| Section Ⅰ The Establishment of Controlled Ovarian Hyperstimulation Mouse Model and Effect of Controlled Ovarian Hyperstimulation on Puberty and Estrus in Mice OffspringObjective: To establish the controlled ovarian hyperstimulation(COH)mouse model and use this model to investigate the effect of COH on puberty transition and estrus in female mice offspring.Methods: Forty female Kunming mice were randomly divided into two groups.In the COH group,7.5 IU pregnant horse serum gonadotropin(PMSG)and 7.5 IU human chorionic gonadotropin(HCG)were used to stimulate the female mice which showed stable estrus to get the first generation of female offspring mice.In the control group,an equal amount of physiological saline was injected intraperitoneally at the same time point as a control,and caged with the male mice during estrus.After the female offspring mice were weaned,the mice from each group which had similar body mass index(BMI)were screened for the observation of vaginal opening and first estrus,and then we evaluated serum levels of E2,anti-Mullerian hormone(AMH),FSH and LH,m RNA expressions of Esr1,Amhr2,Fshr and Lhcgr in ovaries,number of follicles and ovarian histology.We also investigated the apoptosis of follicles by TUNEL;the m RNA expressions of Fas,Fas L,Bax,Bcl2,and caspase 3,8 and 9 by quantitative real-time PCR;and the protein expressions of cleaved-caspase(CASP)3,8 and 9 by Western blot.Moreover,we further observed estrous cyclicity in young adult offspring,performed follicle counting and measured the level ofAMH in both serum and ovary.Results: Compared with the normal pregnancy,COH could lead to poor pregnancy outcome in mice,manifested as prolonged gestational period,a large number of stillbirth,and increased pregnancy loss.Compared to female offspring mice in the control group,the female offspring in the COH group showed a delayed onset of their first estrus,and follicle counting showed more atretic follicles,with significantly enhanced apoptosis of the granulosa cells.In the ovary of COH female offspring,Fas L,Bax,Caspase3,8,9 m RNA levels were increased significantly,and Cleaved-caspase 8 protein level was elevated significantly as well.At the same time,we found that the serum LH and E2 levels of 28-day-old COH female mice offspring were significantly lower than those of the normal female mice offspring,and the m RNA levels of the hormone receptors such as Amhr2,Fshr,Lhcgr and Esr1 were significantly reduced.In addition,we continuously observed the estrus cyclicity of adult female offspring(56 days old)in both groups.Although the average time length of estrus cycles was the same,however,when compared with control group,the young adult female offspring in model group(56 days old)exhibited a growing percentage of days they spent in estrus and a decreased percentage of days in proestrus.Follicle counting showed that the number of primordial follicles and primary follicles in 56-day-old female offspring mice in the COH group was significantly reduced,while AMH levels in the serum and ovaries were significantly elevated.Conclusion: COH could lead to poor pregnancy outcomes and adversely affect follicular development in female offspring mice,resulting in delayed pubertal transition and subsequent disordered estrus cycle in adulthood.The mechanism may be related to hormone insufficiency,increased apoptosis of granulosa cells,and the down-regulated expression of ovarian hormone receptors.Section Ⅱ The Effect of Bushen Huoxue Recipe on Early Pregnancy Loss in Controlled Ovarian Hyperstimulation Mouse Model and the Exploration of the MechanismObjective: To observe the effect of Bushen Huoxue Recipe on the early pregnancy loss in controlled ovarian hyperstimulation mouse and to explore its potential mechanism.Methods: 200 female Kunming mice were randomly divided into four groups: Control group,Model group,BSHXR1 group and BSHXR2 group.The female mice in the model group and BSHXR groups were stimulated with 7.5 IU pregnant horse serum gonadotropin(PMSG)and 7.5 IU human chorionic gonadotropin(HCG)and then mated with male mice.The normal control group was given the same amount of normal saline intraperitoneally as the control at the same time point and mated with male mice during estrus.For all groups,a vaginal plug on the next morning was taken as evidence of successful mating and that day was considered to be day 1 of gestation(D1).Starting from D1,the mice in control group and model group were given 0.3 ml normal saline daily by oral gavage,while in BSHXR1 and BSHXR2 groups,0.3 ml normal and low dose of BSHXR were given by oral gavage.A part of pregnant mice was sacrificed on D5,D6 and D8 to obtain the serum,ovary and uterus,while some pregnant mice were housed in a single cage for the delivery to observe their pregnancy outcomes.The serum levels of estrogen and progesterone were determined by radioimmunoassay.The morphology of ovary and uterus,the formation of decidual blood vessels,the expression of ERα,PR and pro-angiogenic factors such as VEGFA,ANGPT2 and FGF2 were observed by immunohistochemistry.Real-time q PCR and Western blot methods were used to detect the m RNA and protein expressions of relatedfactors in the uterus.Results: Compared with the control group,COH mice showed significantly increased rate of early pregnancy loss after implantation,and there was a large number of stillbirths with a significant prolonged gestational period.BSHXR could reduce the pregnancy loss and maintain the survival of embryos.COH would cause the increase of ovarian volume and weight,serum levels of estrogen and progesterone,the impaired decidualization,as well as the down-regulation of hormone receptor ERα and PR in the uterus,however,BSHXR could control the hyperstimulation status of ovaries,reducing the supraphysiological levels of serum estrogen and progesterone,and upregulate the expression of ERα and PR in the epithelium and stroma,thus promoting the progress of pregnancy.In addition,BSHXR could increase the density of decidual blood vessels and promote the development and maturation of those by up-regulating the expression of FGF2 in the vascular sinus folding,thereby providing adequate blood supply to embryos and supporting the formation of placenta.Conclusion: COH could lead to the hyperstimulation status of ovary,causing supraphysiological levels of serum estrogen and progesterone,disturbing their action on their corresponding receptors in the uterus,impairing the development and maturation of decidual blood vessels,thus leading to poor pregnancy outcomes,but BSHXR could effectively reduce the early pregnancy loss after implantation and promote the establishment and maintenance of pregnancy.The mechanism may be related to the control of estrogen and progesterone,the enhancement of the expression of ERα and PR in epithelium and stroma,as well as the upregulation of FGF2 expression in the vascular sinus folding.Thus,our research provides scientific evidence for clinical application of BSHXR as the effective treatment for earlypregnancy loss,and lays the foundation for the further investigation of BSHXR. |
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