大、小鼠卵泡培养方法及其在雌性生殖毒性研究中的应用
|
摘要
生殖和发育功能障碍是当前严重影响人体健康的主要公共卫生问题之一。外源性化学物作用于雌性生殖系统可以导致卵巢周期紊乱或不育,致使人群中自发性流产率、子代发育异常、生育力下降等显著增加。同时,雌性所有的配子在出生前就已经形成,出生时卵泡的数量是固定的,如果破坏了,无额外的配子补偿,后果较雄性更严重。因此,筛选和鉴定具有雌性生殖毒性的化学物,阐明其毒作用机制,对防治化学物引起的生殖系统危害具有重要意义。
近年来由于组合化学、计算机辅助药物设计等的快速发展带来新化学物的快速增长,现有毒理学评价方法已远远不能满足化学物快速增长的需求。因此,国际上的发展趋势是在方法学上要求发展符合“4R”原则(替代、减少、优化和可靠性)、具有高通量、所需受试样品量少的试验方法。目前雌性生殖毒性评价方法主要采用体内试验,存在的主要问题是整体动物实验不敏感、周期长、所需受试样品量多。体内试验难以揭示毒作用位点和毒作用机制。为此,近几年国内外已经建立了多种体外雌性生殖毒性试验方法,包括卵巢器官和卵巢皮质薄片培养方法和大鼠颗粒细胞和黄体细胞体外毒性试验方法。但这些体外方法无法对卵泡发育和卵子生成做出客观评价,而这应是雌性生殖毒性研究的关键所在。因此,迫切需要发展既能快速检测雌性生殖毒性又能更好地研究其作用机制的新方法。
卵泡体外培养方法是近年来发展的将早期卵泡不成熟卵母细胞(GV期或GV前期)在体外发育到可正常受精的成熟卵母细胞(MⅡ)的方法。由于该方法能对卵巢的主要功能即卵泡发育、激素生成和卵子发生进行动态观察,可在卵泡不同发育阶段对卵泡每一组分进行分析,因此受到生殖生物学和生殖毒理学界的关注。目前国外已经成功建立或基本建立绵羊、猪、小鼠腔前卵泡体外培养模型,并已应用于生殖生物学和生理学对卵泡发育和调控的研究。已有学者尝试将小鼠模型用于生殖毒性研究,但尚未发展成为毒性试验系统。有关大鼠腔前卵泡体外研究极少,仅见大鼠卵泡体外短期阶段性培养的个别研究,目前国内外尚未见类似小鼠可比较完整地观察大鼠卵泡发育过程和卵子形成的体外培养系统的报道。
本研究拟借鉴国外的经验,首先建立小鼠腔前卵泡体外培养方法,在此基础上尝试建立大鼠腔前卵泡的体外培养方法;然后选择已知卵巢毒物探讨大小鼠腔前卵泡体外培养方法用于雌性生殖毒物鉴定和机制研究的可行性,目的是为建立一个既可对雌性生殖毒物进行筛选或鉴定,又可对生殖毒作用机制进行研究的体外毒性试验系统提供依据。
一、小鼠腔前卵泡体外培养方法的建立
采用机械性方法分离出生后(post-natal day,PND)PND12～14(C5781/6JxCBA/Ca)小鼠卵巢内直径为100～130μm的腔前卵泡,96孔板单个卵泡在石蜡油覆盖的M1培养基内连续培养12d后诱导排卵16h,观察卵泡发育、激素分泌和卵子形成,并将体外卵母细胞的成熟情况与体内卵母细胞的生长情况(in vivo growth and in vitromaturation of oocytes,IVM)进行比较,以判断体外培养方法是否成功。
1.小鼠卵泡体外发育特征
7只小鼠分离出419个卵泡,机械性分离完整卵泡率为376/419(89.74%),卵泡和卵母细胞直径分别从118.51±10.40μm和50.75±1.67μm增加到培养d12的452.03±110.25μm和69.54±1.55μm;培养d12卵泡存活率为86.87%(364/419),有腔形成率为31.03%(130/419);诱导排卵后COCs排出率为50.60%(212/419)。
小鼠卵泡体外发育形态学变化:体外培养的部分卵泡经历从腔前卵泡、有腔卵泡、排卵前卵泡、卵丘细胞—卵母细胞复合体(COCs)排出、卵母细胞成熟和极体排出的形态学变化,也有部分卵泡不发生腔样结构改变,在卵泡中央可见卵丘—卵母细胞复合体,周围颗粒细胞平铺在培养板上,间隙不明显。
2.小鼠卵泡体外激素分泌的变化
收集d10、d12、d13更换的培养基测定卵泡激素P,每个时间点收集1-3个样品;收集d4、6、8、10、12更换的培养基测定卵泡激素E_2。E_2和P的检测采用磁性酶联免疫法,E_2抗体用17β-oestradiol,质控管在600～900pg/ml;P质控管在7～13ng/ml。体外培养卵泡从d4天开始检测到E_2增加,并且随着培养时间延长持续增加,d10～12维持在较高水平;d12诱导排卵刺激后P分泌量急剧增加,体外培养卵泡分泌E_2和P激素与体内分泌特征一致。
3.小鼠卵母细胞体外成熟
研究结果显示,IVG组4.61%(10/217)卵母细胞不能恢复成熟(停止在GV期),76.50%(166/217)停止减数分裂在GVBD期,18.89%(41/217)卵泡发出PB;而IVM组1.24%(3/241)的卵母细胞不能恢复成熟(停止在GV期),45.23%(109/241)停止减数分裂在GVBD,53.53%(41/241)卵泡发出PB;能完全成熟过渡到MⅡ期(PB排出)卵母细胞的比例在各个试验组之间有差异;没有一个卵母细胞完全发育成染色体不分离的超倍体。
综上可见,从卵泡发育、激素合成和卵母细胞成熟三方面显示本研究建立的小鼠腔前卵泡体外培养方法基本是成功的,与国内上海计生所汪玉宝等报道实验结果基本一致,但本研究卵母细胞PB形成率低于国外Sun F等微滴三维培养结果,有待进一步优化和完善培养体系。
二、大鼠腔前卵泡体外培养方法的建立
1.大鼠卵泡体外培养条件的确定
(1)不同大小卵泡培养效果的比较:分别收集了直径为100～130μm,131～150μm,151～160μm卵泡在体外培养11d,观察卵泡发育情况,结果表明:100～130μm大小的卵泡在培养dll有腔卵泡形成率、卵泡存活率、COCs排出率和成熟阶段卵母细胞明显较131～150μm组和151～160μm组减少,差异有统计学意义(P＜0.05),而131～150μm组和151～160μm组之间无显著性差异。因此选择直径为131～150μm大小的腔前卵泡进行体外培养比较合适。
(2)不同鼠龄分离出的不同大小卵泡比例的差异:采用机械性分离不同大鼠鼠龄的卵泡,直径为130～150μm卵泡在不同鼠龄中所占比重存在差异:PND13所占比例最大,约94.87%,PND12和PND14分别为17.54%和小于29.58%。
(3)诱导起始排卵时间的选择:分别于d10、d11、d12诱导卵泡排卵后观察COCs的排出,结果显示这三个时间点COCs的排出率无显著性差异(P＞0.05),因此d10、d11、d12都可以作为诱导卵泡排卵的时间。
(4)COCs排出观察时间的确定:大鼠卵泡在培养d11诱导排卵刺激16～22h观察COCs的排出情况,结果显示COCs排出率随着时间延长而增加,诱导刺激后20hCOCs排出率最高,达47.84%,因此选择大鼠卵泡COCs排出的观察时间为20h。
(5)每组卵泡数的确定:鉴于各观察指标的变异情况与小鼠卵泡培养的结果近似,故参照国外Val'erie Van Merris等报告结果,确定每组卵泡样本数为16～20个。
2.大鼠卵泡体外发育特征
4只PND13 SD大鼠共分离出296个腔前卵泡。机械性分离完整卵泡率为96.28%(285/296),卵泡和卵母细胞直径分别从141.26±8.82μm和51.25±1.37μm增加到培养dayll 543.21±131.11μm和70.48±2.89μm,培养d11卵泡存活率为92.23%(273/296),d6有腔形成率为67.91%(201/296),d11有腔形成率为75.67%(224/296),COCs排出率为44.93%(133/296)。
大鼠卵泡体外发育形态学变化:体外培养大鼠卵泡形态学上经历了腔前卵泡、有腔卵泡、排卵前卵泡、卵丘细胞—卵母细胞复合体(COCs)排出、卵母细胞成熟和极体排出的形态学变化,与体内卵泡发育特征一致。
3.大鼠卵泡体外激素分泌情况
收集D4、6、8、10、12更换的培养基测定卵泡激素E_2;D10、D12、D13更换的培养基测定卵泡激素P,每个时间点收集1-3个样品。E_2和P的检测采用磁性酶联免疫法,E_2抗体用17β-oestradiol,质控管在600～900pg/ml;P质控管在7～13ng/ml。体外培养卵泡从d4天开始检测到E_2增加,并且随着培养时间延长持续增加,d8～11维持在较高水平;d11诱导排卵刺激后P分泌量急剧增加。体外培养卵泡分泌E_2和P激素与体内卵泡激素分泌特征一致。
4.大鼠卵母细胞体外成熟
研究显示,IVG组7.44%(9/121)卵母细胞不能恢复成熟,停止在GV期,57.02%(69/121)停止减数分裂在GVBD期,35.53%(43/121)卵泡发出PB,而IVM组1.86%(6/322)的卵母细胞不能恢复成熟,停止在GV期,40.06%(129/322)停止在减数分裂GVBD期,56.21%(181/322)卵泡发出PB;没有一个卵母细胞完全发育成染色体不分离的超倍体。
从卵泡发育、激素合成和卵母细胞成熟三方面以及参照小鼠培养方法的判断方法,显示本研究建立的大鼠腔前卵泡体外培养方法是成功的。
三、大鼠卵泡体外方法作为毒性检测系统的初步验证
由于本研究建立的小鼠卵泡体外培养方法在卵母细胞PB形成率上尚达不到国外水平,有待改进。另一方面,本研究建立的大鼠体外卵泡培养方法多项指标明显较小鼠卵泡培养效果好,加上大鼠是生殖毒性实验中最常使用的动物。因此,本研究在目前的条件下首先选择大鼠卵泡体外培养方法用于化学物雌性生殖毒性及其机制的研究。
本研究选择氯化镉、DEHP、MEHP和噻氨酯哒唑Nocodazole四种卵巢毒物,根据受试物作用特征,观察其对大鼠卵泡的体外毒性。
1.氯化镉
镉是一种常见的工业毒物和环境污染物,动物实验表明镉可干扰卵巢内分泌功能,使大鼠动情周期延长,卵巢组织形态结构发生改变;可以抑制卵泡的正常生长发育,抑制雌性动物排卵,造成暂时性不育;可以通过促使体内颗粒细胞凋亡而诱导卵泡闭锁,进而对卵母细胞的成熟、排卵等造成影响。体外实验表明氯化镉可以抑制大鼠颗粒细胞孕酮的合成。本研究从氯化镉对卵泡发育、激素合成和不同发育阶段卵泡的影响三个方面研究氯化镉的卵巢毒性及其可能的作用机制。
1.1氯化镉对卵泡发育影响
实验设氯化镉0.1～1.6μg/mL 5个浓度和溶剂对照,每组16～20个卵泡,三次重复。在培养d2暴露氯化镉后卵泡在体外连续培养11d,隔天换1/2液,收集隔天更换的培养液d4、8、11检测E_2激素,d11、12检测P。观察卵泡发育的形态学变化、测定d11卵泡存活率、有腔形成率和异常卵泡形成率,诱导排卵后测定COCs排出率。结果显示:形态学上可以观察到1.6μg/ml氯化镉出现较为明显细胞毒性,细胞呈圆形不能贴壁,突出培养板底部,基底膜模糊、缺损或消失,颗粒细胞松散或游离到卵泡外,卵泡轮廓不规则卵泡内出现黑色坏死区,卵母细胞逸出卵泡、变形、萎缩或崩解退化表现。暴露0.8-1.6μg/ml氯化镉对卵泡分泌E_2含量无明显影响,但可以明显降低P的分泌量,与对照组比较差异有统计学意义(P＜0.05),说明氯化镉可以抑制卵泡合成孕激素P。暴露1.2μg/mL以上氯化镉后d11卵泡存活率(66.44±3.95%)出现下降,与对照组(91.57±1.32%)比较差异有统计学意义(P＜0.05),剂量与卵泡存活率之间存在一定的相关性,相关系数R~2为0.90;异常卵泡出现率(33.57±3.95%)明显升高,与对照组(8.44±1.32%)比较差异有统计学意义(P＜0.05);暴露1.6μg/mL氯化镉后d11有腔卵泡形成率(42.51±6.66%)明显减少,与对照组(79.68±6.62%)比较差异有统计学意义(P＜0.05);COCs排出率下降(24.99±3.95%),与对照组(52.78±8.45%)比较差异有统计学意义(P＜0.05)。
1.2氯化镉对不同发育阶段卵泡的影响
本实验根据大鼠卵泡体外发育形态学变化,以卵泡不同阶段发育阶段d2(腔前卵泡阶段)、d6(有腔卵泡阶段)、d11(排卵前卵泡阶段)为暴露时间点,研究氯化镉影响卵泡发育和卵母细胞成熟的敏感阶段;根据第一部分实验选择氯化镉0.8、1.2和1.6μg/ml三个浓度组和溶剂对照组,暴露氯化镉后卵泡在体外继续培养到d11,隔天换1/2液,每组16～20个卵泡,三次重复。结果显示:d2暴露1.6ug/mL氯化镉,培养到d11有腔卵泡形成率(42.51±6.66%)、存活率(52.51±7.84%)比d6有腔卵泡形成率(74.24±6.98%)、存活率(81.75±8.13%)减少,而异常卵泡率(47.49±7.48%)比day6异常卵泡率(18.23±8.17%)增加,差异有统计学意义(P＜0.05);卵母细胞发育停止在GV阶段(58.34±11.79%),明显比d6(15.66±9.28%)、d11(20.2±2.86%)时间点数量增加,差异有统计学意义(P＜0.05)。
d6暴露氯化镉对卵泡发育、排卵和卵母细胞发育成熟无明显影响;虽然在相同浓度下,三个时间点暴露氯化镉对PB影响无明显差异,但d11暴露0.8-1.6μg/ml氯化镉对PB形成率的影响有一定剂量反应趋势,而IVM组1.6μg/mL氯化镉使PB减少(34.11±0.34%),与对照组(67.98±6.57%)比较差异有统计学意义(P＜0.05)。
以上结果说明卵泡不同发育阶段暴露一定剂量氯化镉对卵泡发育和卵母细胞成熟有一定影响:腔前卵泡阶段暴露氯化镉可以影响卵泡发育和卵母细胞的发育成熟(处于GV期),有腔卵泡阶段暴露氯化镉对卵泡发育和卵母细胞成熟无明显的影响,排卵前卵泡阶段暴露氯化镉可以影响卵泡排卵和卵母细胞PB形成。
2.邻苯二甲酸二(2一乙基己基)酯(DEHP)和邻苯二甲酸单(2一乙基己基)酯(MEHP)
DEHP和MEHP属于邻苯二甲酸酯类。动物实验发现DEHP的雌性生殖毒性作用主要是通过其代谢产物MEHP影响卵巢功能,作用位点主要是卵巢颗粒细胞。MEHP可显著抑制排卵前期颗粒细胞产生雌二醇,MEHP与大鼠颗粒细胞共同培养24小时后在降低孕酮分泌量的同时,也可以降低FSH诱导的cAMP蓄积量40%,孕酮分泌量的下降与MEHP存在剂量一效应关系。
本研究中MEHP设2.5～80μg/mL 6个浓度,DEHP设33.7～1000nM 6个浓度,同时设DMSO溶剂对照和阴性对照。每组16～20个卵泡,三次重复。在培养d2分别暴露化学物后卵泡在体外连续培养11天,隔天换1/2液,测定d11天卵泡存活率、有腔形成率、异常卵泡形成率和COCs排出率。结果显示DEHP对d11天卵泡存活率、有腔形成率、异常卵泡形成率和COCs排出率无明显的影响,与溶剂对照无显著性差异(P＞0.05)。对照组和DMSO组也无统计学差异。d2暴露＞10μg/mL以上剂量的MEHP 48h后,d11卵泡存活率明显下降(54.76±3.37%),与对照组(91.57+1.32%)比较差异有统计学意义(P＜0.05),剂量与卵泡存活率之间存在一定相关性,相关系数R~2为0.92;COCs排出(24.99±3.95%)明显减少,与对照组(52.78±8.45%)比较差异有统计学意义(P＜0.05);异常卵泡出现率(45.24±3.37%)明显升高,与对照组比较差异有统计学意义(P＜0.05);暴露＞20μg/mL MEHP有腔形成率(46.18±1.67%)减少,与对照组(85.91±5.03%)比较差异有统计学意义(P＜0.05),且有明显剂量反应关系。
本研究结果还说明大鼠卵泡体外培养方法不具有代谢活化能力,对需经代谢的间接生殖毒物可能需要加体外代谢活化系统,有待进一步研究。
3.噻氨酯哒唑(Nocodazole)
噻氨酯哒唑是一种干扰微管组装合成的抗肿瘤药物。和秋水仙素在β-tubulin上Arg-390竞争结合同一结合位点,直接影响微管聚合作用和微管依赖性进程。可以导致细胞周期永久性或短暂阻滞,以往研究证实噻氨酯哒唑可以明显地增加抑制在第一次减数分裂的卵泡数。本研究在卵泡培养d11加含终浓度为10、20、40nM噻氨酯哒唑暴露20h后观察卵泡排卵和卵母细胞成熟情况。结果显示:ⅣM组,COCs暴露100nM噻氨酯哒唑20h后出现卵母细胞细胞毒性,＞5nM出现PB排出(23.19±14.35%)明显下降,与对照(67.96±6.57%)组比较差异有显著意义(P＜0.05):腔前卵泡培养d11天暴露40nM噻氨酯哒唑后卵泡排出COCs数减少,与对照组比较差异无统计学意义,但存在剂量反应关系,说明噻氨酯哒唑可抑制卵泡排出COCs;同时,噻氨酯哒唑可以抑制减数分裂恢复,PB排出减少(19.35%),与对照组(37.78%)比较差异有统计学意义。
本研究用大鼠卵泡体外培养方法进一步揭示了四种卵巢毒物对卵泡发育、激素形成和卵母细胞成熟的影响,以及对不同发育阶段卵泡的毒作用特征,初步验证了大鼠腔前卵泡体外培养方法用于雌性生殖毒物鉴定和机制研究的可行性。
1、基本建立了小鼠腔前卵泡体外培养方法。
2、建立了大鼠腔前卵泡体外培养方法,国内外文献尚未报道比较完整地观察大鼠卵泡发育过程和卵子形成的体外培养系统。
3、用大鼠腔前卵泡培养方法观察了四种化学物对卵泡发育、激素合成和卵子形成的影响,以及对不同发育阶段卵泡的毒作用。进一步揭示了氯化镉、DEHP、MEHP、噻氨酯哒唑四种卵巢毒物的雌性生殖毒作用特征,为阐明其毒作用机制提供了某些依据。
4、初步提出一个以卵泡形态学改变、卵泡分化阶段、卵泡存活率、卵丘细胞粘液化、激素(E2、P)分泌量、卵母细胞直径、极体形成率为评价指标的大鼠腔前卵泡体外毒性研究系统。该系统可有效鉴定卵巢毒物,并可用于毒物作用阶段、作用特征等机制的研究,具有良好的潜在应用前景。由于该试验系统周期短、所用实验动物少、简便、受试物用量少,待进一步完善试验条件、优化评价指标和不同类型雌性生殖毒物验证后有望成为一种雌性生殖毒性的体外试验系统或替代方法。
Reproductive and developmental dysfunction is one of the major public health problems which imposes a significant impact on human health.The effect of chemicals on female reproductive system will result in ovarian cycle disorder or infertility,which increases significantly the spontaneous abortion rate and the incidence of offspring developmental anomaly and fertility,and so on.Simultaneously,all female gametes have been formed in prenatal,the number of follicles are immovable at birth,if destruction,no supernumerary gametes to retrieve,the consequence is more serious than male.Therefore, it is extremely important to screen and identify chemicals which have female reproductive toxicity and to elucidate the mechanism of their toxic action.
Nevertheless,new chemical entities are produced in their thousands each year by combinatorial chemistry and computer-assisted drug design.Efforts are being made to identify and develop alternative methodologies,such as high-throughput and few testing samples.The European Commission and the EU Member States must actively encourage and support the development,validation and acceptance of methods which could reduce, refine or replace the use of laboratory animals.Currently,the guidelines produced by ICH and OECD for reproductive testing are still performed by in-vivo animal studies,mainly in rodents.Evaluation of the femal reproductive function by in vivo studies is currently carried out on the basis of parameters related to fertility outcome,these parameters do not allow elucidation the site of action and the mechanism of toxic damage to the ovary.Since in-vivo animal studies are less sensitivity,require longer testing cycle and more test samples than in-vitro bioassay,so it is necessary to develop in-vitro bioassay for testing femal reproductive toxicity.As yet,no adequate in-vitro models have been validated to identify potentially hazardous products with regard to the reproductive system,including ovarian follicle,oocyte,oogenesis which is the key to evaluate femal reproductive toxicity.
In rescent years,in-vitro follicle culture systems have been developed with the aim of growing immature oocytes from early follicle stages to fertilizable oocytes.They are also often used as a tool to study the process of folliculogenesis and oogenesis in vitro.Several research groups have described the conditions for in-vitro culture of mouse ovarian follicles.In-depth study of the culture system demonstrates that the whole in-vitro process mimics normal(in-vivo)physiology from the point of view of morphogenesis, defferentiantion,hormone production.Ovarian follicle function can be analyzed simultaneously on folliculogenesis,hormonogenesis,and oogenesis on variant developmental stages.These systems have been applied succeedly to reproductive biology and physiology studies for the folliculogenesis and follicle regulation.Nevertheless,in-vitro follicle bioassay for reproductive toxicity is are not produced except for individual toxicity parameters.As yet,in-vitro rat preantral follicle culture system were not developed.
On basis of the mouse follicle culture system described according to literatures,in-vitro rat preantral follicle culture systems were developed in this study and had never been reported before.The aim of this study is to develop pre-antral mouse/rat follicle culture in-vitro assay in repriductive toxicology testing to provide information regarding the potential mechanism of action and the identification of chemicals which have female reproductive toxicity.
1.Establishment of in-vitro pre-antral mouse follicle culture system
The ovaries were from prepubertal mice(C57B1/6JxCBA/Ca)(aged 12～14 days).Only follicles with 2-3 layers of granulose cells,a centrally located oocyte and a diameter in the range 100～130μm,which were enclosed by an intact basal membrane and had some attached theca cells,were mechanically dissected and randomly allocated to 96-well culture plates.The preantral follicles were subsequently cultured individually for 12 days within culture medium covered by mineral oil.On day 12 of culture,the follicles were induced ovulation for 16h.The in-vitro developmental characteristics of the follicles were observed including hormonogenesis,folliculogenesis and oogenesis,by comparing in vitro growth and maturation of oocytes(IVG) with in vivo growth and in vitro maturation of oocytes(IVM).
1.1 Developmental characteristics of mouse preantral follicles in vitro
A total of 419 from 7 prepubertal mice,preantral follicles and oocyte with an average diameter of 118.51±10.40μm and 50.75±1.67μm,respectively,376/419(89.74%) of follicles were visibly associated with theca cells and possessed a close follicle(granulosa) cell/oocyte apposition.At day 12 of culture,follicles and oocyte with an average diameter of 118.51±10.40μm and 50.75±1.67μm were measured.31.03%of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity,86.87%was survival and 50.60%COCs were released by maturation induction in this study.
The morphological observation showed that developmental characteristics of mouse preantral follicles in vitro were similar to those in vivo,including the formation of antral follicle and preovulatory follicle,the proliferation and differentiation of granulosa cell, cumulus cell mucification,releasingof cumulus-oocyte complexes(COCs),oocyte maturation and the formation of first polar body.
1.2 Hormone secretion characteristics of in-vitro cultured follicles
Estradiol and progesterone was detected using electrochemiluminescence immunoassay. The results showed that the follicle cultures were characterized by a consistent increase in the secretion of oestradiol as measured in the conditioned medium throughout the in vitro culture period from day 4 onwards.Progesterone secretion increased characteristically only after the in-vitro ovulatory stimulus from day 12 to 13.In a word,the patterns of hormone secretion observed in preantral follicle culture were similar to those characteristic for in-vivo follicle hormone secretion.
1.3 The oocyte maturation in vitro
The numbers of oocytes with germinal vesicle(GV) did not differ between IVG and IVM groups,18.89%oocytes form the first polar body in IVG groups and 53.53%oocytes form the first polar body in IVM groups.The results were not found abnormality chromosome segregation in this study.
In conclusion,judging from the three aspects of folliculogenesis,hormonogenesis,and oogenesis,the establishment of in-vitro pre-antral mouse follicle culture is successful.Our results were in accordance with that of Wang Yubao.However,the PB formation rate was relatively lower than that reported by Fengyun Sun.
2.Establishment of in-vitro pre-antral SD rat follicle culture system
2.1 Determination of in-vitro pre-antral SD rat follicle culture conditions
(1) The size of follicle for culture:Mimicing in-vitro pre-antral mouse follicle culture system,we studied follicle size for culture.The results displayed that 100～130μm,131～150μm and 151～160μm groups of the follicle diameters increase gradually up to344.29±105.40μm,535.76±105.41μm,587.65±138.69μm,respectively,showing statistical significance comparing to pre-culture(p＜0.05).Antral follicles and surviving follicles in 100～130μm groups were declined significantly comparing to 131～150μm and 151～160μm group.
(2) The rat years of age:The ovaries were from prepubertal SD rat aged PND12,13 and 14.The follicles with variant diameter in the range of 100～160μm were mechanically dissected.In PND13 rat,94.87%follicles were 130～150μm in diameters in all dissected follicles,PND12 rat(17.54%) and PND14 rat(about 29.58%).
(3) The time of starting to induce ovulation:Observation of the respective releasing of COCs in d10,d11 and d12 of the cultured ovarian follicle showed that there was not significant difference in three groups.Hence,d10,d11 and d12 of culture could be chose as the time of starting to induce ovulation.
(4) The time of COCs released from follicles:After inducing ovulation for 16h,18h,20h and 22h respectively,the numbers of COCs released from follicles increased in a time-dependent way.After inducing ovulation for 20h,the rate of COCs released from follicles reached 47.84%.Therefore the best observing time of COCs releasing is 20h.
(5) The numbers of ovarian follicle each group:Taking into consideration the fact that the variation of the indexes of rats has are similar to that of mice,16-20 ovarian follicles in each group were chose for examination,according to the report of Val'erie Van Merris.
2.2 Developmental characteristics of early rat preantral follicles in vitro
Of a total of 296 preantral follicles and oocytes with an average diameter of 141.26±8.82μm and 51.25±1.37μm,respectively.96.28%follicles possessed theca cells and a close follicle(granulosa) cell/oocyte apposition.The size of the follicles increased dramatically up to543.21±131.11μm and 70.48±2.89μm at day11of culture.75.67%of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity and 92.23%follicles were survival at day 11 of culture.Mucification with expansion of cumulus cells(44.93%COCs)was observed 20h after the in vitro ovulatory stimulus in this study.
The morphological observation showed that developmental characteristics of rat preantral follicles in vitro were similar to those in vivo,including the formation of antral follicle and preovulatory follicle,the proliferation and differentiation of granulosa cell, cumulus cell mucification,releasingof cumulus-oocyte cell complexes(COCs),oocyte maturation and the formation of first polar body.
2.3 Hormone secretion characteristics of in vitro cultured follicles
Estradiol and progesterone was measured using electrochemiluminescence immunoassay.The results showed the follicle cultures were characterized by a consistent increase in the secretion of oestradiol as measured in the conditioned medium throughout the in vitro culture period from day 4 onwards.Progesterone secretion increased characteristically only after the in vitro ovulatory stimulus from day 11 to day12.
2.4 Oocyte maturation in vitro
7.44%(9/121)oocytes was blocked germinal vesicle(GV) stage,57.02%(69/121) oocytes initiated resumption of maturation(GVBD),35.53%(43/121) oocytes formed a first polar bodyin IVG groups,1.86%(6/322)oocytes blocked germinal vesicle(GV) stages,40.06%(129/322)oocytes initiated resumption of maturation(GVBD),56.21% (181/322) oocytes formed a first polar body in IVM groups.The results were not found abnormality chromosome segregation in this study.
3.Application of in-vitro SD rat follicle culture system for femal reproductive toxicity testing
The PB formation rate was lower than that reported by Fengyun Sun.On the other hand, rats are the most common experimental animals in reproductive toxicity testing.Therefore, in-vitro rat follicle culture system was applied for femal reproductive toxicity testing in this study.
Some chemicals including cadmium chloride(CdCL_2),DEHP,MEHP and Nocodazole were assessed for hormonogenesis,folliculogenesis and oogenesis using the in-vitro SD rat follicle screen system.
3.1 Follicle toxicity of cadmium chloride(CdCL_2) in vitro
Cadmium chloride is a common industrial poison and environment contaminant.As some animal experiments show,cadmium chloride can interrupt the function of ovaries, extend the oestrous cycle of rats,cause histological and structural alteration,inhibit the growth of ovary follicle,and increase the number of atretic follicle.Moreover,it impairs the ovulation and causes infertility.Some in-vitro bioassays show that the progesterone production was significantly decreased by cadmium chloride.
3.1.1 Effects of CdCL_2 on follicle development in vitro
CdCL_2 with concentrations＞1.2μg/ml had been reduced significantlysurvival follicles rate(66.44±3.95%)compared to the control group(91.57±1.32%) on day11 (p＜0.05),wherea33.57±3.95%degenerated or underwent premature oocyte release This dose dependent decrease showed a coefficient of correlation(R~2) of 0.90.CdCL_2 with the concentrations of 1.6μg/ml impaired follicle growth and differentiation,significantly reduced(p＜0.05)the rate of antral follicles(42.51±6.66%)compared to the control(79.68±6.62%).
CdCL_2 did not influence the17β-E_2 secretion in the concentration range of 0.8～1.6μg /ml CdCL_2.The basal progesterone production in the treated groups was significantly decreased for＞0.8ug/ml CdCL_2 compared to the control after hCG-administration on days 11 and 12.
3.1.2 Effects of CdCL_2 on different developmental stage follicle in vitro
To determine sensitive follicle stage of CdCL_2 exposure,the culture follicles exposed to CdCL_2 on day2(pre-antral follicle),day 6(antral follicle),day11(pre-avulation follicle) for 48h,respecfively.CdCL_2 with concentrations 1.6ug/ml on day 2 had a significantly reduced(p＜0.05) survival rate(52.51±7.84),the rate of antral follicles(42.51±6.66%),compared to follicles exposed to same concentrations CdCL_2 on day 6(81.75±8.13%), (74.24±6.98%),respectively.Anormal follicle rate(47.49±7.48%) increase significantly on day 2,compared to day6(47.49±7.48%).The numbers of GV oocyte(58.34±11.79%) increase significantly on day2,compared to day6(15.66±9.28%),day 11(20.2±2.86%).There are no significant adverse effects of CdCL_2 exposure on day6 upon follicle development and oocyte maturation.In IVM group,COCs treated with concentrations 1.6ug/ml CdCL_2 reduced(p＜0.05) PB-formation rate(34.11±0.34%),compared to control (67.98±6.57%).These results demondated that CdCL_2 would inhibit both follicle development and oocyte maturation from pre-antral follicle and oocyte maturation and PB-formation from pre-ovulation follicle.
3.2 Effects of DEHP and MEHP on follicle development in vitro
Ai-2-ethylhexyl phttmlate(DEHP) and mono-2-ethylhexyl phthalate(MEHP) belongs to phthalicacidesters(PAEs).Animal experiments showed that the toxicity on reproduction of DEHP result from its metabolite MEHP,which affected granulose cells of ovaries and remarkably inhibited the production of estrogen during preovulatory stage.Some in-vitro bioassays show that MEHP decreased the production of progesterone as well as the FSH-induced cAMP production.
Follicles treated with concentrations＞10μg/mLMEHP had a significantly reduced(p＜0.05) survival rate(54.76±3.37%) compared to the control group(91.57±1.32%)on day11. wherea 45.24±3.37%follicles degenerated or underwent premature oocyte release.This dose dependent decrease showed a coefficient of correlation(R~2) of 0.92. Concentrations＞20ug/mL MEHP dose dependently impaired follicle growth and differentiation,antral follicles(46.18±1.67%) had a significantly reduced(p＜0.05) compared to the control group(85.91±5.03%).DEHP showed no adverse effects upon follicle development and there were no significant defference between DMSO and control.
3.3 Effects of Nocodazole on oocyte maturation in vitro
Nocodazole,methyl[5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl]carbamate,a synthetic anti-tumour drug interfering with microtubule assembly,competes with colchicine for the same binding site on the Arg-390 of b-tubulin and directly affects microtubule polymerization and microtubuledependent processes.
When COCs exposed to Nocodazole concentrations of 100nM for 20h in IVM,there existing oocyte cytotoxicity.The COCs exposed to Nocodazole concentrations of＞5nM, We observed signicant reduced in PB-formation rate compared to its control group. Although the preovulation follicles exposed to Nocodazole concentrations of 10～40nM showed no statistical difference in COCs rate isolated from follicle compared to the controls,there existed a trend.Concentrations of 40nM Nocodazole dose significantly reduced(p＜0.05)PB-formation rate(19.35%) compared to the control group(37.78%)
Summary
1.This study developed in-vitro mouse pre-antral follicle culture system.
2.In-vitro rat preantral follicle culture system were developed and this system had never been reported before
3.Rat preantral follicle culture as a novel in-vitro assay was applied to detect the effects of four chemicals on folliculogenesis,steroidogenesis and oogenesis,and on variant developmental stages,which further revealed that the bioassay is a efficient tool to study ovarian toxicity and their mechanisms of chemical.
4.A preliminary protocol of in-vitro rat preantral follicle culture system for the study on female reproductive toxicity was proposed,including the morphological observation, stages of follicle differentiation,hormone secretion profiles,oocyte diameters,the formatiom of PB and the genetoxic of oocyte.
近年来由于组合化学、计算机辅助药物设计等的快速发展带来新化学物的快速增长,现有毒理学评价方法已远远不能满足化学物快速增长的需求。因此,国际上的发展趋势是在方法学上要求发展符合“4R”原则(替代、减少、优化和可靠性)、具有高通量、所需受试样品量少的试验方法。目前雌性生殖毒性评价方法主要采用体内试验,存在的主要问题是整体动物实验不敏感、周期长、所需受试样品量多。体内试验难以揭示毒作用位点和毒作用机制。为此,近几年国内外已经建立了多种体外雌性生殖毒性试验方法,包括卵巢器官和卵巢皮质薄片培养方法和大鼠颗粒细胞和黄体细胞体外毒性试验方法。但这些体外方法无法对卵泡发育和卵子生成做出客观评价,而这应是雌性生殖毒性研究的关键所在。因此,迫切需要发展既能快速检测雌性生殖毒性又能更好地研究其作用机制的新方法。
卵泡体外培养方法是近年来发展的将早期卵泡不成熟卵母细胞(GV期或GV前期)在体外发育到可正常受精的成熟卵母细胞(MⅡ)的方法。由于该方法能对卵巢的主要功能即卵泡发育、激素生成和卵子发生进行动态观察,可在卵泡不同发育阶段对卵泡每一组分进行分析,因此受到生殖生物学和生殖毒理学界的关注。目前国外已经成功建立或基本建立绵羊、猪、小鼠腔前卵泡体外培养模型,并已应用于生殖生物学和生理学对卵泡发育和调控的研究。已有学者尝试将小鼠模型用于生殖毒性研究,但尚未发展成为毒性试验系统。有关大鼠腔前卵泡体外研究极少,仅见大鼠卵泡体外短期阶段性培养的个别研究,目前国内外尚未见类似小鼠可比较完整地观察大鼠卵泡发育过程和卵子形成的体外培养系统的报道。
本研究拟借鉴国外的经验,首先建立小鼠腔前卵泡体外培养方法,在此基础上尝试建立大鼠腔前卵泡的体外培养方法;然后选择已知卵巢毒物探讨大小鼠腔前卵泡体外培养方法用于雌性生殖毒物鉴定和机制研究的可行性,目的是为建立一个既可对雌性生殖毒物进行筛选或鉴定,又可对生殖毒作用机制进行研究的体外毒性试验系统提供依据。
一、小鼠腔前卵泡体外培养方法的建立
采用机械性方法分离出生后(post-natal day,PND)PND12～14(C5781/6JxCBA/Ca)小鼠卵巢内直径为100～130μm的腔前卵泡,96孔板单个卵泡在石蜡油覆盖的M1培养基内连续培养12d后诱导排卵16h,观察卵泡发育、激素分泌和卵子形成,并将体外卵母细胞的成熟情况与体内卵母细胞的生长情况(in vivo growth and in vitromaturation of oocytes,IVM)进行比较,以判断体外培养方法是否成功。
1.小鼠卵泡体外发育特征
7只小鼠分离出419个卵泡,机械性分离完整卵泡率为376/419(89.74%),卵泡和卵母细胞直径分别从118.51±10.40μm和50.75±1.67μm增加到培养d12的452.03±110.25μm和69.54±1.55μm;培养d12卵泡存活率为86.87%(364/419),有腔形成率为31.03%(130/419);诱导排卵后COCs排出率为50.60%(212/419)。
小鼠卵泡体外发育形态学变化:体外培养的部分卵泡经历从腔前卵泡、有腔卵泡、排卵前卵泡、卵丘细胞—卵母细胞复合体(COCs)排出、卵母细胞成熟和极体排出的形态学变化,也有部分卵泡不发生腔样结构改变,在卵泡中央可见卵丘—卵母细胞复合体,周围颗粒细胞平铺在培养板上,间隙不明显。
2.小鼠卵泡体外激素分泌的变化
收集d10、d12、d13更换的培养基测定卵泡激素P,每个时间点收集1-3个样品;收集d4、6、8、10、12更换的培养基测定卵泡激素E_2。E_2和P的检测采用磁性酶联免疫法,E_2抗体用17β-oestradiol,质控管在600～900pg/ml;P质控管在7～13ng/ml。体外培养卵泡从d4天开始检测到E_2增加,并且随着培养时间延长持续增加,d10～12维持在较高水平;d12诱导排卵刺激后P分泌量急剧增加,体外培养卵泡分泌E_2和P激素与体内分泌特征一致。
3.小鼠卵母细胞体外成熟
研究结果显示,IVG组4.61%(10/217)卵母细胞不能恢复成熟(停止在GV期),76.50%(166/217)停止减数分裂在GVBD期,18.89%(41/217)卵泡发出PB;而IVM组1.24%(3/241)的卵母细胞不能恢复成熟(停止在GV期),45.23%(109/241)停止减数分裂在GVBD,53.53%(41/241)卵泡发出PB;能完全成熟过渡到MⅡ期(PB排出)卵母细胞的比例在各个试验组之间有差异;没有一个卵母细胞完全发育成染色体不分离的超倍体。
综上可见,从卵泡发育、激素合成和卵母细胞成熟三方面显示本研究建立的小鼠腔前卵泡体外培养方法基本是成功的,与国内上海计生所汪玉宝等报道实验结果基本一致,但本研究卵母细胞PB形成率低于国外Sun F等微滴三维培养结果,有待进一步优化和完善培养体系。
二、大鼠腔前卵泡体外培养方法的建立
1.大鼠卵泡体外培养条件的确定
(1)不同大小卵泡培养效果的比较:分别收集了直径为100～130μm,131～150μm,151～160μm卵泡在体外培养11d,观察卵泡发育情况,结果表明:100～130μm大小的卵泡在培养dll有腔卵泡形成率、卵泡存活率、COCs排出率和成熟阶段卵母细胞明显较131～150μm组和151～160μm组减少,差异有统计学意义(P＜0.05),而131～150μm组和151～160μm组之间无显著性差异。因此选择直径为131～150μm大小的腔前卵泡进行体外培养比较合适。
(2)不同鼠龄分离出的不同大小卵泡比例的差异:采用机械性分离不同大鼠鼠龄的卵泡,直径为130～150μm卵泡在不同鼠龄中所占比重存在差异:PND13所占比例最大,约94.87%,PND12和PND14分别为17.54%和小于29.58%。
(3)诱导起始排卵时间的选择:分别于d10、d11、d12诱导卵泡排卵后观察COCs的排出,结果显示这三个时间点COCs的排出率无显著性差异(P＞0.05),因此d10、d11、d12都可以作为诱导卵泡排卵的时间。
(4)COCs排出观察时间的确定:大鼠卵泡在培养d11诱导排卵刺激16～22h观察COCs的排出情况,结果显示COCs排出率随着时间延长而增加,诱导刺激后20hCOCs排出率最高,达47.84%,因此选择大鼠卵泡COCs排出的观察时间为20h。
(5)每组卵泡数的确定:鉴于各观察指标的变异情况与小鼠卵泡培养的结果近似,故参照国外Val'erie Van Merris等报告结果,确定每组卵泡样本数为16～20个。
2.大鼠卵泡体外发育特征
4只PND13 SD大鼠共分离出296个腔前卵泡。机械性分离完整卵泡率为96.28%(285/296),卵泡和卵母细胞直径分别从141.26±8.82μm和51.25±1.37μm增加到培养dayll 543.21±131.11μm和70.48±2.89μm,培养d11卵泡存活率为92.23%(273/296),d6有腔形成率为67.91%(201/296),d11有腔形成率为75.67%(224/296),COCs排出率为44.93%(133/296)。
大鼠卵泡体外发育形态学变化:体外培养大鼠卵泡形态学上经历了腔前卵泡、有腔卵泡、排卵前卵泡、卵丘细胞—卵母细胞复合体(COCs)排出、卵母细胞成熟和极体排出的形态学变化,与体内卵泡发育特征一致。
3.大鼠卵泡体外激素分泌情况
收集D4、6、8、10、12更换的培养基测定卵泡激素E_2;D10、D12、D13更换的培养基测定卵泡激素P,每个时间点收集1-3个样品。E_2和P的检测采用磁性酶联免疫法,E_2抗体用17β-oestradiol,质控管在600～900pg/ml;P质控管在7～13ng/ml。体外培养卵泡从d4天开始检测到E_2增加,并且随着培养时间延长持续增加,d8～11维持在较高水平;d11诱导排卵刺激后P分泌量急剧增加。体外培养卵泡分泌E_2和P激素与体内卵泡激素分泌特征一致。
4.大鼠卵母细胞体外成熟
研究显示,IVG组7.44%(9/121)卵母细胞不能恢复成熟,停止在GV期,57.02%(69/121)停止减数分裂在GVBD期,35.53%(43/121)卵泡发出PB,而IVM组1.86%(6/322)的卵母细胞不能恢复成熟,停止在GV期,40.06%(129/322)停止在减数分裂GVBD期,56.21%(181/322)卵泡发出PB;没有一个卵母细胞完全发育成染色体不分离的超倍体。
从卵泡发育、激素合成和卵母细胞成熟三方面以及参照小鼠培养方法的判断方法,显示本研究建立的大鼠腔前卵泡体外培养方法是成功的。
三、大鼠卵泡体外方法作为毒性检测系统的初步验证
由于本研究建立的小鼠卵泡体外培养方法在卵母细胞PB形成率上尚达不到国外水平,有待改进。另一方面,本研究建立的大鼠体外卵泡培养方法多项指标明显较小鼠卵泡培养效果好,加上大鼠是生殖毒性实验中最常使用的动物。因此,本研究在目前的条件下首先选择大鼠卵泡体外培养方法用于化学物雌性生殖毒性及其机制的研究。
本研究选择氯化镉、DEHP、MEHP和噻氨酯哒唑Nocodazole四种卵巢毒物,根据受试物作用特征,观察其对大鼠卵泡的体外毒性。
1.氯化镉
镉是一种常见的工业毒物和环境污染物,动物实验表明镉可干扰卵巢内分泌功能,使大鼠动情周期延长,卵巢组织形态结构发生改变;可以抑制卵泡的正常生长发育,抑制雌性动物排卵,造成暂时性不育;可以通过促使体内颗粒细胞凋亡而诱导卵泡闭锁,进而对卵母细胞的成熟、排卵等造成影响。体外实验表明氯化镉可以抑制大鼠颗粒细胞孕酮的合成。本研究从氯化镉对卵泡发育、激素合成和不同发育阶段卵泡的影响三个方面研究氯化镉的卵巢毒性及其可能的作用机制。
1.1氯化镉对卵泡发育影响
实验设氯化镉0.1～1.6μg/mL 5个浓度和溶剂对照,每组16～20个卵泡,三次重复。在培养d2暴露氯化镉后卵泡在体外连续培养11d,隔天换1/2液,收集隔天更换的培养液d4、8、11检测E_2激素,d11、12检测P。观察卵泡发育的形态学变化、测定d11卵泡存活率、有腔形成率和异常卵泡形成率,诱导排卵后测定COCs排出率。结果显示:形态学上可以观察到1.6μg/ml氯化镉出现较为明显细胞毒性,细胞呈圆形不能贴壁,突出培养板底部,基底膜模糊、缺损或消失,颗粒细胞松散或游离到卵泡外,卵泡轮廓不规则卵泡内出现黑色坏死区,卵母细胞逸出卵泡、变形、萎缩或崩解退化表现。暴露0.8-1.6μg/ml氯化镉对卵泡分泌E_2含量无明显影响,但可以明显降低P的分泌量,与对照组比较差异有统计学意义(P＜0.05),说明氯化镉可以抑制卵泡合成孕激素P。暴露1.2μg/mL以上氯化镉后d11卵泡存活率(66.44±3.95%)出现下降,与对照组(91.57±1.32%)比较差异有统计学意义(P＜0.05),剂量与卵泡存活率之间存在一定的相关性,相关系数R~2为0.90;异常卵泡出现率(33.57±3.95%)明显升高,与对照组(8.44±1.32%)比较差异有统计学意义(P＜0.05);暴露1.6μg/mL氯化镉后d11有腔卵泡形成率(42.51±6.66%)明显减少,与对照组(79.68±6.62%)比较差异有统计学意义(P＜0.05);COCs排出率下降(24.99±3.95%),与对照组(52.78±8.45%)比较差异有统计学意义(P＜0.05)。
1.2氯化镉对不同发育阶段卵泡的影响
本实验根据大鼠卵泡体外发育形态学变化,以卵泡不同阶段发育阶段d2(腔前卵泡阶段)、d6(有腔卵泡阶段)、d11(排卵前卵泡阶段)为暴露时间点,研究氯化镉影响卵泡发育和卵母细胞成熟的敏感阶段;根据第一部分实验选择氯化镉0.8、1.2和1.6μg/ml三个浓度组和溶剂对照组,暴露氯化镉后卵泡在体外继续培养到d11,隔天换1/2液,每组16～20个卵泡,三次重复。结果显示:d2暴露1.6ug/mL氯化镉,培养到d11有腔卵泡形成率(42.51±6.66%)、存活率(52.51±7.84%)比d6有腔卵泡形成率(74.24±6.98%)、存活率(81.75±8.13%)减少,而异常卵泡率(47.49±7.48%)比day6异常卵泡率(18.23±8.17%)增加,差异有统计学意义(P＜0.05);卵母细胞发育停止在GV阶段(58.34±11.79%),明显比d6(15.66±9.28%)、d11(20.2±2.86%)时间点数量增加,差异有统计学意义(P＜0.05)。
d6暴露氯化镉对卵泡发育、排卵和卵母细胞发育成熟无明显影响;虽然在相同浓度下,三个时间点暴露氯化镉对PB影响无明显差异,但d11暴露0.8-1.6μg/ml氯化镉对PB形成率的影响有一定剂量反应趋势,而IVM组1.6μg/mL氯化镉使PB减少(34.11±0.34%),与对照组(67.98±6.57%)比较差异有统计学意义(P＜0.05)。
以上结果说明卵泡不同发育阶段暴露一定剂量氯化镉对卵泡发育和卵母细胞成熟有一定影响:腔前卵泡阶段暴露氯化镉可以影响卵泡发育和卵母细胞的发育成熟(处于GV期),有腔卵泡阶段暴露氯化镉对卵泡发育和卵母细胞成熟无明显的影响,排卵前卵泡阶段暴露氯化镉可以影响卵泡排卵和卵母细胞PB形成。
2.邻苯二甲酸二(2一乙基己基)酯(DEHP)和邻苯二甲酸单(2一乙基己基)酯(MEHP)
DEHP和MEHP属于邻苯二甲酸酯类。动物实验发现DEHP的雌性生殖毒性作用主要是通过其代谢产物MEHP影响卵巢功能,作用位点主要是卵巢颗粒细胞。MEHP可显著抑制排卵前期颗粒细胞产生雌二醇,MEHP与大鼠颗粒细胞共同培养24小时后在降低孕酮分泌量的同时,也可以降低FSH诱导的cAMP蓄积量40%,孕酮分泌量的下降与MEHP存在剂量一效应关系。
本研究中MEHP设2.5～80μg/mL 6个浓度,DEHP设33.7～1000nM 6个浓度,同时设DMSO溶剂对照和阴性对照。每组16～20个卵泡,三次重复。在培养d2分别暴露化学物后卵泡在体外连续培养11天,隔天换1/2液,测定d11天卵泡存活率、有腔形成率、异常卵泡形成率和COCs排出率。结果显示DEHP对d11天卵泡存活率、有腔形成率、异常卵泡形成率和COCs排出率无明显的影响,与溶剂对照无显著性差异(P＞0.05)。对照组和DMSO组也无统计学差异。d2暴露＞10μg/mL以上剂量的MEHP 48h后,d11卵泡存活率明显下降(54.76±3.37%),与对照组(91.57+1.32%)比较差异有统计学意义(P＜0.05),剂量与卵泡存活率之间存在一定相关性,相关系数R~2为0.92;COCs排出(24.99±3.95%)明显减少,与对照组(52.78±8.45%)比较差异有统计学意义(P＜0.05);异常卵泡出现率(45.24±3.37%)明显升高,与对照组比较差异有统计学意义(P＜0.05);暴露＞20μg/mL MEHP有腔形成率(46.18±1.67%)减少,与对照组(85.91±5.03%)比较差异有统计学意义(P＜0.05),且有明显剂量反应关系。
本研究结果还说明大鼠卵泡体外培养方法不具有代谢活化能力,对需经代谢的间接生殖毒物可能需要加体外代谢活化系统,有待进一步研究。
3.噻氨酯哒唑(Nocodazole)
噻氨酯哒唑是一种干扰微管组装合成的抗肿瘤药物。和秋水仙素在β-tubulin上Arg-390竞争结合同一结合位点,直接影响微管聚合作用和微管依赖性进程。可以导致细胞周期永久性或短暂阻滞,以往研究证实噻氨酯哒唑可以明显地增加抑制在第一次减数分裂的卵泡数。本研究在卵泡培养d11加含终浓度为10、20、40nM噻氨酯哒唑暴露20h后观察卵泡排卵和卵母细胞成熟情况。结果显示:ⅣM组,COCs暴露100nM噻氨酯哒唑20h后出现卵母细胞细胞毒性,＞5nM出现PB排出(23.19±14.35%)明显下降,与对照(67.96±6.57%)组比较差异有显著意义(P＜0.05):腔前卵泡培养d11天暴露40nM噻氨酯哒唑后卵泡排出COCs数减少,与对照组比较差异无统计学意义,但存在剂量反应关系,说明噻氨酯哒唑可抑制卵泡排出COCs;同时,噻氨酯哒唑可以抑制减数分裂恢复,PB排出减少(19.35%),与对照组(37.78%)比较差异有统计学意义。
本研究用大鼠卵泡体外培养方法进一步揭示了四种卵巢毒物对卵泡发育、激素形成和卵母细胞成熟的影响,以及对不同发育阶段卵泡的毒作用特征,初步验证了大鼠腔前卵泡体外培养方法用于雌性生殖毒物鉴定和机制研究的可行性。
1、基本建立了小鼠腔前卵泡体外培养方法。
2、建立了大鼠腔前卵泡体外培养方法,国内外文献尚未报道比较完整地观察大鼠卵泡发育过程和卵子形成的体外培养系统。
3、用大鼠腔前卵泡培养方法观察了四种化学物对卵泡发育、激素合成和卵子形成的影响,以及对不同发育阶段卵泡的毒作用。进一步揭示了氯化镉、DEHP、MEHP、噻氨酯哒唑四种卵巢毒物的雌性生殖毒作用特征,为阐明其毒作用机制提供了某些依据。
4、初步提出一个以卵泡形态学改变、卵泡分化阶段、卵泡存活率、卵丘细胞粘液化、激素(E2、P)分泌量、卵母细胞直径、极体形成率为评价指标的大鼠腔前卵泡体外毒性研究系统。该系统可有效鉴定卵巢毒物,并可用于毒物作用阶段、作用特征等机制的研究,具有良好的潜在应用前景。由于该试验系统周期短、所用实验动物少、简便、受试物用量少,待进一步完善试验条件、优化评价指标和不同类型雌性生殖毒物验证后有望成为一种雌性生殖毒性的体外试验系统或替代方法。
Reproductive and developmental dysfunction is one of the major public health problems which imposes a significant impact on human health.The effect of chemicals on female reproductive system will result in ovarian cycle disorder or infertility,which increases significantly the spontaneous abortion rate and the incidence of offspring developmental anomaly and fertility,and so on.Simultaneously,all female gametes have been formed in prenatal,the number of follicles are immovable at birth,if destruction,no supernumerary gametes to retrieve,the consequence is more serious than male.Therefore, it is extremely important to screen and identify chemicals which have female reproductive toxicity and to elucidate the mechanism of their toxic action.
Nevertheless,new chemical entities are produced in their thousands each year by combinatorial chemistry and computer-assisted drug design.Efforts are being made to identify and develop alternative methodologies,such as high-throughput and few testing samples.The European Commission and the EU Member States must actively encourage and support the development,validation and acceptance of methods which could reduce, refine or replace the use of laboratory animals.Currently,the guidelines produced by ICH and OECD for reproductive testing are still performed by in-vivo animal studies,mainly in rodents.Evaluation of the femal reproductive function by in vivo studies is currently carried out on the basis of parameters related to fertility outcome,these parameters do not allow elucidation the site of action and the mechanism of toxic damage to the ovary.Since in-vivo animal studies are less sensitivity,require longer testing cycle and more test samples than in-vitro bioassay,so it is necessary to develop in-vitro bioassay for testing femal reproductive toxicity.As yet,no adequate in-vitro models have been validated to identify potentially hazardous products with regard to the reproductive system,including ovarian follicle,oocyte,oogenesis which is the key to evaluate femal reproductive toxicity.
In rescent years,in-vitro follicle culture systems have been developed with the aim of growing immature oocytes from early follicle stages to fertilizable oocytes.They are also often used as a tool to study the process of folliculogenesis and oogenesis in vitro.Several research groups have described the conditions for in-vitro culture of mouse ovarian follicles.In-depth study of the culture system demonstrates that the whole in-vitro process mimics normal(in-vivo)physiology from the point of view of morphogenesis, defferentiantion,hormone production.Ovarian follicle function can be analyzed simultaneously on folliculogenesis,hormonogenesis,and oogenesis on variant developmental stages.These systems have been applied succeedly to reproductive biology and physiology studies for the folliculogenesis and follicle regulation.Nevertheless,in-vitro follicle bioassay for reproductive toxicity is are not produced except for individual toxicity parameters.As yet,in-vitro rat preantral follicle culture system were not developed.
On basis of the mouse follicle culture system described according to literatures,in-vitro rat preantral follicle culture systems were developed in this study and had never been reported before.The aim of this study is to develop pre-antral mouse/rat follicle culture in-vitro assay in repriductive toxicology testing to provide information regarding the potential mechanism of action and the identification of chemicals which have female reproductive toxicity.
1.Establishment of in-vitro pre-antral mouse follicle culture system
The ovaries were from prepubertal mice(C57B1/6JxCBA/Ca)(aged 12～14 days).Only follicles with 2-3 layers of granulose cells,a centrally located oocyte and a diameter in the range 100～130μm,which were enclosed by an intact basal membrane and had some attached theca cells,were mechanically dissected and randomly allocated to 96-well culture plates.The preantral follicles were subsequently cultured individually for 12 days within culture medium covered by mineral oil.On day 12 of culture,the follicles were induced ovulation for 16h.The in-vitro developmental characteristics of the follicles were observed including hormonogenesis,folliculogenesis and oogenesis,by comparing in vitro growth and maturation of oocytes(IVG) with in vivo growth and in vitro maturation of oocytes(IVM).
1.1 Developmental characteristics of mouse preantral follicles in vitro
A total of 419 from 7 prepubertal mice,preantral follicles and oocyte with an average diameter of 118.51±10.40μm and 50.75±1.67μm,respectively,376/419(89.74%) of follicles were visibly associated with theca cells and possessed a close follicle(granulosa) cell/oocyte apposition.At day 12 of culture,follicles and oocyte with an average diameter of 118.51±10.40μm and 50.75±1.67μm were measured.31.03%of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity,86.87%was survival and 50.60%COCs were released by maturation induction in this study.
The morphological observation showed that developmental characteristics of mouse preantral follicles in vitro were similar to those in vivo,including the formation of antral follicle and preovulatory follicle,the proliferation and differentiation of granulosa cell, cumulus cell mucification,releasingof cumulus-oocyte complexes(COCs),oocyte maturation and the formation of first polar body.
1.2 Hormone secretion characteristics of in-vitro cultured follicles
Estradiol and progesterone was detected using electrochemiluminescence immunoassay. The results showed that the follicle cultures were characterized by a consistent increase in the secretion of oestradiol as measured in the conditioned medium throughout the in vitro culture period from day 4 onwards.Progesterone secretion increased characteristically only after the in-vitro ovulatory stimulus from day 12 to 13.In a word,the patterns of hormone secretion observed in preantral follicle culture were similar to those characteristic for in-vivo follicle hormone secretion.
1.3 The oocyte maturation in vitro
The numbers of oocytes with germinal vesicle(GV) did not differ between IVG and IVM groups,18.89%oocytes form the first polar body in IVG groups and 53.53%oocytes form the first polar body in IVM groups.The results were not found abnormality chromosome segregation in this study.
In conclusion,judging from the three aspects of folliculogenesis,hormonogenesis,and oogenesis,the establishment of in-vitro pre-antral mouse follicle culture is successful.Our results were in accordance with that of Wang Yubao.However,the PB formation rate was relatively lower than that reported by Fengyun Sun.
2.Establishment of in-vitro pre-antral SD rat follicle culture system
2.1 Determination of in-vitro pre-antral SD rat follicle culture conditions
(1) The size of follicle for culture:Mimicing in-vitro pre-antral mouse follicle culture system,we studied follicle size for culture.The results displayed that 100～130μm,131～150μm and 151～160μm groups of the follicle diameters increase gradually up to344.29±105.40μm,535.76±105.41μm,587.65±138.69μm,respectively,showing statistical significance comparing to pre-culture(p＜0.05).Antral follicles and surviving follicles in 100～130μm groups were declined significantly comparing to 131～150μm and 151～160μm group.
(2) The rat years of age:The ovaries were from prepubertal SD rat aged PND12,13 and 14.The follicles with variant diameter in the range of 100～160μm were mechanically dissected.In PND13 rat,94.87%follicles were 130～150μm in diameters in all dissected follicles,PND12 rat(17.54%) and PND14 rat(about 29.58%).
(3) The time of starting to induce ovulation:Observation of the respective releasing of COCs in d10,d11 and d12 of the cultured ovarian follicle showed that there was not significant difference in three groups.Hence,d10,d11 and d12 of culture could be chose as the time of starting to induce ovulation.
(4) The time of COCs released from follicles:After inducing ovulation for 16h,18h,20h and 22h respectively,the numbers of COCs released from follicles increased in a time-dependent way.After inducing ovulation for 20h,the rate of COCs released from follicles reached 47.84%.Therefore the best observing time of COCs releasing is 20h.
(5) The numbers of ovarian follicle each group:Taking into consideration the fact that the variation of the indexes of rats has are similar to that of mice,16-20 ovarian follicles in each group were chose for examination,according to the report of Val'erie Van Merris.
2.2 Developmental characteristics of early rat preantral follicles in vitro
Of a total of 296 preantral follicles and oocytes with an average diameter of 141.26±8.82μm and 51.25±1.37μm,respectively.96.28%follicles possessed theca cells and a close follicle(granulosa) cell/oocyte apposition.The size of the follicles increased dramatically up to543.21±131.11μm and 70.48±2.89μm at day11of culture.75.67%of the cultured follicles reached the pre-ovulatory stage with a large antrum-like cavity and 92.23%follicles were survival at day 11 of culture.Mucification with expansion of cumulus cells(44.93%COCs)was observed 20h after the in vitro ovulatory stimulus in this study.
The morphological observation showed that developmental characteristics of rat preantral follicles in vitro were similar to those in vivo,including the formation of antral follicle and preovulatory follicle,the proliferation and differentiation of granulosa cell, cumulus cell mucification,releasingof cumulus-oocyte cell complexes(COCs),oocyte maturation and the formation of first polar body.
2.3 Hormone secretion characteristics of in vitro cultured follicles
Estradiol and progesterone was measured using electrochemiluminescence immunoassay.The results showed the follicle cultures were characterized by a consistent increase in the secretion of oestradiol as measured in the conditioned medium throughout the in vitro culture period from day 4 onwards.Progesterone secretion increased characteristically only after the in vitro ovulatory stimulus from day 11 to day12.
2.4 Oocyte maturation in vitro
7.44%(9/121)oocytes was blocked germinal vesicle(GV) stage,57.02%(69/121) oocytes initiated resumption of maturation(GVBD),35.53%(43/121) oocytes formed a first polar bodyin IVG groups,1.86%(6/322)oocytes blocked germinal vesicle(GV) stages,40.06%(129/322)oocytes initiated resumption of maturation(GVBD),56.21% (181/322) oocytes formed a first polar body in IVM groups.The results were not found abnormality chromosome segregation in this study.
3.Application of in-vitro SD rat follicle culture system for femal reproductive toxicity testing
The PB formation rate was lower than that reported by Fengyun Sun.On the other hand, rats are the most common experimental animals in reproductive toxicity testing.Therefore, in-vitro rat follicle culture system was applied for femal reproductive toxicity testing in this study.
Some chemicals including cadmium chloride(CdCL_2),DEHP,MEHP and Nocodazole were assessed for hormonogenesis,folliculogenesis and oogenesis using the in-vitro SD rat follicle screen system.
3.1 Follicle toxicity of cadmium chloride(CdCL_2) in vitro
Cadmium chloride is a common industrial poison and environment contaminant.As some animal experiments show,cadmium chloride can interrupt the function of ovaries, extend the oestrous cycle of rats,cause histological and structural alteration,inhibit the growth of ovary follicle,and increase the number of atretic follicle.Moreover,it impairs the ovulation and causes infertility.Some in-vitro bioassays show that the progesterone production was significantly decreased by cadmium chloride.
3.1.1 Effects of CdCL_2 on follicle development in vitro
CdCL_2 with concentrations＞1.2μg/ml had been reduced significantlysurvival follicles rate(66.44±3.95%)compared to the control group(91.57±1.32%) on day11 (p＜0.05),wherea33.57±3.95%degenerated or underwent premature oocyte release This dose dependent decrease showed a coefficient of correlation(R~2) of 0.90.CdCL_2 with the concentrations of 1.6μg/ml impaired follicle growth and differentiation,significantly reduced(p＜0.05)the rate of antral follicles(42.51±6.66%)compared to the control(79.68±6.62%).
CdCL_2 did not influence the17β-E_2 secretion in the concentration range of 0.8～1.6μg /ml CdCL_2.The basal progesterone production in the treated groups was significantly decreased for＞0.8ug/ml CdCL_2 compared to the control after hCG-administration on days 11 and 12.
3.1.2 Effects of CdCL_2 on different developmental stage follicle in vitro
To determine sensitive follicle stage of CdCL_2 exposure,the culture follicles exposed to CdCL_2 on day2(pre-antral follicle),day 6(antral follicle),day11(pre-avulation follicle) for 48h,respecfively.CdCL_2 with concentrations 1.6ug/ml on day 2 had a significantly reduced(p＜0.05) survival rate(52.51±7.84),the rate of antral follicles(42.51±6.66%),compared to follicles exposed to same concentrations CdCL_2 on day 6(81.75±8.13%), (74.24±6.98%),respectively.Anormal follicle rate(47.49±7.48%) increase significantly on day 2,compared to day6(47.49±7.48%).The numbers of GV oocyte(58.34±11.79%) increase significantly on day2,compared to day6(15.66±9.28%),day 11(20.2±2.86%).There are no significant adverse effects of CdCL_2 exposure on day6 upon follicle development and oocyte maturation.In IVM group,COCs treated with concentrations 1.6ug/ml CdCL_2 reduced(p＜0.05) PB-formation rate(34.11±0.34%),compared to control (67.98±6.57%).These results demondated that CdCL_2 would inhibit both follicle development and oocyte maturation from pre-antral follicle and oocyte maturation and PB-formation from pre-ovulation follicle.
3.2 Effects of DEHP and MEHP on follicle development in vitro
Ai-2-ethylhexyl phttmlate(DEHP) and mono-2-ethylhexyl phthalate(MEHP) belongs to phthalicacidesters(PAEs).Animal experiments showed that the toxicity on reproduction of DEHP result from its metabolite MEHP,which affected granulose cells of ovaries and remarkably inhibited the production of estrogen during preovulatory stage.Some in-vitro bioassays show that MEHP decreased the production of progesterone as well as the FSH-induced cAMP production.
Follicles treated with concentrations＞10μg/mLMEHP had a significantly reduced(p＜0.05) survival rate(54.76±3.37%) compared to the control group(91.57±1.32%)on day11. wherea 45.24±3.37%follicles degenerated or underwent premature oocyte release.This dose dependent decrease showed a coefficient of correlation(R~2) of 0.92. Concentrations＞20ug/mL MEHP dose dependently impaired follicle growth and differentiation,antral follicles(46.18±1.67%) had a significantly reduced(p＜0.05) compared to the control group(85.91±5.03%).DEHP showed no adverse effects upon follicle development and there were no significant defference between DMSO and control.
3.3 Effects of Nocodazole on oocyte maturation in vitro
Nocodazole,methyl[5-(2-thienylcarbonyl)-1H-benzimidazol-2-yl]carbamate,a synthetic anti-tumour drug interfering with microtubule assembly,competes with colchicine for the same binding site on the Arg-390 of b-tubulin and directly affects microtubule polymerization and microtubuledependent processes.
When COCs exposed to Nocodazole concentrations of 100nM for 20h in IVM,there existing oocyte cytotoxicity.The COCs exposed to Nocodazole concentrations of＞5nM, We observed signicant reduced in PB-formation rate compared to its control group. Although the preovulation follicles exposed to Nocodazole concentrations of 10～40nM showed no statistical difference in COCs rate isolated from follicle compared to the controls,there existed a trend.Concentrations of 40nM Nocodazole dose significantly reduced(p＜0.05)PB-formation rate(19.35%) compared to the control group(37.78%)
Summary
1.This study developed in-vitro mouse pre-antral follicle culture system.
2.In-vitro rat preantral follicle culture system were developed and this system had never been reported before
3.Rat preantral follicle culture as a novel in-vitro assay was applied to detect the effects of four chemicals on folliculogenesis,steroidogenesis and oogenesis,and on variant developmental stages,which further revealed that the bioassay is a efficient tool to study ovarian toxicity and their mechanisms of chemical.
4.A preliminary protocol of in-vitro rat preantral follicle culture system for the study on female reproductive toxicity was proposed,including the morphological observation, stages of follicle differentiation,hormone secretion profiles,oocyte diameters,the formatiom of PB and the genetoxic of oocyte.
引文
[1]顾祖维主编.现代毒理学概论.北京:化学工业出版社,2005;254
[2]Kunmar AS,Ghadir N.Eskandri.and Decherney AH.DeCherney,Infertility.In Current Diagnosis & Treatmen tObstetrics & Gynecology,edited by AH.DeCherney and L.Nathan,Mcgraw-Hill,New York.2007;pp.917-925
[3]Huggins J.Alternatives to animal testing:research,trends,validation,regulatory acceptance.ALTEX.2003;20(Suppl 1):3-61.
[4]Schechtman LM,Stokes WS.ECVAM-ICCVAM:prospects for future collaboration.Altern Lab Anita.2002;30(Suppl 2):227-36.
[5]张天宝.毒理学的挑战、机遇和发展趋势.卫生毒理学杂志.2003;17(1):2-5.
[6]张天宝.大鼠卵巢细胞体外培养及生殖毒理研究中的应用.卫生毒理学杂志.1997;11(2):128.
[7]汪玉宝.卵母细胞体外培养成熟技术在人类生殖中的应用.生殖与避孕.2003;23(3):184-188.
[8]Sun F,Betzendahl I,Pacchierotti F,et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitroin preantral follicle culture to nocodazole.Mutagenesis.2005;20(1):65-75.
[9]Sun F,Betzendahl I,Shen Y,et al..Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.Mutagenesis.2004;19(1):13-25.
[10]Thomas FH,Waiters KA,Telfer EE,et al.How to make a good oocyte:an update on in-vitro models to study follicle regulation,Hum Reprod Update.2003;9(6):541-555.
[11]Loret de Mola JR,Bamhart K,Kopf GS,et al.Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles.Clin Exp Obstet Gyneeoi.2004;31(1):15-19.
[12]Cortvrindt RG,.Smitz JEJ.Follicle culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod Update 2002;8(3):243-254.
[13]Capacchietti G,Cecconi S,Colantoni E,et al.In vitro growth of sheep preantral follicles:which perspectives? Vet Res Commum.2003;27(Suppl 1):583-6.
[14] Cecconi S, Capacchietti G, Russo V, et al. In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue. Biol Reprod. 2004;70(1):12-17.
[15] Park JY, Su YQ, Ariga M,, et al. EGF-Like Growth Factors As Mediators of LH Action in the Ovulatory Follicle. Science. 2004; 303 (5658): 682-684.
[16] Spears N, Baker S, Srsen V,et al. Mouse Ovarian Follicles Secrete Factors Affecting the Growth and Development of Like-Sized Ovarian Follicles In Vitro.Biol Reprod.2002; 67(6):1726-1733.
[17] 王红玲,刘铁铮,禚艳书.小鼠腔前卵泡体外培养研究进展. Prog Veterin Med.2005; 26(6): 28 —31.
[18] Alm H, Katska-Ksiazkiewicz L, Ryn'ska B, Tuchscherer A.Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology 2006; 65(7): 1422-1434.
[19] Cecconi S, Rossi G, Bianchi S, Stallone T, Macchiarelli G, Nottola S.In vitro culture of mammalian early ovarian follicles. Morphofunctional correlates and future perspectives. Ital J Anat Embryol.2005;110(2 Suppl 1):189-194.
[20] Van Wemmel K, Gobbers E, Eichenlaub-Ritter U, et al. Ovarian follicle bioassay reveals adverse effects of diazepam exposure upon follicle development and oocyte quality . Reprod Toxicol . 2005; 20(2): 183-193
[21] Merris VV, Wemmel KV, Cortvrindt R.In vitro effects of dexamethasone on mouse ovarian function and pre-implantation embryo development. Reprod Toxicol. 2007 ;23(1):32-41.
[22] Jacquet P, Adriaens I, Buset J, Neefs M, et al.Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system . Mutat Res. 2005 ;583(2):168-177
[23] Rossi G , Palmerini MG , Macchiarelli G .Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.Reprod Toxicol .2006 ;22(1):51-55.
[24] Hartshotne G M. In Vitro Culture of Ovarian Follicles. Rev Reprod. 1997, 2: 94-104.
[25] Demeestere I, Delbaere A, Gervy C,et al. Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice. Hum Reprod.2002; 17(8): 2152-2159.
[26]Roy SK,Greenwald GS.Meththods of separation and in vitro culture of pre-antral follicles from mammalian ovaries.Hum Reprod Update.1996;2(3):236-245
[27]Cortvrindt R,Smitz J,Van Steirteghem AC.In-vitro maturation,fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system.Hum Reprod.1996;11(12):2656-2666.
[28]Nayudu PL,Osborn SM.Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro.J Reprod Fertil.1992;95(2):349-362.
[29]汪玉宝,高敏芝,顾敦瑜等.小鼠腔前卵泡的体外培养.复旦学报(医学版).2004;31(1):42-45
[30]杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社.2005;73-115.
[31]Bishonga C,Takahashiy,katagiri S,et al.In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to denelop to the blastoeyst stage.J Vet Meal Set.2001;63(6):619-624.
[32]膨宇红,谢守珍,程翼平等.小鼠腔前卵泡体外培养的研究.华南国防医学杂志.2006;20(2):22-25.
[33]Li MG,Ieuan M,Joyce.RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199
[34]Adriaens I,Cortvdndt R,Smitz J.Differential FSH exposure in preantral follicle culture has marked effects on folliculogenesis and oocyte developmental competence.Hum Reprod.2004;19(2):398-408,
[35]李蝗元,吴思莫,张文昌.镉的雌性性腺生殖毒性研究现状.中国公共卫生.2002;18(3):379-381.
[36]吴思英,田俊审.镉的生殖毒性流行病学研究进展.现代预防学.2002:29(3):396-397.
[37]夏品苍,张文昌,汪家梨等.镉的雌性性腺毒性与子宫、卵巢雌激素受体表达.海峡预防医学杂志.2005;11(6):1-2.
[38]Henson MC,Chedrese PJ.Endocrine disruption by cadmium,a common environmental toxicant with paradoxical effects on reproduction.Exp Biol Med(Maywood).2004;229(5):383-392.
[39]贾海梅,张文昌,陈昱等.镉对大鼠卵巢颗粒细胞孕激素合成的影响研究.中国预防医学杂 志.2007:8(4):345-347.
[40]Zhang W,Jia H.Effect and mechanism of cadmium on the progesterone synthesis of ovaries.Toxicology.2007;239(3):204-12.
[41]Smida AD,Valderrama XP,Agostini MC,et al.Cadmium stimulates transcription of the cytochrome p450 side chain cleavage gene in genetically modified stable porcine granulosa cells.Biol Reprod.2004;70(1):25-31.
[42]Leoni G,Bogliolo L,Deiana G,et al.Influence of cadmium exposure on in vitro ovine gamete dysfunction.Reprod Toxicol.2002;16(4):371-377.
[43]Lienesch LA,Dumont JN,Bantle JA.The effect of cadmium on oogenesis in Xenopus laevis.Chemosphere.2000;41(10):1651-1658.
[44]Nampoothiri LP,Agarwal A,Gupta S.Effect of co-exposure to lead and cadmium on antioxidant status in rat ovarian granulose cells.Arch Toxicol.2007;81(3):145-50.
[45]Eppig JJ.Oocyte control of ovarian follicular development and function in mammals.Reproduction.2001;122:829-38.
[46]刁书永,袁慧.镉对小鼠卵巢颗粒细胞凋亡研究.畜牧兽医学报.2005;36(11):1155-1158.
[47]Li MG,Joyce IM..RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199.
[48]赵海波,何亚绒,李爱莉等.卵泡液干细胞因子水平对卵母细胞发育、受精和卵裂影响的实验研究.中国计划生育学杂志.2006;130(8):475-476.
[49]Gallardo TD,John GB,Shirley L,Contreras CM,et al.Genomewide discovery and classification of candidate ovarian fertility genes in the mouse.Genetics.2007;177(1):179-194
[50]Sun F,Betzendahl I,Shen Y,et al..Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.Mutagenesis.2004;19(1):13-25.
[51]Cortvrindt RG.and J.E.J.Smitz.Follicle culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod update.2002;8(3):243-254.
[52]Hodges CA,Ilagan A,Jennings D,et al.Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation,Hum Reprod.2002;17(5):1171-1180.
[53]Leoni G,Bogliolo L,Deiana G,et al.Influence of cadmium exposure on in vitro ovine gamete dysfunction.Reprod Toxicol.2002;16(4):371-377.
[54]Barr DB,Silva MJ,Kato K,et al.Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers.Environ Health Perspect.2003;111(9):1148-1151
[55]Latini G,Verrotti A,De Felice C.et al.DI.2.ethylhexyl phthalate and endocrine disruption:a review.Curr Drug Targets Immune Endocr Metabol Disord.2004;4(1):37-40.
[56]Davis BJ,Maronpot RR,Heindel JJ.Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats.Toxicol Appl Pharmacol.1994;128(2):216-223.
[57]Laskey JW,Berman E.Steroidogenic assessment using ovary culture in cycling rats:effects of bis(2-diethylhexyl)phthalate on ovarian steroid production.Reprod Toxicol.1993;7(1):25-33.
[58]Treinen KA,Dodson WC,Heindel JJ.Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulosa cell cultures.Toxicol Appl Pharmacol.1990;106(2):334-40.
[59]Lovekamp TN,Davis BJ.Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells.Toxicol Appl Pharmacol.2001;172(3):217-24.
[60]Shen Y,Betzendahl I,Sun F,et al.Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes.Reprod Toxicol.2005;19(4):459-471.
[61]Sun F,Betzendahl I,Pacchierotti F,et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole.Mutagenesis.2005;20(1):65-75.
[62]Li MG,Ieuan M,Joyce.RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199.
[63]Bishonga C,Takahashi Y,Katagiri S,et al.In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage.J Vet Med Sct.2001;63(6):619-624.
[64]张晓晖,黄高焺.有丝分裂阻滞剂Nocodazole诱导HL-60细胞的凋亡.第四军医大学学报.2001;22(20):1855-1858.
[1]顾祖维主编.现代毒理学概论.北京:化学工业出版社.2005;254
[2]Kunmar AS,Ghadir N.Eskandri.and Decherney AH.DeCherney,Infertility.2007;pp.917-925.in Current Diagnosis&TreatmentObstetrics&Gynecology,edited by AH.DeCherney and L.Nathan,Mcgraw-Hill,New York..
[3] Eichenlaub-Ritter,U. Ageing and aneuploidy in oocytes. Ernst Schering Res. Found. Workshop, 2002, (41), 111-136.
[4] Eichenlaub-Ritter,U. Aneuploidy in ageing oocytes and after toxic insult. In Trounson,A.O. and Gosden,R.G. (eds), Biology and Pathology of the Oocyte. Cambridge University Press, Cambridge, (2003). 220-257.
[5] Hassold,T. and Hunt,P. To err (meiotically) is human: the genesis of human aneuploidy. Nature Rev. Genet. 2001; 2 (4) :280-291.
[6] Demeestere I, Centner J, Gervy C, et al.Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction. 2005;130(2):147-56.
[7] Hutt KJ, Albertini DF.An oocentric view of folliculogenesis and embryogenesis. Reprod Biomed Online.2007;14(6):758-64.
[8] 杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社.2005, 73-115.
[9] Hutt KJ, Albertini DF.An oocentric view of folliculogenesis and embryogenesis. Reprod Biomed Online. 2007; 14(6):758-64.
[10] Sun F, Betzendahl I, Pacchierotti F, et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitroin preantral follicle culture to nocodazole. Mutagenesis. 2005;20(1): 65-75.
[11] Sun F, Betzendahl I, Shen Y,et al.. Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes. Mutagenesis. 2004; 19(1): 13-25.
[12] Thomas FH, Walters KA, Telfer EE, et al. How to make a good oocyte: an update on in-vitro models to study follicle regulation. Hum Reprod Update. 2003;9(6):541-555.
[13] Loret de Mola JR, Barnhart K, Kopf GS, et al.Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles. Clin Exp Obstet Gynecol. 2004;31(1):15-19.
[14] Cortvrindt RG,.Smitz JEJ.Follicle culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod Update .2002;8(3):243-254.
[15] Comment in: Aardema MJ, Albertini S, Ami P, et al.Aneuploidy: a report of an ECETOC task force. Mutat Res. 1998;410(1): 117-20.
[16] Jacquet P, Adriaens I, Buset J,et al. Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system. Mutat Res. 2005;583(2):168-77.
[17]Rossi G,Palmerini MG,Macchiarelli G.Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.Reprod Toxicol.2006;22(1):51-55.
[18]Bremer S,Pellizzer C,Hoffmann S,et al.The development of new concepts for assessing reproductive toxicity applicable to large scale toxicological programmes.Curt Pharm Des.2007;13(29):3047-3058.
[19]Combes R,Grindon C,Cronin MT,Roberts DW,Garrod J.Proposed integrated decision-tree testing strategies for mutagenicity and carcinogenicity in relation to the EU REACH legislation.Aitern Lab Anim.2007;35(2):267-287.
[20]张天宝.大鼠卵巢细胞体外培养及生殖毒理研究中的应用.卫生毒理学杂志.1997;11(2):128
[21]Koes.RD,Jaccaridd FJ.Magaril RA,et al.Perfusion ofihe rat ovary in vitro:methology,induction of ovulation and pattern ofstermdogenesis.Bio Reprod,1984.30(5):1135
[22]Todoroff EC,Sevcik M,Villeneuve DC,et al.The effect of photomirex on the in vitro perfused ovary of the rat.Reprod Toxicol.1998;12(3):305-16.
[23]Kemp B,Winkler M,Hauptmann S,Rath W.Cervical dilatation:induction by antigestagens via adhesion molecules.An in vitro examination in endothelial cell cultures.Gyneeol Obstet Invest.1998;45(2):116-20.
[24]Wilmut I,A E Schnieke,J Mcwhir,et al.Viable ofspring deriver form fetal and adult mammalian cell.Nature.1997;385(6619):810-813.
[25]Barash I,Faeman A,Richensteiu M,et al.In vivo and in vitro expression of human serum albumin genemic sequence～in mammary epithelial cells with β—lactoblobumin and whey acidic protein promoters.Mol Reprod Dev.1999;52(3):241-252.
[26]Vanessa Lollivierl,Pierre-Guy Marnetl,Serge Delpal,et al.Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells.J Physiol.2006;570(1):125-140.
[27]孙丽翠,刘朋删,杨国庆等.小鼠和兔原代乳腺上皮细胞的培养.生物技术.2004;14(2):33-34.
[28]Fleming JM,Leibowitz BJ,Kerrl DE et al.IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells.Journal of Endocrinology.2005;186(5):165-178.
[29]Freshney RI,Pragnell IB,Fr eshney MG.Culture of epithelial cells.Second edition.New York:Wiley-Liss,2002.3.
[30]叶媚娜,陈红风.温肾中药对原代培养正常人乳腺上皮细胞增殖的影响中西医结合学报.2006;4(4):397-401.
[31] Spielmann H.Reproduction and development. Environ Health Perspect. 1998 ;106 (Suppl 2) :571-576.
[32] Huggins J. Alternatives to animal testing: research, trends, validation, regulatory acceptance. ALTEX. 2003;20(Suppl 1):3-61.
[33] Schechtman LM, Stokes WS. ECVAM-ICCVAM: prospects for future collaboration.Altern Lab Anim. 2002;30 (Suppl 2) :227-236.
[1]Hutt KJ,Albertini DF.An oocentric view of folliculogenesis and embryogenesis.Reprod Biomed Online.2007;14(6):758-64.
[2]Li MG,Ieuan M,Joyce.RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199.
[3]Demeestere I,Centner J,Gervy C,Englert Y,Delbaere A.Impact of 4.various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents.Reproduction.2005;130(2):147-156.
[4]Oocyte-secreted factors:regulators of cumulus cell function and oocyte quality.Hum Reprod Update.2008;14(2):159-177.
[5]杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社.2005;73-115
[6]Capacchietti G,Cecconi S,Colantoni E,et al.In vitro growth of sheep preantral follicles:which perspectives? Vet Res Commun.2003;27(Suppl 1):583-586.
[7]Cecconi S,Capacchietti G,Russo V,et al.In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue.Biol Reprod.2004;70(1):12-17.
[8]Park JY,Su YQ,Ariga M,,et al.EGF-Like Growth Factors As Mediators of LH Action in the Ovulatory Follicle.Science.2004;303(5658):682-684.
[9]Spears N,Baker S,Srsen V,et al.Mouse Ovarian Follicles Secrete Factors Affecting the Growth and Development of Like-Sized Ovarian Follicles In Vitro.Bioi Reprod.2002;67(6):1726-1733.
[10]王红玲,刘铁铮,禚艳书.小鼠腔前卵泡体外培养研究进展.Progrvet Med.2005;26(6):28-31.
[11]Alm H,Katska-Ksiazkiewicz L,Rynska B,et al.Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology. 2006 15;65(7): 1422-1434.
[12] Cecconi S, Rossi G, Bianchi S, Stallone T, Macchiarelli G, Nottola S.In vitro culture of mammalian early ovarian follicles. Morphofunctional correlates and future perspectives. Ital J Anat Embryol.2005; 110(2 Suppl 1):189-194.
[13] Telfer EE. In vitro models for oocyte development. Theriogenology. 1998; 15;49(2):451-460.
[14] Nayudu PL, Fehrenbach A, Kiesel P,et al.Progress toward understanding follicle development in vitro: appearances are not deceiving. Arch Med Res. 2001 ;32(6):587-594.
[15] Devine PJ, Rajapaksa KS, Hoyer PB.In vitro ovarian tissue and organ culture: a review.Front Biosci. 2002;7:d1979-89.
[16] Dole G, Nilsson E, Skinner M.Glial derived neurotrophic factor promotes ovarian primordial follicle development and cell-cell interactions during folliculogenesis. Reproduction. 2008 Feb 27 [Epub ahead of print]
[17] Fortune JE . Kito S. Warldji S-A Srsen V.Activation of bovine and baboon primordial follicles ln vitro.Theriogenology I998;49 (2): 441-449
[18] Hovatta O, Wright C, Krausz T,et al.Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum Reprod. 1999; 14(10):2519-2524.
[19] Grob HS. Enzymatic dissection of the mammalian ovary. Science. 1964 Oct 2;146:73-4.
[20] Nicosia SV, Evangelista I, Batta SK.Rabbit ovarian follicles. I. Isolation technique and characterization at different stages of development. Biol Reprod. 1975 ;13(4):423-47.
[21] Figueiredo J R et al. Development of a combined new mechanical and enzymatticmethod for the isolation of intact preantral follicle from fetal calf and adult bovine ovaries. Theriogenology .1993, (40): 789-799.
[22] Demeestere I,Delbaere A,Gervy C,et al .Effect of preantral follicle isolation technique on in vitro follicular growth,oocyte maturation and embryo development in mice.Hum Reprod.2001;7(8):2152
[23] Hartshotne G M. In Vitro Culture of Ovarian Follicles. Rev Reprod. 1997, 2 (2): 94—104.
[24] Erin R. West, Min Xu, Teresa K,Woodruff,et al.Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials. 2007 ; 28(30): 4439-4448.
[25] Cortvrindt R, Smitz J, Van Steirteghem AC. In vitro maturation. fertilization and embryo development of immature oocytes from early preantral folheles from prepuberal mice in asimpliedculture system. Hum Reprod. 1996;11(12):2656-66.
[26] Nayudu PL, Osborn SM.Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro. J Reprod Fertil. 1992;95(2):349-62.
[27] Jhohan E,Smitz J,Cortvrindt RG.The earliest stages of follieuligenesis in vitro.Rproduction.2002; 123(2): 185—202.
[28] Cortvrindt RG,.Smitz JEJ.FollicIe culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod Update 2002;8(3):243-254.
[29] Sun F, Betzendahl I, Shen Y,et al. Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.Mutagenesis. 2004;19(1): 13-25.
[30] Dela Pe(n|~)a EC, Takahashi Y, Katagiri S,et al.Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles.Reproduction. 2002;123(4):593-600.
[31] Roberts R, Stark J, Iatropoulou A,et al.Energy substrate metabolism of mouse cumulus-oocyte complexes: response to follicle-stimulating hormone is mediated by the phosphatidylinositol 3-kinase pathway and is associated with oocyte maturation. Biol Reprod. 2004;71(1): 199-209.
[32 Ptak A, Ludewig G, Robertson L, et al.In vitro exposure of porcine prepubertal follicles to 4-chlorobiphenyl (PCB3) and its hydroxylated metabolites: effects on sex hormone levels and aromatase activity. Toxicol Lett. 2006; 164(2): 113-122.
[33] Sun F, Betzendahl I, Pacchierotti F,et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole. Mutagenesis . 2005;20 (1):65—75.
[34] Shen Y, Betzendahl I, Sun F, et al. Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes.Reprod Toxicol. 2005;19(4):459-71.
[35] Rossi G, Palmerini MG, Macchiarelli G, et al.Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes. Reprod Toxicol. 2006;22(1):51 -55.
[36] Nayudu PL, Fehrenbach A, Kiesel P,et al.Progress toward understanding follicle development in vitro:appearances are not deceiving.Arch Med Res. 2001 ;32(6):587-94.
[37] Van Wemmel K, Gobbers E, Eichenlaub-Ritter U,et al. Ovarian follicle bioassay reveals adverse effects of diazepam exposure upon follicle development and oocyte quality. Reprod Toxicol. 2005;20(2):183-193.
[38] Van Merris V, Van Wemmel K, Cortvrindt R.In vitro effects of dexamethasone on mouse ovarian function and pre-implantation embryo development. Reprod Toxicol. 2007 ;23(1):32-41.
[2]Kunmar AS,Ghadir N.Eskandri.and Decherney AH.DeCherney,Infertility.In Current Diagnosis & Treatmen tObstetrics & Gynecology,edited by AH.DeCherney and L.Nathan,Mcgraw-Hill,New York.2007;pp.917-925
[3]Huggins J.Alternatives to animal testing:research,trends,validation,regulatory acceptance.ALTEX.2003;20(Suppl 1):3-61.
[4]Schechtman LM,Stokes WS.ECVAM-ICCVAM:prospects for future collaboration.Altern Lab Anita.2002;30(Suppl 2):227-36.
[5]张天宝.毒理学的挑战、机遇和发展趋势.卫生毒理学杂志.2003;17(1):2-5.
[6]张天宝.大鼠卵巢细胞体外培养及生殖毒理研究中的应用.卫生毒理学杂志.1997;11(2):128.
[7]汪玉宝.卵母细胞体外培养成熟技术在人类生殖中的应用.生殖与避孕.2003;23(3):184-188.
[8]Sun F,Betzendahl I,Pacchierotti F,et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitroin preantral follicle culture to nocodazole.Mutagenesis.2005;20(1):65-75.
[9]Sun F,Betzendahl I,Shen Y,et al..Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.Mutagenesis.2004;19(1):13-25.
[10]Thomas FH,Waiters KA,Telfer EE,et al.How to make a good oocyte:an update on in-vitro models to study follicle regulation,Hum Reprod Update.2003;9(6):541-555.
[11]Loret de Mola JR,Bamhart K,Kopf GS,et al.Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles.Clin Exp Obstet Gyneeoi.2004;31(1):15-19.
[12]Cortvrindt RG,.Smitz JEJ.Follicle culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod Update 2002;8(3):243-254.
[13]Capacchietti G,Cecconi S,Colantoni E,et al.In vitro growth of sheep preantral follicles:which perspectives? Vet Res Commum.2003;27(Suppl 1):583-6.
[14] Cecconi S, Capacchietti G, Russo V, et al. In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue. Biol Reprod. 2004;70(1):12-17.
[15] Park JY, Su YQ, Ariga M,, et al. EGF-Like Growth Factors As Mediators of LH Action in the Ovulatory Follicle. Science. 2004; 303 (5658): 682-684.
[16] Spears N, Baker S, Srsen V,et al. Mouse Ovarian Follicles Secrete Factors Affecting the Growth and Development of Like-Sized Ovarian Follicles In Vitro.Biol Reprod.2002; 67(6):1726-1733.
[17] 王红玲,刘铁铮,禚艳书.小鼠腔前卵泡体外培养研究进展. Prog Veterin Med.2005; 26(6): 28 —31.
[18] Alm H, Katska-Ksiazkiewicz L, Ryn'ska B, Tuchscherer A.Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology 2006; 65(7): 1422-1434.
[19] Cecconi S, Rossi G, Bianchi S, Stallone T, Macchiarelli G, Nottola S.In vitro culture of mammalian early ovarian follicles. Morphofunctional correlates and future perspectives. Ital J Anat Embryol.2005;110(2 Suppl 1):189-194.
[20] Van Wemmel K, Gobbers E, Eichenlaub-Ritter U, et al. Ovarian follicle bioassay reveals adverse effects of diazepam exposure upon follicle development and oocyte quality . Reprod Toxicol . 2005; 20(2): 183-193
[21] Merris VV, Wemmel KV, Cortvrindt R.In vitro effects of dexamethasone on mouse ovarian function and pre-implantation embryo development. Reprod Toxicol. 2007 ;23(1):32-41.
[22] Jacquet P, Adriaens I, Buset J, Neefs M, et al.Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system . Mutat Res. 2005 ;583(2):168-177
[23] Rossi G , Palmerini MG , Macchiarelli G .Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.Reprod Toxicol .2006 ;22(1):51-55.
[24] Hartshotne G M. In Vitro Culture of Ovarian Follicles. Rev Reprod. 1997, 2: 94-104.
[25] Demeestere I, Delbaere A, Gervy C,et al. Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice. Hum Reprod.2002; 17(8): 2152-2159.
[26]Roy SK,Greenwald GS.Meththods of separation and in vitro culture of pre-antral follicles from mammalian ovaries.Hum Reprod Update.1996;2(3):236-245
[27]Cortvrindt R,Smitz J,Van Steirteghem AC.In-vitro maturation,fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system.Hum Reprod.1996;11(12):2656-2666.
[28]Nayudu PL,Osborn SM.Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro.J Reprod Fertil.1992;95(2):349-362.
[29]汪玉宝,高敏芝,顾敦瑜等.小鼠腔前卵泡的体外培养.复旦学报(医学版).2004;31(1):42-45
[30]杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社.2005;73-115.
[31]Bishonga C,Takahashiy,katagiri S,et al.In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to denelop to the blastoeyst stage.J Vet Meal Set.2001;63(6):619-624.
[32]膨宇红,谢守珍,程翼平等.小鼠腔前卵泡体外培养的研究.华南国防医学杂志.2006;20(2):22-25.
[33]Li MG,Ieuan M,Joyce.RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199
[34]Adriaens I,Cortvdndt R,Smitz J.Differential FSH exposure in preantral follicle culture has marked effects on folliculogenesis and oocyte developmental competence.Hum Reprod.2004;19(2):398-408,
[35]李蝗元,吴思莫,张文昌.镉的雌性性腺生殖毒性研究现状.中国公共卫生.2002;18(3):379-381.
[36]吴思英,田俊审.镉的生殖毒性流行病学研究进展.现代预防学.2002:29(3):396-397.
[37]夏品苍,张文昌,汪家梨等.镉的雌性性腺毒性与子宫、卵巢雌激素受体表达.海峡预防医学杂志.2005;11(6):1-2.
[38]Henson MC,Chedrese PJ.Endocrine disruption by cadmium,a common environmental toxicant with paradoxical effects on reproduction.Exp Biol Med(Maywood).2004;229(5):383-392.
[39]贾海梅,张文昌,陈昱等.镉对大鼠卵巢颗粒细胞孕激素合成的影响研究.中国预防医学杂 志.2007:8(4):345-347.
[40]Zhang W,Jia H.Effect and mechanism of cadmium on the progesterone synthesis of ovaries.Toxicology.2007;239(3):204-12.
[41]Smida AD,Valderrama XP,Agostini MC,et al.Cadmium stimulates transcription of the cytochrome p450 side chain cleavage gene in genetically modified stable porcine granulosa cells.Biol Reprod.2004;70(1):25-31.
[42]Leoni G,Bogliolo L,Deiana G,et al.Influence of cadmium exposure on in vitro ovine gamete dysfunction.Reprod Toxicol.2002;16(4):371-377.
[43]Lienesch LA,Dumont JN,Bantle JA.The effect of cadmium on oogenesis in Xenopus laevis.Chemosphere.2000;41(10):1651-1658.
[44]Nampoothiri LP,Agarwal A,Gupta S.Effect of co-exposure to lead and cadmium on antioxidant status in rat ovarian granulose cells.Arch Toxicol.2007;81(3):145-50.
[45]Eppig JJ.Oocyte control of ovarian follicular development and function in mammals.Reproduction.2001;122:829-38.
[46]刁书永,袁慧.镉对小鼠卵巢颗粒细胞凋亡研究.畜牧兽医学报.2005;36(11):1155-1158.
[47]Li MG,Joyce IM..RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199.
[48]赵海波,何亚绒,李爱莉等.卵泡液干细胞因子水平对卵母细胞发育、受精和卵裂影响的实验研究.中国计划生育学杂志.2006;130(8):475-476.
[49]Gallardo TD,John GB,Shirley L,Contreras CM,et al.Genomewide discovery and classification of candidate ovarian fertility genes in the mouse.Genetics.2007;177(1):179-194
[50]Sun F,Betzendahl I,Shen Y,et al..Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.Mutagenesis.2004;19(1):13-25.
[51]Cortvrindt RG.and J.E.J.Smitz.Follicle culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod update.2002;8(3):243-254.
[52]Hodges CA,Ilagan A,Jennings D,et al.Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation,Hum Reprod.2002;17(5):1171-1180.
[53]Leoni G,Bogliolo L,Deiana G,et al.Influence of cadmium exposure on in vitro ovine gamete dysfunction.Reprod Toxicol.2002;16(4):371-377.
[54]Barr DB,Silva MJ,Kato K,et al.Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers.Environ Health Perspect.2003;111(9):1148-1151
[55]Latini G,Verrotti A,De Felice C.et al.DI.2.ethylhexyl phthalate and endocrine disruption:a review.Curr Drug Targets Immune Endocr Metabol Disord.2004;4(1):37-40.
[56]Davis BJ,Maronpot RR,Heindel JJ.Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats.Toxicol Appl Pharmacol.1994;128(2):216-223.
[57]Laskey JW,Berman E.Steroidogenic assessment using ovary culture in cycling rats:effects of bis(2-diethylhexyl)phthalate on ovarian steroid production.Reprod Toxicol.1993;7(1):25-33.
[58]Treinen KA,Dodson WC,Heindel JJ.Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulosa cell cultures.Toxicol Appl Pharmacol.1990;106(2):334-40.
[59]Lovekamp TN,Davis BJ.Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells.Toxicol Appl Pharmacol.2001;172(3):217-24.
[60]Shen Y,Betzendahl I,Sun F,et al.Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes.Reprod Toxicol.2005;19(4):459-471.
[61]Sun F,Betzendahl I,Pacchierotti F,et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole.Mutagenesis.2005;20(1):65-75.
[62]Li MG,Ieuan M,Joyce.RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199.
[63]Bishonga C,Takahashi Y,Katagiri S,et al.In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage.J Vet Med Sct.2001;63(6):619-624.
[64]张晓晖,黄高焺.有丝分裂阻滞剂Nocodazole诱导HL-60细胞的凋亡.第四军医大学学报.2001;22(20):1855-1858.
[1]顾祖维主编.现代毒理学概论.北京:化学工业出版社.2005;254
[2]Kunmar AS,Ghadir N.Eskandri.and Decherney AH.DeCherney,Infertility.2007;pp.917-925.in Current Diagnosis&TreatmentObstetrics&Gynecology,edited by AH.DeCherney and L.Nathan,Mcgraw-Hill,New York..
[3] Eichenlaub-Ritter,U. Ageing and aneuploidy in oocytes. Ernst Schering Res. Found. Workshop, 2002, (41), 111-136.
[4] Eichenlaub-Ritter,U. Aneuploidy in ageing oocytes and after toxic insult. In Trounson,A.O. and Gosden,R.G. (eds), Biology and Pathology of the Oocyte. Cambridge University Press, Cambridge, (2003). 220-257.
[5] Hassold,T. and Hunt,P. To err (meiotically) is human: the genesis of human aneuploidy. Nature Rev. Genet. 2001; 2 (4) :280-291.
[6] Demeestere I, Centner J, Gervy C, et al.Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction. 2005;130(2):147-56.
[7] Hutt KJ, Albertini DF.An oocentric view of folliculogenesis and embryogenesis. Reprod Biomed Online.2007;14(6):758-64.
[8] 杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社.2005, 73-115.
[9] Hutt KJ, Albertini DF.An oocentric view of folliculogenesis and embryogenesis. Reprod Biomed Online. 2007; 14(6):758-64.
[10] Sun F, Betzendahl I, Pacchierotti F, et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitroin preantral follicle culture to nocodazole. Mutagenesis. 2005;20(1): 65-75.
[11] Sun F, Betzendahl I, Shen Y,et al.. Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes. Mutagenesis. 2004; 19(1): 13-25.
[12] Thomas FH, Walters KA, Telfer EE, et al. How to make a good oocyte: an update on in-vitro models to study follicle regulation. Hum Reprod Update. 2003;9(6):541-555.
[13] Loret de Mola JR, Barnhart K, Kopf GS, et al.Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles. Clin Exp Obstet Gynecol. 2004;31(1):15-19.
[14] Cortvrindt RG,.Smitz JEJ.Follicle culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod Update .2002;8(3):243-254.
[15] Comment in: Aardema MJ, Albertini S, Ami P, et al.Aneuploidy: a report of an ECETOC task force. Mutat Res. 1998;410(1): 117-20.
[16] Jacquet P, Adriaens I, Buset J,et al. Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation, by use of an early preantral follicle culture system. Mutat Res. 2005;583(2):168-77.
[17]Rossi G,Palmerini MG,Macchiarelli G.Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes.Reprod Toxicol.2006;22(1):51-55.
[18]Bremer S,Pellizzer C,Hoffmann S,et al.The development of new concepts for assessing reproductive toxicity applicable to large scale toxicological programmes.Curt Pharm Des.2007;13(29):3047-3058.
[19]Combes R,Grindon C,Cronin MT,Roberts DW,Garrod J.Proposed integrated decision-tree testing strategies for mutagenicity and carcinogenicity in relation to the EU REACH legislation.Aitern Lab Anim.2007;35(2):267-287.
[20]张天宝.大鼠卵巢细胞体外培养及生殖毒理研究中的应用.卫生毒理学杂志.1997;11(2):128
[21]Koes.RD,Jaccaridd FJ.Magaril RA,et al.Perfusion ofihe rat ovary in vitro:methology,induction of ovulation and pattern ofstermdogenesis.Bio Reprod,1984.30(5):1135
[22]Todoroff EC,Sevcik M,Villeneuve DC,et al.The effect of photomirex on the in vitro perfused ovary of the rat.Reprod Toxicol.1998;12(3):305-16.
[23]Kemp B,Winkler M,Hauptmann S,Rath W.Cervical dilatation:induction by antigestagens via adhesion molecules.An in vitro examination in endothelial cell cultures.Gyneeol Obstet Invest.1998;45(2):116-20.
[24]Wilmut I,A E Schnieke,J Mcwhir,et al.Viable ofspring deriver form fetal and adult mammalian cell.Nature.1997;385(6619):810-813.
[25]Barash I,Faeman A,Richensteiu M,et al.In vivo and in vitro expression of human serum albumin genemic sequence～in mammary epithelial cells with β—lactoblobumin and whey acidic protein promoters.Mol Reprod Dev.1999;52(3):241-252.
[26]Vanessa Lollivierl,Pierre-Guy Marnetl,Serge Delpal,et al.Oxytocin stimulates secretory processes in lactating rabbit mammary epithelial cells.J Physiol.2006;570(1):125-140.
[27]孙丽翠,刘朋删,杨国庆等.小鼠和兔原代乳腺上皮细胞的培养.生物技术.2004;14(2):33-34.
[28]Fleming JM,Leibowitz BJ,Kerrl DE et al.IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells.Journal of Endocrinology.2005;186(5):165-178.
[29]Freshney RI,Pragnell IB,Fr eshney MG.Culture of epithelial cells.Second edition.New York:Wiley-Liss,2002.3.
[30]叶媚娜,陈红风.温肾中药对原代培养正常人乳腺上皮细胞增殖的影响中西医结合学报.2006;4(4):397-401.
[31] Spielmann H.Reproduction and development. Environ Health Perspect. 1998 ;106 (Suppl 2) :571-576.
[32] Huggins J. Alternatives to animal testing: research, trends, validation, regulatory acceptance. ALTEX. 2003;20(Suppl 1):3-61.
[33] Schechtman LM, Stokes WS. ECVAM-ICCVAM: prospects for future collaboration.Altern Lab Anim. 2002;30 (Suppl 2) :227-236.
[1]Hutt KJ,Albertini DF.An oocentric view of folliculogenesis and embryogenesis.Reprod Biomed Online.2007;14(6):758-64.
[2]Li MG,Ieuan M,Joyce.RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice.Biol Reprod.2005;72(9):195-199.
[3]Demeestere I,Centner J,Gervy C,Englert Y,Delbaere A.Impact of 4.various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents.Reproduction.2005;130(2):147-156.
[4]Oocyte-secreted factors:regulators of cumulus cell function and oocyte quality.Hum Reprod Update.2008;14(2):159-177.
[5]杨增明,孙青原,夏国良主编.生殖生物学.北京:科学出版社.2005;73-115
[6]Capacchietti G,Cecconi S,Colantoni E,et al.In vitro growth of sheep preantral follicles:which perspectives? Vet Res Commun.2003;27(Suppl 1):583-586.
[7]Cecconi S,Capacchietti G,Russo V,et al.In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue.Biol Reprod.2004;70(1):12-17.
[8]Park JY,Su YQ,Ariga M,,et al.EGF-Like Growth Factors As Mediators of LH Action in the Ovulatory Follicle.Science.2004;303(5658):682-684.
[9]Spears N,Baker S,Srsen V,et al.Mouse Ovarian Follicles Secrete Factors Affecting the Growth and Development of Like-Sized Ovarian Follicles In Vitro.Bioi Reprod.2002;67(6):1726-1733.
[10]王红玲,刘铁铮,禚艳书.小鼠腔前卵泡体外培养研究进展.Progrvet Med.2005;26(6):28-31.
[11]Alm H,Katska-Ksiazkiewicz L,Rynska B,et al.Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology. 2006 15;65(7): 1422-1434.
[12] Cecconi S, Rossi G, Bianchi S, Stallone T, Macchiarelli G, Nottola S.In vitro culture of mammalian early ovarian follicles. Morphofunctional correlates and future perspectives. Ital J Anat Embryol.2005; 110(2 Suppl 1):189-194.
[13] Telfer EE. In vitro models for oocyte development. Theriogenology. 1998; 15;49(2):451-460.
[14] Nayudu PL, Fehrenbach A, Kiesel P,et al.Progress toward understanding follicle development in vitro: appearances are not deceiving. Arch Med Res. 2001 ;32(6):587-594.
[15] Devine PJ, Rajapaksa KS, Hoyer PB.In vitro ovarian tissue and organ culture: a review.Front Biosci. 2002;7:d1979-89.
[16] Dole G, Nilsson E, Skinner M.Glial derived neurotrophic factor promotes ovarian primordial follicle development and cell-cell interactions during folliculogenesis. Reproduction. 2008 Feb 27 [Epub ahead of print]
[17] Fortune JE . Kito S. Warldji S-A Srsen V.Activation of bovine and baboon primordial follicles ln vitro.Theriogenology I998;49 (2): 441-449
[18] Hovatta O, Wright C, Krausz T,et al.Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum Reprod. 1999; 14(10):2519-2524.
[19] Grob HS. Enzymatic dissection of the mammalian ovary. Science. 1964 Oct 2;146:73-4.
[20] Nicosia SV, Evangelista I, Batta SK.Rabbit ovarian follicles. I. Isolation technique and characterization at different stages of development. Biol Reprod. 1975 ;13(4):423-47.
[21] Figueiredo J R et al. Development of a combined new mechanical and enzymatticmethod for the isolation of intact preantral follicle from fetal calf and adult bovine ovaries. Theriogenology .1993, (40): 789-799.
[22] Demeestere I,Delbaere A,Gervy C,et al .Effect of preantral follicle isolation technique on in vitro follicular growth,oocyte maturation and embryo development in mice.Hum Reprod.2001;7(8):2152
[23] Hartshotne G M. In Vitro Culture of Ovarian Follicles. Rev Reprod. 1997, 2 (2): 94—104.
[24] Erin R. West, Min Xu, Teresa K,Woodruff,et al.Physical properties of alginate hydrogels and their effects on in vitro follicle development. Biomaterials. 2007 ; 28(30): 4439-4448.
[25] Cortvrindt R, Smitz J, Van Steirteghem AC. In vitro maturation. fertilization and embryo development of immature oocytes from early preantral folheles from prepuberal mice in asimpliedculture system. Hum Reprod. 1996;11(12):2656-66.
[26] Nayudu PL, Osborn SM.Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro. J Reprod Fertil. 1992;95(2):349-62.
[27] Jhohan E,Smitz J,Cortvrindt RG.The earliest stages of follieuligenesis in vitro.Rproduction.2002; 123(2): 185—202.
[28] Cortvrindt RG,.Smitz JEJ.FollicIe culture in reproductive toxicology:a tool for in-vitro testing of ovarian function? Hum Reprod Update 2002;8(3):243-254.
[29] Sun F, Betzendahl I, Shen Y,et al. Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes.Mutagenesis. 2004;19(1): 13-25.
[30] Dela Pe(n|~)a EC, Takahashi Y, Katagiri S,et al.Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles.Reproduction. 2002;123(4):593-600.
[31] Roberts R, Stark J, Iatropoulou A,et al.Energy substrate metabolism of mouse cumulus-oocyte complexes: response to follicle-stimulating hormone is mediated by the phosphatidylinositol 3-kinase pathway and is associated with oocyte maturation. Biol Reprod. 2004;71(1): 199-209.
[32 Ptak A, Ludewig G, Robertson L, et al.In vitro exposure of porcine prepubertal follicles to 4-chlorobiphenyl (PCB3) and its hydroxylated metabolites: effects on sex hormone levels and aromatase activity. Toxicol Lett. 2006; 164(2): 113-122.
[33] Sun F, Betzendahl I, Pacchierotti F,et al.Aneuploidy in mouse metaphase Ⅱ oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole. Mutagenesis . 2005;20 (1):65—75.
[34] Shen Y, Betzendahl I, Sun F, et al. Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes.Reprod Toxicol. 2005;19(4):459-71.
[35] Rossi G, Palmerini MG, Macchiarelli G, et al.Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes. Reprod Toxicol. 2006;22(1):51 -55.
[36] Nayudu PL, Fehrenbach A, Kiesel P,et al.Progress toward understanding follicle development in vitro:appearances are not deceiving.Arch Med Res. 2001 ;32(6):587-94.
[37] Van Wemmel K, Gobbers E, Eichenlaub-Ritter U,et al. Ovarian follicle bioassay reveals adverse effects of diazepam exposure upon follicle development and oocyte quality. Reprod Toxicol. 2005;20(2):183-193.
[38] Van Merris V, Van Wemmel K, Cortvrindt R.In vitro effects of dexamethasone on mouse ovarian function and pre-implantation embryo development. Reprod Toxicol. 2007 ;23(1):32-41.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |