KDM1A在牦牛卵泡发育过程中的表达

doi:10.3864/j.issn.0578-1752.2019.24.016

摘要/Abstract

摘要：

【目的】分析牦牛卵泡发育过程中组蛋白赖氨酸脱甲基酶1（lysine-specific histone demethylase 1A,KDM1A）基因的表达谱,探讨KDM1A对牦牛卵泡发育和卵母细胞成熟的影响。【方法】以牦牛卵泡为研究对象,根据卵泡直径的大小,将卵泡分为3组：大（6.0—9.0 mm）、中（3.0—5.9 mm）、小（1.0—2.9 mm）卵泡,收集各组别卵泡中卵丘-卵母细胞复合体（cumulus-oocyte complex, COCs）进行体外培养,对不同发育阶段卵泡中卵母细胞的体外成熟率进行统计并分析;分别提取各组卵泡卵母细胞及壁层颗粒细胞总RNA,通过实时荧光定量PCR（quantitative real-time RCR, RT-qPCR）方法,检测KDM1A基因在不同发育阶段的卵泡中mRNA相对表达量的变化;采用免疫组织化学技术检测KDM1A在牦牛卵泡发育过程中的细胞定位及表达规律,并结合体外成熟与RT-qPCR结果进行关联性分析。【结果】各组卵母细胞体外成熟率与卵泡直径的大小呈显著正相关关系,其伴随卵泡发育的进行呈明显的上升趋势,其中大、中、小卵泡组的体外成熟率依次分别为90.53 %、88.10 %、55.14 %。RT-qPCR结果显示,KDM1A基因在牦牛卵泡发育过程中均有表达,且不同发育阶段的mRNA表达水平差异显著,其中大、中卵泡时期的卵母细胞中的mRNA相对表达量显著低于小卵泡时期(P ＜ 0.05),而在小卵泡时期的壁层颗粒细胞中的相对表达量极显著低于大、中卵泡时期 (P ＜ 0.01),在大、中卵泡时期的卵母细胞及壁层颗粒细胞中该基因mRNA表达水平均无差异(P ＞ 0.05)。免疫组织化学研究发现KDM1A在大、中、小卵泡壁层颗粒细胞以及膜细胞中均表达,其蛋白表达趋势与RT-qPCR结果吻合,即随着发育时间的进行KDM1A表达水平呈上升趋势,其中在大卵泡时期KDM1A的表达量最高。【结论】在牦牛不同发育时期卵泡的卵母细胞及壁层颗粒细胞中KDM1A mRNA及蛋白的表达水平呈动态变化,提示KDM1A在卵泡发育及卵母细胞成熟过程中发挥重要作用。这可能与卵母细胞的减数分裂及颗粒细胞的增殖分化有关,本研究为进一步研究该基因在牦牛卵母细胞减数分裂过程中的作用机制提供基础数据。

关键词: 牦牛, KDM1A, 免疫组化, 卵泡, 表达

Abstract:

【Objective】 The aim of this study was to analyze the role of the lysine-specific histone demethylase 1A (KDM1A) in follicle development and oocyte maturation of yak. 【Method】 Taking yak follicles as research objects and according to the size of the follicles, they were divided into three groups: large-sized (6.0-9.0 mm), medium-sized (3.0-5.9 mm), and small-sized (1.0-2.9 mm) follicles. And the cumulus-oocyte complexes (COCs) were collected from each group and cultured in vitro. The maturation rate of oocytes was counted and analyzed. The total RNA was extracted from oocyte and granulosa cells in each group of follicles. The real time quantitative PCR (RT-qPCR) was used to detect the relative expression of KDM1A during follicular development. The cell localization and expression of KDM1A in yak follicle were detected by immunohistochemistry, and the correlation analysis was performed by in vitro maturation and RT-qPCR. 【Result】 The maturation rate of oocytes in vitro maturation was positively related to the size of follicles and showed a rising trend with progressing of follicular development. Meanwhile, the oocyte maturation rates of large-sized, medium-sized and small-sized follicle oocytes were 90.53 %, 88.10 % and 55.14 %, respectively. The result of RT-qPCR was found that KDM1A gene of yak was widely expressed during the development of follicles, and its expression level was significantly different in developmental stages of follicles. The relative expression of mRNA in the oocytes of the large and medium-sized follicle was significantly lower than small-sized follicles (P ＜ 0.05), but the relative expression of granulosa cells in the small-sized follicle was significantly lower than large and medium-sized (P ＜ 0.01), and there was no difference in mRNA expression levels between oocytes and granulosa cells in the large and middle-sized (P ＞ 0.05). The results of immunohistochemistry showed that KDM1A was expressed in granulosa cells and membrane cells of follicles, and its expression trend was consistent with RT-qPCR. The expression of KDM1A was the highest in the large follicle and increased with the development of follicles. 【Conclusion】 The expression levels of KDM1A mRNA and protein in oocytes and granulosa cells at the development of yak follicles were dynamic, which indicated that KDM1A played an important role in follicular development and oocyte maturation. It might be related to meiosis of oocytes and proliferation and differentiation of granulosa cells, and these results of study would provide a basic data for further research of the mechanism of KDM1A in the meiosis of yak oocytes.

Key words: Yak, KDM1A, immunohistochemistry, follicle, expression

黄向月,熊显荣,韩杰,杨显英,王艳,王斌,李键. KDM1A在牦牛卵泡发育过程中的表达[J]. 中国农业科学, 2019, 52(24): 4624-4631.

XiangYue HUANG,XianRong XIONG,Jie HAN,XianYing YANG,Yan WANG,Bin WANG,Jian LI. Expression Pattern of KDM1A in the Development of Yak Follicles[J]. Scientia Agricultura Sinica, 2019, 52(24): 4624-4631.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.24.016

https://www.chinaagrisci.com/CN/Y2019/V52/I24/4624

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参考文献 32

[1]	王汝, 余四九, 崔燕 . 幼龄牦牛甲状腺的显微结构和超微结构观察. 中国兽医科学, 2009,39(4):357-361.
	WANG R, YU S J, CUI Y . Observation of microstructure and ultrastructure of the thyroid gland in juvenile yak. Veterinary Science in China, 2009,39(4):357-361. (in Chinese)
[2]	兰道亮, 熊显荣, 位艳丽, 徐通, 钟金城, 字向东, 王永, 李键 . 基于RNA-Seq高通量测序技术的牦牛卵巢转录组研究: 进一步完善牦牛基因结构及挖掘与繁殖相关新基因. 中国科学, 2014,44(3):307-317.
	LAN D L, XIONG X R, WEI Y L, XU T, ZHONG J C, ZI X D, WANG Y, LI J . RNA-Seq analysis of yak ovary: improving yak gene structure information and mining reproduction-related genes. Scientia Sinica, 2014,44(3):307-317. (in Chinese)
[3]	SINHA P B, TESFAYE D, RINGS F, HOSSIEN M, HOELKER M, HELD E, NEUHOFF C, THOLEN E, SCHELLANDER K, SALILEW- WONDIM D . MicroRNA-130b is involved in bovine granulosa and cumulus cells function, oocyte maturation and blastocyst formation. Journal of Ovarian Research, 2017,10(1):37.
[4]	HUNT P A, HASSOLD T J . Human female meiosis: what makes a good egg go bad ? Trends in Genetics, 2008,24(2):86-93.
[5]	PETRO E M, LEROY J L, COVACI A, FRANSEN E, DE NEUBOURG D, DIRTU A C, DE PAUW I, BOLS P E . Endocrine- disrupting chemicals in human follicular fluid impair in vitro oocyte developmental competence. Human Reproduction, 2012,27(4):1025-1033.
[6]	HAYASHI K, YOSHIDA K, MATSUI Y . A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature, 2005,438(7066):374-378.
[7]	TACHIBANA M, NOZAKI M, TAKEDA N, SHINKAI Y . Functional dynamics of H3K9 methylation during meiotic prophase progression. Embo Journal, 2014,26(14):3346-3359.
[8]	SHI Y, LAN F, MATSON C, MULLIGAN P, WHETSTINE J R, COLE P A, CASERO R A, SHI Y . Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell, 2004,119(7):941-953.
[9]	FEI L, NOTTKE A C, YANG S . Mechanisms involved in the regulation of histone lysine demethylases. Current Opinion in Cell Biology, 2008,20(3):316-325.
[10]	邵根宝, 黄晓佳, 龚爱华, 张志坚, 陆荣柱, 桑建荣 . 组蛋白去甲基化酶LSD1及其生物学功能. 遗传, 2010,04:331-338.
	SHAO G B, HUANG X J, GONG A H, ZHANG Z J, LU R Z, SANG J R . Histone to methylase LSD1 and its biological functions. Hereditas, 2010,04:331-338. (in Chinese)
[11]	GARCIA-BASSETS I, KWON Y S, TELESE F, PREFONTAINE G G, HUTT K R, CHENG C S, JU B G, OHGI K A, WANG J, ESCOUBET-LOZACH L, ROSE D W, GLASS C K, FU X D, ROSENFELD M G . Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell, 2007,128(3):505-518.
[12]	METZGER E, WISSMANN M, YIN N, MÜLLER J M, SCHNEIDER R, PETERS A H, GÜNTHER T, BUETTNER R, SCHÜLE R . LSD1 demethylates repressive histone marks to promote androgen-receptor- dependent transcription. Nature, 2005,437(7057):436-439.
[13]	JEESUN K, KUMAR S A, YOKO T, LIN K, SHEN J, LU Y, KERENYI M A, ORKIN S H, CHEN T . LSD1 is essential for oocyte meiotic progression by regulating CDC25B expression in mice. Nature, 2015,6:10116.
[14]	SHAO G, WANG J, LI Y . Lysine-specific demethylase1 mediates epidermal growth factor signaling to promote cell migration in ovarian cancer cells. Scientific Reports, 2015,5:15344.
[15]	ZHENG Y C, MA J, WANG Z, LI J, JIANG B, ZHOU W, SHI X, WANG X, ZHAO W, LIU H M . A systematic review of histone lysine-specific demethylase 1 and its inhibitors. Medicinal Research Reviews, 2015,35(5):1032-1071.
[16]	LYNCH J T, HARRIS W J, SOMERVAILLE T C . LSD1 inhibition: a therapeutic strategy in cancer? Expert Opinion on Therapeutic Targets, 2012,16(12):1239-1249.
[17]	OMBRA M N, DI S A, ABBONDANZA C, MIGLIACCIO A, AVVEDIMENTO E V, PERILLO B . Retinoic acid impairs estrogen signaling in breast cancer cells by interfering with activation of LSD1 via PKA. Biochimica et Biophysica Acta, 2013,1829(5):480-486.
[18]	CICCONE D N, SU H, HEVI S, GAY F, LEI H, BAJKO J, XU G, LI E, CHEN T . KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature, 2009,461(7262):415-418.
[19]	JOHNSON M T, FREEMAN E A, GARDNER D K, HUNT PA . Oxidative metabolism of pyruvate is required for meiotic maturation of murine oocytes in vivo. Biology of Reproduction, 2007,77(1):2-8.
[20]	SANCHEZLAZO L, BRISARD D, ELIS S, MAILLARD V, UZBEKOV R, LABAS V, DESMARCHAIS A, PAPILLIER P, MONGET P, UZBEKOVA S . Fatty acid synthesis and oxidation in cumulus cells support oocyte maturation in bovine. Molecular Endocrinology, 2014,28(9):1502-1521.
[21]	USONGO M, RIZK A, FAROOKHI R . β-Catenin/Tcf signaling in murine oocytes identifies nonovulatory follicles. Reproduction, 2012,144(6):669-676.
[22]	HUANG Z, LI S, WEI S, LI X, LI Q, ZHANG Z, HAN Y, ZHANG X, MIAO S, DU R, WANG L . Lysine-specific demethylase 1 (LSD1/ KDM1A) contributes to colorectal tumorigenesis via activation of the Wnt/β-catenin pathway by down-regulating Dickkopf-1 (DKK1). PLoS One, 2013,8(7):70077.
[23]	ANCELIN K, SYX L, BORENSZTEIN M, RANISAVLJEVIC N, VASSILEV I, BRISEÑO-ROA L, LIU T, METZGER E, SERVANT N, BARILLOT E1, CHEN C J, SCHÜLE R, HEARD E . Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. Elife, 2016,2(5):e08851.
[24]	WASSON J A, SIMON A K, MYRICK D A, WOLF G, DRISCOLL S, PFAFF SL, MACFARLAN T S, KATZ D J . Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally. Elife, 2016,27(5):e08848.
[25]	YOKOYAMA A, IGARASHI K, SATO T, TAKAGI K, OTSUKA I M, SHISHIDO Y, BABA T, ITO R, KANNO J, OHKAWA Y, MOROHASHI K, SUGAWARA A . Identification of myelin transcription factor 1 (MyT1) as a subunit of the neural cell type-specific lysine-specific demethylase 1 (LSD1) complex. Journal of Biological Chemistry, 2014,289(26):18152-18162.
[26]	SU O J, JIN H S, MARCO C . Wee1B, Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption. Journal of Cell Biology, 2010,188(2):199-207.
[27]	ABDI S, SALEHNIA M, HOSSEINKHANI . Quality of oocytes derived from vitrified ovarian follicles cultured in two- and three-dimensional culture system in the presence and absence of kit ligand. Biopreservation and Biobanking, 2016,14(4):279-288.
[28]	WIGGLESWORTH K, LEE K B, O'BRIEN M J, PENG J, MATZUK M M, EPPIG J J . Bidirectional communication between oocytes and ovarian follicular somatic cells is required for meiotic arrest of mammalian oocytes. Proceedings of the National Academy of Sciences of the United States of America, 2013,110(39):3723-3729.
[29]	ALM H, KATSKAKSIAZKIEWICZ L, RYŃSKA B, TUCHSCHERER A . Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles. Theriogenology, 2006,65(7):1422-1434.
[30]	LEQUARRE A S, VIGNERON C, RIBAUCOUR F, HOLM P, DONNAY I DALBIÈS-TRAN R CALLESEN H MERMILLOD P . Influence of antral follicle size on oocyte characteristics and embryo development in the bovine. Theriogenology, 2005,63(3):841-859.
[31]	禹学礼, 昝林森, 邓雯, 庞有志, 王新庄 . 卵泡大小及卵泡液对牛卵母细胞体外受精后发育的影响. 中国农业科学, 2005,38(8):1664-1668.
	YU X L, ZAN L S, DENG W, PANG Y Z, WANG X Z . Effects of follicle size and bovine follicular fluid on developmental competence of bovine oocytes following maturation, fertilization and culture in vitro. Scientia Agricultural Sinica, 2005,38(8):1664-1668. (in Chinese)
[32]	KIM J H, PARK U H, MOON M, UM S J, KIM E J . Negative regulation of ERa by a novel protein CAC1 through association with histone demethylase LSD1. Febs Letters, 2013,587(1):17-22.

基因 Gene	引物序列(5'-3') Sequence of primer	退火温度 Tm(℃)	产物长度 Product size (bp)
GAPDH	F: TGCTGGTGCTGAGTATGTGGTG	60	293
GAPDH	R: TCTTCTGGGTGGCAGTGATGG	60	293
KDM1A	F: CGCAAAGGAAACTATGTAG	60	245
KDM1A	R: ATTATTGAGGACGTTGAAGTC	60	245

卵泡类型 Types of follicles/cm	重复数 NO. of replicates	卵母细胞数 NO. of COCs	MII期卵母细胞数 NO. of COCs in MII	成熟率 Maturation rate (%)
小 Small（1.0-2.9 mm）	3	380	209	55.14%±1.52
中 Medium（3.0-5.9 mm）	3	285	251	88.10%±0.94**
大 Large（6.0-9.0 mm）	3	169	153	90.53%±1.55**