TNF-α对牦牛卵母细胞HIF-1α和HSP70的表达及后续胚胎发育能力的影响
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摘要
旨在研究肿瘤坏死因子(TNF-α)对牦牛卵母细胞体外成熟和对低氧诱导因子1α(HIF-1α)、热休克蛋白70(HSP70)的表达以及后续胚胎发育能力的影响。在牦牛卵母细胞体外成熟培养液中添加不同浓度的TNF-α(最终浓度分别为0、10、25、50 ng·mL~(-1)),牦牛卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs)培养成熟后,统计其卵母细胞成熟率及体外受精后早期胚胎的发育率,采用RT-PCR扩增牦牛HIF-1α、HSP70基因,采用实时荧光定量PCR(qRT-RCR)、蛋白免疫印迹法(Western blotting)和免疫荧光染色技术检测HIF-1α、HSP70在基因和蛋白水平的表达情况。结果发现:1)成熟液中加入TNF-α,卵母细胞的成熟率提高,且随着TNF-α浓度的升高,卵母细胞的成熟率、卵裂率和囊胚率逐渐升高,当TNF-α浓度为25 ng·mL~(-1)时,卵母细胞的成熟率和卵裂率达到最高,分别为84.98%和66.85%,而囊胚率也达到了21.48%,均显著高于对照组(P<0.05);2)随着TNF-α浓度的升高,HIF-1α的表达量逐渐降低,对照组HIF-1α的表达量极显著高于其他组(P<0.01),50 ng·mL~(-1) TNF-α组HIF-1α的表达量极显著低于其他组(P<0.01),HIF-1α在卵丘细胞和卵母细胞上均有表达;3)25 ng·mL~(-1)TNF-α组的HSP70表达量最高,极显著高于其他组(P<0.01),而当TNF-α达到50 ng·mL~(-1)时,HSP70的表达量最低。综上表明,在牦牛卵母细胞体外成熟过程中,TNF-α明显提高了卵母细胞的发育能力,同时还诱导了HSP70的表达,抑制了HIF-1α的表达,为今后探讨TNF-α、HIF-1α和HSP70在牦牛生殖过程中发挥的作用以及对胚胎发育的影响提供理论依据。
The aim of this study was to investigate the effects of tumor necrosis factor-α(TNF-α) on yak oocytes maturation in vitro, the expression of epidermal growth factor(HIF-1α) and heat shock protein receptor(HSP70) in yak oocytes and the subsequent embryo development. In this experiment, TNF-α with different concentrations(0, 10, 25, 50 ng·mL~(-1)) were added to in vitro maturation medium of yak oocytes. After the cumulus-oocyte complexes(COCs) matured, the oocyte maturation rate and the development rate of early embryos after in vitro fertilization were counted. The HIF-1α and HSP70 genes of yak were amplified by RT-PCR. The mRNA and protein expression of HIF-1α and HSP70 were detected by real-time PCR(qRT-RCR), Western blotting and immunofluorescence staining. The results showed that: 1) The addition of TNF-α to the oocyte maturation medium promoted the maturation rate of oocytes. With the increase of TNF-α concentration, the maturation rate, cleavage rate and blastocyst rate of oocytes increased gradually. When the concentration of TNF-α was 25 ng·mL~(-1), the maturation rate and cleavage rate of oocytes reached the highest, 84.98% and 66.85%, respectively, and the blastocyst rate also reached 21.48%, which were significantly higher than that in the control group(P<0.05). 2) With the increase of TNF-α concentration, the expression level of HIF-1α decreased gradually. When the concentration of TNF-α was 0 ng·mL~(-1), the expression level of HIF-1α was the highest, which was significantly higher than that in other groups(P<0.01). The expression level of HIF-1α was the lowest when the concentration of TNF-α was 50 ng·mL~(-1), which was sig-nificantly lower than that in other groups(P<0.01). Immunofluorescence assay showed that HIF-1α was expressed in both cumulus cells and oocytes. 3) When the concentration of TNF-α was 25 ng·mL~(-1), the expression level of HSP70 was the highest, which was significantly higher than that in other groups(P<0.01), and when the concentration of TNF-α reached 50 ng·mL~(-1), the expression level of HSP70 was the lowest. The results showed that TNF-α significantly increased the developmental capacity of oocytes during maturation of yak oocytes in vitro, and also induced the expression of HSP70 and inhibited the expression of HIF-1α. These would provide a theoretical basis for exploring the role of TNF-α, HIF-1α and HSP70 in the reproductive process of yak and the impact on embryo development.
The aim of this study was to investigate the effects of tumor necrosis factor-α(TNF-α) on yak oocytes maturation in vitro, the expression of epidermal growth factor(HIF-1α) and heat shock protein receptor(HSP70) in yak oocytes and the subsequent embryo development. In this experiment, TNF-α with different concentrations(0, 10, 25, 50 ng·mL~(-1)) were added to in vitro maturation medium of yak oocytes. After the cumulus-oocyte complexes(COCs) matured, the oocyte maturation rate and the development rate of early embryos after in vitro fertilization were counted. The HIF-1α and HSP70 genes of yak were amplified by RT-PCR. The mRNA and protein expression of HIF-1α and HSP70 were detected by real-time PCR(qRT-RCR), Western blotting and immunofluorescence staining. The results showed that: 1) The addition of TNF-α to the oocyte maturation medium promoted the maturation rate of oocytes. With the increase of TNF-α concentration, the maturation rate, cleavage rate and blastocyst rate of oocytes increased gradually. When the concentration of TNF-α was 25 ng·mL~(-1), the maturation rate and cleavage rate of oocytes reached the highest, 84.98% and 66.85%, respectively, and the blastocyst rate also reached 21.48%, which were significantly higher than that in the control group(P<0.05). 2) With the increase of TNF-α concentration, the expression level of HIF-1α decreased gradually. When the concentration of TNF-α was 0 ng·mL~(-1), the expression level of HIF-1α was the highest, which was significantly higher than that in other groups(P<0.01). The expression level of HIF-1α was the lowest when the concentration of TNF-α was 50 ng·mL~(-1), which was sig-nificantly lower than that in other groups(P<0.01). Immunofluorescence assay showed that HIF-1α was expressed in both cumulus cells and oocytes. 3) When the concentration of TNF-α was 25 ng·mL~(-1), the expression level of HSP70 was the highest, which was significantly higher than that in other groups(P<0.01), and when the concentration of TNF-α reached 50 ng·mL~(-1), the expression level of HSP70 was the lowest. The results showed that TNF-α significantly increased the developmental capacity of oocytes during maturation of yak oocytes in vitro, and also induced the expression of HSP70 and inhibited the expression of HIF-1α. These would provide a theoretical basis for exploring the role of TNF-α, HIF-1α and HSP70 in the reproductive process of yak and the impact on embryo development.
引文
[1] 何翃闳,潘阳阳,张慧珠,等.FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响[J].畜牧兽医学报,2018,49(9):1899-1907.HE H H,PAN Y Y,ZHANG H Z,et al.The effects of follicle-stimulating hormone (FSH) on the expression of EGF and EGFR in yak oocytes and apoptosis[J].Acta Veterinaria et Zootechnica Sinica,2018,49(9):1899-1907.(in Chinese)
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[16] YU X,DENG L Y,WANG D,et al.Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes:initiated by hypoxia inducible factor 1α,presented by exosomes[J].J Mol Cell Cardiol,2012,53(6):848-857.
[17] H?GELE S,KüHN U,B?NING M,et al.Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1α mRNA 3′-UTR and modulate HIF-1α protein expression[J].Biochem J,2009,417(1):235-246.
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[19] TSAPOURNIOTI S,MYLONIS I,HATZIEFTHIMIOU A,et al.TNFα induces expression of HIF-1α mRNA and protein but inhibits hypoxic stimulation of HIF-1 transcriptional activity in airway smooth muscle cells[J].J Cell Physiol,2013,228(8):1745-1753.
[20] VAN MOLLE W,WIELOCKX B,MAHIEU T,et al.HSP70 protects against TNF-induced lethal inflammatory shock[J].Immunity,2002,16(5):685-695.
[21] YANG R C,JAO H C,HUANG L J,et al.The essential role of PKCα in the protective effect of heat-shock pretreatment on TNFα-induced apoptosis in hepatic epithelial cell line[J].Exp Cell Res,2004,296(2):276-284.
[22] PANG Q S,KEEBLE W,CHRISTIANSON T A,et al.FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-γ/TNF-α-mediated cytotoxicity[J].EMBO J,2014,20(16):4478-4489.
[23] HUANG W,YANDELL B S,KHATIB H.Transcriptomic profiling of bovine IVF embryos revealed candidate genes and pathways involved in early embryonic development[J].BMC Genomics,2010,11:23.
[24] 李小红,罗珊,樊伟,等.HSP70在不同应激程度CUMS昆明小鼠胚胎中的表达及作用[J].四川大学学报:医学版,2017,48(4):515-519.LI X H,LUO S,FAN W,et al.Expression of HSP70 in Kunming mouse embryos stimulated by chronic mild anticipatory stress[J].Journal of Sichuan University:Medical Science Edition,2017,48(4):515-519.(in Chinese)
[25] KONG Q Q,WANG J,XIAO B,et al.Cumulus cell-released tumor necrosis factor (TNF)-α promotes post-ovulatory aging of mouse oocytes[J].Aging,2018,10(7):1745-1757.
[26] DEB G K,DEY S R,BANG J I,et al.9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression[J].J Anim Sci,2011,89(9):2759-2767.
[27] TORCHINSKY A,SHEPSHELOVICH J,ORENSTEIN H,et al.TNF-α protects embryos exposed to developmental toxicants[J].Am J Reprod Immunol,2003,49(3):159-168.
[28] XIA L M,MO P,HUANG W J,et al.The TNF-α/ROS/HIF-1-induced upregulation of FoxMI expression promotes HCC proliferation and resistance to apoptosis[J].Carcinogenesis,2012,33(11):2250-2259.
[29] TANG M J,TIAN Y H,LI D L,et al.TNF-α mediated increase of HIF-1α inhibits VASP expression,which reduces alveolar-capillary barrier function during acute lung injury (ALI)[J].PLoS One,2014,9(7):e102967.
[30] MASSEY A J,WILLIAMSON D S,BROWNE H,et al.A novel,small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells[J].Cancer Chemother Pharmacol,2010,66(3):535-545.
[31] MENG X Z,HARKEN A H.The interaction between Hsp70 and TNF-α expression:a novel mechanism for protection of the myocardium against post-injury depression[J].Shock,2002,17(5):345-353.
[32] MITSUHASHI M,YAMAGUCHI M,KOJIMA T,et al.Effects of HSP70 on the compression force-induced TNF-α and RANKL expression in human periodontal ligament cells[J].Inflamm Res,2011,60(2):187-194.
[2] XU X M,DUAN X,LU C F,et al.Dynamic distribution of NuMA and microtubules in human fetal fibroblasts,developing oocytes and somatic cell nuclear transferred embryos[J].Hum Reprod,2011,26(5):1052-1060.
[3] BINELLI M,MURPHY B D.Coordinated regulation of follicle development by germ and somatic cells[J].Reprod Fertil Dev,2009,22(1):1-12.
[4] KAPADIA S,LEE J,TORRE-AMIONE G,et al.Tumor necrosis factor-alpha gene and protein expression in adult feline myocardium after endotoxin administration[J].J Clin Invest,1995,96(2):1042-1052.
[5] VARFOLOMEEV E E,ASHKENAZI A.Tumor necrosis factor:an apoptosis JuNKie?[J].Cell,2004,116(4):491-497.
[6] MARCINKIEWICZ J L,BALCHAK S K,MORRISON L J.The involvement of tumor necrosis factor-alpha (TNF) as an intraovarian regulator of oocyte apoptosis in the neonatalrat.[J].Front Biosci,2002,7:d1997-2005.
[7] HUNT J S,CHEN H L,HU X L,et al.Normal distribution of tumor necrosis factor-α messenger ribonucleic acid and protein in the uteri,placentas,and embryos of osteopetrotic (op/op) mice lacking colony-stimulating factor-1[J].Biol Reprod,1993,49(3):441-452.
[8] NAZ R K,ZHU X L,MENGE A C.Expression of tumor necrosis factor-α and its receptors type I and type II in human oocytes[J].Mol Reprod Dev,1997,47(2):127-133.
[9] SOTO P,NATZKE R P,HANSEN P J.Actions of tumor necrosis factor-α on oocyte maturation and embryonic development in cattle[J].Am J Reprod Immunol,2003,50(5):380-388.
[10] YAMAMOTO Y,KUWAHARA A,TANIGUCHI Y,et al.Tumor necrosis factor alpha inhibits ovulation and induces granulosa cell death in rat ovaries[J].Reprod Med Biol,2015,14(3):107-115.
[11] JOHNSON M L,MURDOCH J,VAN KIRK E A,et al.Tumor necrosis factor α regulates collagenolytic activity in preovulatory ovine follicles:relationship to cytokine secretion by the oocyte-cumulus cell complex[J].Biol Reprod,1999,61(6):1581-1585.
[12] 刘汝宏.TNF系统与自身免疫性疾病[J].医学综述,2003,9(4):200-202.LIU R H.TNF system and autoimmune diseases [J].Medical Recapitulate,2003,9(4):200-202.(in Chinese)
[13] ROBY K F,WEED J,LYLES R,et al.Immunological evidence for a human ovarian tumor necrosis factor-α[J].J Clin Endocrinol Metab,1990,71(5):1096-1102.
[14] SANCHO-TELLO M,PéREZ-ROGER I,IMAKAWA K,et al.Expression of tumor necrosis factor-α in the rat ovary[J].Endocrinology,1992,130(3):1359-1364.
[15] MARCINKIEWICZ J L,KRISHNA A,CHEUNG C M Y,et al.Oocytic tumor necrosis factor alpha:localization in the neonatal ovary and throughout follicular development in the adult rat[J].Biol Reprod,1994,50(6):1251-1260.
[16] YU X,DENG L Y,WANG D,et al.Mechanism of TNF-α autocrine effects in hypoxic cardiomyocytes:initiated by hypoxia inducible factor 1α,presented by exosomes[J].J Mol Cell Cardiol,2012,53(6):848-857.
[17] H?GELE S,KüHN U,B?NING M,et al.Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1α mRNA 3′-UTR and modulate HIF-1α protein expression[J].Biochem J,2009,417(1):235-246.
[18] VAN UDEN P,KENNETH N S,ROCHA S.Regulation of hypoxia-inducible factor-1α by NF-κB[J].Biochem J,2008,412(3):477-484.
[19] TSAPOURNIOTI S,MYLONIS I,HATZIEFTHIMIOU A,et al.TNFα induces expression of HIF-1α mRNA and protein but inhibits hypoxic stimulation of HIF-1 transcriptional activity in airway smooth muscle cells[J].J Cell Physiol,2013,228(8):1745-1753.
[20] VAN MOLLE W,WIELOCKX B,MAHIEU T,et al.HSP70 protects against TNF-induced lethal inflammatory shock[J].Immunity,2002,16(5):685-695.
[21] YANG R C,JAO H C,HUANG L J,et al.The essential role of PKCα in the protective effect of heat-shock pretreatment on TNFα-induced apoptosis in hepatic epithelial cell line[J].Exp Cell Res,2004,296(2):276-284.
[22] PANG Q S,KEEBLE W,CHRISTIANSON T A,et al.FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-γ/TNF-α-mediated cytotoxicity[J].EMBO J,2014,20(16):4478-4489.
[23] HUANG W,YANDELL B S,KHATIB H.Transcriptomic profiling of bovine IVF embryos revealed candidate genes and pathways involved in early embryonic development[J].BMC Genomics,2010,11:23.
[24] 李小红,罗珊,樊伟,等.HSP70在不同应激程度CUMS昆明小鼠胚胎中的表达及作用[J].四川大学学报:医学版,2017,48(4):515-519.LI X H,LUO S,FAN W,et al.Expression of HSP70 in Kunming mouse embryos stimulated by chronic mild anticipatory stress[J].Journal of Sichuan University:Medical Science Edition,2017,48(4):515-519.(in Chinese)
[25] KONG Q Q,WANG J,XIAO B,et al.Cumulus cell-released tumor necrosis factor (TNF)-α promotes post-ovulatory aging of mouse oocytes[J].Aging,2018,10(7):1745-1757.
[26] DEB G K,DEY S R,BANG J I,et al.9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression[J].J Anim Sci,2011,89(9):2759-2767.
[27] TORCHINSKY A,SHEPSHELOVICH J,ORENSTEIN H,et al.TNF-α protects embryos exposed to developmental toxicants[J].Am J Reprod Immunol,2003,49(3):159-168.
[28] XIA L M,MO P,HUANG W J,et al.The TNF-α/ROS/HIF-1-induced upregulation of FoxMI expression promotes HCC proliferation and resistance to apoptosis[J].Carcinogenesis,2012,33(11):2250-2259.
[29] TANG M J,TIAN Y H,LI D L,et al.TNF-α mediated increase of HIF-1α inhibits VASP expression,which reduces alveolar-capillary barrier function during acute lung injury (ALI)[J].PLoS One,2014,9(7):e102967.
[30] MASSEY A J,WILLIAMSON D S,BROWNE H,et al.A novel,small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells[J].Cancer Chemother Pharmacol,2010,66(3):535-545.
[31] MENG X Z,HARKEN A H.The interaction between Hsp70 and TNF-α expression:a novel mechanism for protection of the myocardium against post-injury depression[J].Shock,2002,17(5):345-353.
[32] MITSUHASHI M,YAMAGUCHI M,KOJIMA T,et al.Effects of HSP70 on the compression force-induced TNF-α and RANKL expression in human periodontal ligament cells[J].Inflamm Res,2011,60(2):187-194.