A comparative study of cyclophosphamide with different doses and withdrawal cycles in constructing animal model of premature ovarian failure in mice
Objective:To compare the animal models of premature ovarian failure(POF)in mice constructed by cyclophosphamide(CTX)with different doses and withdrawal cycles for exploring the best model method,so as to provide a more suitable experimental model for further research on premature ovarian failure.Methods:Female C57BL/6 mice with a regular estrous cycle in 8-week-old were randomly divided into five groups:CON group(100 mg·kg-1·d-1),CTX80 group(80 mg·kg-1·d-1),CTX100 group(100 mg·kg-1·d-1),CTX120 group(120 mg·kg-1·d-1),CTX150 group(150 mg·kg-1·d-1).The mice underwent intraperitoneal injection for 10 days,those of the CON group were injected with saline and those in other groups were injected with CTX.The body weight,estrous cycle and ovarian index of the mice were recorded,and the optimal exposure dose of CTX in the model of CTX-induced POF was determined by comparison.Subsequently,female C57BL/6 mice with a regular estrous cycle in 8-week-old that were remodeled with the optimal exposure dose of CTX and randomly divided into the model group and the control group.In the 1st,2nd,3rd,and 4th weeks after drug withdrawal,serum levels of sex hormones were measured,and follicles at all levels were counted by HE staining of the ovaries,which were compared to derive the optimal withdrawal cycle for the CTX-induced POF model.Results:(1)Two mice in CTX150 group died during the modeling process,those in CTX80 group still had a full estrous cycle,which indicating that these two doses were not optimal.Mice in other CTX groups showed a progressive decrease in body weight during the exposure process.The body weight of the mice began to rebound after the cessation of the exposure,but they were still lower than that of CON group on the 7th day of the cessation of cyclophosphamide(P＜0.05).The ovarian indexes in CTX groups were significantly lower than that of CON group(P＜0.01)and that of CTX100 was the lowest on day 7 of drug cessation(P＜0.001),but the differences among CTX groups were not statistically significant(P＞0.05).The ethical requirement of minimizing the harm of the drug to the experimental animal was taken into account,which made a conclusion that the optimal cyclophosphamide exposure dose for the cyclophosphamide-induced premature ovarian failure model was 100 mg·kg-1·d-1× 10 d.(2)Compared with the control group,serum levels of anti-Mullerian hormone,estradiol,primordial follicle,and primordial/secondary follicle in the model group were significantly lower than that in the control group in the same period at the 1st,2nd,3rd,and 4th weeks after discontinuation of the drug(P＜0.05).The levels of follicular stimulating hormone were significantly higher(P＜0.05)in the 1st,3rd,and 4th weeks than those of the control group in the same period,while atretic follicle counts were significantly higher(P＜0.05)in the 1st,2nd,and 4th weeks than those of the control group in the same period,which all showed the typical characteristics of POF.With the prolongation of CTX withdrawal cycle,the counts of primordial follicles,primary/secondary follicles and mature follicles in the model group of mice showed a progressive decrease,and the counts of atretic follicles increased(P＜0.05),indicating that the damage of ovarian function caused by cyclophosphamide was progressively aggravated.The results showed that cyclophosphamide cessation in the 1st week was the optimal modeling cycle for inducing premature ovarian failure.Conclusions:The evidence suggests that 100 mg·kg-1·d-1 of cyclophosphamide using for 10 d is the optimal modeling dose to establish a cyclophosphamide-induced premature ovarian failure model in mice,and the optimal modeling cycle is the 1st week after rescission of the cyclophosphamide.
万方数据
维普
