SPATA3基因克隆及其在牦牛和犏牛睾丸中的差异表达研究

doi:10.11843/j.issn.0366-6964.2020.02.009

摘要/Abstract

摘要： 旨在克隆牦牛精子发生蛋白3（spermatogenesis associated 3，SPATA3）基因，并检测其在牦牛不同组织及在不同发育时期牦牛和犏牛睾丸中的表达水平，探讨SPATA3对睾丸发育和精子成熟的影响，为进一步研究该基因在精子形成过程中的作用机制提供理论依据。本试验以牦牛为研究对象，利用RT-PCR克隆技术获取牦牛SPATA3 cDNA序列，使用生物信息软件分析其结构和功能，并检测其在牦牛心、肝、脾、肺、肾、脑、小肠、卵巢、子宫和睾丸组织中的表达谱。通过实时荧光定量PCR（quantitative real-time PCR，RT-qPCR）检测SPATA3基因在牦牛和犏牛睾丸不同发育时期的表达规律；采用免疫组化技术检测SPATA3在牦牛和犏牛睾丸中的细胞定位及表达差异。结果显示，SPATA3基因CDS区为693 bp，编码230个氨基酸，与黄牛、野牦牛和水牛的同源性较高；SPATA3仅在耗牛睾丸组织中特异性表达，其它组织未见其表达。RT-qPCR结果显示，SPATA3基因在牦牛睾丸发育过程中均有表达，其中成年期（4~5岁）睾丸中的表达极显著高于胎牛时期（5~6月，P<0.01），且其在牦牛不同发育时期睾丸中的表达均极显著高于犏牛（P<0.01）。免疫组化结果发现，SPATA3在圆形精子、长形精子细胞胞浆中均表达，且成年期牦牛睾丸中该基因的表达水平极显著高于犏牛（P<0.01）。综上表明，SPATA3在遗传进化中高度保守，且在牦牛和犏牛睾丸组织中差异表达，该基因低表达可能引起精子形成障碍，从而参与精子成熟进程，但具体功能有待进一步的研究。

关键词: 牦牛, 犏牛, SPATA3, 免疫组化, 精子发生, 表达

Abstract: The aim of this research was to clone the spermatogenesis associated 3 (SPATA3) gene in yak, identify its expression pattern in various tissues of yak, and in different developmental stages in yak and cattle-yak testis, respectively, explore the effects of SPATA3 on testicular development and sperm maturity, and provide reliable basis data for studying the mechanism of SPATA3 in spermatogenesis of yak. The samples of yak heart, liver, spleen, lung, kidney, small intestine, brain, ovary, uterus and testis were collected after slaughtering. The total RNAs in different samples were extracted. The coding sequence of SPATA3 gene was cloned and the expression of SPATA3 in different tissues was detected by RT-PCR. The structure and function of SPATA3 were analyzed by bioinformatics softwares. The mRNA expression of SPATA3 gene in testicular different development stages of yak and cattle-yak was compared by quantitative real-time PCR (RT-qPCR). Then, the localization and expression of SPATA3 in yak and cattle-yak testis were detected by immunohistochemistry. The results showed that the CDS region of SPATA3 gene was 693 bp, encoding 230 amino acids; It had high homology with SPATA3 sequence of cattle, wild yak and buffalo. SPATA3 gene was specifically expressed in testis tissue, and no expression was observed in other tissues of yak. The results of RT-qPCR showed that SPATA3 gene was expressed during testicular development of yak, and the expression level in testis of adulthood yak(4-5 years old) was extremely significantly higher than that of fetal period yak(5-6 months old, P<0.01). And its expression in testis in different developmental stages of yak were all extremely significantly higher than that of cattle-yak (P<0.01). The results of immunohistochemistry showed that SPATA3 was expressed in cytoplasm of round spermatid and elongating spermatid, and its expression level in testis of adulthood yak was extremely significantly higher than that in testis of adulthood cattle-yak (P<0.01). The above results show that SPATA3 is highly conserved during evolution, and it is differentially expressed in testis tissues of yak and cattle-yak. Its low expression may cause sperm formation disorder and participate in sperm maturation process, but the specific function should be researched further.

Key words: yak, cattle-yak, SPATA3, immunohistochemistry, spermatogenesis, expression

中图分类号:

S823.85

黄向月, 熊显荣, 张磊, 马鸿程, 海卓, 李键. SPATA3基因克隆及其在牦牛和犏牛睾丸中的差异表达研究[J]. 畜牧兽医学报, 2020, 51(2): 279-287.

HUANG Xiangyue, XIONG Xianrong, ZHANG Lei, MA Hongcheng, HAI Zhuo, LI Jian. Cloning of SPATA3 Gene and Its Differential Expression Analysis in Yak and Cattle-yak Testis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 279-287.

参考文献 35

[1]	LAN D L,XIONG X R,MIPAM T D,et al.Genetic diversity,molecular phylogeny,and selection evidence of Jinchuan yak revealed by whole-genome resequencing[J].G3(Bethesda),2018,8(3):945-952.
[2]	QIU Q,ZHANG G J,MA T,et al.The yak genome and adaptation to life at high altitude[J].Nat Genet,2012,44(8):946-949.
[3]	韩杰,熊显荣,王艳,等.牦牛KDM1A基因克隆及其在不同发育时期睾丸中的表达规律[J].畜牧兽医学报,2018, 49(12):2777-2785.HN J,XIONG X R,WANG Y,et al.Cloning of yak KDM1A gene and its expression profile in testis during different developmental stages[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2777-2785.(in Chinese)
[4]	HERMO D L,BARIN K,ROBAIRE B.Structural differentiation of the epithelial cells of the testicular excurrent duct system of rats during postnatal development[J].Anat Rec,1992,233(2):205-228.
[5]	GRISWOLD M D.Spermatogenesis:the commitment to meiosis[J].Physiol Rev,2016,96(1):1-17.
[6]	BELLEANNÉE C,THIMON V,SULLIVAN R.Region-specific gene expression in the epididymis[J].Cell Tissue Res,2012,349(3):717-731.
[7]	BJÖRKGREN I,ALVAREZ L,BLANK N,et al.Targeted inactivation of the mouse epididymal beta-defensin 41 alters sperm flagellar beat pattern and zona pellucida binding[J].Mol Cell Endocrinol,2016,427:143-154.
[8]	ZHAMG Z B,SHEN X N,GUDE D R,et al.MEIG1 is essential for spermiogenesis in mice[J].Proc Natl Acad Sci U S A,2009, 106(40):17055-17060.
[9]	KRAUSZ C,RIERA-ESCAMILLA A.Genetics of male infertility[J].Nat Rev Urol,2018,15(6):369-384.
[10]	MALCHER A,ROZWADOWSKA N,STOKOWY T,et al.Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis[J].Fertil Steril,2013,100(6):1686-1694.
[11]	温丽敏.Spata3基因参与小鼠生殖细胞发育及其初步功能研究[D].山西:山西医科大学,2017:1-80.WN L M.The preliminary function of Spata3 during germ cell development in mouse[D].Shanxi:Shanxi Medical University, 2017:1-80.(in Chinese)
[12]	YANG H M,LIU G,NIE Z Y,et al.Molecular cloning of a novel rat gene Tsarg1,a member of the DnaJ/HSP40 protein family[J]. DNA Seq,2005,16(3):166-172.
[13]	CAI L,HALES B F,ROBAIRE B.Induction of apoptosis in the germ cells of adult male rats after exposure to cyclophosphamide[J]. Biol Reprod,1997,56(6):1490-1497.
[14]	LI L,LIU G,FU J J,et al.Molecular cloning and characterization of a novel transcript variant of Mtsarg1 gene[J].Mol Biol Rep, 2009, 36(5):1023-1032.
[15]	WIESER V,TSIBULAK I,DEGASPER C,et al.Tumor necrosis factor receptor modulator spermatogenesis-associated protein 2 is a novel predictor of outcome in ovarian cancer[J].Cancer Sci,2019,110(3):1117-1126.
[16]	ABU-HALIMA M,AYESH B M,HART M,et al.Differential expression of miR-23a/b-3p and its target genes in male patients with subfertility[J].Fertil Steril,2019,112(2):323-335.
[17]	FU J J,LU G X,LI L Y,et al.Molecular cloning for testis spermatogenesis cell apoptosis related gene TSARG1 and Mtsarg1 and expression analysis for Mtsarg1 gene[J]. Acta Genetica Sinica,2003,30(1):25-29.
[18]	OTA T,SUZUKI Y,NISHIKAWA T,et al.Complete sequencing and characterization of 21,243 full-length human cDNAs[J].Nat Genet,2004,36:40-45.
[19]	ROLLAND T,TAŞAN M,CHARLOTEAUX B,et al.A proteome-scale map of the human interactome network[J].Cell, 2014, 159(5):1212-1226.
[20]	王玉晶,温丽敏,白欣艳,等.小鼠精子发生相关蛋白3基因在小鼠生精细胞的特异表达及对HEK293T细胞凋亡和自噬的影响[J].解剖学报,2018,49(1):41-48.WNG Y J,WEN L M,BAI X Y,et al.Novel expression of spermatogenesis-associated protein 3 gene in mouse spermatogenic cells and its influence upon apoptosis and autophagy in HEK 293T cells[J].Acta Anatomica Sinica,2018,49(1):41-48.(in Chinese)
[21]	LI H Y,ZHANG W H,ZHANG H,et al.Mitochondrial proteomics reveals the mechanism of spermatogenic cells apoptosis induced by carbon ion radiation in zebrafish[J].J Cell Physiol,2019,234(12):22439-22449.
[22]	XU Y R,DONG H S,YANG W X.Regulators in the apoptotic pathway during spermatogenesis:killers or guards?[J]. Gene, 2016, 582(2):97-111.
[23]	XIAO B,LI X,FENG X Y,et al.Restraint stress of male mice induces apoptosis in spermatozoa and spermatogenic cells:role of the FasL/Fas system[J].Biol Reprod,2019,101(1):235-247.
[24]	BRVNNERT D,LANGER C,ZIMMERMANN L,et al.The heat shock protein 70 inhibitor VER155008 suppresses the expression of HSP27,HOP and HSP90β and the androgen receptor,induces apoptosis,and attenuates prostate cancer cell growth[J]. J Cell Biochem,2019,121(1):407-417.
[25]	XU Y,YU S S,SHU Q M,et al.Upregulation of CREM-1 relates to retinal ganglion cells apoptosis after light-induced damage in vivo[J].J Mol Neurosci,2014,52(3):331-338.
[26]	YAO C P,CAO X J,FU Z,et al.Boschniakia rossica polysaccharide triggers laryngeal carcinoma cell apoptosis by regulating expression of Bcl-2,Caspase-3,and P53[J].Med Sci Monit,2017,23:2059-2064.
[27]	WANG J H,WANG C,YU H B,et al.Bacterial quorum-sensing signal IQS induces host cell apoptosis by targeting POT1-p53 signalling pathway[J].Cell Microbiol,2019,21(10):e13076.
[28]	ZHOU Y J,JI J H,ZHUANG J,et al.Nanoparticulate TiO2 induced suppression of spermatogenesis is involved in regulatory dysfunction of the cAMP-CREB/CREM signaling pathway in mice[J].J Biomed Nanotechnol,2019,15(3):571-580.
[29]	CHALMEL F,ROLLAND A D.Linking transcriptomics and proteomics in spermatogenesis[J].Reproduction, 2015, 150(5):R149-R157.
[30]	ZHOU B X,WEI C L,KHAN M A,et al.Characterization and molecular cloning of novel isoforms of human spermatogenesis associated gene SPATA3[J].Mol Biol Rep,2019,46(4):3827-3834.
[31]	HUANG Y F,YANG P,LIU T F,et al.Subcellular evidence for biogenesis of autophagosomal membrane during spermiogenesis in vivo[J].Front Physiol,2016,7:470.
[32]	LIU C,SONG Z H,WANG L N,et al.Sirt1 regulates acrosome biogenesis by modulating autophagic flux during spermiogenesis in mice[J].Development,2017,144(3):441-451.
[33]	WANG H N,WAN H F,LI X X,et al.Atg7 is required for acrosome biogenesis during spermatogenesis in mice[J].Cell Res,2014, 24(7):852-869.
[34]	YIN J,NI B,TIAN Z Q,et al.Regulatory effects of autophagy on spermatogenesis[J].Biol Reprod,2017,96(3):525-530.
[35]	MUKHOPADHYAY S,PANDA P K,SINHA N,et al.Autophagy and apoptosis:where do they meet?[J]. Apoptosis, 2014,19(4):555-566.