FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响

doi:10.11843/j.issn.0366-6964.2018.09.011

摘要/Abstract

摘要：

旨在研究促卵泡素（FSH）对牦牛卵母细胞体外成熟，以及对表皮生长因子（EGF）、表皮生长因子受体（EGFR）的表达影响和与细胞凋亡的关系。在牦牛卵母细胞体外成熟培养液中添加不同浓度FSH （终浓度分别为0、2、5、10 μg·mL^-1），体外成熟培养后，运用实时荧光定量PCR （qRT-RCR）和免疫荧光染色技术检测EGF、EGFR表达情况，采用qRT-RCR和蛋白免疫印迹法检测Bax、Bcl-2表达变化。结果表明：1）成熟培养液中加入FSH可提高COCs成熟率，当成熟液中FSH为5 μg·mL^-1时，成熟率最高，达到76.84%，显著高于其他组（P<0.05）；2）随着FSH浓度的增加，EGF和EGFR表达逐渐升高，其中在5 μg·mL^-1FSH组中，EGF和EGFR的表达最高，显著高于其他组（P<0.05），而在10 μg·mL^-1FSH组中，EGF和EGFR的表达水平降低；EGF主要位于卵丘细胞中，而EGFR在卵丘细胞和卵母细胞均有表达；3）随着FSH浓度的增加，Bax和Bcl-2的表达显示出明显的逆向模式，Bax的表达水平逐渐降低，Bcl-2的表达水平逐渐升高，在5 μg·mL^-1FSH组中Bax的表达量最低，Bcl-2的表达量最高，而在10 μg·mL^-1FSH组中，Bax的表达水平升高。综上表明，在牦牛卵母细胞体外成熟过程中，FSH提高了卵母细胞发育能力，诱导EGF和EGFR表达，而且可能通过调控Bax、Bcl-2等凋亡相关因子的表达，抑制卵母细胞凋亡。

Abstract:

The aim of this study was to investigate the specific contribution of follicle-stimulating hormone (FSH) on maturation of yak oocytes in vitro, the effect of FSH on the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in yak oocytes, and the relationship between FSH and apoptosis. In this experiment, different concentrations of FSH (final concentration:0,2,5,10 μg·mL^-1) were added into the medium of the yak oocytes in vitro. The mRNA and protein expression of EGF and EGFR were detected by real-time PCR (qRT-RCR) and immunofluorescence staining, respectively. Meanwhile, the expression of Bax and Bcl-2 were detected by qRT-RCR and Western blotting. The results showed that:1) It was noteworthy that FSH promoted the maturation of oocytes, and the optimal concentration of FSH was 5 μg·mL^-1, and the rate of maturation oocytes was 76.84%, which was significantly higher than other groups (P<0.05). 2) The expression levels of EGF and EGFR depended on the doses of FSH. The expressions of EGF and EGFR were upregulated during the doses of FSH raised from 0 to 5 μg·mL^-1, which was significantly higher in 5 μg·mL^-1 FSH group than other groups (P<0.05). When the concentration of FSH was 10 μg·mL^-1, the expression level of EGF and EGFR was reduced. Immunofluorescence assay showed that EGF was mainly located in cumulus cells, while EGFR was expressed in cumulus cells and oocytes. 3) With the increase of FSH concentration, the expression of Bax and Bcl-2 showed the obvious reverse pattern, the expression level of Bcl-2 increased gradually, while the expression level of Bax decreased gradually. The expression of Bax was the lowest, and the expression of Bcl-2 was the highest when the dose of FSH was 5 μg·mL^-1, but the expression level of Bax increased in 10 μg·mL^-1 FSH group. The results showed that FSH increased the ability of oocyte development and induced the expression of EGF and EGFR during the process of yak oocyte maturation in vitro, and inhibited the apoptosis of oocytes by regulating the expression of apoptosis related factors such as Bax and Bcl-2.

中图分类号:

何翃闳, 潘阳阳, 张慧珠, 李秦, 王萌, 崔燕, 樊江峰, 余四九. 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 Hong-hong, PAN Yang-yang, ZHANG Hui-zhu, LI Qin, WANG Meng, CUI Yan, FAN Jiang-feng, YU Si-jiu. 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.

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参考文献

[1] 何翃闳,崔燕,潘阳阳,等.牦牛HSP27基因的克隆及其在雌性生殖器官中的表达[J].中国农业科学,2015,48(20):4178-4187.
HE H H,CUI Y,PAN Y Y,et al.Cloning of bos grunniens HSP27 gene and its expression in the female yak reproductive organs[J].Scientia Agricultura Sinica,2015,48(20):4178-4187.(in Chinese)
[2] 潘阳阳,李秦,崔燕,等.EGF、EGFR在牦牛卵母细胞中的表达及对胚胎发育能力的作用[J].中国农业科学,2015,48(12):2439-2448.
PAN Y Y,LI Q,CUI Y,et al.The expression of EGF and EGFR in yak oocyte and its function on development competence of embryo[J].Scientia Agricultura Sinica,2015,48(12):2439-2448.(in Chinese)
[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] OKTEM O,URMAN B.Understanding follicle growth in vivo[J].Hum Reprod,2010,25(12):2944-2954.
[5] DEMEESTERE I,STREIFF A K,SUZUKI J,et al.Follicle-stimulating hormone accelerates mouse oocyte development in vivo[J].Biol Reprod,2012,87(1):1-11.
[6] SU J M,WANG Y S,ZHANG L,et al.Oocyte-secreted factors in oocyte maturation media enhance subsequent development of bovine cloned embryos[J].Mol Reprod Dev,2014,81(4):341-349.
[7] HUNZICKER-DUNN M,MAIZELS E T.FSH signaling pathways in immature granulosa cells that regulate target gene expression:branching out from protein kinase A[J].Cell Signal,2006,18(9):1351-1359.
[8] SINGH B,MENG L,RUTLEDGE J M,et al.Effects of epidermal growth factor and follicle-stimulating hormone during in vitro maturation on cytoplasmic maturation of porcine oocytes[J].Mol Reprod Dev,1997,46(3):401-407.
[9] VERAGUAS D,GALLEGOS P F,VELASQUEZ A E,et al.FSH stimulation of anestrous cats improves oocyte quality and development of parthenogenetic embryos[J].Theriogenology,2016,87:25-35.
[10] HIRADATE Y,INOUE H,KOBAYASHI N,et al.Neurotensin enhances sperm capacitation and acrosome reaction in mice[J].Biol Reprod,2014,91(2):53.
[11] PROCHAZKA R,BLAHA M,NEMCOVA L.Signaling pathways regulating FSH-and amphiregulin-induced meiotic resumption and cumulus cell expansion in the pig[J].Reproduction,2012,144(5):535-546.
[12] SÁNCHEZ F,ADRIAENSSENS T,ROMERO S,et al.Different follicle-Stimulating hormone exposure regimens during antral follicle growth alter gene expression in the cumulus-oocyte complex in mice[J].Biol Reprod,2010,83(4):514-524.
[13] XU H,DENG K,LUO Q,et al.High serum FSH is associated with brown oocyte formation and a lower pregnacy rate in human IVF parctice[J].Cell Physiol Biochem,2016,39(2):677-684.
[14] FRANCIOSI F,MANANDHAR S,CONTI M.FSH regulates mRNA translation in mouse oocytes and promotes developmental competence[J].Endocrinology,2016,157(2):872-882.
[15] LI H J,LIU D J,CANG M,et al.Early apoptosis is associated with improved developmental potential in bovine oocytes[J].Anim Prod Sci,2009,114(1-3):89-98.
[16] PALMERINI M G,NOTTOLA S A,TUNJUNG W A S,et al.EGF-FSH supplementation reduces apoptosis of pig granulosa cells in co-culture with cumulus-oocyte complexes[J].Biochem Biophys Res Commun,2016,481(1-2):159-164.
[17] 徐庚全,樊江峰,杨世华,等.FSH、LH对体外培养的牦牛卵泡颗粒细胞凋亡及E₂、P分泌功能的影响[J].畜牧兽医学报,2015,46(6):932-939.
XU G Q,FAN J F,YANG S H,et al.The effects of FSH,LH on apoptosis and E₂,P secreting of yak's granulosa cells cultured in vitro[J].Acta Veterinaria et Zootechnica Sinica,2015,46(6):932-939.(in Chinese)
[18] YANG M Y,RAJAMAHENDRAN R.Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro[J].Anim Prod Sci,2002,70(3-4):159-169.
[19] 张译夫,潘阳阳,温泽星,等.表皮生长因子对牦牛卵丘细胞低氧诱导因子-1α表达的影响及与凋亡的关联性分析[J].畜牧兽医学报,2016,47(6):1154-1161.
ZHANG Y F,PAN Y Y,WEN Z X,et al.The effect of epidermal growth factor on the expression of hypoxia inducible factor-1α in cumulus cells of yak (Bos grunniens) and its correlation analysis with apoptosis[J].Acta Veterinaria et Zootechnica Sinica,2016,47(6):1154-1161.(in Chinese)
[20] CHEN X F,ZHOU B,YAN J,et al.Epidermal growth factor receptor activation by protein kinase C is necessary for FSH-induced meiotic resumption in porcine cumulus-oocyte complexes[J].J Endocrinol,2008,197(2):409-419.
[21] UHM S J,GUPTA M K,YANG J H,et al.Epidermal growth factor can be used in lieu of follicle-stimulating hormone for nuclear maturation of porcine oocytes in vitro[J].Theriogenology,2010,73(8):1024-1036.
[22] HUSSEIN T S,THOMPSON J G,GILCHRIST R B.Oocyte-secreted factors enhance oocyte developmental competence[J].Dev Biol,2006,296(2):514-521.
[23] CALDER M D,CAVENEY A N,SMITH L C,et al.Responsiveness of bovine cumulus-oocyte-complexes (COC) to porcine and recombinant human FSH,and the effect of COC quality on gonadotropin receptor and Cx43 marker gene mRNAs during maturation in vitro[J].Reprod Biol Endocrinol,2003,1:14.
[24] LIANG C G,HUO L J,ZHONG Z S,et al.Cyclic adenosine 3',5'-monophosphate-dependent activation of mitogen-activated protein kinase in cumulus cells is essential for germinal vesicle breakdown of porcine cumulus-enclosed oocytes[J].Endocrinology,2005,146(10):4437-4444.
[25] PALERMO R.Differential actions of FSH and LH during folliculogenesis[J].Reprod Biomed Online,2007,15(3):326-337.
[26] WU J,DENT P,JELINEK T,et al.Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate[J].Science,1993,262(5136):1065-1069.
[27] DU X H,ZHOU X L,CAO R,et al.FSH-induced p38-MAPK-mediated dephosphorylation at serine 727 of the signal transducer and activator of transcription 1 decreases Cyp1b1,expression in mouse granulosa cells[J].Cell Signal,2015,27(1):6-14.
[28] 王怡,张姣姣,汪勇,等.FSH和17β-雌二醇联合作用对Cyclin D1 mRNA和Cyclin E1 mRNA表达的影响[J].畜牧兽医学报,2013,44(6):858-865.
WANG Y,ZHANG J J,WANG Y,et al.Effect of combination of FSH and 17β-estradiol on the mRNA expression of Cyclin D1 and Cyclin E1 in the cultured immature boar sertoli cell[J].Acta Veterinaria et Zootechnica Sinica,2013,44(6):858-865.(in Chinese)
[29] YAMASHITA Y,KAWASHIMA I,YANAI Y,et al.Hormone-induced expression of tumor necrosis factor α-converting enzyme/A disintegrin and metalloprotease-17 impacts porcine cumulus cell oocyte complex expansion and meiotic maturation via ligand activation of the epidermal growth factor receptor[J].Endocrinology,2007,148(12):6164-6175.
[30] TOYODA H,KOMURASAKI T,UCHIDA D,et al.Epiregulin:a novel epidermal growth factor with mitogenic activity for rat primary hepatocytes[J].J Biol Chem,1995,270(13):7495-7500.
[31] LINSE S,THULIN E,GIFFORD L K,et al.Domain organization of calbindin D_28k as determined from the association of six synthetic EF-hand fragments[J].Protein Sci,1997,6(11):2385-2396.
[32] ZHANG C H,QIAN W P,QI S T,et al.Maternal diabetes causes abnormal dynamic changes of endoplasmic reticulum during mouse oocyte maturation and early embryo development[J].Reprod Biol Endocrinol,2013,11:31.
[33] KAWAMURA K,CHEN Y,SHU Y M,et al.Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors[J].PLoS One,2012,7(11):e49328.
[34] FENG Y J,TEITELBAUM D H.Epidermal growth factor/TNF-α transactivation modulates epithelial cell proliferation and apoptosis in a mouse model of parenteral nutrition[J].Am J Physiol Gastrointest Liver Physiol,2012,302(2):G236-G249.