The Expression of TNF-α in Yak (Bos grunniens) Oocytes and Preimplantation Embryos Produced by In vitro Fertilization
XU Tao, PAN Yang-Yang, HE Hong-Hong, LI Gu-Yue, CHEN Ping, WEN Ze-Xing, CUI Yan, YU Si-Jiu*
Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Tumor necrosis factor α (TNF-α) is a pro-inflammatory Th1 cytokine that is involved in the regulation of mammalian ovulation, embryonic development and implantation. The aim of this study was to investigate the role of TNF-α in the development of oocyte maturation and in vitro fertilization (IVF) embryos by detecting the expression of TNF-α in yak (Bos grunneins) oocyte maturation and early IVF embryo development. In this study, the cumulus-oocyte complex (COCs) were collected and matured in vitro and fertilized to obtain IVF embryos, and to evaluate the developmental ability of embryos at different stages of implantation. Methods of qRT-PCR, indirect immunofluorescence staining and Western blot were used to detect the expression and distribution of TNF-α in immature oocytes, mature oocytes and embryos at different stages. The results showed as follows: (1) The rates of embryos developed to stages of 2~4 cells, 5~8 cells, morulae were (61.59±0.31)%, (53.76±0.22)% and (26.88±0.32)%, respectively, and the blastocyst rate was (16.12±0.23)%. (2) qRT-PCR results showed that, TNF-α mRNA could be detected in immature COCs, mature COCs, morulae and blastocysts, the relative expression levels in the order of high to low was mature COCs, immature COCs, blastocysts and morulae. 2~4 cells and 5~8 cells embryos had no expression. (3) The result of indirect immunofluorescence and Western blot showed that the protein of TNF-α was mainly distributed in the cytoplasm of COCs and embryos, which was weaker in the nucleus. The protein of TNF-α could be detected in COCs, morula and blastocyst, the relative expression levels in the order of high to low was mature COCs, immature COCs, blastocysts and morulae, but 2~4 cells and 5~8 cells embryos had no expression. The results in this study indicated that TNF-α might play a role in the process of the maturation and senescence of the yak oocytes and might be involved in the process of blastocyst cell apoptosis and embryo implantation, which provides a theoretical basis for revealing the mechanism of TNF-α in yak reproduction.
许涛, 潘阳阳, 何翃闳, 李谷月, 陈平, 温泽星, 崔燕, 余四九. TNF-α在牦牛卵母细胞及早期体外受精胚胎中的表达[J]. 农业生物技术学报, 2019, 27(8): 1423-1433.
XU Tao, PAN Yang-Yang, HE Hong-Hong, LI Gu-Yue, CHEN Ping, WEN Ze-Xing, CUI Yan, YU Si-Jiu. The Expression of TNF-α in Yak (Bos grunniens) Oocytes and Preimplantation Embryos Produced by In vitro Fertilization. 农业生物技术学报, 2019, 27(8): 1423-1433.
[1] 边淑玲, 金宏波, 王淑珍, 等. 2007. 干扰素-γ和肿瘤坏死因子-α对精子受精能力的影响及机制初探[J]. 中华男科学杂志, 13(8): 681-684. (Bian S L, Jin H B,Wang S Z, et al.2007. Effect and mechanism of interferon-γ and tumor necrosis factor-α on sperm fertilization ability[J]. Chinese Journal of male Science, 13(8): 681-684) [2] 刘汝宏, 丛志强. 2003. TNF系统与自身免疫性疾病[J]. 医学综述, 9(4): 200-202. (Liu R H, Cong Z Q.2003. Tnf system and autoimmune diseases[J]. Medical Review, 9(4): 200-202.) [3] 陶慧英. 2014. 导致胚胎停育的原因分析及预防措施探讨[J]. 医学信息, 27(23): 618-619. (Tao H Y.2014. Cause analysis and preventive measures of embryo termination[J]. Medical Information, 27(23): 618-619.) [4] 田世河. 2002. 肿瘤坏死因子α mRNA及其受体在人精子表达的研究[D]. 硕士学位论文, 山西医科大学, 导师: 郝素珍, pp 19-24. (Tian S H.2002. Expression of tumor necrosis factor α mRNA and its receptor in human spermatozoa[D]. Thesis for M.S., Shanxi Medical University, Supervisor: Hao S Z, pp 19-24.) [5] 王文珊, 傅冷西, 叶君健. 2005. TNF-α信号传导通路的研究进展[J]. 福建医科大学学报, 39(增刊): 27-31. (Wang W S, Fu L X, YE J J.2005. Research progress of TNF-α signal transduction pathway[J]. Journal of Fujian Medical University, 39(Supplement): 27-31.) [6] 谢宝国, 朱伟杰. 2013. Wnt/β-catenin信号通路在胚胎着床和发育中的作用[J]. 生殖与避孕, 33(5): 328-332. (Xie B G, Zhu W J.2013. Role of Wnt/b-catenin signaling pathway in embryo implantation and development[J]. Reproduction and Contraception, 33(5): 328-332.) [7] 许颂华. 2016. 雌激素受体α在小鼠植入前胚合子型基因激活中的作用机制探讨[D]. 博士学位论文, 福建医科大学, 导师: 王世鄂, pp 124. (XU S H.2014. The roles of estrogen receptor α in regulating zygotic gene activation of mouse preimplantation embryos[D]. Thesis for Ph.D., Fujian Medical University, Suppervisor: Wang S E, pp 124.) [8] 赵天, 李谷月, 潘阳阳, 等. 2017. OCT 4在牦牛卵母细胞及早期胚胎发育过程中的表达[J]. 畜牧兽医学报, 48(3): 577-584. (Zhao T, Li G Y, Pan Y Y, et al.2017. The expression of octamer-binding transcription factor 4 gene (Oct 4) in yak (Bos grunniens) oocytes and preimplantation embryos produced by in vitro fertilization[J].Acta Veterinaria Et Zootechnica Sinica, 48(3): 577-584.) [9] Alijotas-Reig J, Esteve-Valverde E, Ferrer-Oliveras R, et al.2017. Tumor necrosis factor-alpha and pregnancy: Focus on biologics. An updated and comprehensive review[J]. Clinical Reviews in Allergy & Immunology, 53(1): 40-53. [10] Alijotas-Reig J, Llurba E, Gris J M.2014. Potentiating maternal immune tolerance in pregnancy: A new challenging role for regulatory T cells[J]. Placenta, 35(4): 241-248. [11] Bell C E, Calder M D, Watson A J.2008. Genomic RNA profiling and the programme controlling preimplantation mammalian development[J]. Molecular Human Reproduction, 14(12): 691-701. [12] Beutler B, Mahoney J, Trang N L, et al.1985. Purification of cachectin, alipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells[J]. Journal of Experimental Medicine, 161(5): 984-995. [13] Casey M L,Cox S M, Beutler B, et al.1989. Cachectin/tumor necrosis factor-α formation in human deciduas: Potential role of cytokines in infection induced preterm labor[J]. Journal of Clinical Investigation, 83(2): 430-436. [14] Desai N, Scarrow M, Lawson J, et al.1999. Evaluation of the effect to finterleukin-6 and human ex tracellular matrix on embryo nic development[J]. Human Reproduction, 14(6): 1588-1592. [15] Duncan F E, Schultz R M.2010. Gene expression profiling of mouse oocytes and preimplantation embryos[J]. Methods in Enzymology, 477(477): 457-480. [16] Ferrero E, Zocchi M R, Maghi E, et al.200l. Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced vascular permeability[J]. American Journal of Physiology-cell Physiology, 28l(7): 1173-1179. [17] Gottsch M L, vaan Kirk E A, Murdoch W J.2000. Tumor necrosis factor alpha up regulates matrix metalloproteinase-2 activity in periovulatory ovine follicles: Metamo rphic and endo crine implications[J]. Reproduction, Fertility and Development, 12(12): 75-80. [18] Hiyama A, Yokoyama K, Nukaga T, et al.2013. A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells[J]. Arthritis research & therapy, 15(6): R189. [19] Hunt J S, Chen H L, Hu X L, et al.1992. Tumor necrosis factor-alpha messenger ribonucleic acid and protein in human endometrium[J]. Biology of Reproduction, 47(1):141-147. [20] Hunt J S, Chen H L, Hu X L, et al.1993. Normal distribution of tumor necrosis factor-alpha messenger ribonucleic acid and protein in the uteri, placentas, and embryos of osteopetrotic (op/op) mice lacking colony-stimulating factor-1[J]. Biology of Reproduction, 49(3): 441-452. [21] Johnson M L, Murdoch J, Van Kirk E A, et al.1999. Tumor necrosis factor alpha regulates collagenolytic activity in preovulatory ovine follicles: Relationship to cytokine secretion by the oocyte-cumulus cell complex[J]. Biology of Reproduction, 61(6): 1581-1585. [22] Kong Q Q, Wang J, Xiao B, et al.2018. Cumulus cell-released tumor necrosis facto r (TNF)-α promotes post-ovulatory aging of mouse oocytes[J]. Aging, 10(7): 1745-1757. [23] Lachappelle M H, Miron P, Hemmings R, et al.1993. Embryonic resistance to tumour necrosis factor-a mediated cytotoxicity: Novel mechanismunderlying maternal immunological tolerance to the fetal allograft[J]. Human Reproduction, 8(7): 1032-1038. [24] Li E.1999. The mojo of methy lation[J]. Nature Genetics, 23(1): 5-6. [25] Liu N, Wu Y G, Lan G C, et al.2009. Pyruvate prevents aging of mouseoocytes[J]. Reproduction, 138(2): 223-234. [26] Marcinkiewicz J L.1994. Oocytic tumor necrosis factor alpha: Localization in the neonatal ovary and throughout follicular development in the adult rat[J]. Biology of Reproduction, 50(6): 1251-1260. [27] Marcinkiewicz J L, Balchak S K, Morrison L J.2002.The involvement of tumor necrosis factor-alpha (TNF) as an intraovarian regulator of oocyte apoptosis in the neonatal rat[J]. Frontiers in Bioscience, 7(38): 1997-2005. [28] Mason M C, Copeland J, Cuadra E J, et al.2014. Dynamics of progesterone, TNF-α, and a metabolite of PGF2α in blood plasma of beef cows following embryo transfer[J]. Veterinary Medicine International, 2014: 1-8. [29] Miao Y L.2005. Cumulus cells accelerate aging of mouse oocytes[J]. Biology of Reproduction, 73(5): 1025-1031. [30] Mourik M S M V, Macklon N S, Heijnen C J.2009. Embryonic implantation: Cytokines, adhesion molecules,and immune cells in establishing an implantation environment[J]. Journal of Leukocyte Biology, 85(1): 4. [31] Ng H H, Zhang Y, Hendrich B, et al.1999. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex[J]. Nature Genetics, 23(1): 58-61. [32] Okuda K, Sakumoto R.2003. Multiple roles of TNF super family members in corpus luteum function[J]. Reproductive Biology and Endocrinology, 1(1): 95. [33] Pampfer S, Vanderheyden I, Vesela J, et al.1995. Neutralization of tumor necrosis factor alpha (TNF alpha) action on cell proliferation in rat blastocysts by antisence oligodeoxyribonucleotides directed against TNF alpha p60 receptor[J]. Biology of Reproduction, 52(6): 1316-1326. [34] Payan-Carreira R, Pires M A, Holst B S, et al.2011.Tumour necrosis factor in the canine endometrium: An immunohistochemical study[J]. Reproduction in Domestic Animals, 46(3): 410-418. [35] Prabhala R H,Wira C R.1995. Sex hormone and IL -6 regulation of antigen presentation in the female reproductive tract mucosal tissues[J]. Journal of Immunology, 155(12): 5566-5573. [36] Reik W, Dean W, Walt er J.2001. Epigenetic reprogramming in mammalian development[J]. Science, 293(5532): 1089-1093. [37] Robertson S A, Chin P Y, Femia J G, et al.2018. Embryotoxic cytokines-potential roles in embryo loss and fetal programming[J]. Journal of Reproductive Immunology, 125: 80-88. [38] Roby K F, Weed J, Lyles R, et al.1990. Immunological evidence for a human ovarian tumor necrosis factor-α[J]. The Journal of Clinical Endocrinology & Metabolism, 71(5): 1096-1102. [39] Rosario G X, Sachdeva G, Manjramkar D D, et al.2005. Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys[J]. Cytokine, 31(6): 459-464. [40] Saito S, Nakashima A, Shima T, et al.2010. Th1/Th2/Th17 andregulatory T-cell paradigm in pregnancy[J]. American Journal of Reproductive Immunology 63(6): 601-610. [41] Sancho-Tello M, I Pérez-Roger, Imakawa K, et al.1992. Expression of tumor necrosis factor-α in the rat ovary[J]. Endocrinology, 130(3): 1359-1364. [42] Sharkey A M, Dellow K, Blayney M, et al.1995. Stage-specific expression of cytokine and receptor messenger ribonucleic acids in human preimplantaion embryos[J]. Biology of Reproduction, 53(4): 955-962. [43] Soto P, Natzke R P, Hansen P J.2003. Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle[J]. American Journal of Reproductive Immunology, 50(5): 380-388. [44] Straczkowski M, Kowalska M, Dzienis-Straczkowska S, et al.2001. Changes in tumor necrosis factor-α system and insulin sensitivity during an exercise training program in obese women with normal and impaired glucose tolerance[J]. European Journal of Endocrinology, 145(3): 273-280. [45] Tadros W, Lipshitz H D.2009. The maternal-to-zygotic transition: A play in two acts[J]. Development, 136(18): 3033-3042. [46] Tarín J J, Ten J, Vendrell F J, et al.1998. Dithiothreitolprevents age-associated decrease in oocyte/conceptus viability in vitro[J]. Human Reproduction, 13(2): 381-386. [47] Tarín J J, Pérez-Albalá S, Aguilar A, et al.1999. Long-term effects of postovulatory aging of mouse oocytes on offspring: A two-generational study[J]. Biology of Reproduction, 61(5): 1347-1355. [48] Tarín J J, Pérez-Albalá S, Pérez-Hoyos S, et al.2002. Postovulatory aging of oocytes decreases reproductive fitness and longevity of offspring[J]. Biology of Reproduction, 66(2): 495-499. [49] Torchinsky A, Shepshelovich J, Orenstein H, et al.2015. TNF-alpha protects embryos exposed to developmental toxicants[J]. American Journal of Reproductive Immunology, 49(3): 159-168. [50] Vialard F, Sirkasi M E, Tronchon V, et al.2013. Tumor necrosis factor-308 polymorphism increases the embryo implantation rate in women undergoing in vitro fertilization[J]. Human Reproduction, 28(10): 2774-2783. [51] Walser C B, Lipshitz H D.2011. Transcript clearance during the maternal-to-zygotic transition[J]. Current Opinion in Genetics & Development, 21: 431. [52] Whittingham D G, Siracusa G.1978. The involvement of calcium in the activation of mammalian oocytes[J]. Experimental Cell Research, 113(2): 311-317. [53] Yanagimachi R, Chang M C.1961. Fertilizable life of goldenhamster ova and their morphological changes at thetime of losing fertilizability[J]. Journal of Experimental Zoology, 148(3): 185-203. [54] Yelavarthi K K, Chen H L, Yang Y, et al.1991. Tumor necrosis factor alpha mRNA and protein in rat uterineand placental cells[J]. Journal of Immunology, 146(11): 3840-3848. [55] Yu S J, Huang Y M, Chen B X.1993. Reproductive patterns of the yak.Ⅲ.Levels of progesterone and oesteadiol-17beta during pregnancy and the periparturient period[J]. British Veterinary Journal, 149(6): 595-602. [56] Zhu J, Zhang J, Li H, et al.2015. Cumulus cells accelerate oocyte aging byreleasing soluble fas ligand in mice[J]. Scientific Reports, 5(3): 8683-8690.
