米非司酮对小鼠卵泡的影响
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摘要
目的:以不同方式给小鼠服用不同剂量米非司酮,研究停药后不同时间,米非司酮对小鼠卵泡发育、动情周期及形态学的影响,探讨其引起细胞凋亡的机制,并研究其对卵巢局部生长因子对卵泡调节的影响。
方法:105只小鼠随机分组,灌胃方法以不同方式给小鼠不同剂量米非司酮,按照不同停药时间取小鼠卵巢,分组如下:长期小剂量用药组(A1、A2、A3各10只),间断小剂量用药组(B1、B2、B3各10只),间断大剂量用药组(C1、C2、C3各10只)。对照组(D1、D2、D3各5只)。利用光镜、电镜、免疫组化、末端标记、荧光定量PCR以及小鼠阴道细胞涂片等方法,观察实验组和对照组小鼠动情周期,卵泡病理结构及超微结构特征,细胞凋亡及相关蛋白Bcl-2/Bax,生长因子IGF-1、IGF-1R,IGF-1R mRNA,GDF-9,BMPR-ⅡmRNA在卵巢的表达。
结果:
1.动情周期:实验组A1仅有两只小鼠出现动情周期,其余动情周期消失。与对照组D组相比,A2组动情周期明显延长,差异有统计学意义(P＜0.001)。其余各实验组与对照组相比差异无统计学意义。
2.卵泡发育:
2.1长期小剂量米非司酮用药组:与对照组相比,A1组各级正常卵泡数量显著减少,闭锁率显著增高(P＜0.001);
与对照组相比,A2组各级生长卵泡数量显著减少(P＜0.001),闭锁率较高,除窦前卵泡以外,其余两型卵泡闭锁率显著增高(P＜0.001)。
与对照组相比,A3组正常窦状卵泡无明显差异,其余两型卵泡的数量较少,(P＜0.001)。
2.2间断小剂量米非司酮用药组:
与对照组相比,B1组原始-初级卵泡数量下降明显(P＜0.001);窦前和窦状正常卵泡差异无统计学意义;窦前卵泡闭锁率显著增高(P＜0.001),其余两型卵泡的闭锁率差异无统计学意义。
与对照组相比,B2、B3组正常卵泡数和闭锁卵泡率差异无统计学意义。
2.3间断大剂量米非司酮用药组:
与对照组相比,C1组各级正常卵泡数量均减少(P＜0.001);其中窦前和窦状卵泡闭锁率显著增高(P＜0.001)。
与对照组相比,C2、C3组各级正常卵泡数量和闭锁率差异无统计学意义。
3.病理结构:
3.1 A1组卵巢结构松散紊乱,原始及初级卵泡少见,绝大部分卵泡内的卵母细胞、大量颗粒细胞坏死,大面积颗粒细胞黄素化出现。A2组见少量结构正常的各级卵泡,各级闭锁卵泡亦多见,可见大面积的或局部的纤维化。部分血管壁增厚扩张,呈显著玻璃样变或纤维素样坏死。A3组见各级卵泡的数量仍较少,但闭锁卵泡较少。仍可见局部的纤维化,部分血管壁仍呈玻璃样变。
3.2 B1组各级卵泡数量略少,部分卵泡闭锁。皮质区未见明显纤维化,血管无明显病变。B2、B3组未见明显异常
3.3 C1组各级卵泡数量较少,部分卵泡闭锁。皮质区未见明显纤维化,血管无明显病变。C2、C3组:光镜和电镜未见明显异常。
4.超微结构:
4.1 A1组透射电镜下偶见形态正常的原始-初级卵泡。大部分各级卵泡卵母细胞的胞浆细胞器、微绒毛以及围绕其周围的颗粒细胞出现了一系列变性、坏死。A2组电镜下可见少量形态正常的各级卵泡,多数卵母细胞胞浆细胞器不丰富,线粒体少;颗粒细胞凋亡多见。A3组电镜下可见到少量形态正常的各级卵泡,部分卵泡细胞胞浆细胞器仍不丰富,核仁浓缩。
4.2 B1组透射电镜下可见部分形态正常的各级卵泡,部分卵泡胞浆细胞器不丰富,线粒体空泡样变常见。卵母细胞表面微绒毛减少。B2、B3组未见明显异常。
4.3 C1组透射电镜下可见部分生长卵泡的卵母细胞核固缩,异染色质极浓密,透明带不规则,间质腺较少,腺细胞核大色浅,胞质内出现较多的空泡。C2、C3未见明显异常。
5.除A1组未见典型的TUNEL阳性细胞外,其余组小鼠卵巢部分颗粒细胞表现TUNEL阳性。与对照组相比,A2、B1、C1组凋亡指数增高(P＜0.001),其余组小鼠颗粒细胞的凋亡指数差异无统计学意义。
6.对各组原始-初级卵泡卵母细胞Bax表达的结果发现,各组与对照组相比无显著差异。
对各组窦状前卵泡卵母细胞和颗粒细胞Bcl-2/BaX的表达结果发现,与对照组相比,无论在卵母细胞还是颗粒细胞,A2组Bcl-2表达降低,Bax表达升高(P＜0.001)。其余各组Bcl-2和Bax的表达和各自的对照组相比,差异无统计学意义。
对各组窦状卵泡卵母细胞Bcl-2和Bax的表达结果发现,与对照组相比,A2组Bcl-2表达降低,Bax表达升高,A3组Bcl-2表达升高(P＜0.001),其余各组卵泡卵母细胞Bcl-2和Bax的表达差异无统计学意义。
对各组窦状卵泡颗粒细胞Bcl-2和Bax的表达结果发现,与对照组相比,A2组Bax表达升高,差异有统计学意义(P＜0.001),其余各组颗粒细胞Bcl-2和Bax的表达差异无统计学意义。
7.IGF-1、IGF-1R在长期用药组小鼠卵巢颗粒细胞的表达,A1组显著低于对照组(P＜0.001),其余各组与对照组相比,差异无统计学意义。IGF-1RmRNA在长期用药组小鼠卵巢相对定量结果显示,与对照组相比,A1、A2组表达量明显降低(P＜0.001),A3组差异统计学意义。
8.GDF-9在长期用药组小鼠卵巢卵母细胞的表达,在窦前卵泡的卵母细胞,GDF-9在A2组卵母细胞的表达明显低于对照组(P＜0.001),A3组的表达与对照组相比差异无统计学意义。在窦状卵泡,GDF-9在A2组卵母细胞的表达明显低于对照组(P＜0.001),A3组的表达和对照组相比差异无统计学意义。BMPR-ⅡmRNA在长期用药组小鼠卵巢相对定量结果显示,与对照组相比,A1组表达量明显降低(P＜0.001),A2、A3组差异无统计学意义。
结论
1.长期小剂量米非司酮对小鼠卵巢功能的影响不仅表现为抑制卵泡的发育,引起内分泌功能的紊乱,还表现为对卵泡细胞的损伤,这种损伤随着停药时间的延长会得到不同程度的恢复。小剂量米非司酮间断用药及大剂量间断用药对小鼠卵泡发育的影响表现为暂时的抑制作用,而非不可逆的损伤作用。
2.米非司酮可能通过影响凋亡相关蛋白的表达干扰卵泡的正常生长和卵巢功能,米非司酮导致卵泡闭锁(尤其在晚期卵泡)的主要原因是引起颗粒细胞的凋亡。随停药时间的延长,不同组别颗粒细胞凋亡指数的下降及凋亡相关蛋白表达的变化,说明米非司酮抑制卵巢功能在一定程度上是可复的,但是时间依赖性的。
3.在长期小剂量用药组,米非司酮可通过抑制IGF-I及其受体IGF-IR,干扰卵泡的正常生长,影响颗粒细胞增殖。但随停药时间延长,受抑制的IGF-I及其受体的表达可恢复正常,卵泡受到的抑制和损害也可以得到不同程度的恢复。
4.在长期小剂量用药组,米非司酮可通过干扰GDF-9及其受体的表达与调控,影响卵母细胞的生长与成熟,并影响卵母细胞对卵泡生长的调节作用。随停药时间的延长,GDF-9及其受体的表达可恢复正常。
Objective:Female mice were administrated mifepristone by different doses in different way,the main purpose of the study is to examine the effect of mifepristone on mice follicular development,estrus cycle,morphological characteristics,the apoptotic mechanism of follicular cells and the regulation of intra-ovarian growth factors.
Methods:105 mices were assigned into different groups randomly,administrated different doses of mifepristone,and the mices' ovaries were taken according to the time when the mifepristone was stopped to apply.The groups included the group with long-term small dose(10 each A1,A2,A3),the group with separating small dose(10 each B1,B2,B3),group with separating large dose(10 each C1,C2,C3),and control group(5 each D1,D2,D3).Using vaginal cell smear,light microscope,transmission electron microscope,real-time PCR,immunohistochemical and TUNEL methods,the following aspects were observed as:estrus cycle,pathological and ultrastructural characteristics,cell apoptosis,the expressions of apoptosis related proteins Bcl-2/Bax, IGF-1,IGF-1R,IGF-1R mRNA,GDF-9 and BMPR-ⅡmRNA.
Results:
1.Estrus cycle.There were only two mice emerged estrus cycle in group A1 and the estrus cycle was disappeared for others.Comparing with control group D,estrus cycle in group A2 was significantly extended(P＜0.001),in other groups there was no significantly difference.
2.Follicle development.
2.1 group with long-term small dose:
Comparing with control group,the number of normal follicles at each type in group A1 was decreased significantly(P＜0.001).Meanwhile,the ratio of follicle atresia at each type was increased significantly(P＜0.001).
Comparing with control group,the numbers of normal follicles at each type in group A2 were decreased significantly(P＜0.001).Except the preantral follicle,the ratio of follicle atresia in other two types was high(P＜0.001).
Comparing with control group,the number of normal antral follicles in group A3 was similar,and the difference was no significant.The number of other two type follicles was decreased significantly(P＜0.001).
2.2 the group with separating small dose:
Comparing with control group,the number of primordial-primary follicles in group B1 was decreased significantly(P＜0.001),and number of other two types follicles was no significant difference.The atretic rate of preantral follicles was significantly increased(P＜0.001) and that of other two types follicles was no significant difference.
As far as the number of normal follicles and the ratio of follicle atresia,there was no significant difference between control group and group B2 and B3.
2.3 group with separating large dose:
Comparing with control group,the number of normal follicles at each type was decreased significantly in group C1(P＜0.001).The ratio follicle atresia was significantly high in preantral and antral follicles(P＜0.001).
As far as the number of normal follicles and the ratio of follicle atresia,there was no significant difference between control group and C2 and C3.
3.The structure of pathology
3.1 The structure of ovary in group A1 was incompact and disorganized,and it was difficult to find the primordial-primary follicles.Most oocytes and large number of granulosa cells were necrosis,and the large number of granulosa cells was luteinized. The small number of follicles with normal structure at each type could be found in group A2,large number of follicle atresia could be observed too,and partial area of fibrosis could be found.Partial vessel wall became thick and dilated,which showed significant hyalinization or necrosis.The number of follicles at each type in group A3 was still small,and the ratio of follicle atresia was small.However,the partial fibrosis still could be found,and partial the vessel wall was showed as hyalinization.
3.2 The number of follicles at each type in group B1 was slightly small,and partial follicles were atretic.There were no obvious fibrosis in cortex area and no obvious pathological changes in blood vessel.There was no significant abnormality in group B2 and group B3.
3.3 The number of follicles at each type was relative small in group C1,and partial follicles were atretic.There was no significant fibrosis in cortex area and no definite pathological changes in blood vessel.There were no significant abnormality in group C2 and C3.
4.ultrastructure
4.1 The normal primordial-primary follicles could be occasionally found with transmission microscope in group A1.Most follicles at each type showed a degeneration and necrosis in cell organelle and microvilli of oocytes,and granulosa cells around them.The small number of normal follicles at each type could be found in group A2.Most cell organelles of oocytes were not rich,with small number of mitochondria.Apoptosis was often seen in granulosa cells.A small number of normal follicles at each type could be found in group A3.Condensed nucleoli could be seen in patial oocytes and cell organelle in cytoplasm of partial follicles was still not rich.
4.2 The partial normal follicles at each type could be found in group B1.Cell organelle in cytoplasm of partial follicles was not rich and vacuolated mitochondria could be found easily.The microvilli of oocytes were decreased.No significant abnormality was found in group B2 and group B3.
4.3 The nucleus of partial oocytes could be found pyknosis in group C1 and heterochromatin is extremely thickening,zona pellucida was irregular,lipid droplets with low density were often seen in cytoplasm of granulose cells.No significant abnormality was found in group C2 and group C3.
5.Index of apoptosis:No typical TUNEL masculine cells could be found in group A1.Partial granulosa cells of follicles presented TUNEL masculine in other groups. Comparing with control group,the index of apoptosis was increased significantly in group A2,B1,and C1(P＜0.001).There was no significant difference for the index of apoptosis in the rest groups.
6.Bcl-2/Bax expression:
The results of Bax expression in primordial-primary oocytes of follicles indicated that there was no significant difference between experimental groups and control group.
The results of Bcl-2/Bax expression of preantral follicles indicated whatever in oocytes of follicles and granulosa cell,the Bcl-2 expression was decreased and the Bax expression was increased in group A2.Comparing with control group,the difference was significant(P＜0.001).Bcl-2 and Bax expression were no difference in other groups.
Comparing with control groups,the results of Bcl-2/Bax expression in oocvtes of preantral follicles indicated the Bcl-2 expression was decreased and the Bax expression was increased in group A2(P＜0.001).Bcl-2 expression in A3 was increased significantly(P＜0.001).The Bcl-2 and Bax expression in oocytes were no difference in other groups.
Comparing with control groups,the results of Bcl-2/Bax expression of granulosa cell of antral follicles indicated the Bax expression was decreased significantly in group A1(P＜0.001).The Bcl-2 and Bax expression of granulosa cell was no difference in other groups.
7.IGF-1/IGF-1R expression:Comparing with control group,the expression of IGF-1 and IGF-1R in granulosa cell in group with long-term small dose was significantly lower(P＜0.001).There was no difference in other groups.The expression of IGF-1RmRNA in mice's ovary was definitely decreased in group A1 and group A2,and there was no difference in group A3.
8.In group with long-term small dose,comparing with control group,the expression of GDF-9 in oocytes of preantral follicles was definitely lower in group A2(P＜0.001), and there was no significant difference in group A3.The expression of GDF-9 in antral follicles was definitely lower in group A2 but significantly higher in group A3 (P＜0.001).The relative quantitative result of BMPR-ⅡmRNA of mice's ovary was obviously decreased in group A1(P＜0.001),and there was no difference in group A2 and group A3.
Conclusions:
1.The impact of mifepristone on group with long-term small dose to mice's ovary is not only demonstrating as the growth inhibition of follicles and resulting in the disorder of endocrinal secretion,but also showing as the damage of follicles which could be recovered gradually if there is no mifepristone applying.Separating small dose and large dose of mifepristone to the growth of mice's follicles demonstrates temporarily inhabitation,rather than permanent damage with non-reversible.
2.Mifepristone might disturb the normal growth of follicles and the function of ovary through influencing the expression of the apoptosis related protein.The main reason of mifepristone caused follicle atretsia might be the apoptosis of granulosa cells.With the time for stopping mifepristone being longer,the apoptosis index of granular cell in different groups was decreased and the expression of apoptosis related protein was changed,which shows the ovary function inhibition by mifepristone could be recovered in a certain level.However,it would depend on the time.
3.In the group with long-term small dose,mifepristone could disturb regulation of follicles development and influence the proliferation of granulosa cells through inhibiting IGF-I and its receptor.However,With the time for stopping mifepristone being longer,the expression of inhibited IGF-I and its receptor could be recovered. Meanwhile,the inhibition and damage of follicles could get a certain recovery.
4.In the group with long-term small dose,mifepristone could influence the growth and mature of oocytes through disturbing GDF-9 and its receptor expression.It could also influence the accommodation of oocytes to follicles.The expression of GDF-9 and its receptor could be recovered to normal if the time of stopping mifepristone is getting longer.
方法:105只小鼠随机分组,灌胃方法以不同方式给小鼠不同剂量米非司酮,按照不同停药时间取小鼠卵巢,分组如下:长期小剂量用药组(A1、A2、A3各10只),间断小剂量用药组(B1、B2、B3各10只),间断大剂量用药组(C1、C2、C3各10只)。对照组(D1、D2、D3各5只)。利用光镜、电镜、免疫组化、末端标记、荧光定量PCR以及小鼠阴道细胞涂片等方法,观察实验组和对照组小鼠动情周期,卵泡病理结构及超微结构特征,细胞凋亡及相关蛋白Bcl-2/Bax,生长因子IGF-1、IGF-1R,IGF-1R mRNA,GDF-9,BMPR-ⅡmRNA在卵巢的表达。
结果:
1.动情周期:实验组A1仅有两只小鼠出现动情周期,其余动情周期消失。与对照组D组相比,A2组动情周期明显延长,差异有统计学意义(P＜0.001)。其余各实验组与对照组相比差异无统计学意义。
2.卵泡发育:
2.1长期小剂量米非司酮用药组:与对照组相比,A1组各级正常卵泡数量显著减少,闭锁率显著增高(P＜0.001);
与对照组相比,A2组各级生长卵泡数量显著减少(P＜0.001),闭锁率较高,除窦前卵泡以外,其余两型卵泡闭锁率显著增高(P＜0.001)。
与对照组相比,A3组正常窦状卵泡无明显差异,其余两型卵泡的数量较少,(P＜0.001)。
2.2间断小剂量米非司酮用药组:
与对照组相比,B1组原始-初级卵泡数量下降明显(P＜0.001);窦前和窦状正常卵泡差异无统计学意义;窦前卵泡闭锁率显著增高(P＜0.001),其余两型卵泡的闭锁率差异无统计学意义。
与对照组相比,B2、B3组正常卵泡数和闭锁卵泡率差异无统计学意义。
2.3间断大剂量米非司酮用药组:
与对照组相比,C1组各级正常卵泡数量均减少(P＜0.001);其中窦前和窦状卵泡闭锁率显著增高(P＜0.001)。
与对照组相比,C2、C3组各级正常卵泡数量和闭锁率差异无统计学意义。
3.病理结构:
3.1 A1组卵巢结构松散紊乱,原始及初级卵泡少见,绝大部分卵泡内的卵母细胞、大量颗粒细胞坏死,大面积颗粒细胞黄素化出现。A2组见少量结构正常的各级卵泡,各级闭锁卵泡亦多见,可见大面积的或局部的纤维化。部分血管壁增厚扩张,呈显著玻璃样变或纤维素样坏死。A3组见各级卵泡的数量仍较少,但闭锁卵泡较少。仍可见局部的纤维化,部分血管壁仍呈玻璃样变。
3.2 B1组各级卵泡数量略少,部分卵泡闭锁。皮质区未见明显纤维化,血管无明显病变。B2、B3组未见明显异常
3.3 C1组各级卵泡数量较少,部分卵泡闭锁。皮质区未见明显纤维化,血管无明显病变。C2、C3组:光镜和电镜未见明显异常。
4.超微结构:
4.1 A1组透射电镜下偶见形态正常的原始-初级卵泡。大部分各级卵泡卵母细胞的胞浆细胞器、微绒毛以及围绕其周围的颗粒细胞出现了一系列变性、坏死。A2组电镜下可见少量形态正常的各级卵泡,多数卵母细胞胞浆细胞器不丰富,线粒体少;颗粒细胞凋亡多见。A3组电镜下可见到少量形态正常的各级卵泡,部分卵泡细胞胞浆细胞器仍不丰富,核仁浓缩。
4.2 B1组透射电镜下可见部分形态正常的各级卵泡,部分卵泡胞浆细胞器不丰富,线粒体空泡样变常见。卵母细胞表面微绒毛减少。B2、B3组未见明显异常。
4.3 C1组透射电镜下可见部分生长卵泡的卵母细胞核固缩,异染色质极浓密,透明带不规则,间质腺较少,腺细胞核大色浅,胞质内出现较多的空泡。C2、C3未见明显异常。
5.除A1组未见典型的TUNEL阳性细胞外,其余组小鼠卵巢部分颗粒细胞表现TUNEL阳性。与对照组相比,A2、B1、C1组凋亡指数增高(P＜0.001),其余组小鼠颗粒细胞的凋亡指数差异无统计学意义。
6.对各组原始-初级卵泡卵母细胞Bax表达的结果发现,各组与对照组相比无显著差异。
对各组窦状前卵泡卵母细胞和颗粒细胞Bcl-2/BaX的表达结果发现,与对照组相比,无论在卵母细胞还是颗粒细胞,A2组Bcl-2表达降低,Bax表达升高(P＜0.001)。其余各组Bcl-2和Bax的表达和各自的对照组相比,差异无统计学意义。
对各组窦状卵泡卵母细胞Bcl-2和Bax的表达结果发现,与对照组相比,A2组Bcl-2表达降低,Bax表达升高,A3组Bcl-2表达升高(P＜0.001),其余各组卵泡卵母细胞Bcl-2和Bax的表达差异无统计学意义。
对各组窦状卵泡颗粒细胞Bcl-2和Bax的表达结果发现,与对照组相比,A2组Bax表达升高,差异有统计学意义(P＜0.001),其余各组颗粒细胞Bcl-2和Bax的表达差异无统计学意义。
7.IGF-1、IGF-1R在长期用药组小鼠卵巢颗粒细胞的表达,A1组显著低于对照组(P＜0.001),其余各组与对照组相比,差异无统计学意义。IGF-1RmRNA在长期用药组小鼠卵巢相对定量结果显示,与对照组相比,A1、A2组表达量明显降低(P＜0.001),A3组差异统计学意义。
8.GDF-9在长期用药组小鼠卵巢卵母细胞的表达,在窦前卵泡的卵母细胞,GDF-9在A2组卵母细胞的表达明显低于对照组(P＜0.001),A3组的表达与对照组相比差异无统计学意义。在窦状卵泡,GDF-9在A2组卵母细胞的表达明显低于对照组(P＜0.001),A3组的表达和对照组相比差异无统计学意义。BMPR-ⅡmRNA在长期用药组小鼠卵巢相对定量结果显示,与对照组相比,A1组表达量明显降低(P＜0.001),A2、A3组差异无统计学意义。
结论
1.长期小剂量米非司酮对小鼠卵巢功能的影响不仅表现为抑制卵泡的发育,引起内分泌功能的紊乱,还表现为对卵泡细胞的损伤,这种损伤随着停药时间的延长会得到不同程度的恢复。小剂量米非司酮间断用药及大剂量间断用药对小鼠卵泡发育的影响表现为暂时的抑制作用,而非不可逆的损伤作用。
2.米非司酮可能通过影响凋亡相关蛋白的表达干扰卵泡的正常生长和卵巢功能,米非司酮导致卵泡闭锁(尤其在晚期卵泡)的主要原因是引起颗粒细胞的凋亡。随停药时间的延长,不同组别颗粒细胞凋亡指数的下降及凋亡相关蛋白表达的变化,说明米非司酮抑制卵巢功能在一定程度上是可复的,但是时间依赖性的。
3.在长期小剂量用药组,米非司酮可通过抑制IGF-I及其受体IGF-IR,干扰卵泡的正常生长,影响颗粒细胞增殖。但随停药时间延长,受抑制的IGF-I及其受体的表达可恢复正常,卵泡受到的抑制和损害也可以得到不同程度的恢复。
4.在长期小剂量用药组,米非司酮可通过干扰GDF-9及其受体的表达与调控,影响卵母细胞的生长与成熟,并影响卵母细胞对卵泡生长的调节作用。随停药时间的延长,GDF-9及其受体的表达可恢复正常。
Objective:Female mice were administrated mifepristone by different doses in different way,the main purpose of the study is to examine the effect of mifepristone on mice follicular development,estrus cycle,morphological characteristics,the apoptotic mechanism of follicular cells and the regulation of intra-ovarian growth factors.
Methods:105 mices were assigned into different groups randomly,administrated different doses of mifepristone,and the mices' ovaries were taken according to the time when the mifepristone was stopped to apply.The groups included the group with long-term small dose(10 each A1,A2,A3),the group with separating small dose(10 each B1,B2,B3),group with separating large dose(10 each C1,C2,C3),and control group(5 each D1,D2,D3).Using vaginal cell smear,light microscope,transmission electron microscope,real-time PCR,immunohistochemical and TUNEL methods,the following aspects were observed as:estrus cycle,pathological and ultrastructural characteristics,cell apoptosis,the expressions of apoptosis related proteins Bcl-2/Bax, IGF-1,IGF-1R,IGF-1R mRNA,GDF-9 and BMPR-ⅡmRNA.
Results:
1.Estrus cycle.There were only two mice emerged estrus cycle in group A1 and the estrus cycle was disappeared for others.Comparing with control group D,estrus cycle in group A2 was significantly extended(P＜0.001),in other groups there was no significantly difference.
2.Follicle development.
2.1 group with long-term small dose:
Comparing with control group,the number of normal follicles at each type in group A1 was decreased significantly(P＜0.001).Meanwhile,the ratio of follicle atresia at each type was increased significantly(P＜0.001).
Comparing with control group,the numbers of normal follicles at each type in group A2 were decreased significantly(P＜0.001).Except the preantral follicle,the ratio of follicle atresia in other two types was high(P＜0.001).
Comparing with control group,the number of normal antral follicles in group A3 was similar,and the difference was no significant.The number of other two type follicles was decreased significantly(P＜0.001).
2.2 the group with separating small dose:
Comparing with control group,the number of primordial-primary follicles in group B1 was decreased significantly(P＜0.001),and number of other two types follicles was no significant difference.The atretic rate of preantral follicles was significantly increased(P＜0.001) and that of other two types follicles was no significant difference.
As far as the number of normal follicles and the ratio of follicle atresia,there was no significant difference between control group and group B2 and B3.
2.3 group with separating large dose:
Comparing with control group,the number of normal follicles at each type was decreased significantly in group C1(P＜0.001).The ratio follicle atresia was significantly high in preantral and antral follicles(P＜0.001).
As far as the number of normal follicles and the ratio of follicle atresia,there was no significant difference between control group and C2 and C3.
3.The structure of pathology
3.1 The structure of ovary in group A1 was incompact and disorganized,and it was difficult to find the primordial-primary follicles.Most oocytes and large number of granulosa cells were necrosis,and the large number of granulosa cells was luteinized. The small number of follicles with normal structure at each type could be found in group A2,large number of follicle atresia could be observed too,and partial area of fibrosis could be found.Partial vessel wall became thick and dilated,which showed significant hyalinization or necrosis.The number of follicles at each type in group A3 was still small,and the ratio of follicle atresia was small.However,the partial fibrosis still could be found,and partial the vessel wall was showed as hyalinization.
3.2 The number of follicles at each type in group B1 was slightly small,and partial follicles were atretic.There were no obvious fibrosis in cortex area and no obvious pathological changes in blood vessel.There was no significant abnormality in group B2 and group B3.
3.3 The number of follicles at each type was relative small in group C1,and partial follicles were atretic.There was no significant fibrosis in cortex area and no definite pathological changes in blood vessel.There were no significant abnormality in group C2 and C3.
4.ultrastructure
4.1 The normal primordial-primary follicles could be occasionally found with transmission microscope in group A1.Most follicles at each type showed a degeneration and necrosis in cell organelle and microvilli of oocytes,and granulosa cells around them.The small number of normal follicles at each type could be found in group A2.Most cell organelles of oocytes were not rich,with small number of mitochondria.Apoptosis was often seen in granulosa cells.A small number of normal follicles at each type could be found in group A3.Condensed nucleoli could be seen in patial oocytes and cell organelle in cytoplasm of partial follicles was still not rich.
4.2 The partial normal follicles at each type could be found in group B1.Cell organelle in cytoplasm of partial follicles was not rich and vacuolated mitochondria could be found easily.The microvilli of oocytes were decreased.No significant abnormality was found in group B2 and group B3.
4.3 The nucleus of partial oocytes could be found pyknosis in group C1 and heterochromatin is extremely thickening,zona pellucida was irregular,lipid droplets with low density were often seen in cytoplasm of granulose cells.No significant abnormality was found in group C2 and group C3.
5.Index of apoptosis:No typical TUNEL masculine cells could be found in group A1.Partial granulosa cells of follicles presented TUNEL masculine in other groups. Comparing with control group,the index of apoptosis was increased significantly in group A2,B1,and C1(P＜0.001).There was no significant difference for the index of apoptosis in the rest groups.
6.Bcl-2/Bax expression:
The results of Bax expression in primordial-primary oocytes of follicles indicated that there was no significant difference between experimental groups and control group.
The results of Bcl-2/Bax expression of preantral follicles indicated whatever in oocytes of follicles and granulosa cell,the Bcl-2 expression was decreased and the Bax expression was increased in group A2.Comparing with control group,the difference was significant(P＜0.001).Bcl-2 and Bax expression were no difference in other groups.
Comparing with control groups,the results of Bcl-2/Bax expression in oocvtes of preantral follicles indicated the Bcl-2 expression was decreased and the Bax expression was increased in group A2(P＜0.001).Bcl-2 expression in A3 was increased significantly(P＜0.001).The Bcl-2 and Bax expression in oocytes were no difference in other groups.
Comparing with control groups,the results of Bcl-2/Bax expression of granulosa cell of antral follicles indicated the Bax expression was decreased significantly in group A1(P＜0.001).The Bcl-2 and Bax expression of granulosa cell was no difference in other groups.
7.IGF-1/IGF-1R expression:Comparing with control group,the expression of IGF-1 and IGF-1R in granulosa cell in group with long-term small dose was significantly lower(P＜0.001).There was no difference in other groups.The expression of IGF-1RmRNA in mice's ovary was definitely decreased in group A1 and group A2,and there was no difference in group A3.
8.In group with long-term small dose,comparing with control group,the expression of GDF-9 in oocytes of preantral follicles was definitely lower in group A2(P＜0.001), and there was no significant difference in group A3.The expression of GDF-9 in antral follicles was definitely lower in group A2 but significantly higher in group A3 (P＜0.001).The relative quantitative result of BMPR-ⅡmRNA of mice's ovary was obviously decreased in group A1(P＜0.001),and there was no difference in group A2 and group A3.
Conclusions:
1.The impact of mifepristone on group with long-term small dose to mice's ovary is not only demonstrating as the growth inhibition of follicles and resulting in the disorder of endocrinal secretion,but also showing as the damage of follicles which could be recovered gradually if there is no mifepristone applying.Separating small dose and large dose of mifepristone to the growth of mice's follicles demonstrates temporarily inhabitation,rather than permanent damage with non-reversible.
2.Mifepristone might disturb the normal growth of follicles and the function of ovary through influencing the expression of the apoptosis related protein.The main reason of mifepristone caused follicle atretsia might be the apoptosis of granulosa cells.With the time for stopping mifepristone being longer,the apoptosis index of granular cell in different groups was decreased and the expression of apoptosis related protein was changed,which shows the ovary function inhibition by mifepristone could be recovered in a certain level.However,it would depend on the time.
3.In the group with long-term small dose,mifepristone could disturb regulation of follicles development and influence the proliferation of granulosa cells through inhibiting IGF-I and its receptor.However,With the time for stopping mifepristone being longer,the expression of inhibited IGF-I and its receptor could be recovered. Meanwhile,the inhibition and damage of follicles could get a certain recovery.
4.In the group with long-term small dose,mifepristone could influence the growth and mature of oocytes through disturbing GDF-9 and its receptor expression.It could also influence the accommodation of oocytes to follicles.The expression of GDF-9 and its receptor could be recovered to normal if the time of stopping mifepristone is getting longer.
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1.朱顺法,方马荣,金理正.长期低剂量米非司酮对大鼠卵巢超微结构和内分泌功能的影响[J].解剖学杂志.2005,28(1):14-17.
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