LC3蛋白在小鼠卵泡颗粒细胞中的表达及定位研究
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摘要
目的:探讨自噬相关蛋白微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)在小鼠卵泡颗粒细胞中的表达及定位。方法:昆明小鼠腹腔注射孕马血清促性腺激素(pregnant mare serum gonadotropin,PMSG)前及注射后的第1、2、3、4、5天,采用免疫组织化学染色方法检测自噬相关蛋白LC3和凋亡相关蛋白cleaved caspase-3在卵泡中表达及共定位情况;促卵泡激素(follicle stimulating hormone,FSH)处理颗粒细胞后,Western blot检测LC3和cleaved caspase-3在颗粒细胞中的水平,免疫荧光方法对LC3在颗粒细胞中的亚细胞定位进行研究。结果:在小鼠卵泡发育的各个阶段,LC3蛋白主要在颗粒细胞中表达;免疫荧光显示在闭锁卵泡的颗粒细胞中cleaved caspase-3和LC3的蛋白水平较高;在PMSG腹腔注射后第1、2天,颗粒细胞cleaved caspase-3和LC3-II的蛋白水平减少(P<0.05);在PMSG腹腔注射后第3、4、5天,颗粒细胞的cleaved caspase-3和LC3-II蛋白水平依次增加;LC3-II和cleaved caspase-3的蛋白水平具有相关性(r~2=0.8299,P<0.05)。FSH处理后,LC3-II定位于颗粒细胞胞浆并呈点状分布。结论:LC3主要在卵泡颗粒细胞中表达,其表达具有细胞特异性和区域特异性,提示自噬在卵泡发育过程中主要在颗粒细胞中发生。卵巢颗粒细胞自噬和凋亡具有相关性,均呈促性腺激素依赖性。
AIM: To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3( LC3) at various stages of follicular development and atresia in the mice. METHODS: On0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin( PMSG),expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the method of immunohistochemical staining. The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH. LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence. RESULTS: The LC3 protein expressed in granulosa cells during all developmental stages mainly. Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3. The protein levels of cleaved caspase-3 and LC3-II in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG( P < 0. 05). The protein levels of cleaved caspase-3 and LC3-II in the granulosa cells increased in turn on 3,4 and 5 day after intraperitoneal injection of PMSG. The positive correlation between LC3-II and cleaved caspase-3 protein levels was observed( r2= 0. 8299,P < 0. 05). The LC3-II protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH. CONCLUSION: LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity. Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis. Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.
AIM: To investigate the expression and localization of autophagy related protein microtublule associated protein 1 light chain 3( LC3) at various stages of follicular development and atresia in the mice. METHODS: On0,1,2,3,4 and 5 day after intraperitoneal injection of pregnant mare serum gonadotropin( PMSG),expression and positioning situation of autophagy related protein LC3 and apoptosis related protein cleaved caspase-3 were examined by the method of immunohistochemical staining. The protein levels of cleaved caspase-3 and LC3 were determined by Western blot in cultured mouse granulosa cells after incubation under serum-free conditions in the absence or presence of FSH. LC3 subcellular localization in granulosa cells were studied by the method of immunofluorescence. RESULTS: The LC3 protein expressed in granulosa cells during all developmental stages mainly. Granulosa cells of atretic follicles that showed intense staining of cleaved caspase-3 and LC3. The protein levels of cleaved caspase-3 and LC3-II in the granulosa cells significantly decreased at 1 d and 2 d after intraperitoneal injection of PMSG( P < 0. 05). The protein levels of cleaved caspase-3 and LC3-II in the granulosa cells increased in turn on 3,4 and 5 day after intraperitoneal injection of PMSG. The positive correlation between LC3-II and cleaved caspase-3 protein levels was observed( r2= 0. 8299,P < 0. 05). The LC3-II protein expressed with punctuate structures in granulosa cell cytoplasm cultured under serum-free conditions in the presence of FSH. CONCLUSION: LC3 is expressed in the follicular granulosa cells with cell specificity and regional specificity. Autophagy is induced mainly in granulosa cells during folliculogenesis and shows positive correlation with apoptosis. Ovarian granulosa cell autophagy and apoptosis are gonadotropic hormone dependent.
引文
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[4]Levine B,Klionsky DJ.Development by self-digestion:molecular mechanisms and biological functions of autophagy[J].Dev Cell,2004,6(4):463-477.
[5]Voigt O,P9ggeler S.Self-eating to grow and kill:autophagy in filamentous ascomycetes[J].Appl Microbiol Biotechnol,2013,97(21):9277-9290.
[6]Mattiolo P,Yuste VJ,Boix J,et al.Autophagy exacerbates caspase-dependent apoptotic cell death after short times of starvation[J].Biochem Pharmacol,2015,98(4):573-586.
[7]Delou JM,Biasoli D,Borges HL.The complex link between apoptosis and autophagy promising new role for RB[J].An Acad Bras Cienc,2016,8(4):2257-2275.
[8]Duerrschmidt N,Zabirnyk O,Nowicki M,et al.Lectinlike oxidized low-density lipoprotein receptor-1-mediated autophagy in human granulosa cells as an alternative of programmed cell death[J].Endocrinology,2006,147(8):3851-3860.
[9]Gao H,Lin L,Haq IU,et al.Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells[J].Biochem Biophys Res Commun,2016,473(1):311-316.
[10]张丹,李静,李玉苹,等.自噬减轻模拟缺血/再灌注微环境中肺微血管内皮细胞损伤[J].中国病理生理杂志,2015,31(7):1253-1258.
[11]Qin HD,Tan WG,Zhang Z,et al.15d-prostaglandin JZ protects cortical neurons against oxygen-glucose deprivation/reoxygenation injury:involvement of inhibiting autophagy through up-regulation of Bcl-2[J].Cell Mol Neurobiol,2015,35(3):303-312.
[12]陈艾,童煜,毛萌.小自噬的研究进展[J].中国病理生理杂志,2012,28(5):955-960.
[13]La Rovere RM,Roest G,Bultynck G,et al.Intracellular Ca2+signaling and Ca2+microdomains in the control of cell survival,apoptosis and autophagy[J].Cell Calcium,2016,60(2):74-87.
[14]Tsukamoto S,Kuma A,Murakami M,et al.Autophagy is essential for preimplantation development of mouse embryos[J].Science,2008,321(5885):117-120.
[15]Yang YL,Ren LR,Sun LF,et al.The role of GPR1 signaling in mice corpus luteum[J].J Endocrinol,2016,230(1):55-65.
[16]Rodrigues P,Limback D,Mc Ginnis LK,et al.Multiple mechanisms of germ cell loss in the perinatal mouse ovary[J].Reproduction,2009,137(4):709-720.
[17]Escobar ML,Echeverria OM,Ortiz R,et al.Combined apoptosis and autophagy,the process that eliminates the oocytes of atretic follicles in immature rats[J].Apoptosis,2008,13(10):1253-1266.
[18]Gonzales-Polo R,Boya P,Pauleau A,et al.The apoptosis/autophagy paradox:Autophagic vacuolization before apoptotic death[J].J Cell Sci,2005,118(Pt 14):3091-3102.