Effect Of Mifepristone On Mouse Follicles

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 pathology3.1 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.ultrastructure4.1 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.