溶菌酶1在小鼠超数排卵前后孵化及休眠囊胚中差异表达的研究
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摘要
目的研究溶菌酶1(lysozyme 1,LYZ1)基因在超数排卵前后小鼠孵化囊胚和休眠胚胎中的分布以及表达,探究动物胚胎着床过程中新的调节机制。方法从妊娠5 d ICR小鼠体内获取的正常孵化囊胚和超排囊胚,利用小鼠延迟着床模型于妊娠第8天获取休眠胚胎和超排休眠胚胎。利用免疫荧光和Western Blot方法检测LYZ1蛋白在四组胚胎中的分布和差异表达变化。结果 LYZ1在超数排卵前、后小鼠孵化囊胚和休眠胚胎中均有表达,且主要集中在内细胞团中,滋养层细胞和胞质中少见分布;与未进行超排的小鼠相比,LYZ1蛋白在超排后小鼠胚胎中表达量显著上调,与未营造休眠模型的小鼠相比,LYZ1蛋白在休眠模型小鼠胚胎中的表达量显著上调。结论 LYZ1蛋白在囊胚内细胞团中表达,可能参与调节胚胎内细胞团的发育;LYZ1蛋白在超排-休眠胚胎中的高表达,说明LYZ1蛋白在休眠和超数排卵的双重影响下,会因为抵御不利环境而上调。
Objective To explore the regulation of mammalian embryo implantation, we investigated the expression of Lysozyme 1(LYZ1) in normal hatching murine blastocysts and dormant embryos before and after superovulation. Methods Normal hatched blastocysts were obtained from day 5 pregnant mice, while dormant embryos were collected from delayed implantation models on day 8. LYZ1 protein expression in the embryos was detected by immunofluorescence and western blot. Results LYZ1 protein was expressed in both hatching blastocysts and dormant embryos before and after superovulation, where it was mainly concentrated in the inner cell mass. LYZ1 was rarely distributed in trophoblasts or in their cytoplasm. Compared with untreated mice, LYZ1 protein expression in superovulation embryos was significantly upregulated; moreover, LYZ1 protein expression in the dormancy model was significantly upregulated. Conclusions LYZ1 protein was expressed in the inner cell mass of embryos, where it may be involved in regulating early development. The high LYZ1 protein expression in dormant embryos after superovulation indicated that LYZ1 protein is upregulated in embryos experiencing adverse environmental conditions, such as the dual effects of dormancy and superovulation.
Objective To explore the regulation of mammalian embryo implantation, we investigated the expression of Lysozyme 1(LYZ1) in normal hatching murine blastocysts and dormant embryos before and after superovulation. Methods Normal hatched blastocysts were obtained from day 5 pregnant mice, while dormant embryos were collected from delayed implantation models on day 8. LYZ1 protein expression in the embryos was detected by immunofluorescence and western blot. Results LYZ1 protein was expressed in both hatching blastocysts and dormant embryos before and after superovulation, where it was mainly concentrated in the inner cell mass. LYZ1 was rarely distributed in trophoblasts or in their cytoplasm. Compared with untreated mice, LYZ1 protein expression in superovulation embryos was significantly upregulated; moreover, LYZ1 protein expression in the dormancy model was significantly upregulated. Conclusions LYZ1 protein was expressed in the inner cell mass of embryos, where it may be involved in regulating early development. The high LYZ1 protein expression in dormant embryos after superovulation indicated that LYZ1 protein is upregulated in embryos experiencing adverse environmental conditions, such as the dual effects of dormancy and superovulation.
引文
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[20] Kaizu A,Fagutao FF,Kondo H,et al. Functional analysis of Ctypelysozyme in penaeid shrimp[J].J Biol Chem,2011,286(52):44344-44349.
[21] Lapcharoen P,Komalamisra N,Rongsriyam Y,et al. Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development[J]. Dev Comp Immunol,2012,36(1):104-111.
[22] 黄复生,王兴相. 大劣按蚊血细胞对约氏疟原虫卵囊黑化作用的观察[J]. 中国病原生物学杂志,1995,8(4):284-285.Huang FS,Wang XX. Observation of the effect of the blood cells of the inferior Anopheles albopictus on the egg-follicle blackening of the Plasmodium albicans[J].J Pathol Biol,1995,8(4):284-285.
[2] Lopes FL,Desmarais JA,Murphy BD. Embryonic diapause and its regulation[J]. Reproduction,2004,128(6):669.
[3] Bernard RTF. Reproductive synchrony and annual variation in foetal growth rate in the long‐fingered bat (Miniopterus schreibersii)[J]. J Zool,1994,232(3):485-490.
[4] Ragland SA,Criss AK. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme[J]. Plos Pathogens,2017,13(9):e1006512.
[5] 张士璀,许娜. 动物型溶菌酶研究新进展[J]. 中国海洋大学学报(自然科学版),2014,44(6):46-51.Zhang SC,Xu N. New progress in research on animal lysozyme[J].J Ocean Univ China (Nat Sci Edi),2014,44(6):46-51.
[6] 孙国庆,哈福,苗永旺,等. 动物溶菌酶基因的研究进展[J]. 中国牛业科学,2012,38(3):51-54,59.Sun GQ,Ha H,Miao YW, et al. Advances in the study of animal lysozyme genes[J]. China Cattle Sci,2012,38(3);51-54, 59.
[7] 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):15-19.Zhang SY,Liu YH,Ni HM,et al. Differential analysis of gene expression profiles of dormant embryos in programmed frozen mice[J].Acta Lab Anim Sci Sin,2012,20(5):15-19.
[8] 王力红,郭勇,翟椿东,等. 程序化冷冻前后小鼠休眠胚胎中Apol8蛋白分布和表达[J]. 中国农学通报,2017,33(21):149-153.Wang LH,Guo Y,Zhai CD et al. Dstribution and expression of Apol 8 protein in dormant embryos of mice before and after programmed freezing[J].Chin Agricul Sci Bull,2017,33(21):149-153.
[9] Gu M,Ni H,Sheng X,et al. RhoA phosphorylation mediated by Rho/RhoA-associated kinase pathway improves the anti-freezing potentiality of murine hatched and diapaused blastocysts[J]. Sci Rep,2017,7(1):6705.
[10] 刘迪,倪和民,顾美超,等.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[J].中国畜牧兽医,2017,44(2):514-520.Liu D,Ni HM,Gu MC, et al. Study on the expression of C3 gene in normal incubated blastocysts and dormant embryos of mice before and after programmed freezing[J]. China Anim Husbandry Vet Med,2017,44(2):514-520.
[11] 刘迪,倪和民,顾美超,等. 程序化冷冻前后小鼠孵化囊胚和休眠胚胎的Hba-α蛋白分布和差异表达[J].中国实验动物学报,2016,24(6):579-584.Liu D,Ni HM,Gu MC,et al. The distribution and differential expression of Hba-alpha protein in hatchable blastocysts and dormant embryos of mice before and after freezing procedure[J]. Acta Lab Anim Sci Sin,2016,24(6):579-584.
[12] 赖炳森,孙树秦,叶平,等.不同剂量γ线整体照射后家兔血清溶菌酶含量的改变[J].军事医学,1984,(1):31-36.LAI BS, SUN SQ, YE P, et al. Changes in serum lysozyme content in rabbits after different doses of gamma ray irradiation[J]. Milit Med Sci,1984,(1):31-36.
[13] Leese H J, Baumann C G, Brison D R, et al. Metabolism of the viable mammalian embryo: quietness revisited[J]. Mol Human Reprod,2008,14(12):667-672.
[14] Lee J, Oh HA, Song H, et al. Autophagy regulates embryonic survival during delayed implantation[J]. Endocrinology,2011,152:2067-2075.
[15] Fu Z, Wang B, Wang S, et al. Integral proteomic analysis of blastocysts reveals key molecular machinery governing embryonic diapause and reactivation for implantation in mice[J]. Biol Reprod,2014,90(3):52.
[16] Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy[J]. Dev Cell,2004,6(4):463.
[17] Weitlauf HM, Greenwald GS. Survival of blastocysts in the uteri of ovariectomized mice[J]. J Reprod Fertil,1968,17(3):515-520.
[18] Starling S. Innate immunity: A new way out for lysozyme[J]. Nat Rev Immunol,2017,17(9):532.
[19] Ku SK, Yoon EK, Lee HG,et al. Inhibitory effects of lysozyme on endothelial protein C receptor shedding in vitro and in vivo[J]. BMB Reports,2015,48(11):624-629.
[20] Kaizu A,Fagutao FF,Kondo H,et al. Functional analysis of Ctypelysozyme in penaeid shrimp[J].J Biol Chem,2011,286(52):44344-44349.
[21] Lapcharoen P,Komalamisra N,Rongsriyam Y,et al. Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development[J]. Dev Comp Immunol,2012,36(1):104-111.
[22] 黄复生,王兴相. 大劣按蚊血细胞对约氏疟原虫卵囊黑化作用的观察[J]. 中国病原生物学杂志,1995,8(4):284-285.Huang FS,Wang XX. Observation of the effect of the blood cells of the inferior Anopheles albopictus on the egg-follicle blackening of the Plasmodium albicans[J].J Pathol Biol,1995,8(4):284-285.