A Research On The Construction Of A New Rat Combination Model Of The Disease And Syndrome Of Diminished Ovarian Reserve Induced By Bisphenol A

Objective:We aims to find a new way of establishing a rat model combined with disease and syndromes about diminished ovarian reserve by giving different doses of BPA to SD rats at different ages,the type of this model will be defined for providing a new kind of animal model combined with disease and syndromes that will be benefit for preserving fertility by TCM.Methods:Part 1:Thirty sterile female SD rat aged at 12 weeks with regular estrous cycles were randomly and equally divided into 5 groups according to the different interferes and doses of BPA:3 groups were given low dose(50mg/kg/d),middle dose(100mg/kg/d)and high dose(200mg/kg/d)of BPA intrastate administration and they were defined as low dose group at 3 months(3MLG),middle dose group at 3 months(3MMG)and high dose group at 3 months(3MHG).The fourth group was interrupted with glucosidorum tripterygll totorum(GTT)at 50mg/kg/d while the control group was give corn oil 0.1ml/100g/d(3MCG).The rats given different doses of BPA will be sacrificed when the estrous cycles were irregularly and the rats in control group would sacrificed with the last rat with irregularly estrous cycle.Thirty sterile female SD rat aged at 24 weeks with regular estrous cycles were also randomly and equally divided into 5 groups:3 groups were given low dose(50mg/kg/d),middle dose(100mg/kg/d)and high dose(200mg/kg/d)of BPA by intragastric administration and they were defined as low dose group at 6 months(6MLG),middle dose group at 6 months(6MMG)and high dose group at 6 months(6MHG).The fourth group was interrupted with GTT at 50mg/kg/d while the control group was give corn oil 0.1ml/100g/d(6MCG).The sacrifice time was same as previous.The general performance and vaginal smear was observed everyday.Morris water maze was used to observe the differences in learning and memory ability between different individuals.Serum E2,FSH,LH,INHB and AMH were detected by enzymelinked immunosorbent assay(ELISA).The ovary structure,viscera index and follicle numbers were evaluated by ovary serial section.Part 2:Six sterile female SD rat aged at 12 weeks with regular estrous cycles were included as control group and six sterile female SD rat aged at the same age with irregular estrous cycles founded by the best way concluded from part 1 were included in this part as experimental group.Ovaries from both groups were collected for analysis and Western Blotting was used for evaluating the levels of protein expression,including Phosphatidylinositol 3 kinase(PI3K),Protein kinase B(AKT),Phosphorylated Protein kinase B(p-AKT),Mammalian target of rapamycin(mTOR),Phosphorylated-Mammalian target of rapamycin(p-mTOR)nuclear factor-erythroid2-ralated factor(Nrf),home oxygenase-1(HO-1),Kelch-like ECH-associated protein 1(Keap1).The mRNA transcription levels of these proteins were detected by Real-time PCRPart 3:Eighteen sterile female SD rat aged at 12 weeks with irregular estrous cycles founded by the best way concluded from part 1 were randomly and equally divided into 3groups:control group(CG),Yougui Pill group(YGG)and Yangjingzhongyu Decoction group(YJZYG).Rats were given Chinese material medical prescription granule of Yougui Pill(YGG)or Yangjingzhongyu Decoction twice a day(YJZYG)and equal constant volume of distilled water was given to rats of the control group for 14 days.The general performance and vaginal smear was observed everyday.Serum E2,FSH,LH,INHB and AMH were detected by enzyme-linked immunosorbent assay(ELISA),the ovary structure,viscera index and follicle numbers would be evaluated by ovary serial section.Results:Part 1:1.General Information:The rats in the control groups were in good mental state with free and flexible activities,shiny hair,ruddy mouth,eyes,nose and ears,and normal diet.After BPA intervention,the rats in all does of BPA groups had reduced activity and prone to crouching and bunching with their hair sparsely and listless.At the beginning of the intervention,their body weight decreased to varying degrees,and then recovered by themselves.By the end of the experiment,their body weight did not increase while their drink decreased.2.The comparison of estrous cycle disturbance:The 3MCG showed no disturbance of estrous cycle(P<0.01)and no significant change were showed between 3MLG and 3MHG(P>0.05).There was a slight difference in the 3MGTTG when compared with the 3MCG(P<0.05);The 6MCG showed no disturbance of estrous cycle(P<0.01)and there were no differences between the 6MLG,6MMG and the 6MHG when compared with the 6MGTTG(P>0.05)。3.Morris water maze:The mean incubation period and the ratio of central journey were significantly longer in the 3MLG,3MMG,3MHG groups and the 3MGTTG when compared with the 3MCG and the number of crossing platforms in the 3MMG BPA group decreased significantly(P<0.01).The time of staying at the platform decreased significantly in the 3MHG and the 3MGTTG(P<0.01).Compared with the 3MGTTG,the mean incubation periods of the 3MMG and 3MHG were significantly prolonged(P<0.01).The mean velocity of the 3MLG and the 3MMG was significantly decreased(P<0.01)and the time of staying at the platform of the 3MLG was shortened(P<0.05);Compared with the 6MCG,the average incubation period of 6MLG,6MMG,6MHG and 6MGTTG were significantly prolonged(P<0.01),the average speed and the proportion of center distance in 6MMG,6MHG and 6MGTTG were significantly decreased(P<0.01).The number of crossing platform within 60 seconds in 6MHG and 6MGTTG were decreased(P<0.05).The average velocity of 6MLG,6MMG,6MHG decreased more significantly than the 6MGTTG(P<0.01).4.Ultra structure in ovary:The ovarian structure of rats in control groups was normal.Primary follicles,growing follicles and luteum visible could be seen and many layers of granulosa cells showed in the follicle,the volume of corpus luteum was regular.Decreased ovary volume and primary follicles,irregular growing follicles and increased atresia follicle were observed in the BPA low,middle,high doses and the GTT groups.The ovary index:The ovary index in the 3MMG decreased more obviously than 3MGTTG(P<0.01)and it was also lower than 3MGTTG(P<0.01).The ovary index in the 6MMG decreased more obviously than 6MGTTG(P<0.01),it was also lower than 6MGTTG(P<0.01)。5.The differences of all kinds of follicles:Compared with the 3MCG,the primordial follicles in the 3MLG,3MMG,3MHG and 3MGTTG were significantly decreased(P<0.01)and the prenatal follicles were significantly increased(P<0.01).Sinus follicles decreased significantly in 3MLG and 3MGTTG(P<0.01)while atresia follicles increased significantly in 3MMG,3MHG and 3MGTTG(P<0.01).Compared with the 3MGTTG primordial follicles decreased significantly in the 3MMG(P<0.01),prenatal follicles increased significantly(P<0.01)in the 3MLG and atresia follicles increased in the 3MHG(P<0.05).Compared with the 6MCG,the atresia follicles and the prenatal follicles increased significantly while Sinus follicles decreased significantly in the 6MLG,6MMG,6MHG and 6MGTTG(P<0.01).Compared with the 6MGTTG primordial follicles decreased significantly(P<0.01)and atresia follicles increased significantly in the 6MMG(P<0.01).6.Changes of serum hormone,INHB and AMH The level of FSH in 3MLG and 3MMG increased more than 3MCG(P<0.01).Compared with the 6MCG,the FSH increased in 6MMG,6MHG and 6MGTTG(P<0.05).The level of LH in 3MHG and 3MGTTG decreased more than 6MCG(P<0.01).Compared with the 6MCG,the LH increased obviously in 6MHG(P<0.05).The level of E2 in 3MHG decreased more than 3MCG(P<0.01).The level of E2 in 6MHG decreased much more than 6MCG(P<0.05).The level of INHB in 3MLG decreased obviously when compared with 3MCG(P<0.01).The level of INHB in the 6MLG and 6MGTTG decreased more(P<0.01)than 6MCG(P<0.05).Compared with 3MCG,the level of AMH in 3MGTTG decreased(P<0.05)while they decreased more obviously in 3MLG,3MMG and 3MHG(P<0.01).The level of AMH in 6MMG,6MHG and 6MGTTG decreased when compared with 6MCG(P<0.05).Part 2:1.The levels of the key proteins of PI3K/AKT/mTOR pathway:The level of PI3K,mTOR of the experimental group decreased obviously(P<0.01)and AKT decreased slightly(P<0.05)when compared with the control group,P-AKT in the experimental group increased obviously(P<0.01)and P-mTOR increased slightly when compared with the control group.The levels of PI3K/AKT/mTOR mRNA:The level of PI3K,mTOR mRNA increased obviously(P<0.01)and AKT mRNA increased slightly(P<0.05)in the experimental group when compared with the control group.2.The levels of key proteins of the Nrf2/ARE pathway:The level of HO-1 decreased(P<0.O5)and Keapl increased slightly(P<0.01)in the experimental group when compared with the control group.The Nrf2 showed no differences between the two groups(P>0.05).The levels of the Nrf2/ARE mRNA:The level of HO-1 decreased(P<0.05)and Keap1 mRNA increased(P<0.05)while the Nrf2 showed no difference(P>0.05)in the experimental group when compared with the control group.Part 3:1.The comparison of estrous cycle disturbance:The rats in control group stayed at disorder estrous during the experiment while one rat in Yangjingzhongyu Decoction group and 5 rats in Yougui Pill group returned to the regular estrous cycle.2.The changes of kidney deficiency symptom scores in each group:(1)Weight changes:The weight of rats in each group increased according to the day of experiment while the weight of rats in Yougui Pill group increased significantly(P<0.01).(2)Temperature changes:There was no difference in the anus temperature among all groups and it increased significantly in Yougui Pill group(P<0.01)while the Yangjingzhongyu Decoction group showed no difference at the end.(3)Intake of water:The daily water intake per 100 weight in control group showed no changes while the Yogi Pill group increased significantly(P<0.01)and slightly increase in Yangjingzhongyu Decoction group.(4)Comparison of Bristol score:The Bristol score decreased obviously in the Yougui Pill group(P<0.01)when compared with the other two groups.(5)Water labyrinth experiment:Compared with the control group,the average velocity of Yougui Pill group was significantly increased(P<0.01)and that of Yangjingzhongyu Decoction group was significantly increased(P<0.05).Compared with the control group,the proportion of central distance in Yougui Pill group was significantly increased(P<0.01).(6)The scores of kidney-yang deficiency and kidney-yin deficiency:Before treatment,the score of kidney-yang deficiency syndrome in all groups was significantly higher than kidney-yin deficiency syndrome(P<0.01).There were no changes in the score of kidney-yin deficiency syndrome in all groups and kidney-yang deficiency syndrome in control group and Yangjingzhongyu Decoction group while that of Yougui pill group changed significantly(P<0.01)after treatment.The symptoms of tiredness and body temperature were improved in Yougui Pill group when compared with Yangjingzhongyu Decoction group(P<0.05)while the water intake changed significantly(P<0.01).The score of Bristol score in Yougui pill group improved much more than control group(P<0.05)and the difference was more significantly than that of Yangjingzhongyu Decoction group(P<0.01).3.Ultra structure and ovary index:The rats showed obvious differences in pathological section of ovary.The ovary of control group rats was pale,small and hard.The ovaries of Yougui Pill group were bigger than of control group,they had ruddy color and good elasticity with multiple growth follicles on the surface.The ovaries of Yangjingzhongyu Decoction soup group were ruddy,and the volume increased slightly.There were fewer growth follicles and more poor elasticity when compared with Yougui Pill group.The ovarian index of Yougui Pill group increased more when compared with control group(P<0.05),but no significant difference was found between Yougui Pill group and Yangjingzhongyu Decoction group(P>0.05).4.The primary follicles in the Yougui Pill group increased significantly(P<0.01)and the prenatal follicles decreased significantly(P<0.01)when compared with control group.Sinus follicles increased more significantly in Yougui Pill group and Yangjingzhongyu Decoction group(P<0.01)when compared with control group.Atretic follicles of Yougui Pill group decreased more than control group(P<0.01)and Yangjingzhongyu Decoction group(P<0.05).5.The level of FSH in Yougui Pill group decreased(P<0.05)and the level of LH increased when compared with control group(P<0.05);The INHB and AMH in Yougui Pill group increased(P<0.05)when compared with control group.When compared with Yangjingzhongyu Decoction group the AMH increased less(P<0.01)and the INHB increased more in Yougui Pill group(P<0.05).Conclusions:1.The sterile female SD rat aged at 12 weeks can create a new model of Diminish Ovarian Reserve by intragastric administrating BPA for 16 days at the dose of 100mg/kg/d.2.The diminish ovarian reserve maybe caused by BPA through the PI3K/AKT/MTOR and Nrf2/ARE signal pathway.3.The type of the new model about Diminish Ovarian Reserve was kidney deficiency type with Yang deficiency.