摘要
繁殖力是影响养猪业生产的最重要因素之一,是决定养猪业效益高低的主要限制因素。繁殖力的高低取决于母体卵巢卵泡的生长发育,因为卵泡发育决定了排卵数,是影响繁殖力的关键因素。掌握母猪卵泡发育规律及其影响因素有利于进一步提高母猪繁殖能力,提高猪生产的经济效益。哺乳动物卵泡发育是十分复杂的过程,主要包括:原始卵泡的募集、腔前卵泡的发育、有腔卵泡的选择和生长及卵泡的成熟或闭锁。随着后基因组信息学的发展,人们发现卵泡的生长发育是一个高度协调的、非独立生理生化过程,受多种相同或者不同的细胞内、外因子及信号通路的影响和控制。
卵泡颗粒细胞是卵泡中最重要的体细胞,它对卵母细胞成熟、卵泡发育有至关重要的作用。伴随着哺乳动物卵泡生长和发育,颗粒细胞体积增大、数量增多及逐步分化和成熟。BMPs (bone morphogenetic proteins, BMPs)属于TGF-p超家族成员,近几年,研究人员对BMPs在繁殖上的作用进行了广泛的研究,包括卵泡的生长发育、颗粒细胞和膜细胞的增殖与凋亡、类固醇激素的合成、卵母细胞成熟、排卵和黄体化等。但BMP/Smad信号通路在猪卵泡颗粒细胞中具体作用及机制目前尚未见报道。因此,深入研究BMP/Smad信号通路在卵泡发育过程颗粒细胞中的作用及其调控机制,可以为哺乳动物繁殖性状的选育提供可能的理论基础,在提高家畜繁殖性能方面有重要意义。
本研究以猪卵泡颗粒细胞(GCs)为实验材料,采用real time RT-PCR方法检测BMP/Smad信号通路中关键因子(受体和下游信号分子)在猪卵泡不同发育时期的颗粒细胞中的表达水平;利用免疫组织化学方法就BMP/Smad信号通路核心下游信号分子—Smad4,在猪卵巢中的细胞定位进行研究;用RNAi方法敲减Smad4,以阻抑BMP/Smad信号通路,利用MTT、流式细胞术、CLIA、real time RT-PCR等方法检测阻抑该信号通路对猪卵泡颗粒细胞的作用及其分子机制;在阻抑通路的基础上,额外添加FSH,利用MTT、流式细胞术、CLIA、real time RT-PCR等方法检测阻抑该信号通路对FSH影响猪卵泡颗粒细胞的作用的影响;用BMP配体(BMP-6)激活BMP/Smad信号通路,用RNAi方法敲减Smad4,以阻抑该通路,分别利用MTT、流式细胞术、CLIA等方法检测两种条件下体外培养的猪卵泡颗粒细胞的存活率、细胞周期、类固醇激素分泌水平等,并采用高通量基因芯片技术,对阻抑信号通路和阻抑后激活信号通路的猪卵泡颗粒细胞之间的差异表达基因进行比较研究,
主要研究结果如下:
1用real time RT-PCR法检测不同发育时期猪卵泡颗粒细胞中BMP/Smad信号通路的关键因子(BMP受体和Smads)表达情况,结果表明:BMP受体和Smads在猪卵泡颗粒细胞中均有表达,并且ActR1A和BMPR2 mRNA在大卵泡颗粒细胞中的表达量显著高于小卵泡(P<0.05)。BMPR1A、ActR2、Smad1、Smad5和Smad8在卵泡发育过程中的表达有增加趋势,但差异不显著。相反,BMPR1B、Smad4和Smad7的表达有一定程度的下降。这表明BMP/Smad信号通路关键因子在猪卵泡发育阶段特异性表达,在卵泡颗粒细胞生长与分化过程中起重要的调控作用。
2用免疫组织化学方法检测Smad4在猪卵巢组织中的定位与表达,结果显示,Smad4在猪卵泡的多个发育阶段的细胞中均有表达。在原始卵泡中和初级卵泡中,Smad4在卵母细胞的细胞质中表达,而在颗粒细胞中没有表达;在腔前卵泡中,Smad4蛋白在卵母细胞和颗粒细胞中均有表达,在有腔卵泡中,Smad4蛋白主要被定位到颗粒细胞和卵泡内膜细胞中,而卵母细胞中表达较弱。
3为了研究BMP/Smad信号通路在猪卵泡颗粒细胞中的作用,本研究从猪卵巢卵泡(3-5mm)内分离颗粒细胞(pGCs)进行体外培养。基于Smad4在该通路中的核心地位,采用RNAi技术“沉默”猪Smad4基因,阻抑BMP/Smad信号通路,以检测阻抑该通路对猪卵泡颗粒细胞生长和类固醇激素分泌的影响。结果表明:采用的RNAi技术成功的“敲减”猪Smad4基因,在mRNA水平该基因被下调89%,蛋白水平被下调56.6%。建立了BMP/Smad信号通路阻抑的细胞模型。阻抑该通路能显著抑制细胞的增殖,增加G1期细胞的百分含量,减少S期的细胞,并能显著下调细胞增殖标志因子Cyclin D2和CDK4的mRNA表达。阻抑该通路能显著增加颗粒细胞的凋亡比率,下调Bcl-2的表达,但不影响Bax的表达。阻抑该通路能显著抑制雌二醇的分泌,并下调P450arom mRNA的表达,表明BMP/Smad信号通路在猪卵泡颗粒细胞中有重要的调控作用。
4为研究BMP/Smad信号通路对FSH诱导猪卵泡颗粒细胞的影响,本研究在阻抑BMP/Smad信号通路的细胞中添加FSH。用MTT法检测细胞增殖情况、流式细胞法检测细胞周期变化、CLIA方法检测雌二醇和孕酮的分泌以及real time RT-PCR检测相关基因的mRNA表达。结果表明:阻抑信号通路下调FSH受体的表达,阻抑信号通路使FSH促进细胞增殖的作用减弱,阻抑信号通路能显著增加G1期的细胞比率,即使添加FSH;阻抑通路能降低Cyclin D2的表达,且能抑制FSH促进其表达;阻抑信号通路能抑制CDK4的表达,即使添加FSH;阻抑信号通路能降低FSH诱导细胞分泌雌二醇的能力,但对孕酮没有影响。阻抑信号通路能减少P450arom的表达,并且降低FSH对其表达的影响;阻抑信号通路抑制FSH对P450scc表达的影响。
5为研究BMP/Smad信号通路对猪卵泡颗粒细胞的影响,基于Smad4在该通路中的核心地位,采用RNAi技术“沉默”猪Smad4基因,阻抑BMP/Smad信号通路,另一方面,添加BMP配体(BMP-6)激活该通路,分别观察阻抑和/或激活BMP/Smad信号通路对细胞生物学特性的影响。结果表明:阻抑通路显著抑制细胞的增殖,增加G1期的细胞,减少S期细胞;激活通路则促进细胞生长,减少G1期细胞,增加S期细胞,阻抑信号通路能抑制雌二醇的合成,对孕酮没有显著影响,激活该通路抑制两种类固醇激素的分泌。在阻抑通路与阻抑后再激活通路的不同处理组细胞中筛选到37个差异表达探针,上调基因25个,下调基因12个。结合GO分析,37个基因中24个基因可以在生物过程中找到注释,23个基因在分子功能中找到注释,13个基因在细胞组成中找到注释。
Prolificacy is one of the most important factors which influence the development of pig industry and it is also the main limiting factor to decide the efficiency of pig industry development. Fertility depends on the level of maternal growth and development of ovarian follicles as ovulation follicle development determines the number of ovum ovulated which is the key factor affecting the fecundity. To master sow follicular development and its impact factors are conducive to further improve sow reproductive capacity and enhance the economic benefits of survival pigs. Mammalian follicular development is a very complex process, including recruitment of the primitive follicle, the development of preantral follicles and antral follicle selection, growth and mature or follicle atresia. It was found that follicular growth and development is a highly coordinated and physiological and biochemical cascade processes affected and regulated by acting downstream of complex signaling pathways that integrate signals from the surrounding microenvironment but not an independent event with the development of post-genomics and bioinformatics.
Granulosa cells are key somatic cells in follicules, and play very important roles in oocyte maturation and follicular development. Follicular development is accompanied by growth, proliferation, differentiation, and maturation of granulosa cells. Bone morphogenetic proteins (BMPs) belong to the transforming growth factorβ(TGFβ) superfamily and have implicated in the control and regulation of follicular development and female fertility. Recently, there are lots of research about the role of BMP on reproduction, including to follicular growth and development, granulosa cells proliferation and apotosis, steroidogenesis, oocyte maturation, ovulation and luteinization. However, the roles and the molecular mechanisms of BMP/Smad signaling alters follicular development have not been fully investigated in pigs. To further research the roles and mechanisms of BMP/Smad signaling in follicular granulosa cells provide possible rationale to select of mammalian reproductive trait and have great importance to the improvement of reproductive capacity of domestic animal.
In this study, the time-spatial expression of BMP receptors and Smads in porcine follicular granulosa cells (GCs) in different period were investigated, by quantitative real-time PCR and cellular localization of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was studied in porcine ovaries by immunohistochemistry (IHC). In order to repress BMP/Smad signaling, the gene of Smad4 was silenced by RNAi, the effects of repressed BMP/Smad signaling on porcine follicular granulosa cells and underlying molecular mechanism were detected by MTT, FCM, CLIA and real time RT-PCR. Based on the repressed signaling, cells were treated with FSH, and the effects of repressed BMP/Smad signaling on the role FSH to GCs were investigated by MTT, FCM, CLIA and real time RT-PCR. Cells were treated with BMP-6, a activator of BMP/Smad signaling, as well as the Smad4-RNAi which repressed the BMP/Smad signaling, then the survival rate, proliferation and secretions of E2 and P4 of GCs were detected by MTT, FCM and CLIA; Microarray analysis was performed to capture the difference in gene expression in porcine GCs of between repressed the signaling and repressed then activated signaling.
The main results achieved were as follows:
1 The mRNA expression of BMP receptors and Smads in granulosa cells was detected by real time RT-PCR. The results indicated that endogenous BMP receptors and Smads expressed in porcine GCs, and ActRIA and BMPR2 mRNA levels were significantly higher in DF than that in SF(P<0.05). The mRNA levels of BMPR1A, ActR2, Smadl, Smad5 and Smad8 presented increased tendency, but there's no significant difference in DFs and SFs. Whereas BMPR1B, Smad4 and Smad7 expression had tended to decrease from SF to DF. Together, the stage-specific expression pattern of BMP receptors and Smads suggested that the BMP/Smad signaling might play potential roles in the follicle GCs growth and differentiation.
2 Cellular localization and expression of Smad4 proteins were studied by immunohistochemistry (IHC) in the ovaries of pigs. The results indicated that Smad4 localized in all kinds of cells of follicles at all stages. Smad4 localized in the cytoplasm of oocyte of primordial and primary follicles but not in granulosa cells; in pre-antral follicles, Smad4 localized in the oocytes and granulosa cells; Smad4 mainly localized in granulosa cells and follicular theca cells in antral follicles, and in oocyte, the expression of Smad4 was low.
3 To demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs, granulosa cells were isolated from 3-5mm antral follicles in diameter and cultured in vitro. A strategy of RNAi-mediated 'gene silencing' of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to repress endogenous BMP/Smad signaling in cells. Results showed that siRNA-Smad4 causes specific inhibition of Smad4 mRNA and protein expression after transfection, with the Smad4 transcript level reduced by approximately 89% and the level of Smad4 protein also markedly decreased 56.6%. Repressed endogenous BMP/Smad signaling significantly inhibited growth and induced apoptosis of porcine GCs, and decreased E2 production. In addition, repressed BMP/Smad signaling significantly changed the mRNA expression of Cyclin D2, CDK4, Bcl-2, and P450 aromatase (P450arom). These results implied that BMP/Smad signaling have key regulation in porcine GCs.
4 To demonstrate the effects of BMP/Smad signal on FSH-induced porcine GCs, we repress endogenous BMP/Smad signaling in cells while FSH was supplied in the medium of GCs. The proliferation and cycle of cells, secretions of E2 and P4, and the expression of relating genes were detected by MTT, FCM, CLIA and real time RT-PCR, respectively. The results showed that repressed endogenous BMP/Smad signaling significantly decreased the expression of FSHR, inhibited FSH-induced porcine granulosa cell proliferation, and signaificantly increased the proportion of G1-phase cells even with the existence of FSH; the repressed endogenous BMP/Smad signaling significantly decreased the expression of Cyclin D2, and inhibited FSH-induced augmentation of its expression; the repressed the signaling significantly decreased the expression of CDK4 even with the existence of FSH; besides, the repressed the signaling degraded FSH-induced E2 production, but not P4;the repressed the signaling decreaseed expression of P450 aromatase (P450arom), and inhibited FSH-induced expression of P450arom and cholesterol side-chain cleavage enzyme P450 (P450scc).
5 To demonstrate the effects of BMP/Smad signal on porcine GCs, we repressed endogenous BMP/Smad signaling in cells, besides, we activated the signaling by supplyling BMP (BMP-6) in the medium, investigated the effects of repressed and activated BMP/Smad signaling on porcine GCs. The results showed that repressed endogenous BMP/Smad signaling significantly inhibited granulosa cell proliferation, increased the proportion of G1-phase cells, and decreased the cells of S-phase. Activated signaling significantly decreased the proportion of cells in G1-phase, increased the cells of S-phase, and repressed endogenous BMP/Smad signaling significantly inhibited secretion of E2, but not P4;activated signaling significantly inhibited the two steroid hormone production.37 probes showed significant differences in the GCs of between repressed the signaling and repressed then activated signaling, among which 25 were up-regulated, while 12 were down-regulated. By the GO analysis,24 genes were found in biological process, 23 genes in molecular function, and 13 genes in cellular component.
引文
1. Shimasaki S, Moore RK, Erickson GF, Otsuka F. The role of bone morphogenetic proteins in ovarian function, Reprod Suppl.2003,61:323-37.
2. Feuerstein P, Cadoret V, Dalbies-Tran R, Guerif F, Royere D. Oocyte-cumulus dialog, Gynecol Obstet Fertil,2006,34(9):793-800.
3. Goodenough DA, Simon AM, Paul DL, Gap junctional intercellular communication in the mouse ovarian follicle. Navartis Found Symp,1999,219:226-235.
4. Filion F, Bouchard N, Goff AK, Lussier JG, Sirois J. Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo. J Biol Chem.2001,276(36):34323-30
5. Segi E, Haraguchi K, Sugimoto Y, Tsuji M, Tsunekawa H, Tamba S, Tsuboi K, Tanaka S, Ichikawa A. Expression of messenger RNA for prostaglandin E receptor subtypes EP4/EP2 and cyclooxygenase isozymes in mouse periovulatory follicles and oviducts during superovulation. Biol Reprod.2003 Mar;68(3):804-11.
6. Sun T, Deng WB, Diao HL, Ni H, Bai YY, Ma XH, Xu LB, Yang ZM. Differential expression and regulation of prostaglandin E synthases in the mouse ovary during sexual maturation and luteal development. J Endocrinol.2006 Apr;189(1):89-101.
7. Liu YX. Plasminogen activator/plasminogen activator inhibitors in ovarian physiology. Front Biosci 2004 Sep 1;9:3356-73.
8. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals. Reproduction.2001 Dec;122(6):829-38.
9. Spicer LJ, Aad PY, Allen DT, Mazerbourg S, Payne AH, Hsueh AJ., Growth differentiation factor 9 (GDF9) stimulates proliferation and inhibits steroidogenesis by bovine theca cells:influence of follicle size on responses to GDF9., Biol Reprod,2008,78(2):243-253
] 0. Liu YX, Liu K, Feng Q, Hu ZY, Liu HZ, Fu GQ, Li YC, Zou RJ, Ny T., Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey., Endocrinology,2004,145(4):1767-75
11. Paavola LG, The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells. Am J Anat.1979 Mar;154(3):337-64.
12. Gougeon A. Dynamics of follicular growth in the human:a model from preliminary results. Hum Reprodk,1986,1:81-87
13.杨晓箐,姚红军,王玉凤.哺乳动物卵母细胞凋亡的研究进展.动物学杂志,2004,39(6):101-106
14. Yutaka M, Jonathan L, Tilly. Oocyte Apoptosis:Like Sand through an Hourglass. Developmental Biology,1999,213(1):1-17
15. Tilly JL, Kowalski KI, Johnson AL, et al. Involvement of apoptosis in ovarian follicular atresia and postovulatory regression. Endocrinology.1991,129:2799-2801
16. Hughes FM JR, Gorospe WC. Biochemical identification of apoptosis (programmed cell death) in granulosa cells:evidence for a potential mechanism underlying follicular atresia. Endocrinology, 1991,129:2415-2422
17. Miki S, Noboru M, Yoshihiro K. et al. Ultrastructural changes in granuloa cells in porcine antral follicles undergoing atresia indicate apoptotic cell death. J Reprod Dev 1998,44:7-14
18. Jolly PD, Tisdall DJ, Heath DA, et al. Apoptosis in bovine granulosa cells in relation to steroid synthesis, cyclic adenosine 3',5'-monophosphate response to follicle-stimuating hormone and luteinizing hormone, and follicular atresia. Biol Reprod.1994,51:934-944
19. Wiliam JM. Programmed cell death in preovulatory ovine follicles. Bio Reprod.1995,53:8-12
20.严云勤,李光鹏,郑小民编著.发育生物学原理与胚胎工程[M].哈尔滨:黑龙江科学技术出版社,1995,16-35
21.金华,丁家怡,朱庆文,等.颗粒细胞在未成熟卵体外培养时对未成熟卵母细胞发育的影响[J].齐齐哈尔医学院学报,2006,27(3):309-310.
22.王建辰,张孝荣..动物生殖调控[M].合肥:安徽科学技术出版社,1998,63-68.
23. Wang SB, Xing BS, Yi L, Wang W, Xu YX. Expression of Frizzled 2 in the mouse ovary during oestrous cycle. J Anim Physiol Anim Nutr,2009, Aug 3
24. Le Good JA, Joubin K, Giraldez AJ, Ben-Haim N, Beck S, Chen Y, Schier AF, Constam DB. Nodal stability determines signaling range. Curr Biol,2005,15(1):31-6
25. Thisse B and Thisse C. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol,2005,287(2):390-402
26. BMP/Smad信号通路与哺乳动物卵泡发生,王伟,王少兵,徐银学,遗传,2009,(03),245-254
27. Shimisaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endovr Rev,2004,25(1):72-101
28. Drummond AE. TGF-β signaling in the development of ovarian function. Cell Tissue Res,2005, 322(1):107-115
29. Yokota Y, Mori S. Role of Id family proteins in growth control. J Cell Physiol,2002,190(1):21-28
30. Henningfeld KA, Friedle H, Rastegar S, Knochel W. Autoregulation of Xvent-2, direct interaction and functional cooperation of Xvent-2 and Smadl. J Biol Chem,2002,277(3):2097-2103
31. Onichtchouk D, Chen YG, Dosch R, Gawantka V, Delia's H, Massague J, Niehrs C. Silencing of TGF-P signaling by the pseudo receptor BAMBI. Nature,1999,401(6752):480-485
32. Pouliot, F. and Labrie, C. Expression profile of agonistic Smads in human breast cancer cells: absence of regulation by estrogens. Int J Cancer,1999, (81):98-103
33. Kalkhoven, E., Roelen, B.A., deWinter, J.P., Mummery, C.L., van den Eijnden-van Raaij AJ, van der Saag, P. T., and Van der, B.B. Resistance to transforming growth factor beta and activin due to reduced receptor expression in human breast tumor cell lines. Cell Growth Differ,1995, (6): 1151-1161
34. Sun, L., Wu, G., Willson, J. K., Zborowska, E., Yang, J., Rajkarunanayake, I., Wang, J., Gentry, L.E., Wang, X. F., and Brattain, M.G. Expression of transforming growth factor beta type 2 receptor leads to reduced malignancy in human breast cancer MCF-7 cells. J. Biol Chem,1994, (269): 26449-26455
35. Hahn S A, Schutt M, Hoque A T, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1[J]. Science,1996,271(5247):350-353
36. De Caesteckr MP,Y ahata T, Wang D, et al. The Smad4 activation domain(SAD) is a proline-rich, p300-dependent transcriptional activation domain[J]. J Biol Chem,2000,275:2115-2122.
37. Jens U, Wurthner, David B, et al. Transforming growth factor-receptor-associated protein 1 is a Smad4 chaperone[J]. J Biol Chem,2001,276(22):19495-19499.
38. Massague J. TGFβ signaling:receptors, transducers, and Mad proteins[J]. Cell,1996,85 (7): 947-950
39.Zawei L. G1 cell cycle arrest and apoptosis induction by nuclear Smad4/Dpc4:phenotypes reversed by a tumorigenic mutation[J]. Mol Cell,1998,1:611-617.
40. Suzuki H, Yagi K, Kondo M, et al. c-Ski inhibits the TGF-(3 signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements[J]. Oncogene,2004,23(31):5068-5073.
41. Grau AM, Zhang L, Wang W, et al. Induction of p21wafl expression and growth inhibition by transforming growth factor beta involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells[J]. Cancer Res.1997,57(18):3929-3934.
42. Schwarte-Waldhoff I, Klein S, Blass-Kampmann S, et al. DPC4/SMAD4 mediated tumor suppression of colon carcinoma cells is associated with reduced urokinase expression [J]. Oncogene,1999,18 (20):3152-3158.
43. Barbara A, Hocevar, Thomas L, et al. TGF-beta induces fibronectin synthesis through a c-Jun N-terminal kinase-dependent, Smad4-indepent pathway[J]. EMBO J,1999,18:1345-1356
44. Liu JH, Weis, Burnette PK, et al. Functional association of TGF-beta receptor II with Cyclin B[J]. Oncongene,1999,18:269-275.
45. Erickson GF, Shimasaki S. The spatiotemporal expression pattern of the bone morphogenetic protein family in rat ovary cell types during the estrous cycle. Reprod Biol Endocrinol,2003,1:9
46. Souza CJ, Campbell BK, McNeilly AS, Baird DT. Effect of bone morphogenetic protein 2 (BMP-2) on oestradiol and inhibin Aproduction by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry. Reproduction,2002,123(3):363-369
47. A Pierre, C Pisselet, J Dupont, B Mandon-Pepin, D Monniaux, P Monget and S Fabre. Molecular basis of bone morphogenetic protein-4 inhibitory action on progesterone secretion by ovine granulosa cells. Journal of Molecular Endocrinology.2004 (33):805-817
48. Victoria Brankin, Ruth L. Quinn, Robert Webb, Morag G. Hunter. Evidence for a functional bone morphogenetic protein (bmp) system in the porcine ovary. Domestic Animal Endocrinology,2005 (28):367-379
49. Lee W, Otsuka F, Moore RK, Shimasaki S. The effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat. Biol Reprod,2001,65(4):994-999
50. Kenneth P McNatty, Jennifer L Juengel, Karen L Reader, Stan Lun, Samu Myllymaa, Steve B Lawrence, Andrea Western, Mohamed F Meerasahib, David G Mottershead, Nigel P Groome, Olli Ritvos and Mika P E Laitinen. Bone morphogenetic protein 15 and Growth differentiation factor 9 co-operate to regulate granulosa cell function. Reproduciton,2005 (129):473-4809-13
51. Leon J. Spicer, Pauline Y. Aad, Dustin T. Allen, Sabine Mazerbourg, Anita H. Payne, and Aaron J. Hsueh. Growth Differentiation Factor 9 (GDF9) stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells:Influence of Follicle Size on Responses to GDF9. BIOLOGY OF REPRODUCTION,2008 (78):243-253
52. Victoria Brankin a, Ruth L. Quinn a, Robert Webb b, Morag G. Hunter a. BMP-2 and -6 modulate porcine theca cell function alone and co-cultured with granulosa cells. Domestic Animal Endocrinology,2005,29:593-604
53. Shimasaki S, Zachow RJ, Li D, Kim H, Iemura S, Ueno N, Sampath K, Chang RJ, Erickson GF. A functional bone morphogenetic protein system in the ovary. Proc Natl Acad Sci USA,1999,96: 7282-7287
54. Alice Pierre, Claudine Pisselet,3 Joelle Dupont, Martine Bontoux, and Philippe Monget2. Bone Morphogenetic Protein 5 Expression in the Rat Ovary:Biological Effects on Granulosa Cell Proliferation and Steroidogenesis 1. BIOLOGY OF REPRODUCTION,2005,73:1102-1108
55. Takashi Shimizu, Masaki Yokoo, Yuko Miyake, Hiroshi Sasada, Eimei Sato. Differential expression of bone morphogenetic protein 4-6 (BMP-4,-5, and -6) and growth differentiation factor-9 (GDF-9) during ovarian development in neonatal pigs. Domestic Animal Endocrinology, 2004,27:397-405
56. Tomoko Miyoshi, Fumio Otsuka, Kenichi Inagaki, Hiroyuki Otani, Masaya Takeda, Jiro Suzuki, Junko Goto, Toshio Ogura, and Hirofumi Makino. Differential Regulation of Steroidogenesis by Bone Morphogenetic Proteins in Granulosa Cells:lnvolovement of Extracellularly Regulated Kinase Signaling and Oocyte Actions in Follicle-Stimulating Hormone-Induced Estrogen Production. Endocinology,2007,148(1):337-345
57. Heather E. McMahon, Osamu Hashimoto, Pamela L. Mellon and Shunichi Shimasaki. Oocyte-specific over-expression of mouse BMP-15 leads to accelerated folliculogenesis and an early onset of acyclicity in transgenic mice. Copyright (C),2008, by The Endocrine Society
58. Kenneth P McNatty, Jennifer L Juengel, Karen L Reader, Stan Lun, Samu Myllymaa, Steve B Lawrence, Andrea Western, Mohamed F Meerasahib, David G Mottershead, Nigel P Groome, Olli Ritvos and Mika P E Laitinen. Bone morphogenetic protein 15 and Growth differentiation factor 9 co-operate to regulate granulosa cell function. Reproduciton,2005 (129):473-4809-13
59. Su YQ, Sugiura K, Wigglesworth K, O'Brien MJ, Affourtit JP, Pangas SA, Matzuk MM, Eppig JJ. Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes:BMP 15 and GDF9 control cholesterol biosynthesis in cumulus cells, Development,2008,135(1):111-21
60. Hayashi M, McGee EA, Min G, Klein C, Rose UM, van Duin M, Hsueh AJ. Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles., Endocrinology,1999,140(3):1236-44
61. Jayawardana BC, Shimizu T, Nishimoto H, Kaneko E, Tetsuka M, Miyamoto A., Hormonal regulation of expression of growth differentiation factor-9 receptor type Ⅰ and Ⅱ genes in the bovine ovarian follicle., Reproduction,2006,131(3):545-53
62. Guiyu Zhu, Bingran Guo, Dengke Pan, Yulian Mu, Shutang Feng. Expression of bone morphogenetic proteins and receptors in porcine cumulus-oocyte complexes during in vitro maturation. Animal Reproduction Science,2008 (104):275-283
63. Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc I, Pisselet C, Riquet J, Monniaux D, Callebaut I, Cribiu E, Thimonier J, Teyssier J, Bodin L, Cognie Y, Chitour N, Elsen J M. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes. Proc Natl Acad Sci USA,2001,98(9):5104-5109
64. Wilson T, Wu X Y, Juengel J L, Ross I K, LUmsden J M, Lord E A, Dodds K G, Walling G A, McEwan J C, O'Connell A R, McNatty KP, Montgomery G W. Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells. Biol Reprod.2001,64(4): 1225-1235
65. Souza C J, MacDougall C, Campbell B K, McNeilly A S, Baird D T. The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type IB (BMPRIB) gene. J Endocrinol.2001,169(2):1-6
66. Mulsant P, Lecerf F, Fabre S, Bodin L, Thimonier J, Monget P, Lanneluc 1, Monniaux D, Teyssier J, Elsen JM. Prolificacy genes in sheep:the French genetic programmes., Reprod Suppl, 2003,61:353-9
67. Fatehi AN, van den Hurk R, Colenbrander B, Daemen AJ, van Tol HT, Monteiro RM, Roelen BA, Bevers MM., Expression of bone morphogenetic protein2 (BMP2), BMP4 and BMP receptors in the bovine ovary but absence of effects of BMP2 and BMP4 during IVM on bovine oocyte nuclear maturation and subsequent embryo development., Theriogenology,2005,63(3):872-89
68. Chu GC, Dunn NR, Anderson DC, Oxburgh L, Robertson EJ., Differential requirements for Smad4 in TGFbeta-dependent patterning of the early mouse embryo., Development,2004, 131(15):3501-12
69. Biondi, C. A., D. Das, et al. Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling. Biochem J,2007,404(2):235-245;
70. Lin, X., X. Duan, et al. PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling. Cell,2006,125(5):915-928
71. Dai, F., X. Lin, et al. Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signling. Dev Cell,2009,16(3)345-357
72. Gueripel X, Benahmed M, Gougeon A. Sequential gonadotropin treatment of immature mice leads to amplification of transforming growth factor beta action, via upregulation of receptor-type 1, Smad2 and 4, and downregulation of Smad6. Biol Reprod.2004,70(3):640-648
73. Pangas, S.A., X. Li, et al. Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice. Mol Endocrinol,2006,20(6):1406-1422
74. Gong, X. and E. A. McGee. Smad3 is required for normal follicular follicle-stimulating hormone responsiveness in the mouse. Biol Reprod,2009,81(4):730-738
75. GUO S AND KEMPHUES K J. Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell,1995, 81(4):677-620
76. FIRE A, XU S, MONTGOMERY M K, Kostas SA, Driver SE, Mello CC,. Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans. Nature,1998,391(6669): 806-11
77. Montgomery MK, Xu S, Fire A., RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans., Proc Natl Acad Sci U S A,1998,95(26):15502-7
78. Elabshir SM, Lendeckel W, Tuschl T, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature,2001,411(6836):428-429
79. Hannon GJ. RNA interference. Nature,2002,418(6894):244-251
80. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev,2001,15(2):188-200
81. Sijen T, Fleenor J, Simmer F, et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell,2001,107(4):456-476
82. MacManus MT, Sharp PA. Gene silencing in mammals by small interference RNAs. Nat Rev Genet, 2002,3(10):737-747
83. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev,2002,16(8):948-958
84. Svoboda P, Stein P, Hayashi H, et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development,2000,127(19):4147-4156
85. Winston WM, Molodowitch C, Hunter CP. Systemic RANi in C. elegans requires the putative transmembrane protein SID-1. Science,2002,295(5564):2456-2459
86. Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature,2002,428(6896):430-434
87. Baulcombe D. RNA silencing. Diced defense. Nature,2001,409(6818):295-296
88. Zamore PD, Tuschl T, Sharp PA, et al. RNAi:double-stranded RNA directs the ATP dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell,2000,101(1):25-33
89. Zhang H, Kolb FA, Brondani V, et al. Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J,2002,21(21):5875-5885
90. Billy E, Brondani V, Zhang HD, et al. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA, 2001,98(25):14428-14433
91. Oates AC, Bruce AE, Ho RK. Too much interference:injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Dev Biol.2000,224(1):20-28
92. Hammond SM, Bernstein E, Beach D et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophils cells. Nature,2000,404(6775):293-296
93. Li Y, Xiao LY, Yao G, Li H, Yang ZW, Li WY, Jiang ZH, Li MY, Construction of adenovirus expressing siRNA against hTR and its anti-tumor activity in vitro, Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2008,24 (6):570-3
94. Wianny F, Zernicka-Goetz M., Specific interference with gene function by double-stranded RNA in early mouse development, Nat Cell Biol,2000,2(2):70-5
95. Mehlmann LM., Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes., Dev Biol.2005,288(2):397-404
96. Han SJ, Chen R, Paronetto MP, Conti M., Weel B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse., Curr Biol,2005,15(18):1670-6
97. Hanna CB, Yao S, Patta MC, Jensen JT, Wu X., WEE2 Is an Oocyte-Specific Meiosis Inhibitor in Rhesus Macaque Monkeys., Biol Reprod,2010 Mar 3
98. Li M, Li S, Yuan J, Wang ZB, Sun SC, Schatten H, Sun QY, Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis., PLoS One, 2009,4(11):e7701
99. Khang I, Sonn S, Park JH, Rhee K, Park D, Kim K., Expression of epithin in mouse preimplantation development:its functional role in compaction., Dev Biol,2005,281 (1):134-44
100. Home P, Ray S, Dutta D, Bronshteyn I, Larson M, Paul S., GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression., J Biol Chem.2009,284(42):28729-37
101.Prasanth SG, Prasanth KV, Stillman B., Orc6 involved in DNA replication, chromosome segregation, and cytokinesis., Science,2002,297(5583):1026-31
102. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G., Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes., Nature,2003,421 (6920): 268-72
103. Morris JC, Wang Z, Drew ME, Englund PT., Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library., EMBO J,2002,21(17):4429-38
104. Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K., Identification of essential genes in cultured mammalian cells using small interfering RNAs, J Cel Sci,2001 114(24):4557-65
105. Paddison PJ, Caudy AA, Hannon GJ., Stable suppression of gene expression by RNAi in mammalian cells., Proc Natl Acad Sci U S A,2002,99(3):1443-8
106. Caplen NJ. RNAi as a gene therapy approach. Expert Opin Biol Ther 2003,3(4):575-586
107. Wiannty F, Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol,2000,2(2):70-75
108. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J., Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature,2000, 408(6810):325-330
109. Gonczy P, Echeverri C, Oegema K, Coulson A, Jones SJ, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E, Hannak E, Kirkham M, Pichler S, Flohrs K, Goessen A, Leidel S, Alleaume AM, Martin C, Ozlu N, Bork P, Hyman AA., Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III., Nature,2000,408(6810):331-6
110. Makimura H, Mizuno TM, Mastaitis JW, Agami R, Mobbs CV., Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake., BMC Neurosci,2002,3:18
111. Carmichael GG. Medicine:silencing viruses with RNA. Nature,2002,418(6896):379-380
112. Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol,2001,1:34
113.Qin XF, An DS, Chen IS, Baltimore D., Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc NAtl Acad Sci USA, 2003,100(1):183-188
114. Jacque JM, Triques K, Stevenson M. Modulation of HIV-1 replication by RNA interference. Nature, 2002,418(6896):435-438
115. Zeng Y, Wagner EJ, Cullen BR. Both natural and designed micro-RNAs can inhibit the expression of cognate. Mol Cell,2002,9(6):1327-1333
116. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science,2001, 294(5543):862-864
117. Hamasaki K, Nakao K, Matsumoto K, et al. Short interfering RNA-directed inhibition of hepatitis B virus replication. FEBS Lett,2003,543(1-3):51-54
118. McCaffrey AP, Meuse L, Pham TT, et al. RNA interference in adult mice. Nature,2002,418:38-39
119. Lin SL, Chuong CM, Ying SY., A novel mRNA-cDNA interference phenomenon for silencing bcl-2 expression in human LNCaP cells. Biochem Biophys Res Commun,2001,281(3):639-644
120. Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, Rubin MA, Chinnaiyan AM., The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature,2002,49(6907):624-629
121. Wilda M, Fuchs U, Wossmann W, Borkhardt A.. Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene,2002,21(37):5716-5724
122. Wohlbold L, van der Kuip H, Miething C, Vornlocher HP, Knabbe C, Duyster J, Aulitzky WE, Inhibition of bcr-abl gene expression by small interfering RNA Sensitizes for imatinib mesylate (STI571). Blood,2003,102(6):2236-2239
123. Jiang M, Milner J. Selective silencing of viral gene expression in HPV-postitive human cervical carcinoma cells treated with siRNA, a primer of RNA interference. Oncogene,2002, 21(39):6041-6048
124. Hall AH, Alexander KA. RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells. J Virol,2003,77(10) 6066-6069
125. Yin JQ, Gao J, Shao R, Tian WN, Wang J, Wan Y., siRNA agents inhibit oncogene expression and attenuate human tumor cell growth. J Exp Ther Oncol,2003,3(4):194-204
126. Zhang L, Yang N, Mohamed-Hadley A, Rubin SC, Coukos G., Vector-based RNAi, a novels tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer. Biochem Biophys Res Commun,2003,303(4):1169-1178
127. Makimura H, Mizuno TM, Mastaitis JW, Agami R, Mobbs CV., Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake. BMC Neurosci,2002,3(1):18
128. Grado-Ahuir JA, Aad PY, Ranzenigo G, Caloni F, Cremonesi F, Spicer LJ., Microarray analysis of insulin-like growth factor-I-induced changes in messenger ribonucleic acid expression in cultured porcine granulosa cells:possible role of insulin-like growth factor-I in angiogenesis., J Anim Sci, 2009,87(6):1921-33
129. Bonnet A, Le Cao KA, Sancristobal M, Benne F, Robert-Granie C, Law-So G, Fabre S, Besse P, De Billy E, Quesnel H, Hatey F, Tosser-Klopp G.,In vivo gene expression in granulosa cells during pig terminal follicular development., Reproduction,2008,136(2):211-24
130. Agca C, Ries JE, Kolath SJ, Kim JH, Forrester LJ, Antoniou E, Whitworth KM, Mathialagan N, Springer GK, Prather RS, Lucy MC., Luteinization of porcine preovulatory follicles leads to systematic changes in follicular gene expression., Reproduction,2006,132(1):133-45
131. Caetano AR, Johnson RK, Ford JJ, Pomp D., Microarray profiling for differential gene expression in ovaries and ovarian follicles of pigs selected for increased ovulation rate., Genetics,2004, 168(3):1592-37
1. Shimasaki S, Moore RK, Otsuka F & Erickson GF 2004 The bone morphogenetic protein system in mammalian reproduction. Endocrine Reviews 25 72-101.
2. Juengel JL & McNatty KP 2005 The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development. Human Reproduction Update 11 143-160.
3. Gilchrist RB, Lane M & Thompson JG 2008 Oocyte-secreted factors:regulators of cumulus cell function and oocyte quality. Human Reproduction Update 14 159-177.
4. Livak, K. J. and T. D. Schmittgen. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method. Methods,2001,25(4):402-408
5. Zhu, G., B. Guo, et al.. Expression of bone morphogenetic proteins and receptors in porcine cumulus-oocyte complexes during in vitro maturation. Anim Reprod Sci,2008,104 (2-4):275-83.
6. Quinn, R. L., G. Shuttleworth, et al. (2004). "Immunohistochemical localization of the bone morphogenetic protein receptors in the porcine ovary." J Anat 205(1):15-23.
7. Souza, C. J., B. K. Campbell, et al. (2002). "Effect of bone morphogenetic protein 2 (BMP2) on oestradiol and inhibin A production by sheep granulosa cells, and localization of BMP receptors in the ovary by immunohistochemistry."Reproduction 123(3):363-9.
8. Glister, C., C. F. Kemp, et al. (2004). "Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells:actions of BMP-4,-6 and-7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin." Reproduction 127(2):239-54.
9. Erickson, G. F. and S. Shimasaki (2003). "The spatiotemporal expression pattern of the bone morphogenetic protein family in rat ovary cell types during the estrous cycle." Reprod Biol Endocrinol 1:9.
10. Francois Paradis, Susan Novak, et al. (2009). "Temporal regulation of BMP2, BMP6, BMP 15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA expression in the oocyte, granulosa and theca cells of developing preovulaotry follicles in the pig." Reproduction 138:115-129.
11. Kawabata, M., T. Imamura, et al. (1998). "Signal transduction by bone morphogenetic proteins." Cytokine Growth Factor Rev 9(1):49-61.
1. Hahn S A, Schutt M, Hoque A T, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1[J]. Science,1996,271(5247):350-353
2. Grau AM, Zhang L, Wang W, et al. Induction of p21wafl expression and growth inhibition by transforming growth factor beta involve the tumor suppressor gene DPC4 in human pancreatic adenocarcinoma cells[J]. Cancer Res.1997,57(18):3929-3934.
3. Schwarte-Waldhoff I, Klein S, Blass-Kampmann S, et al. DPC4/SMAD4 mediated tumor suppression of colon carcinoma cells is associated with reduced urokinase expression [J]. Oncogene,1999,18 (20):3152-3158.
4. Shi F, Stewart RL, Pere E, Chen JY, LaPolt PS. Cell-specific expression and regulation of soluble guanylyl cyclase alpha and beta subunit in the rat ovary. Bio Reprod,2004,70:1552-1561
5. Shi F, Perez E, Wang T, et al. Stage- and Cell-Specific Expression of Soluble Guanylyl Cyclase Alpha and Beta Subunits, cGMP-Dependent Protein Kinase I Alpha and Beta, and Cyclic Nucleotide-Gated Channel Subunit 1 in the Rat Testis. J Androl,2005,26(2):258-263
6. Shimasaki S, Moore RK, Otsuka F & Erickson GF 2004 The bone morphogenetic protein system in mammalian reproduction. Endocrine Reviews 25 72-101.
7. Juengel JL & McNatty KP 2005 The role of proteins of the transforming growth factor-beta superfamily in the intraovarian regulation of follicular development. Human Reproduction Update 11143-160.
8. Gilchrist RB, Lane M & Thompson JG 2008 Oocyte-secreted factors:regulators of cumulus cell function and oocyte quality. Human Reproduction Update 14159-177.
9. Knight PG, Glister C. TGF-beta superfamily members and ovarian follicle development. Reproduction 2006;132:191-206.
1. Shimasaki, S., R. Kelly Moore, et al. (2004). "The Bone Morphogenetic Protein System In Mammalian Reproduction." Endocrine Reviews 25 (1):72-101.
2. Biondi, C. A., D. Das, et al. (2007). "Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling." Biochem J 404(2):235-245.
3. Lin, X., X. Duan, et al. (2006). "PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling." Cell 125(5):915-928.
4. Dai, F., X. Lin, et al. (2009). "Nuclear export of Smad2 and Smad3 by RanBP3 facilitates termination of TGF-beta signaling." Dev Cell 16(3):345-357.
5. Pangas, S.A., X. Li, et al. Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice. Mol Endocrinol,2006,20(6):1406-1422
6. Dorsett, Y. and T. Tuschl (2004). "siRNAs:applications in functional genomics and potential as therapeutics." Nat Rev Drug Discov 3(4):318-329.
7. Mittal, V. (2004). "Improving the efficiency of RNA interference in mammals." Nat Rev Genet 5(5): 355-365.
8. Paterson,I. C., M. Davies, et al. (2002). "TGF-betal acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest." Oncogene 21(10):1616-1624.
9. Matsuura, I., N. G. Denissova, et al. (2004). "Cyclin-dependent kinases regulate the antiproliferative function of Smads." Nature 430(6996):226-231.
10. Winters, T. A., J. A. Hanten, et al. (1998). "In situ amplification of the cytochrome P-450 cholesterol side-chain cleavage enzyme mRNA in single porcine granulosa cells by IGF-1 and FSH acting alone or in concert." Endocrine 9(1):57-63.
11. Khamsi, F., S. Roberge, et al. (2001). "Recent discoveries in physiology of insulin-like growth factor-1 and its interaction with gonadotropins in folliculogenesis." Endocrine 16(3):151-165.
12. Shimasaki, S., R. J. Zachow, et al. (1999). "A functional bone morphogenetic protein system in the ovary." Proc Natl Acad Sci U S A 96(13):7282-7287.
13. Lee, W. S., F. Otsuka, et al. (2001). "Effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat." Biol Reprod 65(4):994-999.
14. Otsuka, F., Z. Yao, et al. (2000). "Bone morphogenetic protein-15. Identification of target cells and biological functions." J Biol Chem 275(50):39523-39528.
15. Vitt, U. A., M. Hayashi, et al. (2000). "Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles." Biol Reprod 62(2):370-377.
1. Erickson GF, Shimasaki S. The spatiotemporal expression pattern of the bone morphogenetic protein family in rat ovary cell types during the estrous cycle. Reprod Biol Endocrinol 2003; 1:9.
2. Shimasaki S, Moore RK, Erickson GF, Otsuka F. The role of bone morphogenetic proteins in ovarian function. Reprod Suppl 2003;61:323-337.
3. Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endocr Rev 2004; 25:72-101.
4. Shimasaki S, Zachow RJ, Li D, Kim H, Iemura S, Ueno N, Sampath K, Chang RJ, Erickson GF. A functional bone morphogenetic protein system in the ovary. Proc Natl Acad Sci U S A 1999; 96:7282-7287.
5. Lee W, Otsuka F, Moore RK, Shimasaki S. The effect of bone morphogenetic protein-7 on folliculogenesis and ovulation in the rat. Biology of Reproduction 2001;65:994-999.
6. Otsuka F, Moore RK, Shimasaki S. Biological function and cellular mechanism of bone morphogenetic protein-6 in the ovary. J Biol Chem 2001;276:32889-32895.
7. Otsuka F, Yamamoto S, Erickson GF, Shimasaki S. Bone morphogenetic protein-15 inhibits follicle-stimulating hormone (FSH) action by suppressing FSH receptor expression. J Biol Chem 2001;276:11387-11392.
8. Tomoko Miyoshi, Fumio Otsuka,2 Jiro Suzuki, Masaya Takeda, Kenichi Inagaki, Yoshihiro Kano, Hiroyuki Otani, Yukari Mimura, Toshio Ogura, and Hirofumi Makino. Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells. BIOLOGY OF REPRODUCTION 74,1073-1082(2006)
9. Pangas, S. A., X. Li, et al. (2006). "Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice." Mol Endocrinol 20(6):1406-1422.
10. Paterson, I. C., M. Davies, et al. (2002). "TGF-betal acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest." Oncogene 21(10):1616-1624.
11. Matsuura, I., N. G. Denissova, et al. (2004). "Cyclin-dependent kinases regulate the antiproliferative function of Smads." Nature 430(6996):226-231.
12. Otsuka, F., Z. Yao, et al. (2000). "Bone morphogenetic protein-15. Identification of target cells and biological functions." J Biol Chem 275(50):39523-39528.
13. Vitt, U. A., M. Hayashi, et al. (2000). "Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles." Biol Reprod 62(2):370-377.
1. Law AS, Baxter G, Logue DN, O'Shea T, Webb R. Evidence for the action of bovine follicular fluid factor(s) other than inhibin in suppressing follicular development and delaying estrus in heifers. J Reprod Fertil 1992;96:603-16.
2. LV YE, HONGYING ZHANG, LI ZHANG, GUANGHUA YANG, QIKE, HUAGUO and HONG BU. Effects of RNAi-mediated Smad4 silencing on growth and apoptosis of human rhabdomyosarcoma cells. INTERNATIONAL JOURNAL OF ONCOLOGY,2006,29:1149-1157
3. Shimizu T, Yokoo M, Miyake Y, Sasada H, Sato E. Differential expression of bone morphogenetic protein 4-6 (BMP-4,-5, and -6) and growth differentiation factor-9 (GDF-9) during ovarian development in neonatal pigs. Domest Anim Endocrinol 2004; 27:397-405.
4. Victoria Brankin, Ruth L. Quinn, Robert Webb, Morag G. Hunter. Evidence for a functional bone morphogenetic protein (BMP) system in the porcine ovary. Domestic Animal Endocrinology,2005, 28:367-379
5. Erickson GF, Shimasaki S. The spatiotemporal expression pattern of the bone morphogenic protein family in rat ovary cell types during the estrous cycle. Reprod Biol Endocrinol 2003;1:9.
6. Glister C, Kemp CF, Night PG. Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells:actions of BMP-4,-6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin. Reproduction 2004; 127:239-54.
7. Ying Y, Zhao Y. Cooperation of endoderm derived BMP-2 and extraembryonic ectoderm derived BMP-4 in primordial germ cell generation in the mouse. Dev Biol 2001;232:484-92.
8. Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc 1, et al. Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes. Proc Natl Acad Sci USA 2001;98:5104-9.
9. Souza CJ, MacDougall C, MacDougall C, Campbell BK, McNeilly AS, Baird DT. The Booroola (FecB) phenotype is associated with a mutation in the bone morphogenetic receptor type 1 B (BMPR1B) gene. J Endocrinol 2001; 169:R1-6.
10. Galloway SM, Gregan SM,Wilson T, McNatty KP, Juengel JL, Ritvos O, et al. Bmp15 mutations and ovarian function. Mol Cell Endocrinol 2002;191:15-8.