CART在不同发育阶段牛卵泡颗粒细胞中的表达和定位

doi:10.3864/j.issn.0578-1752.2016.12.014

摘要/Abstract

摘要： 【目的】探讨牛卵泡颗粒细胞中可卡因-苯丙胺调节转录肽（cocaine-and amphetamine-regulated transcript, CART）的表达对牛不同阶段卵泡发育的影响。【方法】通过对牛卵泡转录组ODF1(the largest onset of deviation follicle)和ODF2（the second largest onset of deviation follicle）颗粒细胞（granulesa cells, GCs）构建RNA文库，Illumina HiSeq 2000平台测序；采集屠宰牛双侧卵巢，通过黄体形态特征筛选处于第一卵泡波阶段的最大卵泡和第二大卵泡；分别抽取卵泡液，竞争性ELISA法测定卵泡液雌激素（estrodiol, E₂）和孕酮（progestin, P）浓度，确定优势卵泡（dominant follicles，DF）和从属卵泡（subordinate follicles，SF）；分别分离DF和SF颗粒细胞（granulesa cells, GCs），提取总RNA；设定3个重复样本，以RPLP0作为内参基因，设计引物在牛DF和SF进行qRT-PCR表达量检测，GraphPad Prism 5.0作图并进行显著性分析；兔抗CART一抗检测CART在牛卵泡的表达。【结果】转录组测序结果显示，CART在ODF1的表达量是ODF2的38.2倍；通过黄体形态特征及激素测定共筛选出3头牛的卵泡发育处于第一卵泡波，并获得DF和SF；qRT-PCR结果显示CART在DF与SF的表达量差异极显著（P＜0.01），高达2 310倍，与转录组测序结果的表达差异趋势一致；免疫组化结果表明，CART在牛DF和SF中均有表达，且DF表达量明显高于SF，同时，CART主要在卵泡颗粒细胞层表达。【结论】在牛卵泡发育过程中，卵泡发育选择期有其它抑制因子抑制了大多数卵泡的发育，阻止其成为DF；当出现偏差后，CART的高表达抑制了DF进入排卵期，推测CART可能是通过促进卵泡颗粒细胞的凋亡，进一步抑制了颗粒细胞层E₂的分泌，最终导致DF闭锁。

关键词: 牛, 卵泡, 发育, CART, 表达

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

李鹏飞，毕锡麟，王锴，景炅婕，吕丽华. CART在不同发育阶段牛卵泡颗粒细胞中的表达和定位[J]. 中国农业科学, 2016, 49(12): 2389-2396.

LI Peng-fei, BI Xi-lin, WANG Kai, JING Jiong-jie, Lü Li-hua. Research on the Expression and Localization of CART in Bovine Granulosa Cells at Different Developmental Stages[J]. Scientia Agricultura Sinica, 2016, 49(12): 2389-2396.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.12.014

https://www.chinaagrisci.com/CN/Y2016/V49/I12/2389

参考文献

[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.