TFPI2在鸡卵巢中的表达及其在颗粒细胞中的调控机制

doi:10.1016/j.jia.2023.06.038

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (2): 680-696.DOI: 10.1016/j.jia.2023.06.038

TFPI2在鸡卵巢中的表达及其在颗粒细胞中的调控机制

收稿日期:2023-04-11 接受日期:2023-05-28 出版日期:2025-02-20 发布日期:2025-01-22

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells

Chong Li^{1, 2}, Yangguang Ren^{1, 2}, Yudian Zhao^{1, 2}, Zihao Zhang^{1, 2}, Bin Zhai^{1, 2}, Jing Li^{1, 2}, Qi Li³, Guoxi Li^{1, 2}, Zhuanjian Li^{1, 2}, Xiaojun Liu^{1, 2}, Xiangtao Kang^{1, 2}, Ruirui Jiang^{1, 2}, Yadong Tian^{1, 2#}, Donghua Li^{1, 2#}

¹College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China
²Henan Key Laboratory for Innovation and Utilization of Chicken Germplasm Resources, Zhengzhou 450046, China
³ Department of Animal Science, Henan Zhumadian Agricultural School, Zhumadian 463000, China

Received:2023-04-11 Accepted:2023-05-28 Online:2025-02-20 Published:2025-01-22
About author:Chong Li, E-mail: lc0924ll@163.com; #Correspondence Yadong Tian, E-mail: ydtian111@163.com; Donghua Li, Tel: +86-371-56552517, E-mail: lidonghua6656@126.com
Supported by:
This research was funded by grants from the Program for Innovative Research Team (in Science and Technology) in University of Henan Province, China (21IRTSTHN022) and the Zhongyuan Science and the Technology Innovation Leading Scientist Project, China (214200510003).

摘要/Abstract

摘要：

本研究旨在探讨TFPI2在鸡卵巢中的表达分布以及在培养卵巢颗粒细胞中的调控机制。首先，对海兰褐蛋鸡30周龄和15周龄卵巢组织进行转录组测序，获得差异表达基因TFPI2，通过RACE技术获得了TFPI2的cDNA全长，并对其蛋白序列进行生物信息学分析，成功制备TFPI2多克隆抗体后，利用荧光定量PCR（qRT-PCR）和蛋白质印迹（Western Blot）检测了TFPI2 mRNA和蛋白在不同发育状态卵泡中的表达；然后，通过体外培养卵巢颗粒细胞，进行垂体激素（FSH/LH）处理，探究了FSH/LH处理鸡卵巢颗粒细胞对TFPI2表达调控影响；最后，成功构建了含有目的基因TFPI2的过表达载体TFPI2-pcDNA3.1-EGFP和siRNA探究了TFPI2对鸡卵巢颗粒细胞的调控机制。结果表明：从成年鸡卵巢中获得了TFPI2的cDNA全长，包括5'非翻译区（5´UTR）长55 bp、3'非翻译区（3´UTR）长975 bp、蛋白质编码区（CDS）长720 bp、可编码240个氨基酸残基；通过构建进化树发现TFPI2蛋白与已知的鸟类同源物具有高度保守的氨基酸序列。qRT-PCR 和Western Blot表明TFPI2在成年鸡卵泡组织中广泛表达。免疫组织化学表明，TFPI2蛋白存在于不同发育状态的鸡卵巢卵泡中，如原始卵泡、卵巢基质、等级前卵泡（6-8mm）和排卵前卵泡（F1）的颗粒层和膜层。在体外实验中，促卵泡激素或促黄体生成素（FSH/LH）促进鸡颗粒细胞中TFPI2的表达。其中，FSH/LH诱导的TFPI2 mRNA表达是由信号通路如PKA、PKC、PI3K和mTOR等介导。功能上，TFPI2促进了培养的颗粒细胞的增殖和活力，并降低了孕酮（P4）和雌激素（E2）的分泌，类固醇激素合成相关基因（STAR、Cyp17a1、Cyp19a1和3B-HSD）以及细胞外基质（ECM）溶解相关基因MMPs（MMP2、7、9和11）的mRNA丰度。机制上，TFPI2可以通过Wnt信号通路抑制MMP7的表达。以上结果表明，TFPI2在不同卵泡均有表达，主要的作用靶点在GCs，可通过调控鸡卵泡GCs的增殖分化，以及类固醇激素的合成和降低ECM的溶解进而影响卵泡发育，此外，FSH和LH激素可以诱导不同的通路来调控TFPI2的表达，这些研究结果为明确TFPI2基因在鸡卵泡发育过程以及排卵的分子调控机制提供了理论和试验依据。

Abstract:

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In this study, RNA-seq was performed on ovarian tissues from chickens aged 30 and 15 weeks to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT-PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6–8 mm) and preovulatory follicles (F1). In vitro, follicle stimulating hormone or luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

Key words: layer , TFPI2 , FSH/LH , follicles , granulosa cells

Chong Li, Yangguang Ren, Yudian Zhao, Zihao Zhang, Bin Zhai, Jing Li, Qi Li, Guoxi Li, Zhuanjian Li, Xiaojun Liu, Xiangtao Kang, Ruirui Jiang, Yadong Tian, Donghua Li. TFPI2在鸡卵巢中的表达及其在颗粒细胞中的调控机制[J]. Journal of Integrative Agriculture, 2025, 24(2): 680-696.

Chong Li, Yangguang Ren, Yudian Zhao, Zihao Zhang, Bin Zhai, Jing Li, Qi Li, Guoxi Li, Zhuanjian Li, Xiaojun Liu, Xiangtao Kang, Ruirui Jiang, Yadong Tian, Donghua Li. Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells[J]. Journal of Integrative Agriculture, 2025, 24(2): 680-696.

参考文献

Andresen M S, Ali H O, Myklebust C F, Sandset P M, Stavik B, Iversen N, Skretting G. 2017. Estrogen induced expression of tissue factor pathway inhibitor-2 in MCF7 cells involves lysine-specific demethylase 1. Molecular and Cellular Endocrinology, 443, 80–88.

Bruggeman V, Van As P, Decuypere E. 2002. Developmental endocrinology of the reproductive axis in the chicken embryo. Comparative Biochemistry and Physiology, 131, 839–846.

Casarini L, Crépieux P. 2019. Molecular mechanisms of action of FSH. Frontiers in Endocrinology, 10, 305.

Chand H S, Foster D C, Kisiel W. 2005. Structure, function and biology of tissue factor pathway inhibitor-2. Thrombosis and Haemostasis, 94, 1122–1130.

Couse J F, Yates M M, Deroo B J, Korach K S. 2005. Estrogen receptor-beta is critical to granulosa cell differentiation and the ovulatory response to gonadotropins. Endocrinology, 146, 3247–3262.

Curry Jr T E, Osteen K G. 2003. The matrix metalloproteinase system: Changes, regulation, and impact throughout the ovarian and uterine reproductive cycle. Endocrine Reviews, 24, 428–465.

Demeestere I, Centner J, Gervy C, Englert Y, Delbaere A. 2005. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction, 130, 147–156.

Gilbert A B, Perry M M, Waddington D, Hardie M A. 1983. Role of atresia in establishing the follicular hierarchy in the ovary of the domestic hen (Gallus domesticus). Journal of Reproduction and Fertility, 69, 221–227.

Gu B, Ding Q, Xia G, Fang Z. 2009. EGCG inhibits growth and induces apoptosis in renal cell carcinoma through TFPI-2 overexpression. Oncology Reports, 21, 635–640.

Herman M P, Sukhova G K, Kisiel W, Foster D, Kehry M R, Libby P, Schönbeck U. 2001. Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis. The Journal of Clinical Investigation, 107, 1117–1126.

Izumi H, Takahashi C, Oh J, Noda M. 2000. Tissue factor pathway inhibitor-2 suppresses the production of active matrix metalloproteinase-2 and is down-regulated in cells harboring activated ras oncogenes. FEBS Letters, 481, 31–36.

Jamnongjit M, Gill A, Hammes S R. 2005. Epidermal growth factor receptor signaling is required for normal ovarian steroidogenesis and oocyte maturation. Proceedings of the National Academy of Sciences of the United States of America, 102, 16257–16262.

Jang Y J, Kim H K, Choi B C, Song S J, Park J I, Chun S Y, Cho M K. 2021. Expression of tissue factor and tissue factor pathway inhibitors during ovulation in rats: A relevance to the ovarian hyperstimulation syndrome. Reproductive Biology and Endocrinology, 19, 52.

Johnson A L. 2015. Ovarian follicle selection and granulosa cell differentiation. Poultry Science, 94, 781–785.

Kara E, Crépieux P, Gauthier C, Martinat N, Piketty V, Guillou F, Reiter E. 2006. A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation. Molecular Endocrinology, 20, 3014–3026.

Kenigsberg D, Littman B A, Williams R F, Hodgen G D. 1984. Medical hypophysectomy: II. Variability of ovarian response to gonadotropin therapy. Fertility and Sterility, 42, 116–126.

Kousta E, White D M, Piazzi A, Loumaye E, Franks S. 1996. Successful induction ovulation and completed pregnancy using recombinant human luteinizing hormone and follicle stimulating hormone in a woman with Kallmann’s syndrome. Human Reproduction, 11, 70–71.

Leśniak-Walentyn A, Hrabia A. 2016. Involvement of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct regression and recrudescence. Cell and Tissue Research, 366, 443–454.

Leśniak-Walentyn A, Hrabia A. 2017. Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during pause in laying induced by tamoxifen. Theriogenology, 88, 50–60.

Li C, Li Q, Li J, Zhang N, Li Y J, Li Y F, Li H, Yan F B, Kang X T, Liu X J, Li K, Tian Y D. 2021. Expression and localization of adiponectin and its receptors (AdipoR1 and AdipoR2) in the hypothalamic-pituitary-ovarian axis of laying hens. Theriogenology, 159, 35–44.

Li J, Li C, Li Q, Li G X, Li W T, Li H, Kang X T, Tian Y D. 2020. Novel regulatory factors in the hypothalamic-pituitary-ovarian axis of hens at four developmental stages. Frontiers in Genetics, 11, 591672.

Li J Q, Luo W, Huang T, Gong Y Z. 2019. Growth differentiation factor 9 promotes follicle-stimulating hormone-induced progesterone production in chicken follicular granulosa cells. General and Comparative Endocrinology, 276, 69–76.

Li Z W, Xu Y, Wang Q, Xie C L, Liu Y C, Tu Z G. 2017. Tissue factor pathway inhibitor-2 induced hepatocellular carcinoma cell differentiation. Saudi Journal of Biological Sciences, 24, 95–102.

Liu Z, de Matos D G, Fan H Y, Shimada M, Palmer S, Richards J S. 2009. Interleukin-6: An autocrine regulator of the mouse cumulus cell-oocyte complex expansion process. Endocrinology, 150, 3360–3368.

Lovell T M, Gladwell R T, Groome N P, Knight P G. 2003. Ovarian follicle development in the laying hen is accompanied by divergent changes in inhibin A, inhibin B, activin A and follistatin production in granulosa and theca layers. The Journal of Endocrinology, 177, 45–55.

Mishra S K, Chen B L, Zhu Q, Xu Z X, Ning C Y, Yin H D, Wang Y, Zhao X L, Fan X L, Yang M Y, Yang D Y, Ni Q Y, Li Y, Zhang M W, Li D Y. 2020. Transcriptome analysis reveals differentially expressed genes associated with high rates of egg production in chicken hypothalamic-pituitary-ovarian axis. Scientific Reports, 10, 5976.

Miyagi Y, Koshikawa N, Yasumitsu H, Miyagi E, Hirahara F, Aoki I, Misugi K, Umeda M, Miyazaki K. 1994. cDNA cloning and mRNA expression of a serine proteinase inhibitor secreted by cancer cells: Identification as placental protein 5 and tissue factor pathway inhibitor-2. Journal of Biochemistry, 116, 939–942.

Nivet A L, Vigneault C, Blondin P, Sirard M A. 2013. Changes in granulosa cells’ gene expression associated with increased oocyte competence in bovine. Reproduction, 145, 555–565.

Nowak M, Grzesiak M, Saito N, Kwaśniewska M, Sechman A, Hrabia A. 2017. Expression of aquaporin 4 in the chicken ovary in relation to follicle development. Reproduction in Domestic Animals, 52, 857–864.

Ocón-Grove O M, Poole D H, Johnson A L. 2012. Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles. Reproduction, 143, 825–833.

Page-McCaw A, Ewald A J, Werb Z. 2007. Matrix met alloproteinases and the regulation of tissue remodelling. Nature Reviews Molecular Cell Biology, 8, 221–233.

Puttabyatappa M, Al-Alem L F, Zakerkish F, Rosewell K L, Brännström M, Curry Jr T E. 2017. Induction of tissue factor pathway inhibitor 2 by hCG regulates periovulatory gene expression and plasmin activity. Endocrinology, 158, 109–120.

Puttabyatappa M, Brogan R S, Vandevoort C A, Chaffin C L. 2013. EGF-like ligands mediate progesterone’s anti-apoptotic action on macaque granulosa cells. Biology of Reproduction, 88, 18.

Rao C N, Cook B, Liu Y, Chilukuri K, Stack M S, Foster D C, Kisiel W, Woodley D T. 1998. HT-1080 fibrosarcoma cell matrix degradation and invasion are inhibited by the matrix-associated serine protease inhibitor TFPI-2/33 kDa MSPI. International Journal of Cancer, 76, 749–756.

Rao C N, Liu Y Y, Peavey C L, Woodley D T. 1995. Novel extracellular matrix-associated serine proteinase inhibitors from human skin fibroblasts. Archives of Biochemistry and Biophysics, 317, 311–314.

Rao C N, Mohanam S, Puppala A, Rao J S. 1999. Regulation of ProMMP-1 and ProMMP-3 activation by tissue factor pathway inhibitor-2/matrix-associated serine protease inhibitor. Biochemical and Biophysical Research Communications, 255, 94–98.

Richards J S. 1994. Hormonal control of gene expression in the ovary. Endocrine Reviews, 15, 725–751.

Robker R L, Russell D L, Espey L L, Lydon J P, O’Malley B W, Richards J S. 2000. Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases. Proceedings of the National Academy of Sciences of the United States of America, 97, 4689–4694.

Rodgers R J, Irving-Rodgers H F, Russell D L. 2003. Extracellular matrix of the developing ovarian follicle. Reproduction, 126, 415–424.

Rodgers R J, van Wezel I L, Irving-Rodgers H F, Lavranos T C, Irvine C M, Krupa M. 1999. Roles of extracellular matrix in follicular development. Journal of Reproduction and Fertility, 54, 343–352.

Saeki K, Yuo A, Takaku F. 1999. Cell-cycle-regulated phosphorylation of cAMP response element-binding protein: Identification of novel phosphorylation sites. The Biochemical Journal, 338, 49–54.

Schmidt J, Weijdegård B, Mikkelsen A L, Lindenberg S, Nilsson L, Brännström M. 2014. Differential expression of inflammation-related genes in the ovarian stroma and granulosa cells of PCOS women. Molecular Human Reproduction, 20, 49–58.

Sechman A. 2013. The role of thyroid hormones in regulation of chicken ovarian steroidogenesis. General and Comparative Endocrinology, 190, 68–75.

Shen M, Jiang Y, Guan Z Q, Cao Y, Sun SC, Liu H L. 2016. FSH protects mouse granulosa cells from oxidative damage by repressing mitophagy. Scientific Reports, 6, 38090.

Wang Y Y, Chen Q Y, Liu Z M, Guo X L, Du Y Z, Yuan Z J, Guo M, Kang L, Sun Y, Jiang Y L. 2017. Transcriptome analysis on single small yellow follicles reveals that wnt4 is involved in chicken follicle selection. Frontiers in Endocrinology, 8, 317.

Woods D, Johnson A. 2005. Regulation of follicle-stimulating hormone-receptor messenger RNA in hen granulosa cells relative to follicle selection. Biology of Reproduction, 72, 643–650.

Wolak D, Hrabia A. 2020. Tamoxifen-induced alterations in the expression of selected matrix metalloproteinases (MMP-2, -9, -10, and -13) and their tissue inhibitors (TIMP-2 and -3) in the chicken ovary. Theriogenology, 148, 208–215.

Yang Y, Zhang C, Li S, Liu J L, Qin Y Y, Ge A X. 2021. Tissue factor pathway inhibitor 2 suppresses the growth of thyroid cancer cells through by induction of apoptosis. Asia-Pacific Journal of Clinical Oncology, 17, e48–e56.

Yuan Z J, Chen Y X, Chen Q Y, Guo M, Kang L, Zhu G Y, Jiang Y L. 2016. Characterization of chicken MMP13 expression and genetic effect on egg production traits of its promoter polymorphisms. G3: Genes, Genomes, Genetics, 6, 1305–1312.

Zelinski-Wooten M B, Hutchison J S, Hess D L, Wolf D P, Stouffer R L. 1995. Follicle stimulating hormone alone supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated monkeys. Human Reproduction, 10, 1658–1666.

Zheng L L, Huang J, Su Y, Wang F, Kong H F, Xin H. 2020. Overexpression of tissue factor pathway inhibitor 2 attenuates trophoblast proliferation and invasion in preeclampsia. Human Cell, 33, 512–520.

Zhu G Y, Kang L, Wei Q Q, Cui X X, Wang S Z, Chen Y X, Jiang Y L. 2014. Expression and regulation of MMP1, MMP3, and MMP9 in the chicken ovary in response to gonadotropins, sex hormones, and TGFB1. Biology of Reproduction, 90, 57.

TFPI2在鸡卵巢中的表达及其在颗粒细胞中的调控机制

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells

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