山羊妊娠早期胎儿胎盘血管形成及相关基因的表达

doi:10.11843/j.issn.0366-6964.2020.05.013

摘要/Abstract

摘要： 旨在探究大足黑山羊妊娠早期胎儿胎盘血管发育、分布和血管生成相关因子表达状况，并对血管生成相关因子表达之间和血管发育进行相关性分析。本研究随机选取15只8月龄、体格相近、身体健康的国家级畜禽遗传资源-大足黑山羊青年母羊，经同一种公羊自然交配后（以末次配种当天记为0 d），经剖腹产手术采集妊娠第20、25和30天的胎儿和胎儿胎盘以及经屠宰采集妊娠第45和60天的胎儿和胎儿胎盘作为研究对象，通过免疫组化法评估胎盘血管发育和分布情况，采用qRT-PCR技术检测主要血管生成相关基因的mRNA表达水平，分析血管生成相关基因之间以及与胎盘血管发育分布的相关性。结果显示，胎儿体重和体长随着妊娠的进行逐渐增加，且血管内皮细胞阳性率处于较高水平（>26%）。山羊胎儿胎盘毛细血管总面积/组织区域面积（CAD）逐渐升高；毛细血管总数/单位组织区域面积（CND）在妊娠25~30 d增加，30~60 d降低，毛细血管总面积/毛细血管根数（APC）呈相反趋势；毛细血管总周长/单位组织区域面积（CSD）无显著变化。妊娠早期胎儿胎盘VEGFA的表达量随着妊娠的进行逐渐升高，且与血管分布存在一定的相关性，其受体FLT1和KDR表达量呈先增高后降低的趋势，分别在30和45 d达到峰值。血管生成相关基因ANGPT1/2及其受体TEK和FGF2及其受体FGFR2的表达均在妊娠30 d时较高。综上表明，在妊娠30 d前，胎儿胎盘主要通过毛细血管形成分支促进血管面积的增加；而在30~60 d，主要通过单位血管的增粗促进血管面积的增加，并且血管生成相关基因的表达在妊娠30 d时较高，这可能和子叶开始形成有关。山羊妊娠早期胎儿胎盘中VEGFA可能通过其受体结合与其他血管生成因子共同作用调控胎儿胎盘血管形成。

关键词: 大足黑山羊, 妊娠早期, 胎儿胎盘, 血管生成, 基因表达

Abstract: The study aimed to investigate the development and distribution of blood vessels and the expression of angiogenesis-related factors in fetal placenta of Dazu Black goat during early pregnancy, and to analyze the correlation between the expression of angiogenesis-related factors and vascular development. Fifteen 8-month-old, healthy, young female Dazu Black goats were randomly selected, after natural mating with the same ram (recorded as 0 d on the last breeding day), fetuses and fetal placenta were collected by cesarean operation at 20, 25, and 30 d of pregnancy, and fetuses and fetal placenta were collected after slaughter at 45 and 60 d of pregnancy. Placental vascular development and distribution were investigated by immunohistochemical technology, and the expression level of angiogenesis-related genes was detected by qRT-PCR technique, and the correlation between the expression of angiogenesis-related genes and the placental vascular distribution was analyzed. The results showed that fetal body weight and body length increased gradually with the progress of pregnancy, and the positive rate of vascular endothelial cells was at a high level (>26%). With the progress of pregnancy, goat fetal placenta vascularity area/tissue area(CAD) gradually increased; capillary number/tissue area (CND) increased during 25-30 d and decreased during 30-60 d of pregnancy; Average capillary area (APC) showed an opposite trend to CND; There was no significant change in capillary circumference/tissue area(CSD). The expression of VEGFA in fetal placenta was gradually increased with the progress of pregnancy and correlated with vascular distribution. The expression level of FLT1 and KDR in fetal placenta first increased and then decreased, reaching the peak at 30 and 45 d of pregnancy, respectively. The expression of ANGPT1/2 and its receptor TEK, FGF2 and its receptor FGFR2 were higher at 30 d of pregnancy, which could be related to angiogenesis. In conclusion, before 30 d of pregnancy, fetal placenta mainly formed branches through capillaries to promote the increase of vascular area; at 30-60 d of pregnancy, the increase of vascular area was mainly promoted by the thickening of blood vessels per unit, and the expression of angiogenesis-related genes was higher at 30 d of pregnancy, which might be related to cotyledon formation. VEGFA in goat fetal placenta during early pregnancy could act together with other angiogenesis-related factors to regulate the angiogenesis.

Key words: Dazu Black goat, early pregnancy, fetal placenta, angiogenesis, gene expression

中图分类号:

S827.3

程文强, 王攀, 罗南剑, 徐恢仲, 王炜, 张蓉, 成磊, 赵威, 赵永聚. 山羊妊娠早期胎儿胎盘血管形成及相关基因的表达[J]. 畜牧兽医学报, 2020, 51(5): 1019-1029.

CHENG Wenqiang, WANG Pan, LUO Nanjian, XU Huizhong, WANG Wei, ZHANG Rong, CHENG Lei, ZHAO Wei, ZHAO Yongju. Detection of Fetal Placental Blood Vessels and Expression of Angiogenesis-Related Genes in Dazu Black Goats during Early Pregnancy[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1019-1029.

参考文献 46

[1]	PERAZZOLO S,HIRSCHMUGL B,WADSACK C,et al.The influence of placental metabolism on fatty acid transfer to the fetus[J].J Lipid Res,2017,58(2):443-454.
[2]	REYNOLDS L P,REDMER D A.Utero-placental vascular development and placental function[J].J Anim Sci,1995, 73(6):1839-1851.
[3]	MOFFETT A,LOKE C.Immunology of placentation in eutherian mammals[J].Nat Rev Immunol,2006,6(8):584-594.
[4]	KAUFMANN P,MAYHEW T M,CHARNOCK-JONES D S.Aspects of human fetoplacental vasculogenesis and angiogenesis. II. Changes during normal pregnancy[J].Placenta,2004,25(2-3):114-126.
[5]	MONTEIRO L J,CUBILLOS S,SANCHEZ M,et al.Reduced FOXM1 expression limits trophoblast migration and angiogenesis and is associated with preeclampsia[J].Reprod Sci,2019,26(5):580-590.
[6]	ZHAO Y,ZHENG Y F,LIU X X,et al.Inhibiting trophoblast PAR-1 overexpression suppresses sFlt-1-induced anti-angiogenesis and abnormal vascular remodeling:a possible therapeutic approach for preeclampsia[J].Mol Hum Reprod,2018,24(3):158-169.
[7]	FERGUSON M B,THOMPSON A N,GORDON D J,et al.The wool production and reproduction of Merino ewes can be predicted from changes in liveweight during pregnancy and lactation[J].Anim Prod Sci,2011,51(9):763-775.
[8]	CHEN D B,ZHENG J.Regulation of placental angiogenesis[J].Microcirculation,2014,21(1):15-25.
[9]	GUIMARÃES G C,ALVES L A,BETARELLI R P,et al.Expression of vascular endothelial growth factor (VEGF) and factor VIII in the gilt placenta and its relation to fetal development[J].Theriogenology,2017,92:63-68.
[10]	KALKUNTE S,HUANG Z P,LIPPE E,et al.Polychlorinated biphenyls target Notch/Dll and VEGF R2 in the mouse placenta and human trophoblast cell lines for their anti-angiogenic effects[J].Sci Rep,2017,7:39885.
[11]	REYNOLDS L P,BOROWICZ P P,VONNAHME K A,et al.Animal models of placental angiogenesis[J].Placenta, 2005, 26(10):689-708.
[12]	WANG Q,LASH G E.Angiopoietin 2 in placentation and tumor biology:the yin and yang of vascular biology[J]. Placenta, 2017,56:73-78.
[13]	FEARNLEY G W,SMITH G A,ODELL A F,et al.Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells[J].Methods Enzymol,2014,535:265-292.
[14]	HUH J W,HA H S,KIM D S,et al.Placenta-restricted expression of LTR-derived NOS3[J].Placenta,2008,29(7):602-608.
[15]	MATA-GREENWOOD E,LIAO W X,ZHENG J,et al.Differential activation of multiple signalling pathways dictates eNOS upregulation by FGF2 but not VEGF in placental artery endothelial cells[J].Placenta,2008,29(8):708-717.
[16]	GRAZUL-BILSKA A T,JOHNSON M L,BOROWICZ P P,et al.Placental development during early pregnancy in sheep:cell proliferation,global methylation,and angiogenesis in the fetal placenta[J].Reproduction,2011,141(4):529-540.
[17]	MA W Z,ZHENG X M,HEI C C,et al.Optimal FSH usage in revascularization of allotransplanted ovarian tissue in mice[J].J Ovarian Res,2017,10(1):5.
[18]	GALE N W,THURSTON G,HACKETT S F,et al.Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning,and only the latter role is rescued by Angiopoietin-1[J].Dev Cell,2002,3(3):411-423.
[19]	FENG L,LIAO W X,LUO Q,et al.Caveolin-1 orchestrates fibroblast growth factor 2 signaling control of angiogenesis in placental artery endothelial cell caveolae[J].J Cell Physiol,2012,227(6):2480-2491.
[20]	赵中权,张家骅,李周权,等.大足黑山羊种质特性[J].中国畜牧杂志,2008,44(15):9-10.ZHAO Z Q,ZHANG J H,LI Z Q,et al.Germplasm characteristics of Daze Black goats[J].Chinese Journal of Animal Science, 2008,44(15):9-10.(in Chinese)
[21]	VALLET J L,MCNEEL A K,MILES J R,et al.Placental accommodations for transport and metabolism during intra-uterine crowding in pigs[J].J Anim Sci Biotechnol,2014,5(1):55.
[22]	VONNAHME K A,WILSON M E,FORD S P.Relationship between placental vascular endothelial growth factor expression and placental/endometrial vascularity in the pig[J].Biol Reprod,2001,64(6):1821-1825.
[23]	胡淑君,孙红,邓宇,等.双绒毛膜双胎胎盘血管结构与胎儿体重的关系[J].中国生育健康杂志,2011,22(2):80-82.HU S J,SUN H,DENG Y,et al.Correlation between placental vascular structure and birth weight in dichorionic twins[J].Chinese Journal of Reproductive Health,2011,22(2):80-82.(in Chinese)
[24]	赵艳娇,常青,姜怀志.辽宁绒山羊胎儿发育的初步研究[J].中国草食动物科学,2012,32(5):20-23.ZHAO Y J,CHANG Q,JIANG H Z.A preliminary study on the fetal development of Liaoning cashmere goats[J].China Herbivore Science,2012,32(5):20-23.(in Chinese)
[25]	GRAZUL-BILSKA A T,BOROWICZ P P,JOHNSON M L,et al.Placental development during early pregnancy in sheep:vascular growth and expression of angiogenic factors in maternal placenta[J].Reproduction,2010,140(1):165-174.
[26]	BOROWICZ P P,ARNOLD D R,JOHNSON M L,et al.Placental growth throughout the last two thirds of pregnancy in sheep:vascular development and angiogenic factor expression[J].Biol Reprod,2007,76(2):259-267.
[27]	BLOMBERG L,HASHIZUME K,VIEBAHN C.Blastocyst elongation,trophoblastic differentiation,and embryonic pattern formation[J].Reproduction,2008,135(2):181-195.
[28]	SANCHIS E G,CRISTOFOLINI A L,FIORIMANTI M R,et al.Apoptosis and cell proliferation in porcine placental vascularization[J]. Anim Reprod Sci,2017,184:20-28.
[29]	CORREIA-DA-SILVA G,BELL S C,PRINGLE J H,et al.Patterns of uterine cellular proliferation and apoptosis in the implantation site of the rat during pregnancy[J].Placenta,2004,25(6):538-547.
[30]	KAR M,GHOSH D,SENGUPTA J.Histochemical and morphological examination of proliferation and apoptosis in human first trimester villous trophoblast[J].Hum Reprod,2007,22(11):2814-2823.
[31]	REYNOLDS L P,BOROWICZ P P,CATON J S,et al.Uteroplacental vascular development and placental function:an update[J]. Int J Dev Biol,2010,54(2-3):355-366.
[32]	王攀,赵永聚.胎盘血管形成机制及其对动物繁殖性能的影响[J].江苏农业学报,2019,35(4):986-995.WANG P,ZHAO Y J.Mechanism of placental angiogenesis and its effect on animal reproductive performance[J].Jiangsu Journal of Agricultural Sciences,2019,35(4):986-995.(in Chinese)
[33]	唐毅平,陈秋羊,罗南剑,等.大足黑山羊妊娠不同阶段胎盘线粒体相关基因的表达[J].中国兽医学报,2018,38(11):2193-2200.TANG Y P,CHEN Q Y,LUO N J,et al.Mitochondrial related genes expression in placental tissues of different gestational stages in Dazu black goat[J].Chinese Journal of Veterinary Science,2018,38(11):2193-2200.(in Chinese)
[34]	PALMER B F,CLEGG D J.Oxygen sensing and metabolic homeostasis[J].Mol Cell Endocrinol,2014,397(1-2):51-58.
[35]	PAVLOV O V,NIAURI D A,SELUTIN A V,et al.Coordinated expression of TNFα-and VEGF-mediated signaling components by placental macrophages in early and late pregnancy[J].Placenta,2016,42:28-36.
[36]	MOE K,HEIDECKE H,DECHEND R,et al.Dysregulation of circulating autoantibodies against VEGF-A,VEGFR-1 and PlGF in preeclampsia-A role in placental and vascular health?[J].Pregnancy Hypertens,2017,10:83-89.
[37]	OTROCK Z K,MAKAREM J A,SHAMSEDDINE A I.Vascular endothelial growth factor family of ligands and receptors:review[J]. Blood Cells Mol Dis,2007,38(3):258-268.
[38]	GALE N W,YANCOPOULOS G D.Growth factors acting via endothelial cell-specific receptor tyrosine kinases:VEGFs, angiopoietins, and ephrins in vascular development[J].Genes Dev,1999,13(9):1055-1066.
[39]	SURI C,MCCLAIN J,THURSTON G,et al.Increased vascularization in mice overexpressing angiopoietin-1[J]. Science,1998, 282(5388):468-471.
[40]	KEARNEY J B,KAPPAS N C,ELLERSTROM C,et al.The VEGF receptor flt-1(VEGFR-1) is a positive modulator of vascular sprout formation and branching morphogenesis[J].Blood,2004,103(12):4527-4535.
[41]	DEMIR R,SEVAL Y,HUPPERTZ B.Vasculogenesis and angiogenesis in the early human placenta[J].Acta Histochem, 2007, 109(4):257-265.
[42]	KAPPOU D,SIFAKIS S,KONSTANTINIDOU A,et al.Role of the angiopoietin/Tie system in pregnancy (Review)[J].Exp Ther Med,2015,9(4):1091-1096.
[43]	CARMELIET P.Mechanisms of angiogenesis and arteriogenesis[J].Nat Med,2000,6(4):389-395.
[44]	MAISONPIERRE P C,SURI C,JONES P F,et al.Angiopoietin-2,a natural antagonist for Tie2 that disrupts in vivo angiogenesis[J]. Science,1997,277(5322):55-60.
[45]	WELTER H,WOLLENHAUPT K,EINSPANIER R.Developmental and hormonal regulated gene expression of fibroblast growth factor 2(FGF-2) and its receptors in porcine endometrium[J].J Steroid Biochem Mol Biol,2004,88(3):295-304.
[46]	KUNATH T,YAMANAKA Y,DETMAR J,et al.Developmental differences in the expression of FGF receptors between human and mouse embryos[J].Placenta,2014,35(12):1079-1088.