组织型纤溶酶原激活剂基因在牛卵丘-卵母复合体体外成熟进程中的表达

摘要/Abstract

摘要： 为了初步了解组织型纤溶酶原激活剂(tissue-plasminogen activator,tPA)在牛卵丘－卵母复合体体外成熟进程中的潜在作用,试验运用实时定量RT-PCR技术分别检测体外成熟过程中不同时间段(0、8、16、24 h)以及添加不同浓度FSH成熟培养16 h后的牛卵丘细胞和卵母细胞中tPA基因相对表达变化,观察添加不同浓度FSH成熟培养16 h后卵丘细胞膨胀程度差异,并统计卵母细胞第一极体排出情况。结果显示,卵丘－卵母复合体在体外成熟初期,tPA mRNA相对表达水平在体外成熟16和24 h的卵丘细胞和卵母细胞中显著高于0和8 h组;在添加不同浓度FSH (0、0.01、0.1和1 IU/mL)体外成熟16 h的各处理组,卵丘细胞中tPA mRNA相对表达量随FSH添加浓度的升高而升高,卵丘细胞的扩散程度亦随之增加;同时tPA mRNA相对表达量在0.01 IU/mL FSH添加组卵母细胞中显著高于其他各FSH添加组。综合以上研究结果,本研究认为,牛卵丘细胞中tPA基因的表达受FSH正向调节;tPA可能促进了卵丘细胞在体外成熟过程中的扩散;同时,tPA在卵母细胞中的表达是否与其第一极体排出有关,需进一步试验验证。

关键词: tPA; 牛; 卵丘细胞; 卵母细胞; FSH

Abstract: In the present study,four experiments were conducted to investigate the potential effects of tPA on bovine cumulus and oocytes during in vitro maturation (IVM) (0,8,16,24 h). The expressions of tPA were detected by relative Real-time RT-PCR experiments in bovine cumulus and oocytes both after 0,8,16,24 h of IVM and under different FSH concentrations after 16 h of IVM separately. Meanwhile,the degree of bovine cumulus cell expansion and the percentages of first polar body in oocyte were assessed respectively. TPA mRNA levels were higher in cumulus and oocytes at 16 h and 24 h than those at 0 h and 8 h of IVM significantly (P<0.05). Among those groups that under different FSH concentrations (0,0.01,0.1 and 1 IU/mL) after 16 h of IVM,tPA mRNA levels of cumulus was increased following the elevated FSH concentrations,as well as the cumulus expansion,tPA mRNA of cumulus cells in 1 IU/mL FSH group was significantly higher than others. The results indicated that the expression of tPA in bovine cumulus was regulated positively by FSH,tPA could facilitate the cumulus expansion during IVM. However,it needed to be further investigated that tPA expression might be involved in first polar body excrusion of bovine oocytes.

Key words: tPA; cow; cumulus cells; oocytes; FSH

中图分类号:

S814.8

关洪敏, 米焱, 张伶俐, 李海军, 曹贵方. 组织型纤溶酶原激活剂基因在牛卵丘-卵母复合体体外成熟进程中的表达[J]. 中国畜牧兽医.

GUAN Hong-min, MI Yan, ZHANG Ling-li, LI Hai-jun, CAO Gui-fang. Expression of Tissue-type Plasminogen Activator in Bovine Cumulus-oocytes during in vitro Maturation[J]. .

参考文献

1 曲杨燕,赵雁伟,李凯,等. FSH对绵羊卵母细胞体外核成熟的影响[J]. 中国畜牧兽医,2008,35(12):80~83.

2 刘以训,冯强,邹如金. 促性腺激素诱发猕猴排卵周期中纤溶酶原激活因子与抑制因子的变化[J]. 生理学报,1991,43(5):472~479.

3 李海军,靳木子,张睿彪,等. 卵丘细胞凋亡与增殖对牛卵母细胞体外发育的影响[J].中国农业科学,2011,44(8):1702~1709.

4 郑爱燕,吴曼,尚利青,等. 山羊GDF9基因在卵丘卵母细胞复合体体外成熟过程中的表达[J]. 畜牧兽医学报,2010,41(6):671~677.

5 Andreasen P A,Egelund R,Petersen H H. The plasminogen activation system in tumor growth,invasion,and metastasis[J]. Cell Mol Life Sci, 2000,57: 25~40.

6 Blondin P,Bousquet D,Twagiramungu H,et al. Manipulation of follicular development to produce developmentally competent bovine oocytes[J]. Biol Reprod,2002,66: 38~43.

7 Choi Y H,Carnevale E M,Seidel G E J,et al. Effects of gonadotropins on bovine oocytes matured in TCM-199[J]. Theriogenology,2001,56(4):661~670.

8 Dano K,Andreasen P A,Grondahl-Hansen J,et al. Plasminogen activators,tissue degradation and cancer[J]. Adv Cancer Res,1985,44:139~266.

9 Dow M P,Bakke L J,Cassar C A,et al. Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue[J]. Biol Reprod,2002,66(5):1413~1421.

10Huarte J,Belin D,Vassalli J D. Plasminogen activator in mouse and rat oocytes: induction during meiotic maturation[J]. Cell,1985,43:551~558.

11Huarte J,Belin D,Vassalli A,et al. Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue type plasminogen activator mRNA[J]. Genes Dev,1987,1:1201~1211.

12Keefer C L,Stice S L,Dobrinsky J. Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer[J]. Mol Reprod Dev,1993,36(4):469~474.

13Lee H S,Seo Y I,Yin X J,et al. Effect of follicle stimulation hormone and luteinizing hormone on cumulus cell expansion and in vitro nuclear maturation of canine oocytes[J]. Reprod Dom Anim,2007,42:561~565.

14Li Q,Jimenez-Krassel F,Kobayashi Y,et al. Effect of intrafollicular indomethacin injection on gonadotropin surge-induced expression of select extracellular matrix degrading enzymes and their inhibitors in bovine preovulatory follicles[J]. Reproduction,2006,131(3):533~543.

15Liu Y X,Hsueh A J W. Plasminogen activator activity in cumulus oocyte complexes of gonadotropin treated rats during periovulatory period[J]. Biol Reprod,1987,36: 27~36.

16Liu Y X,Cajander S B,Ny T,et al. Gonadotropin regulation of tissue-type and urokinase-type plasminogen activator in rat granulosa and theca-interstitical cells during periovulatory period[J]. Mol Cell Endocrinol,1987,54: 221~229.

17Macchione E,Epifano O,Stefanini M,et al. Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles[J].Biol Reprod,2000,62:895~903.

18Mlynarciková A,Nagyová E,Ficková M,et al. Effects of selected endocrine disruptors on meiotic maturation,cumulus expansion,synthesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes[J]. Toxicology in Vitro,2009,23(3):371~377.

19Mullertz S. Fibrinolysis: general aspects characteristic features and perspectives[J]. Fibrinolysis,1988,1:3~12.

20Ward F,Enright B,Rizos D,et al. Optimization of in vitro bovine embryo production: Effect of duration of maturation,length of gamete co-incubation,sperm concentration and sire[J]. Theriogenology,2002,57:2105~2117.

21O’Connell M L,Canipari R,Strickland S. Hormonal regulation of tissue plasminogen activator secretion and mRNA levels in rat granulosa cells[J]. J Biol Chem,1987,262:2339~2344.

22Roldán-Olarte M,Jiménez-Diaz M,Miceli D C.Plasminogen detection in oocytes and plasminogen activator activities in the porcine oviduct during the estrous cycle[J]. Zygote,2005,13(2):115~123.

23Schmittgen T D,Zakrajsek B A. Effect of experimental treatment on housekeeping gene expression: validation by real-time,quantitative RT-PCR[J]. J Biochem Biophys Methods,2000,46 (122):69~81.

24Smokovitis A,Kokolis N,Alexaki-T zivanidou E. The plasminogen activator activity is markedly increased mainly at the area of the rupture of the follicular wall at the time of ovulation[J]. Anim Reprod Sci,1988,16:285~294.

25Yang W,Lu C L,Yu F Q,et al. Mitogen-activated protein kinase regulates FSH-induced expression of tissue-type plasminogen activator through an activator protein 1 response element[J]. Endocrine,2008,34(1~3):101~107.

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