PI3K信号通路关键基因在猪卵泡发育中的表达规律
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摘要
【目的】研究不同时期猪卵巢卵泡发生的形态特征及磷脂酰肌醇3–激酶(Phosphoinositide 3-kinase,PI3K)信号通路关键基因在卵泡发生发育中的作用。【方法】通过HE染色观察12日龄、30日龄、70日龄、20月龄卵泡期及黄体期、48月龄的长大二元母猪卵巢卵泡发育的形态变化,应用实时荧光定量PCR检测PI3K通路关键基因在这些时期的表达规律,并通过Western blot法检测PI3K通路重要的下游效应因子p-rpS6在不同时期猪卵巢的表达情况。【结果】12日龄母猪卵巢皮质边缘有大量的原始卵泡,皮质与髓质交界处可见少量初级卵泡及个别的次级卵泡;30日龄母猪卵巢次级卵泡数量增加;70日龄出现3级卵泡;20月龄卵泡期、黄体期分别有大量成熟卵泡、大体积的黄体;48月龄较难观察到原始卵泡。PI3K通路抑制因子PTEN、TSC1、TSC2与激活因子PDK1、AKT1、mTOR的mRNA均在12日龄表达量最高,70日龄、48月龄表达量次之,30日龄和20月龄表达量最低;下游效应基因rpS6的mRNA 30日龄表达量最高,20和48月龄表达量最低,p-rpS6蛋白在12日龄、20月龄、48月龄母猪卵巢高表达,在30日龄、70日龄中几乎不表达。【结论】PI3K通路关键基因在不同时期母猪卵巢中的表达水平不同,说明该通路参与了猪卵泡的早期发生及卵泡成熟的调控过程。
【Objective】To study the morphological characteristics of pig ovarian follicles at different developmental stages and possible roles of key genes of phosphoinositide 3-kinase(PI3K) signaling pathway in follicular development.【Method】The morphological changes in follicles of 12-, 30-, 70-day postpartum, 20-month follicular phase, 20-month luteal phase and 48-month luteal phase of landrace×large white sows were observed using HE staining. The expressions of key genes of PI3K pathway in these periods were detected byq RT-PCR. The expression of p-rpS6, a downstream effector, was detected by Western blot assay in porcine ovaries at different stages.【Result】A large number of primordial follicles were observed at the cortical edges of ovaries for 12-day-old pigs, and a small amount of primary follicles and a few secondary follicles were found at the junction of cortex and medulla. The number of secondary follicles increased in 30-day-old pigs. Tertiary follicles appeared in ovaries of 70-day-old pigs. At 20 months of age, a large number of mature follicles and bulked corpus luteum were found at follicular and luteal phases, respectively. It was difficult to observe primary follicles at 48 months of age. For PI3K pathway, the ovarian mRNA expression of inhibitors PTEN, TSC1, TSC2 and activators PDK1, AKT1, mTOR showed the highest levels at 12 days of age, followed by 70 days and 48 months of ages, and the lowest levels at 30 days and 20 months of ages. The downstream effector gene rpS6 of PI3K pathway exhibited the highest level of ovarian mRNA expression at 30 days of age, and the lowest level at 20 and 48 months of ages, while p-rpS6 protein had higher ovarian expression level at 12 days, 20 and 48 months of ages, and no expression at 30 and 70 days of ages.【Conclusion】The key genes of PI3K pathway have different expression levels at different developmental stages of porcine ovary, which indicates that the pathway is involved in the processes of porcine follicular generation and maturation.
【Objective】To study the morphological characteristics of pig ovarian follicles at different developmental stages and possible roles of key genes of phosphoinositide 3-kinase(PI3K) signaling pathway in follicular development.【Method】The morphological changes in follicles of 12-, 30-, 70-day postpartum, 20-month follicular phase, 20-month luteal phase and 48-month luteal phase of landrace×large white sows were observed using HE staining. The expressions of key genes of PI3K pathway in these periods were detected byq RT-PCR. The expression of p-rpS6, a downstream effector, was detected by Western blot assay in porcine ovaries at different stages.【Result】A large number of primordial follicles were observed at the cortical edges of ovaries for 12-day-old pigs, and a small amount of primary follicles and a few secondary follicles were found at the junction of cortex and medulla. The number of secondary follicles increased in 30-day-old pigs. Tertiary follicles appeared in ovaries of 70-day-old pigs. At 20 months of age, a large number of mature follicles and bulked corpus luteum were found at follicular and luteal phases, respectively. It was difficult to observe primary follicles at 48 months of age. For PI3K pathway, the ovarian mRNA expression of inhibitors PTEN, TSC1, TSC2 and activators PDK1, AKT1, mTOR showed the highest levels at 12 days of age, followed by 70 days and 48 months of ages, and the lowest levels at 30 days and 20 months of ages. The downstream effector gene rpS6 of PI3K pathway exhibited the highest level of ovarian mRNA expression at 30 days of age, and the lowest level at 20 and 48 months of ages, while p-rpS6 protein had higher ovarian expression level at 12 days, 20 and 48 months of ages, and no expression at 30 and 70 days of ages.【Conclusion】The key genes of PI3K pathway have different expression levels at different developmental stages of porcine ovary, which indicates that the pathway is involved in the processes of porcine follicular generation and maturation.
引文
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[25]张春林,李科瑛,刘明兰,等.卵母细胞生发泡破裂(GVBD)发生前后脱卵丘细胞对猪卵母细胞体外成熟的影响[J].农业生物技术学报,2014,22(9):1090-1096.
[2]MABUCHI S,KURODA H,TAKAHASHI R,et al.The PI3K/AKT/m TOR pathway as a therapeutic target in ovarian cancer[J].Gynecol Oncol,2015,137(1):173-179.
[3]CANTLEYL C.The phosphoinositide 3-kinase pathway[J].Science,2002,296(5573):1655-1657.
[4]ENGELMANJ A,LUO J,CANTLEY L C.The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[J].Nat Rev Genet,2006,7(8):606-619.
[5]REDDY P,ZHENG W,LIU K.Mechanisms maintaining the dormancy and survival of mammalian primordial follicles[J].Trends Endocrin Met,2010,21(2):96-103.
[6]ZELEZNIK A J,SAXENA D,LITTLE-IHRIG L.Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation[J].Endocrinology,2003,144(9):3985-3994.
[7]FAN H Y,RICHARDS J S.Minireview:Physiological and pathological actions of RAS in the ovary[J].Mol Endocrinol,2010,24(2):286-298.
[8]GONZALEZ-ROBAYNA I J,FALENDERA E,OCHS-NER S,et al.Follicle-stimulating hormone(FSH)stimulates phosphorylation and activation of protein kinase B(PKB/Akt)and serum and glucocorticoid-lnduced kinase(Sgk):Evidence for a kinase-independent signaling by FSH in granulosa cells[J].Mol Endocrinol,2000,14(8):1283-1300.
[9]ZHENG W,NAGARAJU G,LIU Z,et al.Functional roles of the phosphatidylinositol 3-kinases(PI3Ks)signaling in the mammalian ovary[J].Mol Cell Endocrinol,2012,356(1/2):24-30.
[10]RUVINSKY I,SHARON N,LERER T,et al.Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis[J].Gene Dev,2005,19(18):2199-2211.
[11]BLACK J L,ERICKSON B H.Oogenesis and ovarian development in the prenatal pig[J].Anat Rec,1968,161(1):45-55.
[12]GOSDEN R G,TELFER E.Numbers of follicles and oocytes in mammalian ovaries and their allometric relationships[J].Proceedings of the Zoological Society of London,1987,211(1):169-175.
[13]BLAKE J A,EPPIG J T,RICHARDSON J E,et al.The mouse genome database(MGD):Integration nexus for the laboratory mouse[J].Nucleic Acids Res,2001,29(1):91-94.
[14]RINGWALD M,DAVIS G L,SMITH A G,et al.The mouse gene expression database GXD[J].Nucleic Acids Res,1997,32(1):489-497.
[15]EPPIG J J.Oocyte control of ovarian follicular development and function in mammals[J].Reproduction,2001,122(6):829-838.
[16]徐春琳,路晓琳,闫晓楠,等.PI3K/Akt/NF-κB信号通路在FSH促进卵巢癌细胞增殖与侵袭中的作用[J].中华妇产科杂志,2012,47(2):134-138.
[17]JOHN G B,SHIDLER M J,BESMER P,et al.Kit signaling via PI3K promotes ovarian follicle maturation but is dispensable for primordial follicle activation[J].Dev Biol,2009,331(2):292-299.
[18]BROWN C,LAROCCA J,PIETRUSKA J,et al.Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1[J].Biol Reprod,2010,82(2):246-256.
[19]REDDY P,ADHIKARI D,ZHENG W,et al.PDK1 signaling in oocytes controls reproductive aging and lifespan by manipulating the survival of primordial follicles[J].Mol Genet,2009,18(15):2813-2824.
[20]FAN H Y,LIU Z,CAHILL N,et al.Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells[J].Mol Endocrinol,2008,22(9):2128-2140.
[21]FINGAR D C,BLENIS J.Target of rapamycin(TOR):An integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression[J].Oncogene,2004,23(18):3151-3171.
[22]NYGARD O,NILSSON L.Translational dynamics.Interactions between the translational factors,t RNA and ribosomes during eukaryotic protein synthesis[J].Eur JBiochem,1990,191(1):1-17.
[23]朱士恩.动物生殖生理学[M].北京:中国农业出版社,2006:359.
[24]FOURNIER M J,MAXWELL E S.The nucleolar sn RNAs:Catching up with the spliceosomal sn RNAs[J].Trends Biochem Sci,1993,18(4):131-135.
[25]张春林,李科瑛,刘明兰,等.卵母细胞生发泡破裂(GVBD)发生前后脱卵丘细胞对猪卵母细胞体外成熟的影响[J].农业生物技术学报,2014,22(9):1090-1096.