鸡PAPPA2基因在卵泡中的时空表达特性分析
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摘要
为探讨PAPPA2基因在鸡卵泡生长发育中的调控作用,以150日龄海兰褐蛋鸡为供试材料,采用相对定量RT-PCR方法对PAPPA2基因在鸡卵巢组织不同发育时期卵泡中的mRNA转录水平进行检测分析,并用免疫组织化学方法对PAPPA2蛋白质分子在鸡前等级卵泡中的时空表达模式进行了蛋白定位分析。结果表明:鸡PAPPA2基因mRNA在卵巢间质、各前等级卵泡和不同等级化的卵泡中均有表达,但也表现出其在不同卵泡组织中转录水平的差异性,其中,PAPPA2基因在间质组织、5~6 mm、6~7 mm、F2卵泡中的mRNA转录水平最高(P<0.05),而在4~5 mm前等级卵泡和F3卵泡中的mRNA表达量最低(P<0.05)。由此表明,鸡PAPPA2基因对处于不同阶段的卵泡生长发育发挥着不可或缺的调控功能,且表现出卵泡的不同生长发育程度可能与PAPPA2基因的转录水平之间有着剂量依赖关系。通过免疫组化检测结果可见,鸡PAPPA2蛋白质分子在卵巢间质组织、卵母细胞和颗粒细胞中均有表达,但在膜层细胞中未检测到其表达信号。初步推断,鸡PAPPA2分子可能以旁分泌或自分泌的方式在卵泡的生长发育过程中发挥着重要调节作用。
To investigate the regulatory role of PAPPA2 gene in chicken follicular growth and development,the expression abundances of PAPPA2 gene mRNA in the varied-size developing follicles sampled from the 150 d Hylan brown layers were determined by using relative quantitative RT-PCR method. It was found that PAPPA2 gene mRNA was expressed in the all sized follicles and stroma tissues and differential mRNA expression levels were exhibited among some groups sampled,wherein,the highest expression levels were observed in stroma,5-6 mm and 6-7 mm sized prehierarchal follicles and F2 follicles( P<0. 05),on the contrary,the lowest levels were determined in 4-5 mm sized and F3 follicles( P<0. 05). The current results indicated that the PAPPA2 gene may play an indispensable role in different developmental stages of the varied-sized follicles in a dosage-dependent manner. The expression of PAPPA2 protein was detected in the stroma,oocytes and granulosacells by immunohistochemical analysis,but no positive expression signal was probed in the theca cells. It was inferred that chicken PAPPA2 protein amy exert a critical biological function in regulating the follicular growth and development in a paracrine and/or autocrine manner. The present data provide reference for further exploring the exact roles and molecular mechanisms of PAPPA2 gene in the regulation of follicular growth and development in hen ovary.
To investigate the regulatory role of PAPPA2 gene in chicken follicular growth and development,the expression abundances of PAPPA2 gene mRNA in the varied-size developing follicles sampled from the 150 d Hylan brown layers were determined by using relative quantitative RT-PCR method. It was found that PAPPA2 gene mRNA was expressed in the all sized follicles and stroma tissues and differential mRNA expression levels were exhibited among some groups sampled,wherein,the highest expression levels were observed in stroma,5-6 mm and 6-7 mm sized prehierarchal follicles and F2 follicles( P<0. 05),on the contrary,the lowest levels were determined in 4-5 mm sized and F3 follicles( P<0. 05). The current results indicated that the PAPPA2 gene may play an indispensable role in different developmental stages of the varied-sized follicles in a dosage-dependent manner. The expression of PAPPA2 protein was detected in the stroma,oocytes and granulosacells by immunohistochemical analysis,but no positive expression signal was probed in the theca cells. It was inferred that chicken PAPPA2 protein amy exert a critical biological function in regulating the follicular growth and development in a paracrine and/or autocrine manner. The present data provide reference for further exploring the exact roles and molecular mechanisms of PAPPA2 gene in the regulation of follicular growth and development in hen ovary.
引文
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[10]刘强,徐日福,张英英,等.鸡MYCN基因不同组织mRNA表达及其遗传多态性与免疫性状的关联分析[J].吉林农业大学学报,2014,36(4):469-474.
[11]Qin N,Fan X C,Zhang Y Y,et al.New insights into implication of the SLIT/ROBO pathway in the prehierarchical follicle development of hen ovary[J].Poult Sci,2015,94(9):2235-2246.
[12]Johnson A L,Woods D C.Chapter 6.Ovarian dynamics and follicle development[M]∥Reproductive Biology and Phylogeny of Aves.Jamieson:Science Publishers,Inc.,2009:243-277.
[13]Abe W,Nasu K,Nakada C,et al.miR-196b targets c-myc and Bcl-2 expression,inhibits proliferation and induces apoptosis in endometriotic stromal cells[J].Hum Reprod,2013,28(3):750-761.
[14]Gao F,Zhang J,Wang X,et al.Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation[J].Hum Mol Genet,2014,23(2):333-341.
[2]Overgaard M T,Boldt H B,Laursen L S,et al.Pregnancy-associated plasma protein-A2(PAPP-A2),a novel insulin-like growth factor-binding protein-5 proteinase[J].Biol Chem,2001,276(24):21849-21853.
[3]Onagbesan O M,Metayer S,Tona K,et al.Effects of genotype and feed allowance on plasma luteinizing hormones,folliclestimulating hormones,progesterone,estradiol levels,follicle differentiation,and egg production rates of broiler breeder hens[J].Poult Sci,2006,85:1245-1258.
[4]Luna-Nevarez P,Rincon G,Medrano J F,et al.Single nucleotide polymorphisms in the growth hormone-insulin-like growth factor axis in straightbred and crossbred Angus,Brahman,and Romosinuano heifers:population genetic analyses and association of genotypes with reproductive phenotypes[J].J Anim Sci,2011,89(4):926-934.
[5]Wickramasinghe S,Rincon G,Medrano J F.Variants in the pregnancy-associated plasma protein-A2 gene on Bos taurus autosome 16 are associated with daughter calving ease and productive life in Holstein cattle[J].J Dairy Sci,2011,94(3):1552-1558.
[6]Yan X,Baxter R C,Firth S M.Involvement of pregnancy-associated plasma protein-A2 in insulin-like growth factor(IGF)binding protein-5 proteolysis during pregnancy:a potential mechanism for increasing IGF bioavailability[J].J Clin Endocrinol Metab,2010,95(3):1412-1420.
[7]Kramer A W,Lamale-Smith L,Winn V D.Differential expression of human placental PAPP-A2 over gestation and in preeclampsia[J].Placenta,2016,37:19-25.
[8]Wu W,Xu R F,Xu H,et al.Expression of theβ-catenin gene in the skin of embryonic geese during feather bud development[J].Poult Sci,2008,87(1):204-211.
[9]包艳波,王志贤,秦宁,等.鸡卵巢组织SSH c DNA文库构建及差异表达基因筛选与初步鉴定[J].吉林农业大学学报,2011,33(4):443-449.
[10]刘强,徐日福,张英英,等.鸡MYCN基因不同组织mRNA表达及其遗传多态性与免疫性状的关联分析[J].吉林农业大学学报,2014,36(4):469-474.
[11]Qin N,Fan X C,Zhang Y Y,et al.New insights into implication of the SLIT/ROBO pathway in the prehierarchical follicle development of hen ovary[J].Poult Sci,2015,94(9):2235-2246.
[12]Johnson A L,Woods D C.Chapter 6.Ovarian dynamics and follicle development[M]∥Reproductive Biology and Phylogeny of Aves.Jamieson:Science Publishers,Inc.,2009:243-277.
[13]Abe W,Nasu K,Nakada C,et al.miR-196b targets c-myc and Bcl-2 expression,inhibits proliferation and induces apoptosis in endometriotic stromal cells[J].Hum Reprod,2013,28(3):750-761.
[14]Gao F,Zhang J,Wang X,et al.Wt1 functions in ovarian follicle development by regulating granulosa cell differentiation[J].Hum Mol Genet,2014,23(2):333-341.