Preservation Of GnRHa On Ovarian Function Injury Induced By Cyclophosphamide In Mice

Part Ⅰ Establishment of mouse ovarian function injury model with cyclophosphamideObjective: To explore the appropriate dosage of cyclophosphamide for establishing an ovarian injury model in mice.Methods: Twenty-five 7-week-old Kunming mice with normal estrous cycle were randomly divided into five groups: group A(saline control),group B(CTX 50 mg·kg-1·d-1),and group C(CTX 100 mg·kg-1·d-1),D(CTX 150 mg·kg-1·d-1),and E(CTX 200 mg·kg-1·d-1)were all injected intraperitoneally for 7 days.Mice were examined daily for vaginal cytology,and daily body weights and general conditions such as diet,activity,and hair were recorded.After 10 days of drug withdrawal,the bilateral ovaries of the mice were taken for ovarian histopathology to observe the ovarian tissue morphology and count the follicles.Results: The mice in each group that used cyclophosphamide showed a decrease in body weight(P<0.05,compared with the control group)and the deterioration of general condition,and the estrous cycle became disordered.Cyclophosphamide altered the ovarian structure and increased the number of atresia follicles(P<0.05,comparison between the A,B,and C groups)and showed a dose-related effect.During the experimental period(including during administration and after cessation of administration),the death of the experimental animals occurred in groups D and E.Conclusion: A dose of 100 mg·kg-1·d-1 of cyclophosphamide was injected intraperitoneally for 7 consecutive days to establish a model of ovarian function injury in mice,which was more effective and did not cause the death of the experimental mice.Part Ⅱ Observation of the inhibitory effect of GnRHa on ovarian function in mice at the selected doseObjective: To observe whether the selected dose of GnRHa is sufficient to produce significant ovarian function suppression,which provides a basis for the dose of GnRHa used in subsequent experiments.Methods: Twelve 7-week-old female Kunming mice with normal estrous cycle were randomly divided into two groups: normal saline control group and GnRHa 1mg· kg-1·d-1 group,which were injected subcutaneously for 7 days.Mice were subjected to vaginal cytology every day,and daily body weights and general conditions such as diet and hair were recorded until 10 days after drug withdrawal.On the 7th day of drug administration and the 10 th day after drug withdrawal,3 mice were taken from each group to collect blood by the method of enucleation.The concentrations of FSH and E2 in the serum of the mice were measured by ELISA.Results: There was no significant effect on the body weight and general condition of mice after GnRHa injection.Under the GnRHa dose of 1mg· kg-1·d-1,the estrus cycle of the mice was fully expressed as anestrum,and the concentrations of FSH and E2 were decreased.After discontinuation of the drug,the mice re-entered the estrus cycle and the levels of FSH and E2 recovered.Conclusion: When the dose of GnRHa is 1mg· kg-1·d-1,significant ovarian function inhibition can be produced,and this effect is reversible after drug withdrawal.Part Ⅲ To investigate the protective effect of GnRHa on ovarian function impairment induced by cyclophosphamideObjective: To investigate whether GnRHa has a protective effect on cyclophosphamide-induced impairment of ovarian function in mice by using GnRHa in combination with CTX.Methods: Forty-five 7-week-old female Kunming mice with normal estrous cycle were randomly divided into three groups: group A(saline control group),group B(CTX group),group C(group GnRHa+CTX).In group A,normal saline was injected on days 1-14.On the 8th day of group B,CTX was injected intraperitoneally at a dose of 100 mg·kg-1·d-1 for 7 consecutive days.Group C was given a continuous subcutaneous injection of GnRHa at a dose of 1 mg·kg-1·d-for 14 days,and CTX was injected intraperitoneally with a dose of 100 mg·kg-1·d-1 for 7 days from the 8th day.Mice were examined daily for vaginal cytology,and daily body weights and general conditions such as diet,activity,and hair were recorded.On the 10 th day after discontinuation,blood was collected by enucleation and the concentrations of FSH and E2 in the serum were determined by ELISA,and the bilateral ovaries of the mice were taken for ovarian histopathology to observe the ovarian tissue morphology and count the follicles.Results: In the group B(CTX group),The mice showed a decrease in body weight(P<0.05,compared with group A)and the deterioration of general condition,but they recovered after discontinuation.The estrous cycle was disordered and the number of atresia follicles was increased(P<0.05,compared with group A and C).The levels of E2 decreased and FSH increased(P < 0.05,compared with group A and group C).In the group C(GnRHa+CTX group),general condition deterioration,weight loss(P<0.05)after using CTX,and gradual recovery after withdrawal were also observed,but the number of atresia follicles in the group was lower than that in the group B(CTX group)(P<0.05).The levels of E2 increased while FSH decreased(P < 0.05 compared with group B).Compared with the group A(control group),the number of atresia follicles was higher,the level of E2 was lower and FSH was higher(P < 0.05).Conclusions: CTX can damage the ovarian function of mice,and GnRHa has a protective effect on the damage of ovarian function caused by CTX.