超促排卵方法制备围绝经期大鼠模型
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摘要
目的观察超促排卵方法(枸橼酸氯米芬CC灌胃)制备围绝经期大鼠模型的可行性。方法 30只SD大鼠随机分为5组,每组6只,分别为A、B、C、D、E组。从动情间期开始,A、B组大鼠连续2 d于同一时间分别灌胃枸橼酸氯米芬(CC) 5、10 mg/kg,C组大鼠连续2 d在同一时间腹腔注射孕马血清促性腺激素(PMSG) 40 IU[1],D组大鼠连续2天于同一时间灌胃生理盐水2 m L。A、B、C、D组均间隔2 d后重复同样操作,共重复12个周期。E组为去势对照组,戊巴比妥钠麻醉后开腹剪去两侧卵巢。A、B、C、D组从适应性饲养1周后开始,直至12个周期给药结束后30 d,每日进行阴道脱落细胞涂片,E组在去势术后15 d开始连续15天行阴道脱落细胞涂片,HE染色后光镜下观察并记录各组动情周期所处阶段。A、B、C、D组给药结束后15 d腹主动脉采血,E组于去势手术30 d后取腹主动脉血,ELISA法检测各组大鼠血清雌二醇(E2)、促卵泡成熟素(FSH)、卵巢功能储备(AMH)。腹主动脉采血结束后处死各组大鼠,取双侧卵巢组织,HE染色后镜下观察各组大鼠卵巢组织病理形态。结果 A组4只大鼠动情期、动情周期均延长,B组可见动情后期、动情期或动情间期的延长,以动情后期延长为主,C组主要表现为动情间期和动情后期的延长,以及动情周期逻辑顺序的改变,D组大鼠动情周期仍规律地出现,E组去势组呈持续动情间期。与D组比较,A、B组血清AMH水平均降低(P均<0. 05);与E组比较,A、B、C组血清E2水平均降低(P均<0. 05),D组血清E2水平降低,FSH升高(P均<0. 05);与C组比较,B组血清FSH水平降低(P <0. 05)。A、B组的卵巢可见生长卵泡、成熟卵泡及黄体,相比于D组,成熟卵泡数、黄体数较少,颗粒细胞层次较少; C组卵巢可见成熟卵泡及黄体,相比于D组,黄体数较多,颗粒细胞层次较少,血管较少; D组卵巢可见各级生长卵泡、成熟卵泡及黄体,黄体发育好,颗粒细胞层次多,卵泡液丰富,血管丰富。结论超促排卵方法(5、10 mg/kg的CC灌胃)可成功制备围绝经期大鼠模型。
Objective To observe the feasibility of using clomiphene citrate( CC) to prepare perimenopausal rat models. Methods Thirty SD rats were randomly divided into 5 groups: groups A,B,C,D,and E,with 6 in each. From the estrous interval,rats in the groups A and B were given 5 and 10 mg/kg CC at the same time for 2 days. Rats in the group C were intraperitoneally injected with pregnant horse serum gonadotropin( PMSG) 40 IU[1]at the same time for 2 days.Rats in the group D were given saline 2 m L at the same time for 2 days. We repeated the same operation after 2 days interval for a total of 12 cycles in the groups A,B,C and D. Group E was taken as the control group. After anesthesia with pentobarbital sodium,both ovaries were removed by laparotomy. Vaginal exfoliated cells were smeared daily in the groups A,B,C and D from 1 week after adaptive feeding until 30 days after 12 cycles of administration. Vaginal exfoliated cells were smeared at 15 days after castration in group E. The stages of estrous cycles were observed and recorded under light microscopy after HE staining. Abdominal aorta blood was collected at 15 days after administration in the groups A,B,C and D. Abdominal aorta blood was taken at 30 days after castration in the group E. Serum estradiol( E2),follicle stimulating hormone( FSH) and ovarian functional reserve( AMH) were measured by ELISA. After abdominal aorta blood collection,rats in each group were sacrificed,and their bilateral ovarian tissues were taken. The pathological morphology of ovarian tissues in each group was observed under HE staining and microscopy. Results In the group A,the estrous phase and estrous cycle of 4 rats were prolonged. In the group B,metestrus,estrus or diestrus were prolonged,the prolongation of metestrus is mainly. In the group C,the diestrus and metestrus were prolonged,and the logical sequence of estrous cycle was changed. In the group D,the estrous cycle still appeared regularly. In the group E,there was a continuous diestrus. Compared with the group D,serum AMH levels in the groups A and B decreased( both P < 0. 05); compared with the group E,the serum E2 levels in the groups A,B and C decreased( all P < 0. 05),and the serum E2 level in the group D decreased and FSH increased( both P < 0. 05); compared with the group C,the serum FSH level in the group B decreased( P <0. 05). Growing follicles,mature follicles and corpus luteum were observed in the groups A and B. Compared with the group D,the number of mature follicles and corpus luteum was less in the groups A and B,and the levels of granulosa cellswere less; the number of mature follicles and corpus luteum were observed in the group C. Compared with the group D,the number of corpus luteum was higher in the group C,and both the levels of granulosa cells and blood vessels were less in the group C. Growing follicles,mature follicles,and corpus luteum were observed in the group D. The corpus luteum developed well,and there were many layers of granulosa cells,rich follicular fluid and rich blood vessels. Conclusion The perimenopausal rat models can be successfully established by superovulation( intragastric administration of 5 and 10 mg/kg of CC).
Objective To observe the feasibility of using clomiphene citrate( CC) to prepare perimenopausal rat models. Methods Thirty SD rats were randomly divided into 5 groups: groups A,B,C,D,and E,with 6 in each. From the estrous interval,rats in the groups A and B were given 5 and 10 mg/kg CC at the same time for 2 days. Rats in the group C were intraperitoneally injected with pregnant horse serum gonadotropin( PMSG) 40 IU[1]at the same time for 2 days.Rats in the group D were given saline 2 m L at the same time for 2 days. We repeated the same operation after 2 days interval for a total of 12 cycles in the groups A,B,C and D. Group E was taken as the control group. After anesthesia with pentobarbital sodium,both ovaries were removed by laparotomy. Vaginal exfoliated cells were smeared daily in the groups A,B,C and D from 1 week after adaptive feeding until 30 days after 12 cycles of administration. Vaginal exfoliated cells were smeared at 15 days after castration in group E. The stages of estrous cycles were observed and recorded under light microscopy after HE staining. Abdominal aorta blood was collected at 15 days after administration in the groups A,B,C and D. Abdominal aorta blood was taken at 30 days after castration in the group E. Serum estradiol( E2),follicle stimulating hormone( FSH) and ovarian functional reserve( AMH) were measured by ELISA. After abdominal aorta blood collection,rats in each group were sacrificed,and their bilateral ovarian tissues were taken. The pathological morphology of ovarian tissues in each group was observed under HE staining and microscopy. Results In the group A,the estrous phase and estrous cycle of 4 rats were prolonged. In the group B,metestrus,estrus or diestrus were prolonged,the prolongation of metestrus is mainly. In the group C,the diestrus and metestrus were prolonged,and the logical sequence of estrous cycle was changed. In the group D,the estrous cycle still appeared regularly. In the group E,there was a continuous diestrus. Compared with the group D,serum AMH levels in the groups A and B decreased( both P < 0. 05); compared with the group E,the serum E2 levels in the groups A,B and C decreased( all P < 0. 05),and the serum E2 level in the group D decreased and FSH increased( both P < 0. 05); compared with the group C,the serum FSH level in the group B decreased( P <0. 05). Growing follicles,mature follicles and corpus luteum were observed in the groups A and B. Compared with the group D,the number of mature follicles and corpus luteum was less in the groups A and B,and the levels of granulosa cellswere less; the number of mature follicles and corpus luteum were observed in the group C. Compared with the group D,the number of corpus luteum was higher in the group C,and both the levels of granulosa cells and blood vessels were less in the group C. Growing follicles,mature follicles,and corpus luteum were observed in the group D. The corpus luteum developed well,and there were many layers of granulosa cells,rich follicular fluid and rich blood vessels. Conclusion The perimenopausal rat models can be successfully established by superovulation( intragastric administration of 5 and 10 mg/kg of CC).
引文
[1]王志新,张芳.超排卵对大鼠卵巢组织结构及生殖内分泌的影响[J].中国病理生理杂志,2011,27(1):2295-2296.
[2]Kilic-Okman T,Kucuk M,Altaner S. Comparison of the effects of letrozole and clomiphene citrate on ovarian follicles,endometrium,and hormone levels in the rat[J]. Fertil Steril,2003,80(6):1330-1332.
[3]Cora M,Kooistra L,Travlos G. Vaginal cytology of the laboratory rat and mouse:review and criteria for the staging of the estrous cycle using stained vaginal smears[J]. Toxicol Pathol,2015,43(6):776-793.
[4]邹移海,徐志伟,苏钢强.实验动物学[M].北京:科学出版社,2004:84.
[5] Finch C. The menopause and aging,a comparative perspective[J]. J Steroid Biochem Mol Biol,2014,142(7):132-141.
[6]Walf A,Paris J,Llaneza D,et al. Levels of 5α-reduced progesterone metabolite in the midbrain account for variability in reproductive behavior of middle-aged female rats[J]. Brain Res,2011,1379(3):137-148.
[7]Westwood F. The female rat reproductive cycle:a practical histological guide to staging[J]. Toxicol Pathol,2008,36(3):375-384.
[8]Fernandois D,Lara H,Paredes A. Blocking of beta-adrenergic receptors during the subfertile period inhibits spontaneous ovarian cyst formation in rats[J]. Horm Metab Res,2012,44(9):682-687.
[9]Cruz. G,Fernandois. D,Paredes. A. Ovarian function and reproductive senescence in the rat:role of ovarian sympathetic innervation[J]. Reproduction,2017,153(2):59-68.
[10]Pankhurst M. A putative role for anti-Müllerian hormone(AMH)in optimising ovarian reserve expenditure[J]. J Endocrinol,2017,233(1):1-13.
[11]Lin PY,Huang FJ,Kung FT,et al. Evaluation of serum anti-mullerian hormone as a biomarker of early ovarian aging in young women undergoing IVF/ICSI cycle[J]. Int J Clin Exp Pathol,2014,7(9):6245-6253.
[12]苗明三,田硕,辛卫云,等.围绝经期综合征动物模型制备规范(草案)[J].中华中医药杂志,2018,33(3):996-1000.
[13]Davison S,Bell R,Donath S,et al. Androgen levels in adult females:changes with age,menopause,and oophorectomy[J]. J Clin Endocrinol Metab,2005,90(7):3847-3853.
[2]Kilic-Okman T,Kucuk M,Altaner S. Comparison of the effects of letrozole and clomiphene citrate on ovarian follicles,endometrium,and hormone levels in the rat[J]. Fertil Steril,2003,80(6):1330-1332.
[3]Cora M,Kooistra L,Travlos G. Vaginal cytology of the laboratory rat and mouse:review and criteria for the staging of the estrous cycle using stained vaginal smears[J]. Toxicol Pathol,2015,43(6):776-793.
[4]邹移海,徐志伟,苏钢强.实验动物学[M].北京:科学出版社,2004:84.
[5] Finch C. The menopause and aging,a comparative perspective[J]. J Steroid Biochem Mol Biol,2014,142(7):132-141.
[6]Walf A,Paris J,Llaneza D,et al. Levels of 5α-reduced progesterone metabolite in the midbrain account for variability in reproductive behavior of middle-aged female rats[J]. Brain Res,2011,1379(3):137-148.
[7]Westwood F. The female rat reproductive cycle:a practical histological guide to staging[J]. Toxicol Pathol,2008,36(3):375-384.
[8]Fernandois D,Lara H,Paredes A. Blocking of beta-adrenergic receptors during the subfertile period inhibits spontaneous ovarian cyst formation in rats[J]. Horm Metab Res,2012,44(9):682-687.
[9]Cruz. G,Fernandois. D,Paredes. A. Ovarian function and reproductive senescence in the rat:role of ovarian sympathetic innervation[J]. Reproduction,2017,153(2):59-68.
[10]Pankhurst M. A putative role for anti-Müllerian hormone(AMH)in optimising ovarian reserve expenditure[J]. J Endocrinol,2017,233(1):1-13.
[11]Lin PY,Huang FJ,Kung FT,et al. Evaluation of serum anti-mullerian hormone as a biomarker of early ovarian aging in young women undergoing IVF/ICSI cycle[J]. Int J Clin Exp Pathol,2014,7(9):6245-6253.
[12]苗明三,田硕,辛卫云,等.围绝经期综合征动物模型制备规范(草案)[J].中华中医药杂志,2018,33(3):996-1000.
[13]Davison S,Bell R,Donath S,et al. Androgen levels in adult females:changes with age,menopause,and oophorectomy[J]. J Clin Endocrinol Metab,2005,90(7):3847-3853.