妊娠相关糖蛋白的生物学功能及其在畜牧生产中的研究进展
引用本文
钟朱夏, 胡修忠, 向敏, 余婕, 刘辰晖, 赵胜兰, 万平民, 王定发, 周源, 程蕾. 妊娠相关糖蛋白的生物学功能及其在畜牧生产中的研究进展[J]. 畜牧兽医学报, 2024, 55(3): 874-881.
ZHONG Zhuxia, HU Xiuzhong, XIANG Min, YU Jie, LIU Chenhui, ZHAO Shenglan, WAN Pingmin, WANG Dingfa, ZHOU Yuan, CHENG Lei. Research Progress on Biological Function and Application of Pregnancy Associated Glycoproteins in Livestock Production[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 874-881.
妊娠相关糖蛋白的生物学功能及其在畜牧生产中的研究进展
武汉市农业科学院畜牧兽医研究所, 武汉 430208
摘要：妊娠相关糖蛋白（pregnancy associated glycoproteins，PAGs）属于天冬氨酸蛋白酶基因编码的多基因家族成员，由偶蹄类动物胎盘滋养层巨细胞合成分泌的特异性糖蛋白，可在胎盘附着后的母体循环中检测到。PAGs在胚胎附着、胎盘发育、妊娠维持、胚胎存活、蛋白水解活性和免疫调节等方面具有潜在作用，常作为早期妊娠诊断和胚胎丢失的生物标志物，同时还可作为检测胎儿生存能力和监测胎盘健康的指标。在本文主要综述了PAGs家族的起源及特征、生物学功能及其在畜牧生产上应用的研究进展，以期为深入探究PAGs的生物学功能以及推动其在畜牧业快速发展提供参考依据。
关键词：妊娠相关糖蛋白 胎盘 起源及特征 生物学功能 畜牧生产
Research Progress on Biological Function and Application of Pregnancy Associated Glycoproteins in Livestock Production
ZHONG Zhuxia
, HU Xiuzhong, XIANG Min, YU Jie, LIU Chenhui, ZHAO Shenglan, WAN Pingmin, WANG Dingfa, ZHOU Yuan, CHENG Lei
, HU Xiuzhong, XIANG Min, YU Jie, LIU Chenhui, ZHAO Shenglan, WAN Pingmin, WANG Dingfa, ZHOU Yuan, CHENG Lei
Institute of Animal Husbandry and Veterinary Science, Wuhan Academy of Agricultural Sciences, Wuhan 430208, China
Abstract: Pregnancy-associated glycoproteins (PAGs) belonging to the multigene family encoded by the aspartic protease gene, are a group of specific glycoproteins synthesized and secreted by placental trophoblast giant cells of artiodactyls, which can be detected in the maternal circulation after embryo attachment. It has the potential to serve multiple functions, including embryonic attachment, placental development, pregnancy maintenance, embryo survival, proteolytic activity, and immune regulation, which are most often used as biomarkers for early pregnancy diagnosis and assessment of embryo loss, as well as indicators of evaluation of fetal viability and monitoring placental health. In this paper, the origins and characteristics and biological functions of the PAGs family and its application in livestock production were reviewed, in order to provide reference for further research of PAGs biological functions and its rapid development in animal husbandry.
Key words:
pregnancy-associated glycoproteins placenta origins and characteristics biological function livestock production
尽早确定母畜妊娠与否以及诊断并预测妊娠过程中胚胎丢失，以尽快对未孕母畜进行再授精，可提高母畜繁殖效率，缩短母畜产犊-受孕间隔，对于提高养殖农场的经济效益至关重要。妊娠相关糖蛋白(pregnancy associated glycoproteins，PAGs)是胎盘滋养层细胞合成和分泌的一类庞大的糖蛋白家族，属于天冬氨酸蛋白酶家族成员[1-2]。在妊娠期间，反刍动物胎儿胎盘单核滋养层细胞以无细胞分裂的连续核分裂形成双核滋养层细胞，向母体子宫上皮细胞迁移、融合形成三核细胞所替代，并通过胞吐作用释放包括PAGs等颗粒物质传递到母体血液循环中[3-4]，常作为早期妊娠诊断的生物标志物[5-6]。目前，PAGs在妊娠期间的功能尚不清楚。因此，本文主要综述PAGs的生物学功能及其在家畜生产中的应用研究进展，为进一步深入研究PAGs功能和开发推广PAGs在畜牧生产中应用提供参考。
1 PAGs家族起源及特征Butler等[7]在1982年利用免疫电泳法首次从妊娠牛胎盘中分离纯化得到分子量大小为47~53 ku，等电点为4.0~4.4的妊娠特异性蛋白，并将其命名为妊娠特异性蛋白B(pregnancy specific protein B，PSPB)。随后，1991年Zoli等[8]从妊娠2~6月龄的牛胎盘子叶中分离纯化出分子量大小为67 ku的蛋白，命名为PAG1。PAGs属于天冬氨酸蛋白酶超家族(aspartic proteinases superfamily，AP)的一类糖蛋白[1]，其特征是识别位点周围存在天冬氨酸残基，具有典型的两个双叶的三级结构(酶催化中心位于该结构内)，与胃蛋白酶、肾素、组织蛋白酶D、组织蛋白酶E和凝乳酶氨基酸序列相似性达50%以上[9-10]。Hughes等[11]根据PAGs出现时间将其分为“古代组”和“现代组”两个类群，“古代组”起源于8 700万年前，经进化在5 200万年前形成“现代组”。但是，“现代组”中大多数PAGs的酶催化中心关键碱基发生突变，使其失去了催化活性，如boPAG1中甘氨酸被丙氨酸所取代而不具有酶活性[12-13]；“古代组”PAGs保留了酶催化中心的关键碱基，可能仍具有酶活性[14-15]，而PAGs在母胎界面的蛋白水解酶活性在妊娠期间的功能仍不清楚，值得进一步深入挖掘。
Green等[16]发现，“古代组”PAGs在整个滋养外胚层细胞和子宫内膜的连接处表达包括oPAG-2、bPAG-2、bPAG-8、bPAG-10和bPAG-11，同时发现bPAG-1，bPAG-6和bPAG-7在妊娠中期至晚期都存在，而bPAG-4、bPAG-5和bPAG-9在妊娠第25天开始表达，但不存在于妊娠晚期，而“现代组”PAGs表达局限于双核滋养层巨细胞，表明PAGs在妊娠期间存在时空表达特异性。在反刍动物中，PAGs基因家族数量庞大且复杂。在牛、绵羊、山羊、鹿和水牛中分别鉴定出22个boPAG[1, 16-18]、11个ovPAG[13-17]、12个caPAG[18-19]、10个cePAG[20]和20个wtPAG成员[6, 21]，可能存在更多的PAGs基因有待进一步发现。
2 PAGs的生物学功能 2.1 免疫调节作用“古代组”PAGs主要在母胎界面微绒毛连接处表达，能够促进胚胎滋养层细胞和子宫上皮细胞相连接，建立免疫屏障；“现代组”PAGs主要在滋养外胚层巨细胞中表达，具有调节母体免疫系统的作用[22]。研究发现，使用浓度为2 400 ng ·mL-1的bPAG处理骨髓祖细胞能够抑制其增殖[23]。Roberts等[10]研究表明，PAGs可能能够与肽竞争性被主要组织相容性复合体(major histocompatibility complex，MHC)识别和结合以抑制T淋巴细胞的激活从而在母胎界面发挥免疫调节作用。有研究表明，PAGs在体外能够与绵羊子宫丝氨酸蛋白酶抑制剂(ovine uterine serpin，ovUS-1)结合，可能发挥免疫调节活性[24]。Telugu等[14-15]发现重组表达的poPAG-1和poPAG-2具有蛋白水解活性，可能参与刺激局部生长因子的生成[25-26]。Austin等[27]研究发现，PSPB可以诱导牛子宫内膜细胞中嗜中性粒细胞趋化蛋白2(granulocyte chemotactic protein-2，GCP-2)的分泌，可能参与早期植入过程中胚胎黏附、炎症反应和血管生成。Barbato等[28]利用RT-qPCR研究发现，PAG-2 mRNA在人工授精后18 d妊娠牛外周血单核细胞和粒细胞中表达，并且人工授精后14、18、28和40 d妊娠牛外周血单核细胞和粒细胞中PAG-2 mRNA表达水平高于空怀牛，且妊娠牛粒细胞中PAG-2 mRNA表达水平高于外周血单核细胞。Casano等[29]发现，在围着床期直至妊娠40 d水牛外周血单核细胞和多形核白细胞中IFN-τ和干扰素刺激基因(OAS1、MX2和ISG15)mRNA表达水平高于未妊娠水牛，且与外周血浆中PAGs的浓度呈显著正相关，说明PAGs可能发挥免疫调节作用参与妊娠识别与建立过程。Wallace等[30]从妊娠150 d牛胎盘子叶组织中分离出PAG-4、PAG-6和PAG-9蛋白分别处理人工授精后18 d的妊娠和空怀牛子宫外植体，发现未妊娠和妊娠牛子宫外植体中子宫重塑相关基因(EMMPRIN、MMP1、MMP3、MMP7、MMP9、PLAU和TIMP2)和干扰素刺激基因(IFI6和ISG15)mRNA表达水平在PAGs处理24或96 h后上调表达，同样地，CXCL5和PTGES基因mRNA也上调表达，与Austin等[27]的研究结果相一致，说明PAGs通过影响子宫内膜中细胞外基质周转参与子宫重塑和胎儿侵袭从而形成一个有利于胎盘形成的微环境。Hooshmandabbasi等[31]研究发现，与胎盘正常娩出的奶牛相比，胎盘滞留奶牛个体中PAGs阳性双核滋养层细胞数量和“现代组”PAGs mRNA表达水平更高，而在血清中无显著差异，可能沉积在母体间隔基质中的“现代组”PAGs发挥局部的免疫抑制作用进而延迟胎盘的脱落进程。以上研究表明PAGs可能参与妊娠的识别与建立、妊娠维持以及产犊前后免疫功能的抑制。
2.2 促黄体生成作用研究显示，反刍动物妊娠期间孕酮的来源是黄体和胎盘，前列腺素E2(prostaglandin E2，PGE2)发挥着抗黄体溶解和促黄体生成的作用[32]。Del Vecchio等[33]用PSPB或与催产素一起对发情周期第16天的子宫内膜外植体进行处理，发现前列腺素E2和前列腺素F2α(prostaglandin F2α，PGF2α)的分泌增加[34]，同时研究发现用PSPB处理牛黄体细胞后不影响催产素的产生，但会促进孕酮和PGE2的分泌，PGF2α分泌量也升高[35]。Weems等[36]也发现，PSPB处理妊娠88~90 d绵羊黄体细胞显著提高PGE2和孕酮的分泌，进一步研究发现PSPB可能通过刺激卵巢黄体、胎盘和子宫肉阜组织中PGE2分泌来调节妊娠50~90 d黄体中孕酮分泌[37-39]。上述结果表明，PSPB作为一种促黄体生成因子通过刺激胎盘/子宫肉阜中PGE2分泌进而调节黄体中孕酮的合成以维持妊娠。
综上所述，PAGs可能参与母胎界面的免疫调节以保护胎儿免受免疫排斥以及妊娠终止时胎儿的及时娩出，同时也参与妊娠相关激素的合成来为胚胎提供成功着床的条件和稳定发育的环境。
3 PAGs在家畜生产中的应用 3.1 诊断妊娠与否在规模化、现代化牧场的饲养管理中，提高母畜产犊率、保持理想的产犊间隔决定着养殖企业的经济效益。母畜配种后，及时且准确的妊娠诊断可以有效缩短配种期进而保证较短的空怀周期，从而有效地提高繁殖效率。即使由经验丰富的技术人员进行直肠触诊，而直至人工授精后35~40 d仍难以准确判断妊娠与否。
近年以来，由于分子生物学方法的快速发展，已有许多学者尝试鉴定牛羊等妊娠的生物标志物。PAGs一直是主要的研究对象，且商业化的妊娠诊断试剂盒已经应用于牛羊等早期妊娠诊断[40-42]，目前，美国IDEXX和BioTracking公司均有基于PAGs或PSPB的ELISA商品化试剂盒，均能在配种28 d后检测奶牛是否妊娠。Commun等[43]利用基于2种妊娠相关糖蛋白的ELISA方法对人工授精后16、30、41和53 d奶牛血清、血浆和牛奶进行检测，发现人工授精后30~53 d检测妊娠准确度较高。Barbato等[44]利用RT-qPCR对水牛人工授精后0、18、28、40和75 d血细胞中PAG-2 mRNA进行检测，发现PAG-2 mRNA在妊娠18 d、D28(+)D40(-)和D28(-)D40(-)组中表达，但其水平低于D28(+)D40(+)组，且与PAG-1相比更早地在外周血细胞中被检测到，可用于研究早期妊娠和胚胎死亡。据报道，PAG-1在母体血清中表现出较长的半衰期，可在产后80~100 d检测到，从而使在产后60 d内配种的动物中产生假阳性[45]。Lu等[46]利用血清辅助的指数富集的配体系统进化(systematic evolution of ligands by exponential enrichment，SELEX)获得ovPAG7特异性结合的DNA单链适配体，将其用于人工授精后22和28 d母羊血清检测发现诊断灵敏度、特异性和准确性较高，是检测母羊妊娠的有效方法。李志明等[47-48]通过原核表达方式获得PAG-6蛋白制备了多克隆抗体，建立了一种牛妊娠相关糖蛋白6快速检测试纸条方法，用于奶牛早期妊娠诊断效果良好。
3.2 预测和诊断胚胎附着、胚胎丢失、胚胎数量以及性别研究显示，在胚胎附植至妊娠期间胎盘双核滋养层细胞约占所有滋养层细胞数量的20%，而在妊娠末期数量降低[49]。胎盘发育因胎儿的数量、性别、大小和体重而异，胎盘双核滋养层细胞的数量随着胎盘质量和胎儿数量的增加而增加。Middleton等[50]使用BioPRYN ELISA试剂盒对人工授精后15~35 d的小母牛和泌乳奶牛血清中PSPB进行检测，发现与泌乳奶牛相比，小母牛的PSPB浓度增加时间更早，循环浓度更高，以血清PSPB的OD水平从基线开始升高≥10%且随后2 d从基线继续升高≥10%的那一天作为胚胎附着的标志，与初产或经产奶牛相比，未生育小母牛的胚胎附着时间更早。El Amiri等[51]使用放射免疫系统对人工授精后0~49 d母羊血浆和羊奶中PAGs浓度进行测定，发现在人工授精后20 d的妊娠母羊血浆和羊奶中可检测到PAGs存在且双胎妊娠血浆和羊奶中PAGs浓度显著高于单胎妊娠。Alkan等[52]利用IDEXX试剂盒对179头妊娠40~60 d母羊进行检测，发现在妊娠早期40~50 d双胎妊娠母羊PAGs浓度高于单胎妊娠母羊，而在不同妊娠时间携带雄性和雌性胎儿的母羊中PAGs浓度之间无显著差异，可能是双胎妊娠具有多个附植部位，胎盘体积增大，双核滋养层细胞数量增加，导致PAGs分泌量增加。Ranilla等[53]发现，在怀孕第19、20和21周雄性胎儿的母羊的PAGs浓度高于携带雌性胎儿的母羊，与Alkan等[52]的研究结果不一致，可能与不同的妊娠时期有关。
在妊娠期间，胚胎死亡可能发生在早期或晚期，被认为是造成繁殖失败的主要原因[54]。研究显示，在接受胚胎移植和人工授精后28~30 d妊娠成功建立并维持妊娠至60~72 d的奶牛血清中bPAGs浓度显著高于同一时间妊娠成功建立但未维持妊娠[55]。Pohler等[56]利用逻辑回归模型发现，在肉牛妊娠28 d血清中bPAGs浓度大于7.9或低于0.72 ng ·mL-1时预测胚胎维持至第95天存活或死亡的准确率高达95%以上，可作为牛晚期胚胎死亡的标志物。Peixoto等[57]发现，人工授精后37 d双胎妊娠和胚胎丢失荷斯坦奶牛血浆PAGs的浓度无明显变化且其不能有效地预测荷斯坦奶牛胚胎丢失和双胎妊娠的阈值。以上研究表明，不同的抗体组合能够识别不同的PAGs或不同数量的PAGs异构体，检测方法不同和抗体特异性差异可能会影响妊娠丢失诊断的准确性。此外，Panasiewicz等[58]对杂交母猪pPAG2-Ls基因研究共发现196个SNPs，包括9个外显子和8个(A~H)内含子，其中位于第6外显子双倍型(657C>T/749G>C)导致多肽前体中氨基酸替换(Asp220→Asn和Ser250→Thr)与产活仔猪数量增加有关，内含子F中504G>A与产活仔猪数和断奶仔猪数增加有关，可为猪选种选育提供重要参考。
3.3 监测胎儿的生长发育和胎盘功能PAGs除了作为妊娠诊断的生物标志物外，也可用于监测胎儿的生长发育和胎盘的功能。LÓpez-Gatius等[59]研究发现，感染犬新孢子虫发生流产和木乃伊胎儿的奶牛血清中PAG-1浓度较低或无法检测，说明PAG-1可能是评估胎儿-胎盘状态的有效指征。García-Ispierto等[60]发现，妊娠120 d荷斯坦奶牛血浆PAG-2浓度可以作为感染犬新孢子虫流产风险的指标，当数值< 4.5 ng ·mL-1表明慢性感染的动物流产风险高。Mur-Novales等[61-62]对妊娠110 d怀孕母牛感染犬新孢子虫进行研究，发现未流产母牛在感染14 d内血浆PAG-1和PAG-2水平呈现下降，感染42 d单核和双核滋养层细胞比例减少以及胎儿羊水和尿囊中PAG-1和PAG-2浓度显著高于未感染胎儿，且受感染流产母牛血浆中PAGs浓度急剧降低，说明PAGs可能与感染后不同程度的胎盘损伤和无法维持的妊娠有关。此外，Lear等[63]对妊娠65 d母羊接种非细胞病变性牛病毒性腹泻病毒(bovine viral diarrhea virus，BVDV)株1b，发现妊娠80 d感染BVDV的母羊和胎儿血清中PAG-1浓度明显降低以及滋养细胞损伤，表明PAG-1浓度可作为BVDV感染期间评估胎盘功能的指标。越来越多的研究表明，PAGs浓度变化与胎儿存活和胎盘健康有关，但仍需进一步研究母体-胎儿不良妊娠结局与PAGs产生及功能改变之间的关系进而阐明PAGs作为评估母胎单位的生物标志物。
4 展望PAGs是偶蹄目动物胎盘产生的特异性糖蛋白家族，其作为妊娠诊断的生物标志物具有妊娠早期特异性表达和能够进入母体血液循环等特点，近年来被广泛研究。虽然PAGs在胎盘-子宫界面发挥多种生物学功能，但是大多数PAGs家族成员在妊娠期间的表达表现为复杂的时间和空间表达模式，并且释放到母体血液中为作用于靶组织和靶细胞提供了众多的可能性，并且提纯单一PAGs家族成员难度较大。目前已有多种PAGs分子诊断试剂被开发，但因面临其灵敏度、准确性和特异性不强等问题，制约了PAGs在畜牧生产中的发展应用。针对这一问题，可通过对妊娠不同时期表达的PAGs进行天然提纯，制备并筛选单克隆抗体杂交瘤细胞以获得不同PAGs成员抗体或核酸适配体，对不同PAGs成员抗体或核酸适配体进行组装实现对母畜妊娠诊断和监测胎儿生长发育。随着生物技术的不断创新与发展，可利用基因组、蛋白组、代谢组等多组学技术对双核或多核滋养层细胞进行研究以鉴定新的妊娠诊断标志物；可利用生物信息学等技术丰富PAGs分子调控网络以及探究PAGs家族在妊娠维持和胎盘发育中的功能，这将对于畜牧业的长远发展具有十分深远的意义。
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(编辑 郭云雁)