牦牛NGF基因克隆及其在生殖器官中的表达定位

doi:10.11843/j.issn.0366-6964.2022.03.011

摘要/Abstract

摘要： 旨在探究NGF基因的生物学特性及其在母牦牛生殖器官中的表达特性。本研究收集黄体期母牦牛的心、肝、脾、肺、肾以及胎牛期、卵泡期、黄体期、妊娠期母牦牛的卵巢、子宫和输卵管(n=3),利用RT-PCR克隆牦牛NGF基因,并对其序列进行生物信息学分析,利用RT-qPCR技术分析NGF基因的组织表达特性,利用免疫组化技术(IHC)定位NGF蛋白在牦牛生殖器官中的表达分布。结果显示,牦牛NGF基因CDS区全长为726 bp,共编码241个氨基酸,编码的蛋白质属于碱性不稳定亲水蛋白;NGF氨基酸系统进化树结果显示,牦牛首先与黄牛聚为一类,与其他物种的同源性均高于87%,说明该基因在进化过程中表现出高度保守性。RT-qPCR结果显示,牦牛NGF基因在卵巢中的表达量极显著高于心、肝、肾、脾、肺、子宫和输卵管(P<0.01)。黄体期卵巢中NGF的相对表达量最高,极显著高于胎牛期、卵泡期和妊娠期(P<0.01);妊娠期子宫NGF相对表达量较高,显著高于胎牛期和卵泡期(P<0.05);各时期输卵管的NGF mRNA表达差异不显著。IHC结果显示,NGF蛋白主要在牦牛卵泡的颗粒细胞和卵巢上皮层细胞中表达,且在颗粒层细胞表达最高;在子宫内膜和输卵管的黏膜上皮细胞也有表达。综上,牦牛NGF基因序列在进化过程中较为保守,在卵巢中表达量较高,可能在维持母牦牛卵巢机能和妊娠以及促进黄体功能方面发挥重要的调控作用。

关键词: 牦牛, NGF基因, 卵巢, 组织表达, 免疫组化

Abstract: The purpose of this study was to explore the molecular characteristics of NGF gene and its expression properties in reproductive organs of female yak (Bos grunniens). The heart, liver, spleen, lung and kidney of female yak at luteal phase were collected. In addition, the ovaries, uterus and oviduct of yaks during fetal, follicular, luteal phases and gestation were collected (n=3). NGF gene was cloned by RT-PCR and its sequence was analyzed by bioinformatics method. The mRNA expression was evaluated by RT-qPCR and NGF protein location in the reproductive organs was detected by immunohistochemistry. The results showed that the CDS region of yak NGF gene was 726 bp, which encoded 241 amino acids. NGF protein belongs to alkaline unstable hydrophilic protein. The results of NGF amino acid phylogenetic tree showed that yaks were first grouped with yellow cattle, and the homology with other species was more than 87%, indicating that NGF gene was relatively conserved in the course of animal evolution. The results of RT-qPCR showed that the expression of NGF gene in ovary was extremely significantly higher than that in heart, liver, kidney, spleen, lung, uterus and oviduct (P<0.01). The relative expression of NGF in ovary of luteal phase was the highest, which was significantly higher than that in fetal, follicular stage and pregnancy (P<0.01). In uterus, the relative expression of NGF was significantly higher during pregnancy than that in fetal and follicular stages (P<0.05). There was no significant difference in the expression of NGF in different physiological stages of oviduct. The immunohistochemistry analysis indicated that NGF protein was mainly localized in granulosa cells and ovarian epithelium cells, with the highest expression in granulosa cells. It was also expressed in yak endometrium and epithelial cells of yak oviduct. In conclusion, the sequence of NGF gene in yak is relatively conserved in the course of evolution, and is highly expressed in the ovary. NGF gene may play an important regulatory role in maintaining ovary function, pregnancy and promoting corpus luteam function in female yaks.

Key words: yak, nerve growth factor-β(NGF) gene, ovary, tissue expression, immunohistochemistry

中图分类号:

S823.85

邬建飞, 刘宇, 马瑶, 蒋旭东, 胡双阁, 龚三你, 字向东, 卢建远. 牦牛NGF基因克隆及其在生殖器官中的表达定位[J]. 畜牧兽医学报, 2022, 53(3): 778-790.

WU Jianfei, LIU Yu, MA Yao, JIANG Xudong, HU Shuangge, GONG Sanni, ZI Xiangdong, LU Jianyuan. Cloning of Yak NGF Gene and Its Expression and Localization in Reproductive Organs of Yaks[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 778-790.

参考文献 43

[1]	TESSAROLLO L.Pleiotropic functions of neurotrophins in development[J].Cytokine Growth Factor Rev,1998,9(2):125-137.
[2]	ANGELETTI R H,BRADSHAW R A.Nerve growth factor from mouse submaxillary gland:amino acid sequence[J].Proc Natl Acad Sci U S A,1971,68(10):2417-2420.
[3]	ROCCO M L,SOLIGO M,MANNI L,et al.Nerve growth factor:early studies and recent clinical trials[J].Curr Neuropharmacol, 2018,16(10):1455-1465.
[4]	ZHANG R,LIANG Y,WEI S X.The expressions of NGF and VEGF in the fracture tissues are closely associated with accelerated clavicle fracture healing in patients with traumatic brain injury[J].Ther Clin Risk Manag,2018,14:2315-2322.
[5]	LIN H H,HUANG H J,YU Y H,et al.Nerve growth factor regulates liver cancer cell polarity and motility[J].Mol Med Rep,2021,23(4):288.
[6]	LIU Z X,WU H W,HUANG S Y.Role of NGF and its receptors in wound healing (review)[J].Exp Ther Med,2021,21(6):599.
[7]	LIMA F S,STEWART J L,CANISSO I F.Insights into nerve growth factor-β role in bovine reproduction-review[J]. Theriogenology,2020,150:288-293.
[8]	SILVA M,PAIVA L,RATTO M H.Ovulation mechanism in south American camelids:the active role of β-NGF as the chemical signal eliciting ovulation in llamas and alpacas[J].Theriogenology,2020,150:280-287.
[9]	DISSEN G A,HILL D F,COSTA M E,et al.A role for trkA nerve growth factor receptors in mammalian ovulation[J]. Endocrinology,1996,137(1):198-209.
[10]	DISSEN G A,ROMERO C,HIRSHFIELD A N,et al.Nerve growth factor is required for early follicular development in the mammalian ovary[J].Endocrinology,2001,142(5):2078-2086.
[11]	DISSEN G A,PARROTT J A,SKINNER M K,et al.Direct effects of nerve growth factor on thecal cells from antral ovarian follicles[J].Endocrinology,2000,141(12):4736-4750.
[12]	LEVANTI M B,GERMANÀ A,ABBATE F,et al.TrkA and p75NTR in the ovary of adult cow and pig[J].J Anat,2005,207(1):93-96.
[13]	MARANESI M,PALERMO F A,BUFALARI A,et al.Seasonal expression of NGF and its cognate receptors in the ovaries of grey squirrels (Sciurus carolinensis)[J].Animals (Basel),2020,10(9):1558.
[14]	ABIR R,FISCH B,JIN S,et al.Presence of NGF and its receptors in ovaries from human fetuses and adults[J].Mol Hum Reprod,2005,11(4):229-236.
[15]	杨远潇,字向东.玻璃化冷冻对牦牛未成熟卵母细胞发育能力及COC转录组的影响[J].畜牧兽医学报,2020,51(2):288-298.YANG Y X,ZI X D.Effect of vitrification on developmental competence of immature oocytes and COC transcriptome of yaks[J]. Acta Veterinaria et Zootechnica Sinica,2020,51(2):288-298.(in Chinese)
[16]	王琴,熊燕,字向东.体外成熟液中添加PAF对牦牛卵母细胞发育能力及基因表达的影响[J].畜牧兽医学报,2020,51(3):514-523.WANG Q,XIONG Y,ZI X D.Effects of PAF supplementation during in vitro maturation medium on yak oocyte development competence and gene expression[J].Acta Veterinaria et Zootechnica Sinica,2020,51(3):514-523.(in Chinese)
[17]	XIA Y,WANG Q,HE X D,et al.Cloning and expression analysis of the follicle-stimulating hormone receptor (FSHR) gene in the reproductive axis of female yaks (Bos grunniens)[J].Domest Anim Endocrinol,2020,70:106383.
[18]	LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) method[J].Methods,2001,25(4):402-408.
[19]	LEVI-MONTALCINI R,DAL TOSO R,DELLA VALLE F,et al.Update of the NGF saga[J].J Neurol Sci,1995,130(2):119-127.
[20]	STEWART J L,MERCADANTE V R G,DIAS N W,et al.Nerve growth factor-beta,purified from bull seminal plasma,enhances corpus luteum formation and conceptus development in Bos taurus cows[J].Theriogenology,2018,106:30-38.
[21]	GARCIA-GARCIA R M,ARIAS-ALVAREZ M,SANCHEZ-RODRIGUEZ A,et al.Role of nerve growth factor in the reproductive physiology of female rabbits:a review[J].Theriogenology,2020,150:321-328.
[22]	RYDÉN M,IBÁÑEZ C F.A second determinant of binding to the p75 neurotrophin receptor revealed by alanine-scanning mutagenesis of a conserved loop in nerve growth factor[J].J Biol Chem,1997,272(52):33085-33091.
[23]	KAWA S,FUJIMOTO J,TEZUKA T,et al.Involvement of BREK,a serine/threonine kinase enriched in brain,in NGF signalling[J]. Genes Cells,2004,9(3):219-232.
[24]	AHLUWALIA A,JONES M K,HOA N,et al.NGF protects endothelial cells from indomethacin-induced injury through activation of mitochondria and upregulation of IGF-1[J].Cell Signal,2017,40:22-29.
[25]	EBENDAL T,LARHAMMAR D,PERSSON H.Structure and expression of the chicken beta nerve growth factor gene[J].EMBO J,1986,5(7):1483-1487.
[26]	YUAN B,PAN S,ZHANG W W.Effects of gangliosides on expressions of caspase-3 and NGF in rats with acute spinal cord injury[J].Eur Rev Med Pharmacol Sci,2017,21(24):5843-5849.
[27]	LEE R,KERMANI P,TENG K K,et al.Regulation of cell survival by secreted proneurotrophins[J]. Science, 2001, 294(5548):1945-1948.
[28]	URBAN D,LORENZ J,MEYBORG H,et al.Proprotein convertase furin enhances survival and migration of vascular smooth muscle cells via processing of pro-nerve growth factor[J].J Biochem,2013,153(2):197-207.
[29]	FAGERBERG L,HALLSTRÖM B M,OKSVOLD P,et al.Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[J].Mol Cell Proteomics,2014,13(2):397-406.
[30]	YUE F,CHENG Y,BRESCHI A,et al.A comparative encyclopedia of DNA elements in the mouse genome[J]. Nature, 2014,515(7527):355-364.
[31]	JULIO-PIEPER M,LARA H E,BRAVO J A,et al.Effects of nerve growth factor (NGF) on blood vessels area and expression of the angiogenic factors VEGF and TGFbeta1 in the rat ovary[J].Reprod Biol Endocrinol,2006,4:57.
[32]	ADAMS G P,RATTO M H,HUANCA W,et al.Ovulation-inducing factor in the seminal plasma of alpacas and llamas[J].Biol Reprod,2005,73(3):452-457.
[33]	STEWART J L,MERCADANTE V R G,DIAS N,et al.Nerve growth factor-β increases small luteal cell number and steroidogenic activity in the bovine corpus luteum[J].J Dairy Sci,2019,102(1):344.
[34]	SILVA M,FERNÁNDEZ A,ULLOA-LEAL C,et al.LH release and ovulatory response after intramuscular,intravenous,and intrauterine administration of β-nerve growth factor of seminal plasma origin in female llamas[J].Theriogenology, 2015, 84(7):1096-1102.
[35]	GARCÍA-GARCÍA R M,DEL MAR MASDEU M,RODRIGUEZ A S,et al. β-Nerve growth factor identification in male rabbit genital tract and seminal plasma and its role in ovulation induction in rabbit does[J].Ital J Anim Sci,2018,17:442-453.
[36]	ZHAI Y W,YAO G D,RAO F,et al.Excessive nerve growth factor impairs bidirectional communication between the oocyte and cumulus cells resulting in reduced oocyte competence[J].Reprod Biol Endocrinol,2018,16(1):28.
[37]	MANTI M,PUI H P,EDSTRÖM S,et al.Excess of ovarian nerve growth factor impairs embryonic development and causes reproductive and metabolic dysfunction in adult female mice[J].Faseb J,2020,34(11):14440-14457.
[38]	WRAY S,PRENDERGAST C.The myometrium:from excitation to contractions and labour[J].Adv Exp Med Biol,2019,1124:233-263.
[39]	BARRAZA D E,SARI L M,APICHELA S A,et al.New insights into the role of β-NGF/TrKA system in the endometrium of alpacas during early pregnancy[J].Front Vet Sci,2021,7:583369.
[40]	VAROL F G,DUCHEMIN A M,NEFF N H,et al.Nerve growth factor (NGF) and NGF mRNA change in rat uterus during pregnancy[J].Neurosci Lett,2000,294(1):58-62.
[41]	LI C J,MA Y H,YI K L,et al.The interactions between nerve growth factor and gonadotrophins in bovine oviduct[J].Anim Reprod Sci,2014,149(3-4):117-123.
[42]	MARUCCIO L,CASTALDO L,D'ANGELO L,et al.Neurotrophins and specific receptors in the oviduct tracts of Japanese quail (Coturnix coturnix japonica)[J].Ann Anat,2016,207:38-46.
[43]	HARRIS E A,STEPHENS K K,WINUTHAYANON W.Extracellular vesicles and the oviduct function[J].Int J Mol Sci,2020, 21(21):8280.