玻璃化冻融不同时段rFSH联合rLH干预对小鼠卵巢VEGF及Cx37表达的影响
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摘要
目的通过在玻璃化冻存的不同时段给予小鼠卵巢组织重组人卵泡刺激素(recombinant follicle-stimulating hormone,rFSH)与重组人黄体生成素(recombinant luteinizing hormone,rLH)共同干预,观察对冻融小鼠卵巢组织的形态学、血管内皮生长因子(vascular endothelial growth factor,VEGF)及缝隙连接蛋白(connexin-37,Cx37)表达的影响。方法将4周龄C57BL/6J小鼠卵巢组织随机分为新鲜对照组(FCG)、玻璃化冻存对照组(VCG)、冻存全程添加rFSH+rLH组(OG-rFSH+rLH)、冻存前添加rFSH+rLH组(BG-rFSH+rLH)以及冻存后添加rFSH+rLH组(AG-rFSH+rLH),干预组浓度均为0.15IU·mL~(-1) rFSH+0.15IU·mL~(-1) rLH。通过形态学、免疫组织化学技术观察并分析各组卵巢组织形态结构及VEGF、Cx37蛋白表达量的改变;通过Realtime PCR技术检测并分析各组VEGF及Cx37 mRNA的表达变化。结果正常卵泡百分比在VCG组最低,OG-rFSH+rLH组最高(P<0.05),在BG-rFSH+rLH组低于OG-rFSH+rLH组,高于FCG组、VCG组及AG-rFSH+rLH组(P<均0.05);干预组VEGF蛋白表达量由高到低依次为OG-rFSH+rLH组、BG-rFSH+rLH组、AGrFSH+rLH组,且均高于VCG组(P<0.05);Cx37蛋白表达量及VEGF mRNA表达量均为OG-rFSH+rLH组最高,VCG组最低(P<0.05);Cx37 mRNA表达量在OG-rFSH+rLH组最高,VCG组最低,FCG组高于VCG组,低于BG-rFSH+rLH组和AG-rFSH+rLH组(P均<0.05)。结论玻璃化冻存过程添加rFSH+rLH的干预模式提升卵泡存活率、VEGF、Cx37蛋白及mRNA的表达,最佳干预效果依次为全程干预、冻存前时段干预及冻存后时段干预。
Objective To observe the morphological changes of ovary and the expression of VEGF and Cx37 after rFSH+rLH intervention at different steps of vitrification. Methods Four-week-old C57 BL/6 J mice were randomly divided into five groups:fresh control group(FCG),vitrification control group(VCG),rFSH+rLH intervention in the overall process of vitrification(OG-rFSH+rLH)group,rFSH+rLH intervention in the before process of vitrification(BG-rFSH+rLH) group and rFSH+rLH intervention in the after process of vitrification(AG-rFSH+rLH) group. The intervention concentration was 0.15 IU·m L-1 rFSH +0.15 IU·m L-1 rLH,with 30 ovary samples in each group. The changes of morphological structure of ovary,the expression of VEGF and Cx37 protein and m RNA was observed in each group by immunohistochemicaltechnique and Real-time PCR. Results The percentage of normal follicles wasthe lowest in VCG group,the highest in OG-rFSH+rLH group and the BG-rFSH+rLH group was lower than OG-rFSH+rLH group but higher than FCG group,VCG group and AG-rFSH+rLH group(P<0.05). The expression of VEGFprotein at intervention group from the highest to the lowest in turn was OG-rFSH+rLH group,BG-rFSH+rLH group,AG-rFSH+rLH group and it was respectively higher than VCG group(P<0.05). The expression of Cx37 protein and VEGF m RNA was the highest in OG-rFSH+rLH group and the lowest in VCG group(P<0.05);The expression of Cx37 m RNA is the highest in OG-rFSH+rLH group and the lowest in VCG group,and FCG group wass higher than that of VCG group,lower than BG-rFSH+rLH group and AG-rFSH+rLH group(P<0.05). Conclusion rFSH+ rLH intervention in the process of vitrification is in favor of improving the follicular survival and the expression of protein and m RNA of VEGF and Cx37. And the optimal intervention effect is in order of OG-rFSH+rLH group、BG-rFSH+rLH group and AG-rFSH+rLH group.
Objective To observe the morphological changes of ovary and the expression of VEGF and Cx37 after rFSH+rLH intervention at different steps of vitrification. Methods Four-week-old C57 BL/6 J mice were randomly divided into five groups:fresh control group(FCG),vitrification control group(VCG),rFSH+rLH intervention in the overall process of vitrification(OG-rFSH+rLH)group,rFSH+rLH intervention in the before process of vitrification(BG-rFSH+rLH) group and rFSH+rLH intervention in the after process of vitrification(AG-rFSH+rLH) group. The intervention concentration was 0.15 IU·m L-1 rFSH +0.15 IU·m L-1 rLH,with 30 ovary samples in each group. The changes of morphological structure of ovary,the expression of VEGF and Cx37 protein and m RNA was observed in each group by immunohistochemicaltechnique and Real-time PCR. Results The percentage of normal follicles wasthe lowest in VCG group,the highest in OG-rFSH+rLH group and the BG-rFSH+rLH group was lower than OG-rFSH+rLH group but higher than FCG group,VCG group and AG-rFSH+rLH group(P<0.05). The expression of VEGFprotein at intervention group from the highest to the lowest in turn was OG-rFSH+rLH group,BG-rFSH+rLH group,AG-rFSH+rLH group and it was respectively higher than VCG group(P<0.05). The expression of Cx37 protein and VEGF m RNA was the highest in OG-rFSH+rLH group and the lowest in VCG group(P<0.05);The expression of Cx37 m RNA is the highest in OG-rFSH+rLH group and the lowest in VCG group,and FCG group wass higher than that of VCG group,lower than BG-rFSH+rLH group and AG-rFSH+rLH group(P<0.05). Conclusion rFSH+ rLH intervention in the process of vitrification is in favor of improving the follicular survival and the expression of protein and m RNA of VEGF and Cx37. And the optimal intervention effect is in order of OG-rFSH+rLH group、BG-rFSH+rLH group and AG-rFSH+rLH group.
引文
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[11] Ali A,Sophie AC,Waljit SD. Novel concepts for inducing final oocyte maturation in in vitro fertilization treatment[J]. Endocr Rev.2018,39(5):593-628.
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[13] Trau HA,Brannstrom M,Curry TJ al. Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells[J]. Hum Reprod,2016,31(2):11082-11089.
[14] Camille L,Amir M,Mindy S,et al. Recent advances in the field of ovarian tissue cryopreservation and opportunities for research[J]. J Assist Reprod Genet,2017,34(6):709-722.
[15] Prakash V,Aleksander SP,Feilim M,et al. Extracellular regulation of VEGF:isoforms,proteolysis,and vascular patterning[J]. Cytokine Growth Factor Rev,2014,25(1):1-19.
[16] Li SH,Hwu YM,Lu CH,et al. VEGF and FGF2improve revascularization,survival,and oocyte quality of cryopreserved,subcutaneously-transplanted mouse ovarian tissues[J]. IJMS,2016,17(8):1237.
[17] Penna C,Perrelli MG,Karam JP,et al. Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival[J]. J Cell Mol Med,2013,17(1):192-204.
[18] Rossi V,Lispi M,Longobardi S,et al. LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse[J]. Cell Death Differ,2017,24(1):72-82.
[2] Ma WZ,Zheng XM,Hei CC,et al. Optimal FSH usage in revascularization of allotransplanted ovarian tissue in mice[J]. J Ovarian Res,2017,10:5.
[3] Meirow D,Ra'anani H,Biderman H. Ovarian tissue cryopreservation and transplantation:a realistic,effective technology for fertility preservation[J]. Methods in Molecular Biology,2014,1154:455-473.
[4] Zhang H,Yang Y,Ma W,et al. The revascularization and follicular survival of mouse ovarian grafts treated with FSH during cryopreservation by vitrification[J].Cryo Letters,2016,37(2):88-102.
[5] Kang BJ,Wang Y,Zhang L,et al. bFGF and VEGF improve the quality of vitrified-thawed human ovarian tissues after xenotransplantation to SCID mice[J].Journal of Assisted Reproduction and Genetics,2016,33(2):281-289.
[6] Labied S,Delforge Y,Munaut C,et al. Isoform 111 of vascular endothelial growth factor(VEGF111)improves angiogenesis of ovarian tissue xenotransplantation[J].Transplantation,2013,95(3):426-433.
[7] Hattori K,Orisaka M,Fukuda S,et al. Luteinizing hormone facilitates antral follicular maturation and survival via thecal paracrine signaling in cattle[J].Endocrinology,2018,8(3):128-129.
[8] Anne B,Jerome G,Geri M,et al. A common African variant of human connexin 37 is associated with Caucasian primary ovarian insufficiency and has a deleterious effect in vitro[J]. Int J Mol Med,2017,41(2):640-648.
[9] Wang YR,Chang Q,Sun J,et al. Effects of HMG on revascularization and follicular survival in autotransplant mouse ovarian tissue[J]. Reproductive BioMedicine Online,2012,24(6):646-653.
[10]靳镭,徐蓓.黄体生成素在卵泡发育中的作用及卵泡期黄体生成素预处理[J].生殖医学杂志,2014,23(12):940-943.
[11] Ali A,Sophie AC,Waljit SD. Novel concepts for inducing final oocyte maturation in in vitro fertilization treatment[J]. Endocr Rev.2018,39(5):593-628.
[12]李媛.外源性黄体生成素补治疗在超排卵中的应用[J].生殖医学杂志,2013,22(10):738-742.
[13] Trau HA,Brannstrom M,Curry TJ al. Prostaglandin E2 and vascular endothelial growth factor A mediate angiogenesis of human ovarian follicular endothelial cells[J]. Hum Reprod,2016,31(2):11082-11089.
[14] Camille L,Amir M,Mindy S,et al. Recent advances in the field of ovarian tissue cryopreservation and opportunities for research[J]. J Assist Reprod Genet,2017,34(6):709-722.
[15] Prakash V,Aleksander SP,Feilim M,et al. Extracellular regulation of VEGF:isoforms,proteolysis,and vascular patterning[J]. Cytokine Growth Factor Rev,2014,25(1):1-19.
[16] Li SH,Hwu YM,Lu CH,et al. VEGF and FGF2improve revascularization,survival,and oocyte quality of cryopreserved,subcutaneously-transplanted mouse ovarian tissues[J]. IJMS,2016,17(8):1237.
[17] Penna C,Perrelli MG,Karam JP,et al. Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival[J]. J Cell Mol Med,2013,17(1):192-204.
[18] Rossi V,Lispi M,Longobardi S,et al. LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse[J]. Cell Death Differ,2017,24(1):72-82.