青春期前双酚A暴露对大鼠卵泡发育的影响及其相关基因的表达与调控
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摘要
双酚A（Bisphenol A，BPA）学名2，2-二（4-羟基苯基）丙烷，是世界已知重要的环境雌激素之一。BPA是人工合成的高分子材料的单体，是生产塑料增塑剂、农药、涂料等化工产品的原料。在婴儿奶瓶、饭盒、玩具及自来水管等材料中广泛存在。双酚A单体可从盛装食物或饮料的塑料容器中游离出来，在高温或强酸碱等条件下可加速游离过程。BPA暴露人群非常广泛，流行病学研究表明，超过90%的个体尿中可检测到BPA。BPA的化学结构与乙烯雌酚类似，进入体内可模拟雌激素活性。
目前欧盟、美国和中国等众多国家只是禁止含BPA的塑料用于制造婴儿奶瓶，实际上，青春期前的儿童BPA暴露也是一个值得关注的问题。这个时期是生殖系统发育成熟前的一个关键阶段。这个时期的儿童可能通过多种途径暴露于BPA，特别是发生食品和饮水污染事件时，儿童还可能会短期暴露于较高浓度的BPA。尽管环境雌激素与天然激素相比在体内的浓度较低，但由于处于发育期的儿童下丘脑－垂体－性腺轴尚未发育成熟，体内性激素水平比较低，此时的个体对外源性雌激素的反应较成年敏感。同时，较高浓度的BPA对卵巢发育的毒性效应问题也不容忽视。我们推测，青春期前较高浓度BPA短期暴露很可能会影响雌性卵泡的正常发育过程，并且有可能通过影响卵泡发育相关调控基因而发挥作用。本研究通过观察促进卵泡发育的Kitlg、Figla和H1foo基因以及抑制卵泡发育的AMH基因表达的表达和DNA甲基化模式，探讨卵巢发育相关的基因在大鼠BPA短期暴露所致卵巢发育毒性中的作用及其分子生物学机制，为进一步阐明BPA对儿童生殖系统发育的影响提供实验依据。
第一部分：青春期前BPA暴露对卵巢发育和功能的影响
目的：通过建立青春期前BPA短期暴露的大鼠动物模型，观察BPA对卵巢形态、功能，卵泡细胞的凋亡以及卵巢细胞超微结构等改变，探讨青春期前BPA暴露对卵巢发育和功能的影响。
方法：28日龄雌性wistar大鼠通过腹腔注射BPA（10mg/kg、40mg/kg、160mg/kg），每天染毒1次，连续染毒1周。观察卵巢重量、各级卵泡构成、卵泡凋亡（TUNEL）、阴道开放率、第一次动情期出现率、血清性激素水平、卵巢细胞超微结构。
结果：
1.青春期前大鼠BPA暴露后，染毒组大鼠在各个时间段的体重与对照组比较差异均无统计学意义（P＞0.05）；BPA染毒组的卵巢重量和脏器系数随染毒剂量升高而出现下降，其中40mg/kg和160mg/kgBPA组大鼠卵巢重量与对照组比较有显著性差异（P＜0.01）。
2.各染毒剂量组阴道开放率和和首次动情期出现率对照组比较差异均无统计学意义（P＞0.05）。
3.随染毒剂量增加，各染毒组大鼠血清雌E2水平有下降趋势，与对照组比较无显著性差异（P＞0.05）。而血清P4水平与对照组比较有显著性差异（P＜0.01），其中，40mg/kg和160mg/kg BPA组血清P4水平下降与对照组比较有显著性差异（P＜0.01）。
4.随着染毒剂量升高，各染毒组卵泡数量呈下降趋势，其中10mg/kg、40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义(P＜0.05，P＜0.01和P＜0.01）；始基卵泡、初级卵泡/腔前卵泡、有腔卵泡和黄体构成比均呈下降趋势，而闭锁卵泡构成比有升高趋势，其中40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）。
5.随染毒剂量的增加，各染毒剂量组中，腔前卵泡、有腔卵泡以及黄体/白体凋亡率均呈现上升趋势，与对照组比较差异均有统计学意义。
6.电镜观察卵巢细胞超微结构，与对照组比较，40mg/kg BPA组和160mg/kgBPA组镜下的细胞超微结构显示，细胞内脂肪变性增多，次级溶酶体增多，尤其在160mg/kg BPA组中，大部分细胞胞质中几乎都充斥着脂肪颗粒和次级溶酶体。
第二部分：青春期前BPA暴露对卵泡发育相关基因表达的影响
目的：青春期前BPA暴露影响卵泡发育过程中，对发育相关基因表达的影响，探讨BPA卵巢毒性的分子机制。
方法：整体动物模型同前，用实时荧光定量PCR、Western blot分析以及免疫组化技术检测卵泡发育相关基因：干细胞因子（KITLG）、基础螺旋环转录因子（FIGLA）、卵母细胞特有的H1连结组蛋白(H1FOO)、抗苗勒激素（AMH）的mRNA及其蛋白的表达水平。
结果：
1.各染毒剂量组Kitlg mRNA相对表达量呈下降趋势，其中160mg/kgBPA组与对照组比较差异具有统计学意义(p﹤0.01)；各染毒剂量组Figla mRNA相对表达量呈下降趋势，其中160mg/kgBPA组与对照组比较差异具有统计学意义(p﹤0.05)；各染毒剂量组H1foo mRNA相对表达量呈下降趋势，与对照组比较差异均具有统计学意义(p﹤0.01)；各染毒剂量组AMH mRNA相对表达量呈上升趋势，其中40mg/kgBPA组和160mg/kgBPA组与对照组比较差异具有统计学意义(p﹤0.05和p﹤0.01)。
2. Western blot结果显示，KITLG蛋白表达水平在各BPA染毒组均呈明显下调，其中10mg/kg BPA组和40mg/kg BPA组与对照组比较差异有统计学意义（P＜0.05），160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）；FIGLA蛋白表达水平在160mg/kg BPA组呈明显下调，与对照组比较差异有统计学意义（P＜0.05）；H1FOO蛋白表达水平在各BPA染毒组均呈明显下调，其中10mg/kgBPA组与对照组比较差异有统计学意义（P＜0.05），40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）；AMH蛋白表达水平在各BPA染毒组均呈明显上调，其中10mg/kg BPA组与对照组比较差异有统计学意义（P＜0.05），40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）。
第三部分青春期前BPA暴露影响卵泡发育的表观遗传学调控机制研究
目的：从卵巢细胞DNA总甲基化状态和卵泡发育相关基因启动子区DNA甲基化状态两个方面，初步探讨BPA的表观遗传学的调控机制。
方法：整体动物模型同前，卵巢细胞DNA总甲基化采用免疫组化检测卵巢细胞5-甲基胞嘧啶（5-mC）水平。采用亚硫酸氢盐修饰后测序法（BSP）检测卵巢发育相关基因Kitlg启动子区CpG岛的甲基化状况。
结果：
1.随着染毒剂量的增加，卵巢颗粒细胞5-mC免疫组化的平均光密度值（OD）和积分光密度值（IOD）呈总体上升趋势，其中，40mg/kg BPA组和160mg/kg BPA组与对照组比较差异均有统计学意义(P＜0.05)。
2.采用Methprimer软件对以上全部基因的启动子区域进行CpG岛检测，只有Kitlg基因启动子区存在一个CpG岛，35个甲基化位点。Kitlg基因BSP产物重组T载体质粒的测序结果显示，随着染毒剂量增加，发生Cs的个数呈上升趋势，其中，160mg/kg BPA组Cs的甲基化率与对照组比较差异有统计学意义(P＜0.05)；第7甲基化位点（Cs）在对照组存在甲基化状态，在染毒组中出现较为明显的去甲基化现象；第16甲基化位点在对照组呈现无甲基化状态，而随着染毒剂量增加，在染毒组中出现较为明显的甲基化现象。
结论：
根据以上结果结合讨论可得出如下结论:
1.青春期前BPA较高浓度短期暴露可能对卵巢卵泡的发育和性激素分泌功能具有抑制毒效应，主要表现为卵巢重量及卵巢脏器系数下降、卵泡总数下降、生长卵泡构成比下降及闭锁卵泡构成比升高、卵泡凋亡率升高、血清孕激素水平下降。
2.促进卵泡发育的Kitlg基因、Figla基因、H1foo基因表达水平下调以及抑制卵泡发育的AMH基因表达上调，可能在青春期前BPA暴露抑制卵巢发育过程中发挥了重要作用。
3. DNA甲基化模式的改变在Kitlg、Figla、H1foo及AMH等基因表达调控中的可能作用值得我们重视；Kitlg基因表达下调可能与其启动子区CpG岛甲基化模式的改变有关。
Introduction
Bisphenol A (BPA，CAS:80-05-7) name2,2-two (4-hydroxyphenyl) propane，is recognized as one of several environmental estrogens. BPA is a monomer of thesynthetic polymer material that is used in the production of plasticizers, pesticides,paint, and other chemical products. BPA is used in a wide variety of products, such asbaby bottles, lunch boxes, toys, water pipes, and other materials. BPA monomers canleach from a plastic container that holds food or beverages, and the release process isaccelerated under conditions of high temperature, strong acidity, or strong alkalinity.Human exposure to BPA is extensive; epidemiological studies have shown that thechemical could be detected in the urine of more than90%of tested individuals. Thechemical structure of BPA is similar to that of estradiol (E2); it can therefore mimicestrogenic actions in the body.
The use of BPA in baby products has already been banned in the European Union,the United States, China, and many other countries. However, the prepubertal periodis also an important time in child development, and BPA exposure will occur duringthis period through a variety of pathways. Children are especially vulnerable to higherconcentrations of BPA as a consequence of food and drinking water contamination.Although environmental concentration compared with estrogens in vivo is low，thehypothalamus-pituitary-gonadal axis of children is not yet mature during thepre-puberty stage, and the levels of sex hormones in the body are relatively low;therefore, children’s reproductive systems are more sensitive to environmentalestrogens than those of adults. We speculated that exposure to high concentrations ofBPA during the prepubertal period may affect the normal developmental process of ovarian follicles, and the resulting toxic effects may be mediated by the regulatorygenes involved in follicular development. This study explored the roles andmechanisms of genes that are associated with ovarian development in the toxic effectsof short-term BPA exposure on rat ovarian development. We accomplished this aim bystudying changes in expression among follicle development-promoting genes, such asKitlg, Figla and H1foo, and follicular development inhibitory genes, such as AMH.This study could provide an important experimental evidence for the reproductivesystem developmental effect of children exposure to BPA.
Part Ⅰ: Effect of bisphenol A on ovarian development and function inprepubertal rat.
Objective: The aim of this study was to determine whether BPA could affect ovariandevelopment and function in prepubertal rat.
Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. The effects of BPA on ovarian structure and function were assessed bymeasuring the weight of the ovaries, counting ovarian follicles, apoptosis in ovarianfollicles, ratios of vaginal opening and first estrus, assaying sex hormone levels in theserum, and the ultrastrueture of ovarian tissue use of transmission electronmicroscopy.
Results:
1. Prepubertal rats exposed to BPA for7days, no significant difference wasobserved in body weights between the BPA exposure groups and the control group,whereas the ovarian weights in the BPA exposure groups decreased with increasingBPA doses. The ovarian weights and ovary coefficients of the rats from the40mg/kgand160mg/kg BPA groups were significantly different from those of the controlgroup (P<0.01)
2. The ratios of vaginal opening and first estrus have no significant differencebetween the BPA exposure groups and the control group（P＞0.05）.
3. The levels of serum E2in rats from each BPA group decreased with increasingdoses of BPA, but the results were not significantly different from that of the rats in the control group (P0.05). However, the levels of serum P4in rats from the BPAgroups were significantly different from that of the control group rats (P<0.01); inparticular, the decreases of serum P4in the40mg/kg and160mg/kg BPA groupswere significantly different from that of the control group, respectively (P<0.01).
4. The number of follicles in each BPA exposure group decreased with increasingBPA doses; in comparison to the control group, significant changes were observed inthe10mg/kg,40mg/kg, and160mg/kg BPA groups (P<0.05, P<0.01, and P<0.01,respectively); In addition, the constituent ratios of primordial follicles, primaryfollicles/preantral follicles, antral follicles, and corpus luteum (CL) all decreased inresponse to increasing BPA concentrations, but the constituent ratio of atretic folliclesincreased. These changes in the40mg/kg and160mg/kg BPA groups relative to thecontrol group were statistically significant (P<0.01).
5. Compered with control group, the apoptosis ratios of preantral follicles, antralfollicles, and corpus luteum (CL) all increased in response to increasing BPAconcentrations.
6. The ovarian tissue ultrastrueture of steatosis and secondary lysosomes allincreased in response to increasing BPA concentrations, compered with control group.
Part Ⅱ: Effect of ovarian follicle development-related genes expression inprepubertal rat exposed to bisphenol A.
Objective: The aim of this study was to determine whether BPA could affect ovarianfollicle development-related genes expression in prepubertal rat.
Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. The mRNA and protein expression levels of follicle development-relatedgenes: Stem cell factor(Kitlg), Factor In the Germline alpha (Figla), oocyte-specifichistone H1variant (H1foo), and anti-Mullerian hormone (AMH) were analyzed usingreal-time quantitative PCR, western blots and immunohistochemistry.
Results:
1. The real-time PCR revealed that Kitlg gene mRNA expression levels in the160mg/kg BPA group significantly decreased (P<0.01) in comparison to the control. The mRNA expression levels of the Figla gene in all of the BPA groups significantlydecreased (P<0.05) relative to the control. And the mRNA expression levels of theH1foo gene in all of the BPA groups significantly decreased (P<0.01) relative to thecontrol, but the mRNA expression levels of the AMH gene in the160mg/kg BPAgroup significantly increased (P<0.01) relative to the control.
2. Western blot analysis revealed that protein expression levels of KITLG in eachBPA group significantly decreased relative to the control, with P<0.05for the10mg/kg BPA and40mg/kg group and P<0.01for the160mg/kg groups, and theprotein expression levels of FIGLA in the160mg/kg BPA group significantlydecreased (P<0.05) relative to the control, and the protein expression level of H1FOOin each BPA group significantly decreased relative to the control, with P<0.05for the10mg/kg BPA group and P<0.01for the40mg/kg and160mg/kg groups, but theprotein expression level of the AMH gene in each BPA group significantly increasedrelative to the control, with P<0.05for the10mg/kg BPA group and P<0.01for the40mg/kg and160mg/kg groups.
Part Ⅲ: Study on epigenetic regulation mechanism of toxicity during ovarianfollicle development in prepubertal rat exposed to bisphenol A.
Objective: The aim of this study was to determine whether BPA could affect DNAtotal methylation status and gene promoter region DNA methylation status of ovarianfollicle development-related genes in prepubertal rat.
Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. Study on global methylation status, and gene promoter region DNAmethylation status, to explore the epigenetic regulation mechanisms underlying BPAtoxicity during ovarian development.
Results:
1. Compered with control group,the OD and IOD of combined5-mC scores ofovarian granulosa cell increased gradually in response to increasing BPAconcentrations. These changes in the40mg/kg and160mg/kg BPA groups relative tothe control group were statistically significant (P<0.05).
2. All genes test results from the Methprimer software showed only that there isone CpG island in the promoter region of the Kitlg gene, with35methylationcites(Cs). and then use the bisulfite sequencing polymerase chain reaction (BSP)method to test the methylation status of Kitlg gene. The sequencing results showedthat levels of methylation cites increased with increasing doses of BPA. in particular,the increases of methylation cites in the160mg/kg BPA groups was significantlydifferent from that of the control group (P<0.05). and7Cs exists methylation status inthe control group, but demethylation in BPA exposure group;16Cs show nomethylation status in the control group,but obvious methylation in BPA exposuregroup.
Conclusion:
1. Our results indicated that short-term, concentrated BPA exposure during theprepubertal period may have inhibitory effects on ovarian follicle development andhormone secretion, which were observed as the ovarian weight, counting ovarianfollicles were decreased, the constituent ratios of all growing follicles decreased andatretic follicles increased, the apoptosis ratios of follicles increased, and the inhibitionof progesterone secretion.
2. The down-regulation of Kitlg, Figla and H1foo genes and the up-regulation ofthe AMH gene may play important roles in the BPA-induced mechanism of toxicitythat affects ovarian development.
3. The possible role of DNA methylation patterns change in the regulation ofgene expression in Kitlg, Figla, H1foo and AMH genes are clearly worth considering;the mRNA expression levels of the Kitlg gene decreased significantly in the BPAgroups，may associated with promoter region methylation pattern.
目前欧盟、美国和中国等众多国家只是禁止含BPA的塑料用于制造婴儿奶瓶，实际上，青春期前的儿童BPA暴露也是一个值得关注的问题。这个时期是生殖系统发育成熟前的一个关键阶段。这个时期的儿童可能通过多种途径暴露于BPA，特别是发生食品和饮水污染事件时，儿童还可能会短期暴露于较高浓度的BPA。尽管环境雌激素与天然激素相比在体内的浓度较低，但由于处于发育期的儿童下丘脑－垂体－性腺轴尚未发育成熟，体内性激素水平比较低，此时的个体对外源性雌激素的反应较成年敏感。同时，较高浓度的BPA对卵巢发育的毒性效应问题也不容忽视。我们推测，青春期前较高浓度BPA短期暴露很可能会影响雌性卵泡的正常发育过程，并且有可能通过影响卵泡发育相关调控基因而发挥作用。本研究通过观察促进卵泡发育的Kitlg、Figla和H1foo基因以及抑制卵泡发育的AMH基因表达的表达和DNA甲基化模式，探讨卵巢发育相关的基因在大鼠BPA短期暴露所致卵巢发育毒性中的作用及其分子生物学机制，为进一步阐明BPA对儿童生殖系统发育的影响提供实验依据。
第一部分：青春期前BPA暴露对卵巢发育和功能的影响
目的：通过建立青春期前BPA短期暴露的大鼠动物模型，观察BPA对卵巢形态、功能，卵泡细胞的凋亡以及卵巢细胞超微结构等改变，探讨青春期前BPA暴露对卵巢发育和功能的影响。
方法：28日龄雌性wistar大鼠通过腹腔注射BPA（10mg/kg、40mg/kg、160mg/kg），每天染毒1次，连续染毒1周。观察卵巢重量、各级卵泡构成、卵泡凋亡（TUNEL）、阴道开放率、第一次动情期出现率、血清性激素水平、卵巢细胞超微结构。
结果：
1.青春期前大鼠BPA暴露后，染毒组大鼠在各个时间段的体重与对照组比较差异均无统计学意义（P＞0.05）；BPA染毒组的卵巢重量和脏器系数随染毒剂量升高而出现下降，其中40mg/kg和160mg/kgBPA组大鼠卵巢重量与对照组比较有显著性差异（P＜0.01）。
2.各染毒剂量组阴道开放率和和首次动情期出现率对照组比较差异均无统计学意义（P＞0.05）。
3.随染毒剂量增加，各染毒组大鼠血清雌E2水平有下降趋势，与对照组比较无显著性差异（P＞0.05）。而血清P4水平与对照组比较有显著性差异（P＜0.01），其中，40mg/kg和160mg/kg BPA组血清P4水平下降与对照组比较有显著性差异（P＜0.01）。
4.随着染毒剂量升高，各染毒组卵泡数量呈下降趋势，其中10mg/kg、40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义(P＜0.05，P＜0.01和P＜0.01）；始基卵泡、初级卵泡/腔前卵泡、有腔卵泡和黄体构成比均呈下降趋势，而闭锁卵泡构成比有升高趋势，其中40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）。
5.随染毒剂量的增加，各染毒剂量组中，腔前卵泡、有腔卵泡以及黄体/白体凋亡率均呈现上升趋势，与对照组比较差异均有统计学意义。
6.电镜观察卵巢细胞超微结构，与对照组比较，40mg/kg BPA组和160mg/kgBPA组镜下的细胞超微结构显示，细胞内脂肪变性增多，次级溶酶体增多，尤其在160mg/kg BPA组中，大部分细胞胞质中几乎都充斥着脂肪颗粒和次级溶酶体。
第二部分：青春期前BPA暴露对卵泡发育相关基因表达的影响
目的：青春期前BPA暴露影响卵泡发育过程中，对发育相关基因表达的影响，探讨BPA卵巢毒性的分子机制。
方法：整体动物模型同前，用实时荧光定量PCR、Western blot分析以及免疫组化技术检测卵泡发育相关基因：干细胞因子（KITLG）、基础螺旋环转录因子（FIGLA）、卵母细胞特有的H1连结组蛋白(H1FOO)、抗苗勒激素（AMH）的mRNA及其蛋白的表达水平。
结果：
1.各染毒剂量组Kitlg mRNA相对表达量呈下降趋势，其中160mg/kgBPA组与对照组比较差异具有统计学意义(p﹤0.01)；各染毒剂量组Figla mRNA相对表达量呈下降趋势，其中160mg/kgBPA组与对照组比较差异具有统计学意义(p﹤0.05)；各染毒剂量组H1foo mRNA相对表达量呈下降趋势，与对照组比较差异均具有统计学意义(p﹤0.01)；各染毒剂量组AMH mRNA相对表达量呈上升趋势，其中40mg/kgBPA组和160mg/kgBPA组与对照组比较差异具有统计学意义(p﹤0.05和p﹤0.01)。
2. Western blot结果显示，KITLG蛋白表达水平在各BPA染毒组均呈明显下调，其中10mg/kg BPA组和40mg/kg BPA组与对照组比较差异有统计学意义（P＜0.05），160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）；FIGLA蛋白表达水平在160mg/kg BPA组呈明显下调，与对照组比较差异有统计学意义（P＜0.05）；H1FOO蛋白表达水平在各BPA染毒组均呈明显下调，其中10mg/kgBPA组与对照组比较差异有统计学意义（P＜0.05），40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）；AMH蛋白表达水平在各BPA染毒组均呈明显上调，其中10mg/kg BPA组与对照组比较差异有统计学意义（P＜0.05），40mg/kg和160mg/kg BPA组与对照组比较差异有统计学意义（P＜0.01）。
第三部分青春期前BPA暴露影响卵泡发育的表观遗传学调控机制研究
目的：从卵巢细胞DNA总甲基化状态和卵泡发育相关基因启动子区DNA甲基化状态两个方面，初步探讨BPA的表观遗传学的调控机制。
方法：整体动物模型同前，卵巢细胞DNA总甲基化采用免疫组化检测卵巢细胞5-甲基胞嘧啶（5-mC）水平。采用亚硫酸氢盐修饰后测序法（BSP）检测卵巢发育相关基因Kitlg启动子区CpG岛的甲基化状况。
结果：
1.随着染毒剂量的增加，卵巢颗粒细胞5-mC免疫组化的平均光密度值（OD）和积分光密度值（IOD）呈总体上升趋势，其中，40mg/kg BPA组和160mg/kg BPA组与对照组比较差异均有统计学意义(P＜0.05)。
2.采用Methprimer软件对以上全部基因的启动子区域进行CpG岛检测，只有Kitlg基因启动子区存在一个CpG岛，35个甲基化位点。Kitlg基因BSP产物重组T载体质粒的测序结果显示，随着染毒剂量增加，发生Cs的个数呈上升趋势，其中，160mg/kg BPA组Cs的甲基化率与对照组比较差异有统计学意义(P＜0.05)；第7甲基化位点（Cs）在对照组存在甲基化状态，在染毒组中出现较为明显的去甲基化现象；第16甲基化位点在对照组呈现无甲基化状态，而随着染毒剂量增加，在染毒组中出现较为明显的甲基化现象。
结论：
根据以上结果结合讨论可得出如下结论:
1.青春期前BPA较高浓度短期暴露可能对卵巢卵泡的发育和性激素分泌功能具有抑制毒效应，主要表现为卵巢重量及卵巢脏器系数下降、卵泡总数下降、生长卵泡构成比下降及闭锁卵泡构成比升高、卵泡凋亡率升高、血清孕激素水平下降。
2.促进卵泡发育的Kitlg基因、Figla基因、H1foo基因表达水平下调以及抑制卵泡发育的AMH基因表达上调，可能在青春期前BPA暴露抑制卵巢发育过程中发挥了重要作用。
3. DNA甲基化模式的改变在Kitlg、Figla、H1foo及AMH等基因表达调控中的可能作用值得我们重视；Kitlg基因表达下调可能与其启动子区CpG岛甲基化模式的改变有关。
Introduction
Bisphenol A (BPA，CAS:80-05-7) name2,2-two (4-hydroxyphenyl) propane，is recognized as one of several environmental estrogens. BPA is a monomer of thesynthetic polymer material that is used in the production of plasticizers, pesticides,paint, and other chemical products. BPA is used in a wide variety of products, such asbaby bottles, lunch boxes, toys, water pipes, and other materials. BPA monomers canleach from a plastic container that holds food or beverages, and the release process isaccelerated under conditions of high temperature, strong acidity, or strong alkalinity.Human exposure to BPA is extensive; epidemiological studies have shown that thechemical could be detected in the urine of more than90%of tested individuals. Thechemical structure of BPA is similar to that of estradiol (E2); it can therefore mimicestrogenic actions in the body.
The use of BPA in baby products has already been banned in the European Union,the United States, China, and many other countries. However, the prepubertal periodis also an important time in child development, and BPA exposure will occur duringthis period through a variety of pathways. Children are especially vulnerable to higherconcentrations of BPA as a consequence of food and drinking water contamination.Although environmental concentration compared with estrogens in vivo is low，thehypothalamus-pituitary-gonadal axis of children is not yet mature during thepre-puberty stage, and the levels of sex hormones in the body are relatively low;therefore, children’s reproductive systems are more sensitive to environmentalestrogens than those of adults. We speculated that exposure to high concentrations ofBPA during the prepubertal period may affect the normal developmental process of ovarian follicles, and the resulting toxic effects may be mediated by the regulatorygenes involved in follicular development. This study explored the roles andmechanisms of genes that are associated with ovarian development in the toxic effectsof short-term BPA exposure on rat ovarian development. We accomplished this aim bystudying changes in expression among follicle development-promoting genes, such asKitlg, Figla and H1foo, and follicular development inhibitory genes, such as AMH.This study could provide an important experimental evidence for the reproductivesystem developmental effect of children exposure to BPA.
Part Ⅰ: Effect of bisphenol A on ovarian development and function inprepubertal rat.
Objective: The aim of this study was to determine whether BPA could affect ovariandevelopment and function in prepubertal rat.
Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. The effects of BPA on ovarian structure and function were assessed bymeasuring the weight of the ovaries, counting ovarian follicles, apoptosis in ovarianfollicles, ratios of vaginal opening and first estrus, assaying sex hormone levels in theserum, and the ultrastrueture of ovarian tissue use of transmission electronmicroscopy.
Results:
1. Prepubertal rats exposed to BPA for7days, no significant difference wasobserved in body weights between the BPA exposure groups and the control group,whereas the ovarian weights in the BPA exposure groups decreased with increasingBPA doses. The ovarian weights and ovary coefficients of the rats from the40mg/kgand160mg/kg BPA groups were significantly different from those of the controlgroup (P<0.01)
2. The ratios of vaginal opening and first estrus have no significant differencebetween the BPA exposure groups and the control group（P＞0.05）.
3. The levels of serum E2in rats from each BPA group decreased with increasingdoses of BPA, but the results were not significantly different from that of the rats in the control group (P0.05). However, the levels of serum P4in rats from the BPAgroups were significantly different from that of the control group rats (P<0.01); inparticular, the decreases of serum P4in the40mg/kg and160mg/kg BPA groupswere significantly different from that of the control group, respectively (P<0.01).
4. The number of follicles in each BPA exposure group decreased with increasingBPA doses; in comparison to the control group, significant changes were observed inthe10mg/kg,40mg/kg, and160mg/kg BPA groups (P<0.05, P<0.01, and P<0.01,respectively); In addition, the constituent ratios of primordial follicles, primaryfollicles/preantral follicles, antral follicles, and corpus luteum (CL) all decreased inresponse to increasing BPA concentrations, but the constituent ratio of atretic folliclesincreased. These changes in the40mg/kg and160mg/kg BPA groups relative to thecontrol group were statistically significant (P<0.01).
5. Compered with control group, the apoptosis ratios of preantral follicles, antralfollicles, and corpus luteum (CL) all increased in response to increasing BPAconcentrations.
6. The ovarian tissue ultrastrueture of steatosis and secondary lysosomes allincreased in response to increasing BPA concentrations, compered with control group.
Part Ⅱ: Effect of ovarian follicle development-related genes expression inprepubertal rat exposed to bisphenol A.
Objective: The aim of this study was to determine whether BPA could affect ovarianfollicle development-related genes expression in prepubertal rat.
Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. The mRNA and protein expression levels of follicle development-relatedgenes: Stem cell factor(Kitlg), Factor In the Germline alpha (Figla), oocyte-specifichistone H1variant (H1foo), and anti-Mullerian hormone (AMH) were analyzed usingreal-time quantitative PCR, western blots and immunohistochemistry.
Results:
1. The real-time PCR revealed that Kitlg gene mRNA expression levels in the160mg/kg BPA group significantly decreased (P<0.01) in comparison to the control. The mRNA expression levels of the Figla gene in all of the BPA groups significantlydecreased (P<0.05) relative to the control. And the mRNA expression levels of theH1foo gene in all of the BPA groups significantly decreased (P<0.01) relative to thecontrol, but the mRNA expression levels of the AMH gene in the160mg/kg BPAgroup significantly increased (P<0.01) relative to the control.
2. Western blot analysis revealed that protein expression levels of KITLG in eachBPA group significantly decreased relative to the control, with P<0.05for the10mg/kg BPA and40mg/kg group and P<0.01for the160mg/kg groups, and theprotein expression levels of FIGLA in the160mg/kg BPA group significantlydecreased (P<0.05) relative to the control, and the protein expression level of H1FOOin each BPA group significantly decreased relative to the control, with P<0.05for the10mg/kg BPA group and P<0.01for the40mg/kg and160mg/kg groups, but theprotein expression level of the AMH gene in each BPA group significantly increasedrelative to the control, with P<0.05for the10mg/kg BPA group and P<0.01for the40mg/kg and160mg/kg groups.
Part Ⅲ: Study on epigenetic regulation mechanism of toxicity during ovarianfollicle development in prepubertal rat exposed to bisphenol A.
Objective: The aim of this study was to determine whether BPA could affect DNAtotal methylation status and gene promoter region DNA methylation status of ovarianfollicle development-related genes in prepubertal rat.
Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. Study on global methylation status, and gene promoter region DNAmethylation status, to explore the epigenetic regulation mechanisms underlying BPAtoxicity during ovarian development.
Results:
1. Compered with control group,the OD and IOD of combined5-mC scores ofovarian granulosa cell increased gradually in response to increasing BPAconcentrations. These changes in the40mg/kg and160mg/kg BPA groups relative tothe control group were statistically significant (P<0.05).
2. All genes test results from the Methprimer software showed only that there isone CpG island in the promoter region of the Kitlg gene, with35methylationcites(Cs). and then use the bisulfite sequencing polymerase chain reaction (BSP)method to test the methylation status of Kitlg gene. The sequencing results showedthat levels of methylation cites increased with increasing doses of BPA. in particular,the increases of methylation cites in the160mg/kg BPA groups was significantlydifferent from that of the control group (P<0.05). and7Cs exists methylation status inthe control group, but demethylation in BPA exposure group;16Cs show nomethylation status in the control group,but obvious methylation in BPA exposuregroup.
Conclusion:
1. Our results indicated that short-term, concentrated BPA exposure during theprepubertal period may have inhibitory effects on ovarian follicle development andhormone secretion, which were observed as the ovarian weight, counting ovarianfollicles were decreased, the constituent ratios of all growing follicles decreased andatretic follicles increased, the apoptosis ratios of follicles increased, and the inhibitionof progesterone secretion.
2. The down-regulation of Kitlg, Figla and H1foo genes and the up-regulation ofthe AMH gene may play important roles in the BPA-induced mechanism of toxicitythat affects ovarian development.
3. The possible role of DNA methylation patterns change in the regulation ofgene expression in Kitlg, Figla, H1foo and AMH genes are clearly worth considering;the mRNA expression levels of the Kitlg gene decreased significantly in the BPAgroups，may associated with promoter region methylation pattern.
引文
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