育麟方改善老龄雌性小鼠超促排卵作用的机制研究
|
摘要
[目的]观察育麟方对老龄导致卵巢储备功能下降(diminished ovarian reserve,DOR)小鼠卵母细胞数量与体外受精率的影响。[方法]将70只6～7月龄的C57BL/6J雌性小鼠随机分为育麟方高剂量组、育麟方中剂量组、育麟方低剂量组、脱氢表雄酮(dehydroepiandrosterone,DHEA)组和老龄对照组;另外14只C57BL/6J性成熟雌性小鼠作为青年对照组。育麟方高、中、低剂量组分别用25.0g/kg·d、12.0g/kg·d、6.0g/kg·d育麟方浓缩液灌胃,DHEA组用10.2mg/kg·d的DHEA灌胃,青年对照组和老龄对照组每日均采用蒸馏水0.2mL/10g灌胃,共用药40d。用药第38天开始腹腔序贯注射孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)10U、人绒毛膜促性腺激素(human choionic gonadotophin,HCG)10U行控制性超促排卵,每组选择7只小鼠收集并比较卵母细胞数量;余下7只与C57BL/6J青年雄性小鼠行体外受精(in vitro fertilization,IVF),观察体外受精率;收集各组小鼠卵巢组织,并采用Western blot检测β-catenin、Wnt4蛋白表达。[结果]与青年对照组小鼠比较,老龄对照组小鼠卵巢指数、卵母细胞数量和成熟卵率显著下降,差异具有统计学意义(P<0.01),受精率显著下降(P<0.01)。与老龄对照组比较,育麟方高剂量组、DHEA组小鼠卵巢指数明显升高(P<0.01),且育麟方高剂量组高于DEHA组(P<0.01);与老龄对照组比较,育麟方高剂量组、DHEA组卵母细胞数显著上升(P<0.01),且育麟方高剂量组高于DHEA组(P<0.05);育麟方高剂量组小鼠卵母细胞成熟卵率高于老龄对照组(P<0.05)。[结论]高剂量育麟方可以改善老龄所致的卵巢指数下降、增加卵母细胞数量和成熟卵率,从而提高体外受精率。
[Objective] To observe the effect of Yulin formula on the number of oocytes and in vitro fertilization(IVF) rate in aged mice with diminished ovarian reserve(DOR).[Methods] Seventy female C57 BL/6 J mice aged from 6 to 7 months were randomly divided into five groups: Yulin formula high dose group, Yulin formula middle dose group, Yulin formula low dose group, dehydroepiandrosterone(DHEA) group and aged control group; and other 14 mature female C57 BL/6 J mice were used as young control group. Yulin formula high, middle and low dose groups were given Yulin formula 25.0 g/kg·d,12.0 g/kg·d and 6.0 g/kg·d, respectively; while the DHEA group was given DHEA 10.2 mg/kg·d, young control group and aged control group were given0.2 mL/10 g distilled water daily for 40 days. Sequential intraperitoneal injection of pregnant mare serum gonadotrophin(PMSG) and human chorionic gonadotrophin(HCG) were started on the 38 th day. Oocytes were collected and compared in 7 mice in each group; 7 young male mice were fertilized in vitro to observe the fertilization rate in vitro; ovarian tissues were collected from each group and ovarian tissue was collected. The expression of β-catenin and Wnt4 was detected by Western blot. [Results] Compared with young control group, the ovarian index, the number of oocytes and the metaphase Ⅱ(MⅡ) rate of aged control group decreased significantly(P<0.01), and the fertilization rate decreased significantly(P<0.01). Compared with aged control group,the ovarian index of Yulin formula high dose group and DHEA group increased significantly( P<0.01), and it was higher in Yulin formula high dose group than DEHA group(P<0.01); the number of oocyte in Yulin formula high dose group and DHEA group increased significantly( P<0.01), and it was higher in Yulin formula high dose group than DHEA group(P<0.05); the MⅡ rate of Yulin formula high dose group was higher than aged control group(P<0.05).[Conclusion] High dose Yulin formula can increase the ovarian index, the number of oocyte and M Ⅱ rate, raise the fertilization rate of IVF.
[Objective] To observe the effect of Yulin formula on the number of oocytes and in vitro fertilization(IVF) rate in aged mice with diminished ovarian reserve(DOR).[Methods] Seventy female C57 BL/6 J mice aged from 6 to 7 months were randomly divided into five groups: Yulin formula high dose group, Yulin formula middle dose group, Yulin formula low dose group, dehydroepiandrosterone(DHEA) group and aged control group; and other 14 mature female C57 BL/6 J mice were used as young control group. Yulin formula high, middle and low dose groups were given Yulin formula 25.0 g/kg·d,12.0 g/kg·d and 6.0 g/kg·d, respectively; while the DHEA group was given DHEA 10.2 mg/kg·d, young control group and aged control group were given0.2 mL/10 g distilled water daily for 40 days. Sequential intraperitoneal injection of pregnant mare serum gonadotrophin(PMSG) and human chorionic gonadotrophin(HCG) were started on the 38 th day. Oocytes were collected and compared in 7 mice in each group; 7 young male mice were fertilized in vitro to observe the fertilization rate in vitro; ovarian tissues were collected from each group and ovarian tissue was collected. The expression of β-catenin and Wnt4 was detected by Western blot. [Results] Compared with young control group, the ovarian index, the number of oocytes and the metaphase Ⅱ(MⅡ) rate of aged control group decreased significantly(P<0.01), and the fertilization rate decreased significantly(P<0.01). Compared with aged control group,the ovarian index of Yulin formula high dose group and DHEA group increased significantly( P<0.01), and it was higher in Yulin formula high dose group than DEHA group(P<0.01); the number of oocyte in Yulin formula high dose group and DHEA group increased significantly( P<0.01), and it was higher in Yulin formula high dose group than DHEA group(P<0.05); the MⅡ rate of Yulin formula high dose group was higher than aged control group(P<0.05).[Conclusion] High dose Yulin formula can increase the ovarian index, the number of oocyte and M Ⅱ rate, raise the fertilization rate of IVF.
引文
[1]乔杰.生殖医学临床及生殖生物学基础研究促进精准辅助生殖技术发展[J].中华生殖与避孕杂志,2017,37(1):1-4.QIAO Jie.Promoting the development of precision assisted reproductive technology through clinical and fundamental reproductive biology research in reproductive medicine[J].Chin J Reprod Contracep,2017,37(1):1-4.
[2]Kamphuis E I,Van Wely M,Repping S,et al.Should the individual preterm birth risk be incorporated into the embryo transfer policy in in vitro fertilisation?a decision analysis[J].BJOG,2015,122(6):825-833.
[3]Devesa M,Tur R,Rodríguez I,et al.Cumulative live birth rates and number of oocytes retrieved in women of advanced age.a single centre analysis including 4500women≥38 years old[J].Hum Reprod,2018,33(11):2010-2017.
[4]Lin L T,Cheng J T,Wang P H,et al.Dehydroepiandrosterone as a potential agent to slow down ovarian aging[J].J Obstet Gynaecol Res,2017,43(12):1855-1862.
[5]Narkwichean A,Maalouf W,Baumgarten M,et al.Efficacy of dehydroepiandrosterone(DHEA)to overcome the effect of ovarian ageing(DITTO):A proof of principle double blinded randomized placebo controlled trial[J].Eur JObstet Gynecol Reprod Biol,2017,218:39-48.
[6]中国医师协会生殖医学专业委员会.高龄女性不孕诊治指南[J].中华生殖与避孕杂志,2017,37(2):87-100.Chinese Medical Doctor Association Reproductive Medicine Specialized Committee.Guideline for diagnosis and treatment of infertility in advanced age women[J].Chin J Reprod Contracep,2017,37(2):87-100.
[7]何嘉琳,俞佳.育麟方加减治疗不同年龄女性卵巢储备功能下降30例疗效观察[J].中国中医药科技,2011,18(6):521-522.HE Jialin,YU Jia.Effect of Yulin prescription on 30cases of ovarian reserve dysfunction in different age women[J].Chinese Journal of Traditional Medical Science and Technology,2011,18(6):521-522.
[8]张婷,何嘉琳.育麟方对肾阳虚大鼠排卵功能的影响[J].现代中西医结合杂志,2005,14(16):2111-2113.ZHANG Ting,HE Jialin.Influence of Yulin recipe on ovulation function of rats with deficiency of kidney-Yang[J].Modern Journal of Integrated Traditional Chinese and Western Medicine,2005,14(16):2111-2113.
[9]蔡彬彬,何嘉琳,王素霞.育麟方对卵巢储备功能降低大鼠颗粒细胞分泌功能的影响[J].中国实验方剂学杂志,2015,21(15):138-141.CAI Binbin,HE Jialin,WANG Suxia.Effects of Yulin decoction on secretion function of granulosa cells of ovarian reserve declined rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2015,21(15):138-141.
[10]缪晨韵,章勤,马景,等.育麟方改善老龄小鼠卵巢储备功能的实验研究[J].浙江中医杂志,2018,53(7):491-492.MIAO Chenyun,ZHANG Qin,MA Jing,et al.Experimental study of Yulin formula on improving ovarian reserve function in aged mice[J].Zhejiang J Tradit Chin Med,2018,53(7):491-492.
[11]魏伟,吴希美,李元建.药理实验方法学[M].4版.北京:人民卫生出版社,2010:1698.WEI Wei,WU Ximei,LI Yuanjian.Experimental Methodology of Pharmacology[M].4th edition.Beijing:People’s Medical Publishing House,2010:1698.
[12]Chen W C,Chen Y M,Huang C C,et al.Dehydroepiandrosterone supplementation combined with wholebody vibration training affects testosterone level and body composition in mice[J].Int J Med Sci,2016,13(10):730-740.
[13]Huang Y,Li J,Zhang F,et al.Factors affecting the live-birth rate in women with diminished ovarian reserve undergoing IVF-ET[J].Arch Gynecol Obstet,2018,298(5):1017-1027.
[14]王婉萍,陈光盛.竹林寺女科养荣丸加减联合优思敏治疗卵巢低反应不孕症疗效观察[J].中华中医药学刊,2016,34(6):1443-1445.WANG Wanping,CHEN Guangsheng.Yangrong Pill combined with Yasmin in treatment of patients with infertility due to low ovarian response[J].Chinese Archivesof Traditional Chinese Medicine,2016,34(6):1443-1445.
[15]吴清洪,顾为望,张嘉宁,等.SPF级C57BL/6J小鼠生长发育和繁殖性能指标的测定[J].中国比较医学杂志,2006,16(10):606-607.WU Qinghong,GU Weiwang,ZHANG Jianing,et al.Characterization of growth and reproductive performance in SPF C57BL/6J Mice[J].Chinese Journal of Comparative Medicine,2006,16(10):606-607.
[16]徐盛玉,吴德,王定越.卵母细胞质量评定方法[J].中国生物工程杂志,2008,28(7):116-121.XU Shengyu,WU De,WANG Dingyue.The review of assessment of oocyte quality[J].J Chin Biotechnol,2008,28(7):116-121.
[17]武之望.济阴纲目[M].北京:人民卫生出版社,2006:86.WU Zhiwang.Synopsis of Treating Women's Diseases[M].Beijing:People’s Medical Publishing House,2006:86.
[18]伏静,江华峰,李路,等.脱氢表雄酮对卵巢储备功能低下患者的胚胎质量及卵泡液中标志物的作用[J].中华生殖与避孕杂志,2017,37(8):607-613.FU Jing,JIANG Huafeng,LI Lu,et al.Effect of dehydroepiandrosterone supplementation on the embryo quality and follicular fluid markers in patients with diminished ovarian reserve[J].Chin J Reprod Contracep,2017,37(8):607-613.
[19]叶雅萍,欧奇志,覃爱平.脱氢表雄酮在卵巢储备功能低下高龄患者体外受精/卵胞质内单精子注射-胚胎移植中的应用研究[J].中华生殖与避孕杂志,2017,37(7):545-550.YE Yaping,OU Qizhi,QIN Aiping.Application of DHEA to advanced aged patients with diminished ovarian reserve in in vitro fertilization or intracytoplasmic sperm injection and embryo transfer[J].Chin J Reprod Contracep,2017,37(7):545-550.
[20]闫玮,周素,程静,等.β-连环蛋白在小鼠卵泡发育成熟过程中的作用研究[J].中国妇幼保健,2015,30(26):4536-4538.YAN Wei,ZHOU Su,CHENG Jing,et al.Study on the role ofβ-catenin during follicular development and maturation of mice[J].Maternal and Child Health Care of China,2015,30(26):4536-4538.
[21]Prunskaite-hyyryl inen R,Shan J,Railo A,et al.Wnt4,a pleiotropic signal for controlling cell polarity,basement membrane integrity,and antimullerian hormone expression during oocyte maturation in the female follicle[J].FASEBJ,2014,28(4):1568-1581.
[22]Hernandez Gifford J A.The role of WNT signaling in adult ovarian folliculogenesis[J].Reproduction,2015,150(4):R137-148.
[23]Tanwar P S,Zhang L H,Kaneko-Tarui T,et al.Mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated Wnt/β-catenin and PTEN[J].PLoS One,2011,6(6):e20715.
[24]Biason-Lauber A,Chaboissier M C.Ovarian development and disease:The known and the unexpected[J].Semin Cell Dev Biol,2015,45:59-67.
[25]Bothun A M,Woods D C.Dynamics of WNT signaling components in the human ovary from development to adulthood[J].Histochem Cell Biol,2018,2018:1-9.
[26]St-Jean G,Boyer A,Zamberlam G,et al.Targeted ablation of Wnt4 and Wnt5a in Müllerian duct mesenchyme impedes endometrial gland development and causes partial Müllerian agenesis[J].Biol Reprod,2019,100(1):49-60.
[2]Kamphuis E I,Van Wely M,Repping S,et al.Should the individual preterm birth risk be incorporated into the embryo transfer policy in in vitro fertilisation?a decision analysis[J].BJOG,2015,122(6):825-833.
[3]Devesa M,Tur R,Rodríguez I,et al.Cumulative live birth rates and number of oocytes retrieved in women of advanced age.a single centre analysis including 4500women≥38 years old[J].Hum Reprod,2018,33(11):2010-2017.
[4]Lin L T,Cheng J T,Wang P H,et al.Dehydroepiandrosterone as a potential agent to slow down ovarian aging[J].J Obstet Gynaecol Res,2017,43(12):1855-1862.
[5]Narkwichean A,Maalouf W,Baumgarten M,et al.Efficacy of dehydroepiandrosterone(DHEA)to overcome the effect of ovarian ageing(DITTO):A proof of principle double blinded randomized placebo controlled trial[J].Eur JObstet Gynecol Reprod Biol,2017,218:39-48.
[6]中国医师协会生殖医学专业委员会.高龄女性不孕诊治指南[J].中华生殖与避孕杂志,2017,37(2):87-100.Chinese Medical Doctor Association Reproductive Medicine Specialized Committee.Guideline for diagnosis and treatment of infertility in advanced age women[J].Chin J Reprod Contracep,2017,37(2):87-100.
[7]何嘉琳,俞佳.育麟方加减治疗不同年龄女性卵巢储备功能下降30例疗效观察[J].中国中医药科技,2011,18(6):521-522.HE Jialin,YU Jia.Effect of Yulin prescription on 30cases of ovarian reserve dysfunction in different age women[J].Chinese Journal of Traditional Medical Science and Technology,2011,18(6):521-522.
[8]张婷,何嘉琳.育麟方对肾阳虚大鼠排卵功能的影响[J].现代中西医结合杂志,2005,14(16):2111-2113.ZHANG Ting,HE Jialin.Influence of Yulin recipe on ovulation function of rats with deficiency of kidney-Yang[J].Modern Journal of Integrated Traditional Chinese and Western Medicine,2005,14(16):2111-2113.
[9]蔡彬彬,何嘉琳,王素霞.育麟方对卵巢储备功能降低大鼠颗粒细胞分泌功能的影响[J].中国实验方剂学杂志,2015,21(15):138-141.CAI Binbin,HE Jialin,WANG Suxia.Effects of Yulin decoction on secretion function of granulosa cells of ovarian reserve declined rats[J].Chinese Journal of Experimental Traditional Medical Formulae,2015,21(15):138-141.
[10]缪晨韵,章勤,马景,等.育麟方改善老龄小鼠卵巢储备功能的实验研究[J].浙江中医杂志,2018,53(7):491-492.MIAO Chenyun,ZHANG Qin,MA Jing,et al.Experimental study of Yulin formula on improving ovarian reserve function in aged mice[J].Zhejiang J Tradit Chin Med,2018,53(7):491-492.
[11]魏伟,吴希美,李元建.药理实验方法学[M].4版.北京:人民卫生出版社,2010:1698.WEI Wei,WU Ximei,LI Yuanjian.Experimental Methodology of Pharmacology[M].4th edition.Beijing:People’s Medical Publishing House,2010:1698.
[12]Chen W C,Chen Y M,Huang C C,et al.Dehydroepiandrosterone supplementation combined with wholebody vibration training affects testosterone level and body composition in mice[J].Int J Med Sci,2016,13(10):730-740.
[13]Huang Y,Li J,Zhang F,et al.Factors affecting the live-birth rate in women with diminished ovarian reserve undergoing IVF-ET[J].Arch Gynecol Obstet,2018,298(5):1017-1027.
[14]王婉萍,陈光盛.竹林寺女科养荣丸加减联合优思敏治疗卵巢低反应不孕症疗效观察[J].中华中医药学刊,2016,34(6):1443-1445.WANG Wanping,CHEN Guangsheng.Yangrong Pill combined with Yasmin in treatment of patients with infertility due to low ovarian response[J].Chinese Archivesof Traditional Chinese Medicine,2016,34(6):1443-1445.
[15]吴清洪,顾为望,张嘉宁,等.SPF级C57BL/6J小鼠生长发育和繁殖性能指标的测定[J].中国比较医学杂志,2006,16(10):606-607.WU Qinghong,GU Weiwang,ZHANG Jianing,et al.Characterization of growth and reproductive performance in SPF C57BL/6J Mice[J].Chinese Journal of Comparative Medicine,2006,16(10):606-607.
[16]徐盛玉,吴德,王定越.卵母细胞质量评定方法[J].中国生物工程杂志,2008,28(7):116-121.XU Shengyu,WU De,WANG Dingyue.The review of assessment of oocyte quality[J].J Chin Biotechnol,2008,28(7):116-121.
[17]武之望.济阴纲目[M].北京:人民卫生出版社,2006:86.WU Zhiwang.Synopsis of Treating Women's Diseases[M].Beijing:People’s Medical Publishing House,2006:86.
[18]伏静,江华峰,李路,等.脱氢表雄酮对卵巢储备功能低下患者的胚胎质量及卵泡液中标志物的作用[J].中华生殖与避孕杂志,2017,37(8):607-613.FU Jing,JIANG Huafeng,LI Lu,et al.Effect of dehydroepiandrosterone supplementation on the embryo quality and follicular fluid markers in patients with diminished ovarian reserve[J].Chin J Reprod Contracep,2017,37(8):607-613.
[19]叶雅萍,欧奇志,覃爱平.脱氢表雄酮在卵巢储备功能低下高龄患者体外受精/卵胞质内单精子注射-胚胎移植中的应用研究[J].中华生殖与避孕杂志,2017,37(7):545-550.YE Yaping,OU Qizhi,QIN Aiping.Application of DHEA to advanced aged patients with diminished ovarian reserve in in vitro fertilization or intracytoplasmic sperm injection and embryo transfer[J].Chin J Reprod Contracep,2017,37(7):545-550.
[20]闫玮,周素,程静,等.β-连环蛋白在小鼠卵泡发育成熟过程中的作用研究[J].中国妇幼保健,2015,30(26):4536-4538.YAN Wei,ZHOU Su,CHENG Jing,et al.Study on the role ofβ-catenin during follicular development and maturation of mice[J].Maternal and Child Health Care of China,2015,30(26):4536-4538.
[21]Prunskaite-hyyryl inen R,Shan J,Railo A,et al.Wnt4,a pleiotropic signal for controlling cell polarity,basement membrane integrity,and antimullerian hormone expression during oocyte maturation in the female follicle[J].FASEBJ,2014,28(4):1568-1581.
[22]Hernandez Gifford J A.The role of WNT signaling in adult ovarian folliculogenesis[J].Reproduction,2015,150(4):R137-148.
[23]Tanwar P S,Zhang L H,Kaneko-Tarui T,et al.Mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated Wnt/β-catenin and PTEN[J].PLoS One,2011,6(6):e20715.
[24]Biason-Lauber A,Chaboissier M C.Ovarian development and disease:The known and the unexpected[J].Semin Cell Dev Biol,2015,45:59-67.
[25]Bothun A M,Woods D C.Dynamics of WNT signaling components in the human ovary from development to adulthood[J].Histochem Cell Biol,2018,2018:1-9.
[26]St-Jean G,Boyer A,Zamberlam G,et al.Targeted ablation of Wnt4 and Wnt5a in Müllerian duct mesenchyme impedes endometrial gland development and causes partial Müllerian agenesis[J].Biol Reprod,2019,100(1):49-60.