程序化冷冻前后小鼠正常孵化囊胚和休眠胚胎中C3基因表达的研究
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摘要
试验旨在研究程序化冷冻前后小鼠正常孵化囊胚和休眠胚胎中C3蛋白的分布及转录水平的差异表达情况,为阐明哺乳动物胚胎在抗冻过程中的相关调控机制提供理论依据。试验选用ICR系雌性小鼠,从妊娠第5天小鼠子宫回收正常孵化囊胚;构建延迟着床模型获取小鼠休眠胚胎,利用激光共聚焦和实时荧光定量PCR技术对各组胚胎进行细胞免疫荧光和C3mRNA相对表达量的检测。结果发现,程序化冷冻前后的正常孵化囊胚和休眠胚胎中C3在转录和翻译水平均有表达;正常孵化囊胚经冷冻处理后C3mRNA相对表达量显著上调(P<0.05);休眠胚胎冷冻后C3mRNA相对表达量与冷冻前相比显著下调(P<0.05);冷冻前休眠胚胎C3mRNA相对表达量显著高于冷冻前正常孵化囊胚(P<0.05);冷冻后休眠胚胎C3mRNA相对表达量显著低于冷冻后正常孵化囊胚(P<0.05)。结果表明,胚胎中C3基因在转录水平的下调表达对胚胎抗冻具有一定的积极作用,C3基因很可能参与了胚胎抗冻过程的相关调控。
The study was aimed to investigate the protein location and the transcriptional expression of C3 on mouse normal hatched blastocysts and dormant embryos before and after cryopreservation,and to provide theoretical basis for elucidating the regulation mechanism of mammalian embryos during antifreeze.The ICR female mouse were selected,normal hatched blastocysts were collected from 5th pregnant mouse and dormant embryos were obtained from mouse delayed implantation model,then the confocal and Real-time PCR were used to detect ICC and relative expression of C3 mRNA on each group.The results showed that C3 were both expressed on transcriptional and translational levels in mouse normal hatched blastocysts and dormant embryos before and after cryopreservation;The relative expression of C3 mRNA in normal hatched blastocysts was significantly up-regulated after cryopreservation(P<0.05);The relative expression of C3 mRNA in dormant embryos was significantly down-regulated after cryopreservation(P<0.05);The relative expression of C3 mRNA in dormant embryos before cryopreservation was significantly higher thanthe normal hatched blastocysts before cryopreservation(P<0.05);The relative expression of C3 mRNA in dormant embryos after cryopreservation was significantly lower than the normal hatched blastocysts after cryopreservation(P<0.05).The results showed that the transcriptional down-regulated expression of C3 in embryos had a positive effect on the antifreeze of embryos,and C3 gene might be involved in the regulation of embryo freezing-thawing process.
The study was aimed to investigate the protein location and the transcriptional expression of C3 on mouse normal hatched blastocysts and dormant embryos before and after cryopreservation,and to provide theoretical basis for elucidating the regulation mechanism of mammalian embryos during antifreeze.The ICR female mouse were selected,normal hatched blastocysts were collected from 5th pregnant mouse and dormant embryos were obtained from mouse delayed implantation model,then the confocal and Real-time PCR were used to detect ICC and relative expression of C3 mRNA on each group.The results showed that C3 were both expressed on transcriptional and translational levels in mouse normal hatched blastocysts and dormant embryos before and after cryopreservation;The relative expression of C3 mRNA in normal hatched blastocysts was significantly up-regulated after cryopreservation(P<0.05);The relative expression of C3 mRNA in dormant embryos was significantly down-regulated after cryopreservation(P<0.05);The relative expression of C3 mRNA in dormant embryos before cryopreservation was significantly higher thanthe normal hatched blastocysts before cryopreservation(P<0.05);The relative expression of C3 mRNA in dormant embryos after cryopreservation was significantly lower than the normal hatched blastocysts after cryopreservation(P<0.05).The results showed that the transcriptional down-regulated expression of C3 in embryos had a positive effect on the antifreeze of embryos,and C3 gene might be involved in the regulation of embryo freezing-thawing process.
引文
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[2]顾美超.冻融后小鼠休眠胚胎超微结构变化的研究[J].中国实验动物学报,2014,22(3):53-56.
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[7]翟椿东,顾美超,刘迪,等.通过优化超排后合笼时间高效获取小鼠孵化囊胚[J].中国畜牧兽医,2015,42(5):1253-1258.
[8]岳峰.猪PD-1及其配体PD-L1和PD-L2SYBR GreenⅠReal-time PCR检测方法的建立及应用[J].中国畜牧兽医,2016,43(11):2892-2899.
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[13]Buhi W C,Alvarez I M.Identification,characterization and localization of three proteins expressed by the porcine oviduct[J].Theriogenology,2003,60(2):225-238.
[14]Jin M,Larsson A,Nilsson B O.A functionally active complement system is present in uterine secretion of the mouse prior to implantation[J].American Journal of Reproductive Immunology,1991,26(2):53-57.
[15]Price R J,Boettcher B.The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm-immobilizing antibodies[J].Fertility and Sterility,1979,32(1):61-66.
[16]Daffern P J,Pfeifer P H,Ember J A,et al.C3ais a chemotaxin for human eosinophils but not for neutrophils.Ⅰ.C3astimulation of neutrophils is secondary to eosinophil activation[J].Journal of Experimental Medicine,1995,181(6):2119-2127.
[17]Rothman B,Despins A,Webb S,et al.Cytokine regulation of C3and C5production by human corneal fibroblasts[J].Experimental Eye Research,1991,53(3):353-361.
[18]Katz Y,Strunk R C.Synovial fibroblast-like cells synthesize seven proteins of the complement system[J].Arthritis and Rheumatology,1988,31(11):1365-1370.
[19]Welch T R,Beischel L S,Witte D P.Differential expression of complement C3and C4in the human kidney[J].Journal of Clinical Investigation,1993,92(3):1451-1458.
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[21]Ault B H,Colten H R.Cellular specificity of murine renal C3expression in two models of inflammation[J].Immunology,1994,81(4):655-660.
[22]Choy L N,Rosen B S,Spiegelman B M.Adipsin and an endogenous pathway of complement from adipose cells[J].Journal of Biological Chemistry,1992,267(18):12736-12741.
[23]Georgiou A S,Snijders A P,Sostaric E,et al.Modulation of the oviductal environment by gametes[J].Journal of Proteome Research,2007,6(12):4656-4666.
[24]White R T,Damm D,Hancock N,et al.Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue[J].Journal of Biological Chemistry,1992;267(13):9210-9213.
[25]Strey C W,Markiewski M,Mastellos D,et al.The proinflammatory mediators C3aand C5aare essential for liver regeneration[J].Journal of Experimental Medicine,2003,198(6):913-923.
[26]Matthews K W,Drouin S M,Liu C,et al.Expression of the third complement component(C3)and carboxypeptidase N small subunit(CPN1)during mouse embryonic development[J].Developmental and Comparative Immunology,2004,28(6):647-655.
[27]Markiewski M M,Mastellos D,Tudoran R,et al.C3aand C3bactivation products of the third component of complement(C3)are critical for normal liver recovery after toxic injury[J].Journal of Immunology,2004,173(2):747-754.
[28]Circolo A,Garnier G,Fukuda W,et al.Genetic disruption of the murine complement C3promoter region generates deficient mice with extrahepatic expression of C3 mRNA[J].Immunopharmacology,1999,42(1-3):135-149.
[29]Menke T M,Mclaren A.Carbon dioxide production by mouse blastocysts during lactational delay of implantation or after ovariectomy[J].Journal of Endocrinology,1970,47(3):287-294.
[30]Xu C,Mao D,Holers V M,et al.A critical role for murine complement regulator Crry in fetomaternal tolerance[J].Science,2000,287(5452):498-501.
[31]Paria B C,Das S K,Andrews G K,et al.Expression of the epidermal growth factor receptor gene is regulated in mouse blastocysts during delayed implantation[J].Proceedings of the National Academy of Sciences,1993,90(1):55-59.
[32]Paria B C,Huethudson Y M,Dey S K.Blastocyst’s state of activity determines the“window”of implantation in the receptive mouse uterus[J].Proceedings of the National Academy of Sciences,1993,90(21):10159-10162.
[2]顾美超.冻融后小鼠休眠胚胎超微结构变化的研究[J].中国实验动物学报,2014,22(3):53-56.
[3]张劭俣,刘云海,倪和民,等.经程序化冷冻的小鼠休眠胚胎的基因表达谱差异分析[J].中国实验动物学报,2012,20(5):15-20.
[4]Perricone R,Pasetto N,Carolis C D,et al.Functionally active complement is present in human ovarian follicular fluid and can be activated by seminal plasma[J].Clinical Experimental Immunology,1992,89(1):154-157.
[5]Lee Y L,Lee K F,Xu J S,et al.The embryotrophic activity of oviductal cell-derived complement C3band iC3b,a novel function of complement protein in reproduction[J].Journal of Biological Chemistry,2004,279(13):12763-12768.
[6]Katz Y,Strunk R C.Synovial fibroblast-like cells synthesize seven proteins of the complement system[J].Arthritisand Rheumatology,1988,31(11):1365-1370.
[7]翟椿东,顾美超,刘迪,等.通过优化超排后合笼时间高效获取小鼠孵化囊胚[J].中国畜牧兽医,2015,42(5):1253-1258.
[8]岳峰.猪PD-1及其配体PD-L1和PD-L2SYBR GreenⅠReal-time PCR检测方法的建立及应用[J].中国畜牧兽医,2016,43(11):2892-2899.
[9]Kader A,Agarwal A,Abdelrazik H,et al.Evaluation of post-thaw DNA integrity of mouse blastocysts after ultrarapid and slow freezing[J].Fertility and Sterility,2009,91(5):2087-2094.
[10]Nilsson G,Johnell M,Hammer C H,et al.C3aand C5aare chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway[J].Journal of Immunology,1996,157(4):1693-1698.
[11]Mantzavinos T,Dalamanga N,Hassiakos D,et al.Immunoglobulins IgG,IgA,IgM,complement C3,C4and ferritin and transferrin levels in serum and follicular fluid in IVF patients[J].Clinical and Experimental Obstetrics and Gynecology,1993,20(1):32-36.
[12]Rooney I A,Oglesby T J,Atkinson J P,et al.Complement in human reproduction:activation and control[J].Immunologic Research,1993,12(3):276-294.
[13]Buhi W C,Alvarez I M.Identification,characterization and localization of three proteins expressed by the porcine oviduct[J].Theriogenology,2003,60(2):225-238.
[14]Jin M,Larsson A,Nilsson B O.A functionally active complement system is present in uterine secretion of the mouse prior to implantation[J].American Journal of Reproductive Immunology,1991,26(2):53-57.
[15]Price R J,Boettcher B.The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm-immobilizing antibodies[J].Fertility and Sterility,1979,32(1):61-66.
[16]Daffern P J,Pfeifer P H,Ember J A,et al.C3ais a chemotaxin for human eosinophils but not for neutrophils.Ⅰ.C3astimulation of neutrophils is secondary to eosinophil activation[J].Journal of Experimental Medicine,1995,181(6):2119-2127.
[17]Rothman B,Despins A,Webb S,et al.Cytokine regulation of C3and C5production by human corneal fibroblasts[J].Experimental Eye Research,1991,53(3):353-361.
[18]Katz Y,Strunk R C.Synovial fibroblast-like cells synthesize seven proteins of the complement system[J].Arthritis and Rheumatology,1988,31(11):1365-1370.
[19]Welch T R,Beischel L S,Witte D P.Differential expression of complement C3and C4in the human kidney[J].Journal of Clinical Investigation,1993,92(3):1451-1458.
[20]Brooimans R A,Stegmann A P,VanDorp W T,et al.Interleukin 2 mediates stimulation of complement C3biosynthesis in human proximal tubular epithelial cells[J].Journal of Clinical Investigation,1991,88(2):379-384.
[21]Ault B H,Colten H R.Cellular specificity of murine renal C3expression in two models of inflammation[J].Immunology,1994,81(4):655-660.
[22]Choy L N,Rosen B S,Spiegelman B M.Adipsin and an endogenous pathway of complement from adipose cells[J].Journal of Biological Chemistry,1992,267(18):12736-12741.
[23]Georgiou A S,Snijders A P,Sostaric E,et al.Modulation of the oviductal environment by gametes[J].Journal of Proteome Research,2007,6(12):4656-4666.
[24]White R T,Damm D,Hancock N,et al.Human adipsin is identical to complement factor D and is expressed at high levels in adipose tissue[J].Journal of Biological Chemistry,1992;267(13):9210-9213.
[25]Strey C W,Markiewski M,Mastellos D,et al.The proinflammatory mediators C3aand C5aare essential for liver regeneration[J].Journal of Experimental Medicine,2003,198(6):913-923.
[26]Matthews K W,Drouin S M,Liu C,et al.Expression of the third complement component(C3)and carboxypeptidase N small subunit(CPN1)during mouse embryonic development[J].Developmental and Comparative Immunology,2004,28(6):647-655.
[27]Markiewski M M,Mastellos D,Tudoran R,et al.C3aand C3bactivation products of the third component of complement(C3)are critical for normal liver recovery after toxic injury[J].Journal of Immunology,2004,173(2):747-754.
[28]Circolo A,Garnier G,Fukuda W,et al.Genetic disruption of the murine complement C3promoter region generates deficient mice with extrahepatic expression of C3 mRNA[J].Immunopharmacology,1999,42(1-3):135-149.
[29]Menke T M,Mclaren A.Carbon dioxide production by mouse blastocysts during lactational delay of implantation or after ovariectomy[J].Journal of Endocrinology,1970,47(3):287-294.
[30]Xu C,Mao D,Holers V M,et al.A critical role for murine complement regulator Crry in fetomaternal tolerance[J].Science,2000,287(5452):498-501.
[31]Paria B C,Das S K,Andrews G K,et al.Expression of the epidermal growth factor receptor gene is regulated in mouse blastocysts during delayed implantation[J].Proceedings of the National Academy of Sciences,1993,90(1):55-59.
[32]Paria B C,Huethudson Y M,Dey S K.Blastocyst’s state of activity determines the“window”of implantation in the receptive mouse uterus[J].Proceedings of the National Academy of Sciences,1993,90(21):10159-10162.