Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (12): 2389-2396.doi: 10.3864/j.issn.0578-1752.2016.12.014

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Research on the Expression and Localization of CART in Bovine Granulosa Cells at Different Developmental Stages

LI Peng-fei1, BI Xi-lin2, WANG Kai2, JING Jiong-jie2, LÜ Li-hua2   

  1. 1College of Life Science, Shanxi Agricultural University, Taigu 030801, Shanxi
    2College of Animal Science and Technology, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2015-12-01 Online:2016-06-16 Published:2016-06-16

RichHTML

6

PDF

596

Abstract: 【Objective】The objective of this paper is to investigate the effect of CART (cocaine-and amphetamine- regulated transcript) expression in granulosa cells on bovine follicular development at different stages.【Method】 Two RNA libraries from ODF1 (the largest onset of deviation follicle) and ODF2 (the second largest onset of deviation follicle) granulosa cells were prepared and sequenced using Illumina HiSeq 2000 platform. Cows were slaughtered, and both ovaries were collected, then the largest and the second largest follicles were screened through the morphology of corpus luteum in the first follicular wave. Follicular fluid was extracted for the concentration of E2 and P measurement using competitive ELISA method to determine DF (dominant follicles) and SF (subordinate follicles). Granulesa cells were isolated from DF and SF, and total RNA were extracted, respectively. Using RPLP0 as a reference gene, primers were designed. qRT-PCR was performed to detect the expression of CART in DF and SF, GraphPad Prism 5.0 was used for construction and significant analysis. Rabbit anti-CART antibody was used to detect the CART expression in cow follicles.【Result】Deep sequencing results showed that CART amounts in ODF1 were 38.2 times more than that in ODF2. Ovaries of 3 cows were screened out which were in the first follicular wave according to the observation of the morphology of corpus luteum, and the DF and SF were obtained. Real-time PCR results showed that CART expressed in DF was extremely significantly higher than that in SF, up to 2310-fold, which is consistent with the deep sequencing results. Immunohistochemistry results showed that CART expressed both in DF and SF, and the amounts in DF was significantly greater than that in SF, at the same time, CART was mainly expressed in granulosa cell layer.【Conclusion】During the process of follicular development in cattle, other inhibitors have suppressed most of the follicles, and prevented them to develop into DF; when the deviation appears, CART inhibited DF to further develop into ovulatory follicle, so it is considered that CART possibly by promoting granulosa cells apoptosis, and suppresses the E2 secretion in granulosa cell layer, eventually leads to DF atresia.

Key words: bovine, follicle, development, CART, expression

[1]    SEN A, LÜ L, BELLO N, IRELAND J J, SMITH G W. Cocaine-and amphetamine-regulated transcript (CART) accelerates the temination of FSH induced ERK1/2 and AKT activation by regulating the expression and degradation of specific map kinase phosphatases. Molecular Endocrinology, 2008, 22(12): 2655-2676.
[2]    LÜ L, JIMENEZ-KRASSEL F, SEN A, BETTEGOWDA A, MONDAL M, FOLGER J K, LEE K B, IRELAND J J, SMITH G W. Evidence supporting a role for cocaine and amphetamine regulated transcript (CART) in control of granulosa cell estradiol production associated with dominant follicle selection in cattle. Biology of Reproduction, 2009, 81(3):580-586.
[3]    李鹏飞, 岳文斌, 黄洋, 孙晋艳, 李晓明, 庞钰莹, 于学静, 贺俊平, 孟金柱, 任有蛇, 吕丽华. 可卡因-苯丙胺调控转录肽对绵羊卵巢卵泡颗粒细胞雌激素产生的影响. 畜牧兽医学报, 2013, 44(6): 853-857.
LI P F, YUE W B, HUANG Y, SUN J Y, LI X M, PANG Y Y, YU X J, HE J P, MENG J Z, REN Y S, LÜ L H. Effects of CART on estradiol production of sheep ovarian follicular granulosa cells. Acta Veterinaria et Zootechnica Sinica, 2013, 44(6):853-857. （in Chinese）
[4]    李鹏飞, 岳文斌, 李富禄, 黄洋, 孙晋艳, 朱芷薇, 于秀菊, 贺俊平, 范瑞文, 任有蛇, 吕丽华.可卡因-苯丙胺调控转录肽（CART）对猪卵巢卵泡颗粒细胞雌激素产生的影响. 畜牧兽医学报, 2012, 43(12):1879-1886.
LI P F, YUE W B, LI F L, HUANG Y, SUN J Y, ZHU Z W, YU X J, HE J P, FAN R W, REN Y S, LV L H. Effects of CART on estradiol production of pig ovarian follicular granulosa cells in vitro culture. Acta Veterinaria et Zootechnica Sinica, 2012, 43(12): 1879-1886. （in Chinese）
[5]    ROCHE J F, AUSTIN E J, RYAN M, OROURKE M, MIHM M, DISKIN M G. Regulation of follicle waves to maximize fertility in cattle. Journal of Reproduction and Fertility, 1999, 54(Suppl):61-71.
[6]    GINTHER O J, WILTBANK M C, FRICKE P M, GIBBONS J R, KOT K. Selection of the dominant follicle in cattle. Biology of Reproduction, 1996, 55(6):1187-1194.
[7]    WEBB R, ARMSTRONG D G. Control of ovarian function; effect of local interactions and environmental influences on follicular turnover in cattle: a review. Livestock Production Science, 1998, 53(2):95-112.
[8]    卫晓红, 祁丽花, 迟晓春, 董静霞, 杨京京, 徐健. Smad2/Smad3蛋白在大鼠卵巢颗粒细胞中的表达及FSH对其活化的影响. 解剖学报, 2007, 38 (2): 205-208.
WEI X H, QI L H, CHI X C, DONG J X, YANG J J, XU J. The effects of FSH on the phosphorylation of smad2/smad3 protein in rat ovarian granulosa cells. Acta Anatomica Sinica, 2007, 38 (2): 205-208. （in Chinese）
[9]    PEDERSON H G, WATSON E D, TELFER E E. Analysis of atresia in equine follicles using histology, fresh granulosa cell morphology and detection of DNA fragmentation. Reproduction, 2003, 125(3): 417-423.
[10]   ZEUNER A, MÜLLER K, REGUSZYNSKI K, JEWGENOW K. Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation. Theriogenology, 2003, 59(5-6): 1421-1433.
[11]   WONGSRIKEAO P, KANESHIGE Y, OOKI R, TANIGUCHI M, AGUNG B, NII M, OTOI T. Effect of the removal of cumulus cells on the nuclear maturation,fertilization and development of porcine oocytes. Reproduction in Domestic Animals, 2005, 40(2): 166-170.
[12]   BABITHA V, PANDA R P, YADAV V P, CHOUHAN V S, DANGI S S, KHAN F A, SINGH G, BAG S, TARU SHARMA G, SILVIA W J, SARKAR M. Amount of mRNA and localization of vascular endothelial growth factor and its receptors in the ovarian follicle during estrous cycle of water buffalo(Bubalus bubalis). Animal Reproduction Science, 2013, 137(3-4):163-176.
[13]   GUPTA S, CHOI A, YU H Y, CZERNIAK S M, HOLICK E A, PAOLELLA L J, AGARWAL A, COMBELLES C M. Fluctuations in total antioxidant capacity, catalase activity and hydrogen peroxide levels of follicular fluid during bovine folliculogenesis. Reproduction Fertility and Development, 2011, 23(5):673-680.
[14]   KAFI M, MESBAH S F, DAVOODIAN N, KADIVAR A. Fine structures of the oocyte in relation to serum, follicular fluid steroid hormones and IGF-I in the ovulatory-sized follicles in one-humped camel (Camelus dromedarius). Avicenna Journal of Medical Biotechnology, 2014, 6(1):57-61.
[15]   AAD P Y, ECHTERNKAMP S E, SPICER L J. Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births. Domestic Animal Endocrinology, 2013, 45(4):187-195.
[16]   DIAS F C, KHAN M I, ADAMS G P, SIRARD M A, SINGH J. Granulosa cell function and oocyte competence: Super-follicles, super-moms and super-stimulation in cattle. Animal Reproduction Science, 2014, 149(1-2):80-89.
[17]   李鹏飞, 孟金柱, 谢建山, 朱芷葳, 刘岩, 姜晓龙, 陈建伟, 姚晓 磊, 赵妙妙, 吕丽华. 牛卵泡ODF1与ODF2转录组发育相关基因筛选及表达差异分析. 畜牧兽医学报，2015, 46(11):1961-1966.
LI P F, MENG J Z, XIE J S, ZHU Z W, LIU Y, JIANG X L, CHEN J W, YAO X L, ZHAO M M, LÜ L H. Screening and analyse study of genes associated with follicular development in bovine ODF1 and ODF2 transcript. Acta Veterinaria et Zootechnica Sinica, 2015, 46(11):1961-1966. （in Chinese）
[18]   IRELAND J J, MURPHEE R L, COULSON P B. Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. Journal of Dairy Science, 1980, 63(1):155-160.
[19]   AUDIC S, CLAVERIE J M. The significance of digital gene expression profiles. Genome Research, 1997, 7(10):986-995.
[20]   XU Z, GARVERICK H A, SMITH G W, SMITH M F, HAMILTON S A, YOUNGQUIST R S. Expression of messenger ribonucleic acid encoding cytochrome P450 side-chain cleavage, cytochrome p450 17 alpha-hydroxylase, and cytochrome P450 aromatase in bovine follicles during the first follicular wave. Endocrinology, 1995, 136(3): 981-989.
[21]   KOBAYASHI Y, JIMENEZ-KRASSEL F, IRELAND J J, SMITH G W. Evidence of a local negative role for cocaine and amphetamine regulated transcript (CART), inhibins and low molecular weight insulin like growth factor binding proteins in regulation of granulose cell estradiol production during follicular waves in cattle. Reproductive Biology and Endocrinology, 2006, 4(1):22-33.
[22]   IRELAND J J, ROCHE J F. Development of nonovulatory antral follicles in heifers: changes in steroids in follicular fluid and receptors for gonadotropins. Endocrinology, 1983, 112(1):150-156.
[23]   IRELAND J J, ROCHE J F. Development of antral follicles in cattle after prostaglandin-induced luteolysis: changes in serum hormones, steroids in follicular fluid, and gonadotropin receptors. Endocrinology, 1982, 111(6):2077-2086.
[24]   SUNDERLAND S J, CROWE M A, BOLAND M P, ROCHE J F, IRELAND J J. Selection, dominance and atresia of follicles during the oestrous cycle of heifers. Journal of Reproduction and Fertility, 1994, 101(3):547-555.
[25]   GINTHER O J, BERGFELT D R, KULICK L J, KOT K. Selection of the dominant follicle in cattle: establishment of follicle deviation in less than 8 hours through depression of FSH concentrations. Theriogenology,1999, 52(6):1079-1093.
[26]   GOODMAN A L, NIXON W E, JOHNSON D K, HODGEN G D. Regulation of folliculogenesis in the cycling rhesus monkey: selection of the dominant follicle. Endocrinology, 1977, 100(1):155-161.
[27]   IRELAND J J, ROCHE J F. Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers: changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles. Journal of Animal Science, 1983, 57(1): 157-167.
[28]   ILHA G F, ROVANI M T, GASPERIN B G, ANTONIAZZI A Q, GONÇALVES P B, BORDIGNON V, DUGGAVATHI R. Lack of FSH support enhances LIF–STAT3 signaling in granulosa cells of atretic follicles in cattle. Reproduction, 2015, 150(4):395-403.
[29]   GARCÍA GUERRA A, TRIBULO A, YAPURA J, ADAMS G P, SINGH J, MAPLETOFT R J. Lengthened superstimulatory treatment in cattle: Evidence for rescue of follicles within a wave rather than continuous recruitment of new follicles. Theriogenology, 2015, 84(3):467-476.
[30]   ATANASOV B, DE KOSTER J, BOMMELÉ L, DOVENSKI T, OPSOMER G. Pathways of the dominant follicle after exposure to sub-luteal circulating progesterone concentrations are different in lactating dairy cows versus non-lactating heifers. Animal Reproduction Science, 2015, 154(3):8-15.
[31]   SILVA FILHO M L, BEZERRA L R, FERREIRA-SILVA J C, SOUTO F M, OLIVEIRA N R, DE LIMA P F, BARTHOLOMEW C C, DE OLIVEIRA M A. Influence of biostimulation and temporary weaning on follicular dynamics and pregnancy rates in Nelore cows (Bos taurus indicus). Tropical Animal Health and Production, 2015, 47(7): 1285-1291.
[32]   KOBAYASHI Y, JIMENEZ-KRASSEL F, LI Q, YAO J, HUANG R, IRELAND J J, COUSSENS P M, SMITH G W. Evidence that cocaine-and amphetamine-regulated transcript is a novel intraovarian regulator of follicular atresia. Endocrinology, 2004, 145(11): 5373-5383.
[33]   SEN A, BETTEGOWDA A, JIMENEZ-KRASSEL F, IRELAND J J, SMITH G W. Cocaine-and amphetamine-regulated transcript (CART) regulation of follicle stimulating hormone signal transduction in bovine granulosa cells. Endocrinology, 2007, 148(9):4400-4410.
[1] WANG JiaNuo, CHEN GuiPing, LI Pan, WANG LiPing, NAN YunYou, HE Wei, FAN ZhiLong, HU FaLong, CHAI Qiang, YIN Wen, ZHAO LiaoHao. Photo-Physiological Mechanism at Grain Filling Stage of No-Tillage with Plastic Re-Mulching to Increase Maize Yield in Oasis Irrigation Areas [J]. Scientia Agricultura Sinica, 2026, 59(6): 1189-1202.
[2] GERIQIMUGE, PUBUZHANDUI, XU Qing, HOU LingLing. Effects of Hypoxia on Proliferation of Bovine Renal Cells and Mitochondrial Autophagy [J]. Scientia Agricultura Sinica, 2026, 59(6): 1333-1347.
[3] CUI ShiYou, CHEN PengJun, MIAO YuanQing, HAN JiJun, SHEN JunMing. Development and Field Evaluation of Glyphosate-Resistant Wheat Germplasm Generated Through EMS Mutagenesis [J]. Scientia Agricultura Sinica, 2026, 59(4): 723-733.
[4] LUO ZhengYing, HU SiZhen, LIN XiuQin, HU Xin, ZHANG Min, XU ChaoHua, LIU XinLong, ZENG QianChun. Identification and Functional Characterization of the PEBP Gene Family in Regulating Flowering Time in Saccharum spontaneum and Saccharum officinarum [J]. Scientia Agricultura Sinica, 2026, 59(4): 734-749.
[5] LIAO TingLu, SHI YaFei, XIAO DongHao, SHE YangMengFei, GUO FuCheng, YANG JiuJu, TANG HaiJiang, LUO ChengKe. The Effect of Exogenous Nitroprusside on Sugar Metabolism in Rice Seedlings Under Alkaline Stress [J]. Scientia Agricultura Sinica, 2026, 59(2): 265-277.
[6] WEI Ping, PAN JuZhong, ZHU DePing, SHAO ShengXue, CHEN ShanShan, WEI YaQian, GAO WeiWei. The Function of OsDREB1J in Regulating Rice Grain Size [J]. Scientia Agricultura Sinica, 2025, 58(8): 1463-1478.
[7] YANG CaiLi, LI YongZhou, HE LiangLiang, SONG YinHua, ZHANG Peng, LIU ZhaoXian, LI PengHui, LIU SanJun. Genome-Wide Identification and Analysis of TPS Gene Family and Functional Verification of VvTPS4 in the Formation of Monoterpenes in Grape [J]. Scientia Agricultura Sinica, 2025, 58(7): 1397-1417.
[8] TENG MengXin, XU Ya, HE Jing, WANG Qi, QIAO Fei, LI JingYang, LI XinGuo. Identification and Functional Analysis of Ca2+-ATPase Gene Family in Banana [J]. Scientia Agricultura Sinica, 2025, 58(7): 1418-1433.
[9] XIONG JiaNi, LI ZongYue, HU HengLiang, GU TianYu, GAO Yan, PENG JiaShi. Influence of Expressing OsNRAMP5 Under the Driving of the OsLCT1 Promoter on Cadmium Migration to Rice Seeds [J]. Scientia Agricultura Sinica, 2025, 58(7): 1259-1268.
[10] PAN LiYuan, WANG YongJun, LI HaiJun, HOU Fu, LI Jing, LI LiLi, SUN SuYang. Screening Regulatory Genes Related to Wheat Grain Protein Accumulation Based on Transcriptome and WGCNA Analysis [J]. Scientia Agricultura Sinica, 2025, 58(6): 1065-1082.
[11] LIU LuPing, HU XueJie, QI Jin, CHEN Qiang, LIU Zhi, ZHAO TianTian, SHI XiaoLei, LIU BingQiang, MENG QingMin, ZHANG MengChen, HAN TianFu, YANG ChunYan. Cloning of the Promoters and Analysis of Expression Patterns of Maturity Genes E1 and E2 in Soybean [J]. Scientia Agricultura Sinica, 2025, 58(5): 840-850.
[12] ZHANG TianYu, LI Bai, ZANG JinPing, CAO HongZhe, DONG JinGao, XING JiHong, ZHANG Kang. Genome-Wide Identification and Expression Analysis of HMG Family Genes in Botrytis cinerea [J]. Scientia Agricultura Sinica, 2025, 58(4): 704-718.
[13] GUO AoLin, LIN JunXuan, LAI GongTi, HE LiYuan, CHE JianMei, PAN Ruo, YANG FangXue, HUANG YuJi, CHEN GuiXin, LAI ChengChun. Effect of VdF3′5′H2 Overexpression on the Accumulation of Anthocyanin Composition in Spine Grape Cells [J]. Scientia Agricultura Sinica, 2025, 58(4): 802-818.
[14] ZHANG LinLin, GONG Rui, CUI YanLing, ZHONG XiongHui, LI Ye, LI RanHong, QIAN ZongWei. Effect Analysis of SmWRKY30 in Eggplant Resistance to Ralstonia solanacearum by Virus Induced Gene Silencing (VIGS) [J]. Scientia Agricultura Sinica, 2025, 58(3): 548-563.
[15] ZHANG XiangKun, LI JiaYing, QIAO RuMeng, HE JingLei, WANG Li, SHI XiaoXin, DU GuoQiang. Effects of GFabV Under Different Zn Levels on Photosynthetic Efficiency and Photosynthesis-Related Gene Expression of ‘Shine Muscat’ Grapevine [J]. Scientia Agricultura Sinica, 2025, 58(24): 5190-5200.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd