中Magef1基因啟動子區(qū)甲基化水平顯著增加（P0.01）；BPA暴露組F0、F1兩代小鼠精子中Tnnt2基因分別與對照組相比，甲基化水平都發(fā)生下降，而Magef1基因的甲基化水平相對增加。 高劑量BPA暴露導(dǎo)致F0代小鼠睪丸和精子整體DNA甲基化水平下降，睪丸中組蛋白H3K9Me3表達水平增加。 3、F1代小鼠精原細胞表達譜檢測共得到2091個差異基因，BPA引起上調(diào)的基因有1546個，下調(diào)的基因545個。共有101個基因在精子DNA甲基化與精原細胞mRNA表達之間具有負相關(guān)性。 4、50μg/kg低劑量BPA暴露引起小鼠精子數(shù)量顯著下降20.1%（P0.01），而5μg/kg低劑量BPA暴露對精子數(shù)量沒有影響（P0.05）。兩組低劑量BPA暴露對小鼠生殖器官臟器指數(shù)不產(chǎn)生明顯影響（P0.05），但50μg/kg BPA能引起每胎產(chǎn)仔數(shù)減少（P0.05）。50μg/kg BPA能引起睪丸中的Tnnt2、Tectb基因甲基化水平發(fā)生變化。50μg/kg BPA能引起F1代小鼠精子中組蛋白H3K9Me3水平下降，H3K27Me3、H3K36Me3水平顯著增加。 5、10-9M-10-6M BPA能促進精原干細胞C18-4細胞增殖（P0.05）；10-5M BPA能顯著抑制細胞生長（P0.01）；10-5M BPA引起C18-4細胞凋亡率明顯增加（P0.01），基因組整體DNA甲基化水平顯著下降50%（P0.01），H3K27Me3，H3K36Me3的表達水平顯著下降（P0.01）。 結(jié)論： （1）青春期雄性小鼠短暫地暴露于高劑量BPA，能對成年后及其F1子代的生精功能產(chǎn)生不良影響。高劑量BPA能引起F0代小鼠睪丸和精子總體DNA甲基化水平下降；F0代小鼠睪丸組蛋白H3K9Me3水平增加。BPA暴露引起F0、F1兩代小鼠精子中Tnnt2基因甲基化水平發(fā)生相對下降、而Magef1基因甲基化水平相對增加。（2）50μg/kg/day低劑量BPA對小鼠生精功能產(chǎn)生不良影響，引起F1代小鼠精子組蛋白甲基化水平變化。（3）BPA對小鼠精原干細胞C18-4細胞增殖具有非單調(diào)劑量效應(yīng)，10-5M高劑量BPA能影響整體DNA甲基化和組蛋白甲基化水平。
[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偽 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 .

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本文編號：2055398