摘要
比格犬因其性情温顺，体格适中，遗传背景清晰，均质性好，在医药、公共卫生、生命科学和军事医学研究领域，发挥着重要的作用。但目前，我国比格犬种群内的不发情或发情不孕、休情期延长、停发情、窝产仔数少等情况比较常见，严重制约着比格犬的生产效率，因此，针对比格犬生殖调控机理的研究具有重要的理论与实际意义。本研究对不同发情阶段比格犬卵巢与子宫内雌激素受体ERα、ERβ蛋白的表达规律进行了研究，并对犬ERα、ERβ及可能存在的ERβ剪接异构体进行基因扩增，构建真核表达重组质粒，并瞬时转染293T细胞，对其活性进行初步研究。突出的结果是首次发现了比格犬ERβ的四个剪接异构体，为深入进行比格犬ERα、ERβ及其剪接异构体在生殖调控中的作用机制研究奠定了基础。现将实验研究报告如下。
1.间情期与发情期比格犬血清性激素检测及卵巢与子宫正常组织形态的比较
应用病理切片技术对间情期及发情期比格犬卵巢及子宫的正常组织形态进行比较，并对两个发情阶段比格犬血清性激素：E2、PRGE、FSH和LH水平进行测定，分析其性激素变化特点和卵巢、子宫在不同阶段的组织形态学特征。
性激素检测结果表明：在发情期比格犬出现了很明显的E2峰值，而在间情期及发情后期雌激素均维持在极低的水平。而PRGE在间情期未能检测到，在发情期及发情后期均能检出，并且在发情后期表达水平明显升高。
组织形态学比较结果表明：间情期犬子宫及其内膜较薄，间质纤维增生，黄体中以初级卵泡为主，可见1-2个次级卵泡，未见成熟卵泡，卵泡和黄体细胞间纤维较多、血管少。发情期犬子宫及其内膜增厚，内膜腺体腔较大，部分腺体呈分枝状弯曲，腺上皮肿大，胞浆淡染，少数可见核下空泡。卵巢中卵泡数量较多，有初级、次级和1-2个成熟卵泡。黄体细胞数量多，排列规则，境界清楚，间质纤维比较疏松，血管多，未见空泡变性。
2.间情期与发情期比格犬卵巢与子宫内ERα、ERβ的定位与定量研究
运用免疫组织化学和图像灰度分析法对间情期与发情期比格犬卵巢及子宫内ERα、ERβ蛋白的表达进行组织定位和半定量分析，结果如下：
⑴比格犬ERα、ERβ的免疫组织化学定位
ERα主要表达于卵泡颗粒细胞、卵巢间质腺腺上皮细胞胞核内，胞质内有微弱表达，卵母细胞胞核及卵母细胞胞质内也有表达，在卵泡膜内膜的间质细胞及小动脉血管内皮细胞和平滑肌细胞、小静脉内皮细胞的胞核内也有少量表达。在膜黄体细胞的胞核，颗粒黄体细胞的胞核与胞质内均有表达。
ERβ主要表达于卵泡颗粒细胞，卵巢间质腺的腺上皮细胞及颗粒黄体细胞的核膜及胞质内，少量表达于卵巢间质动脉血管内皮细胞和平滑肌细胞、静脉内皮细胞的核膜及胞质内。
ERα主要表达于子宫内膜腺体细胞胞核内，而ERβ主要表达于子宫内膜腺体细胞核膜及胞质，在血管内皮细胞及平滑肌细胞胞质内也有表达。
BCIP/NBT与AEC双染可见ERα、ERβ在子宫内可表达于同一个内膜腺体细胞，但其定位部位不同：ERα主要在核内，而ERβ主要在核膜及胞质内。
⑵ERα、ERβ半定量分析结果
间情期与发情期比格犬卵巢内各组成成份ERα蛋白的表达特点：间情期与发情期比格犬卵巢内腺体、卵泡及周围基质、黄体各组份ERα表达的变化以腺体（0.03297±0.004075vs0.06647±0.008961，P＜0.01）和黄体（0.01286±0.003219vs0.06241±0.007671，P＜0.01）为显著，在发情期的表达水平均较间情期显著增加。表明ERα蛋白主要表达于发情期卵巢腺体及黄体内。
间情期与发情期比格犬卵巢内各组成成份ERβ蛋白的表达特点：由间情期发展至发情期，比格犬卵巢生长卵泡、初级卵泡、黄体、间质血管各组份中，生长卵泡ERβ表达水平显著增加（0.04471±0.004291vs0.1300±0.03970，P＜0.01），黄体内ERβ表达水平显著下降（0.1515±0.02711vs0.1008±0.02247，P＜0.01），但黄体内ERβ的起始表达水平很高，与初级卵泡及间质血管中的表达水平（0.01879±0.001484）比较仍保持在较高的水平。初级卵泡及卵巢间质血管中ERβ保持在相对较低的水平。表明在卵巢内ERβ蛋白主要表达于生长卵泡及黄体内。
间情期与发情期比格犬ERα、ERβ蛋白在卵巢内的表达特点：在间情期及发情期比格犬卵巢内ERβ的表达水平显著高于ERα（间情期：0.08646±0.008002vs0.02398±0.001911，P＜0.01；发情期：0.09007±0.007534vs0.04691±0.003052，P＜0.01）比格犬卵巢内ERα在由间情期发展至发情期时表达水平显著增加（0.02398±0.001911vs0.04691±0.003052，P＜0.01），而ERβ在两个生理阶段基本保持不变（0.08646±0.008002vs0.09007±0.007534，P＞0.05），但与ERα比较，维持在较高的表达水平。说明在间情期与发情期卵巢内均以ERβ的表达占主导地位。
间情期与发情期比格犬ERα、ERβ蛋白在子宫内的表达特点：在比格犬子宫内，在间情期，ERα、ERβ都有表达，但都维持在相对较低的水平，并且两者的表达水平无显著差异（0.02855±0.008855vs0.02908±0.003865，P＞0.05），而发展至发情期时， ERα的表达水平显著增加（0.02855±0.008855vs0.04896±0.006833，P＜0.01），ERβ仍维持在相对较低的水平（0.02170±0.003959）。由此得出结论在发情期比格犬子宫内，ERα为优势表达的雌激素受体亚型。
3. ERα、ERβ及ERβ新剪接异构体的扩增与鉴定
应用RT-PCR扩增了ERα和ERβ的全长编码序列：ERα1791和ERβ1593，并首次筛选出了ERβ的四个选择性剪接异构体：第4外显子完整缺失的一个；第7外显子完整缺失的一个；部分第4和部分第5外显子组合缺失的两个。应用5`3`RACE对第4外显子完整缺失的ERβ1293及第7外显子完整缺失的ERβ1257两个剪接异构体扩增出其全长编码序列。ERα1791分子量为66kD，ERβ1593分子量为59kD；ERβ1293缺失300bp，编码430aa，分子量为48kD；ERβ1257缺失181bp，编码418aa，分子量为47kD，碱基的缺失造成了读码移位，使翻译提前终止，在其终止密码子前插入10个非编码ERβ氨基酸残基。讫今，我们尚未查到关于比格犬ERβ的剪接异构体的研究报道。本实验首次发现比格犬ERβ的四个剪接异构体，扩增出其中两个的全长编码序列，并且证明其在瞬时转染的293T细胞中能够翻译成蛋白质。
4. ERα、ERβ及ERβ剪接异构体真核表达重组质粒的构建及活性的初步研究
构建了pEGFP-n1-ERα1788，pEGFP-n1-ERβ1590，pEGFP-n1-ERβ1290，pEGFP-n1-ERβ1254真核表达重组质粒，瞬时转染293T细胞，提取细胞总蛋白，经WB验证ERα1788、ERβ1590、ERβ1290、ERβ1254在体外均能翻译成目的蛋白质。
Because of its gentle temperament, moderate physique, clear genetic backgroundand high homogeneity, beagle dog has been playing an important role on medicalsciences, public health, life sciences and military medical researches. However, inbeagle dog breeding, some reproductive disorders emerged, such as no estrus, inferti-lity, extended diestrus and small litter size, that has been limited the production ofbeagle dog severely. It is necessary to do some profound researches on caninereproductive regulation. The paper focused on the expression of estrogen receptor α(ERα) and ERβ protein in diestrus and estrus canine ovary and uterus, gene ampli-fication of ERα, ERβ and its splice isoforms, maybe existed, construction of their euk-aryotic expression plasmids and primary study of their activities in transient trans-fected293T cells. The outstanding results were that four canine ERβ novel spliceisoforms were discovered for the first time by our team. It was laid the foundation forthe further study of canine ERα, ERβ and its splice isoforms in the regulation ofreproduction. The experiment elucidated as follow.
1. Detection of serum sexual hormone and comparison of normal ovarianand uterine histologic morphology in diestrus and estrus beagle dogs
Paraffin embedded normal uterus and ovary sections were used for histologycomparison on diestrus and estrus beagle dogs, their serum sexual hormone: E2,PRGE, FSH and LH levels were detected, and the changes of serum sexual hormoneand ovarian, uterine histological features of different stages were analyzed.
Sexual hormone test results showed that: In estrus beagles appeared estrogenpeak obviously, while in diestrus and metestrus estrogen were maintained at a verylow level. But PRGE were undetectable in diestrus, and it could be detected in estrusand metestrus, and the expression level increased significantly in metestrus.
Histology results showed that: The uterus and endometrium of diestrus dog wasthin, interstitial fibrosis, corpora luteum were occupied by primary follicles,1-2visible secondary follicles, and fewer mature follicles. Follicular and lutein cells hadmore fiber, less vascularized. While the uterus and endometrium of estrus canines were more thicker, endometrial cavity was larger, part of the glands are branch-likebended, glandular cells were enlarged, cytoplasm pale dyed, with a small number ofvisible vacuoles under the nuclear. Ovarian follicles were in large numbers, and weremainly primary, secondary and1-2mature follicles. There were numeric luteal cellswhich arranged regularly, state clearly, loose interstitial fibrosis, vascular, and novisible vacuolar degeneration.
2. Localization and quantitative study of ERα and ERβ protein in ovary anduterus of diestrus and estrus beagle dogs
Immunohistochemistry and images gray scale analysis were used to localize andsemi-quantitatively analyze ERα, ERβ protein expression in diestrus and estrus beagledog uterus and ovary. The results are as follow.
(1) Immunolocalization of beagle dog ERα and ERβ proteins
ERα was mainly expressed in the nuclei of follicle granulosa cells, ovarianinterstitial gland epithelial cells with weaker staining in the cytoplasm, whereas in thetheca cells, the stromal cells, arteriolar vascular endothelial cells and smooth musclecells, small vein endothelial cells, it was also expressed sporadically in their nuclei. Intheca lutein cells the positive staining were observed in the nuclei, as to granulosalutein cells both in nuclei and cytoplasms.
ERβ was mainly expressed in the nuclear membrane and cytoplasms of folliclegranulosa cells, ovarian interstitial gland epithelial cells, and granulosa lutein cellswhereas weakly expressed in the nuclear membrane and cytoplasms of arteriolarvascular endothelial cells and smooth muscle cells, small vein endothelial cells in thestroma of ovary.
ERα was mainly expressed in the nuclei of endometrial glandular cells, whileERβ mainly expressed in the nuclear membrane and cytoplasm of endometrialglandular cells, it was also weakly expressed in vascular endothelial cells and smoothmuscle cells.
Coexpression of ERα and ERβ in canine uterus was detected with BCIP/NBTand AEC double staining. Canine ERα and ERβ could coexpressed in same endo-metrial glandular epithelium cells but had different distribution: ERα in nucleus, ERβin nuclear membrane and cytoplasm.
(2) Results of beagle dog ERα and ERβ protein semi-quantitative analysis
Characterisation of ERα protein within ovarian components of diestrus and estrus beagle dogs： In diestrus and estrus beagle dog ovary, ERα protein levels ofthe ovarian components, including glands, follicles and their surrounding stroma,corpora lutea were estimated. ERα protein expression levels in ovarian glands andcorpora luteum were significantly increased in estrus than diestrus (ovarian glands:0.03297±0.004075vs0.06647±0.008961, P＜0.01; corpora luteum:0.01286±0.003219vs0.06241±0.007671, P＜0.01). It was suggested that ERα protein wasmainly expressed in estrus ovarian glands and corpora lutea.
Characterisation of ERβ protein within ovarian components of diestrus andestrus beagle dogs： In diestrus and estrus beagle dog ovary, ERβ protein levels ofthe ovarian components, including growing follicles, primary follicles, corpora luteaand interstitial vascular, were estimated. The expression level of ERβ in growingfollicles was significantly increased in estrus than diestrus (diestrus0.04471±0.004291vs estrus0.1300±0.03970, P <0.01), however, in corpora lutea it wassignificantly decreased (0.1515±0.02711vs0.1008±0.02247, P <0.01). The initialexpression level of ERβ in diestrus and estrus corpora luteum was very high, so whencompared with the primary follicles and interstitial vascular expression level(0.01879±0.001484), it was still maintained at a relatively high condition. Theexpression level of ERβ protein in primary follicles and ovarian stroma blood vesselswas constantly maintained at a relatively lower level. It was suggested that ERβprotein was mainly expressed in growing follicle and corpora luteum of canine ovaryregardless of estrus cycle.
Characterisation of ERα and ERβ protein in ovary of diestrus and estrusbeagle dogs： In beagle dog ovary, ERβ protein expression level was significantlyhigher than ERα protein both in diestrus and estrus (diestrus:0.08646±0.008002vs0.02398±0.001911, P＜0.01; estrus:0.09007±0.007533vs0.04691±0.003052，P＜0.01)； ERα protein expression level was increased significantly from diestrus toestrus (0.02398±0.001911vs0.04691±0.003052, P＜0.01), and ERβ in twophysiological stages remained unchanged (0.08646±0.008002vs0.09007±0.007534, P>0.05), but as contrast to ERα, still maintained at high expression levels.It was suggested that ERβ protein was a dominantly expressed ER subtype in bothdiestrus and estrus canine ovary.
Characterisation of ERα and ERβ protein in uterus of diestrus and estrusbeagle dogs： In beagle dog uterus, in diestrus, ERα and ERβ protein were bothexpressed, but maintained at a relatively low level, and no significant difference of expression level was found between these two subtypes (0.02855±0.008855vs0.02908±0.003856, P＞0.05). The expression level of ERα protein wassignificantly increased in estrus than diestrus (0.02855±0.008855vs0.04896±0.006833, P <0.01), while ERβ remained at a relatively low level (0.02170±0.003959). It is showed that ERα was dominantly expressed in estrus beagle doguterus.
3. Amplification and identification of ERα, ERβ and novel ERβ spliceisoforms genes
The full-length coding sequences of ERα and ERβ: ERα1791and ERβ1593wereattained by RT-PCR. Four alternatively spliced ERβ isoforms of beagle dog werediscovered for the first time by our team, and two of them, ERβ1293and ERβ1257,full-length coding sequences were amplified with5'3' RACE. ERα1791molecularweight was66kD, and ERβ1593molecular weight was59kD; ERβ1293was the4thexoncompletely deleted isoforms,300bp deleted, encoding430aa, the molecular weight of48kD; ERβ1257was the7thexon completely deleted isoforms,181bp deleted, resultedin reading code shift, premature translation termination, inserted10non-coding ERβamino acid residues before the termination codon. ERβ1257encodes418aa, molecularweight of47kD. This is the first study we have ever known that we discovered four ofcanine ERβ isoforms, gained full-length code sequences of two of them, and provedthat they could be translated into proteins in transient transfected293T cells.
4. Construction of ERα, ERβ and ERβ isoforms eukaryotic recombinantplasmids and preliminary study of their activities in vitro.
pEGFP-n1-ERα1788, pEGFP-n1-ERβ1590, pEGFP-n1-ERβ1290, pEGFP-n1-ERβ1254eukaryotic recombinant plasmids were constructed, transiently transfected293Tcells, and the extraction of total cellular protein was confirmed by WB. ERα1788,ERβ1590, ERβ1290, ERβ1254could be translated into the target protein in vitro.
引文
[1]邢华.犬卵母细胞的体内成熟环境与体外成熟培养[J].动物学杂志,2009,44(6):160-165.
[2]孔德强,李爱学,曾林,等.一种比格犬雌二醇β受体剪切异构体的克隆和序列分析[J].中国比较医学杂志,2011,21(7):44-47.
[3]孙兆增,曾林,洪宝庆,等.乙烯雌酚对间情期比格犬发情的诱导[J].中国比较医学杂志,2008,18(11):36-38,43.
[4]孙敬方.动物实验方法学[M].北京:人民卫生出版社,2001:194-195.
[5]仲剑平.医疗护理技术操作常规[M].北京:人民军医出版社,1998:981-989.
[6]张洪海,马承宝,刘国世,等.犬科动物繁殖生物技术研究进展[J].中国畜牧杂志,2005,41(12):54-57.
[7]王树声,付伟,赫广伟,等.母犬不孕症的病因和防治措施[J].当代畜牧,2002,10:29-30.
[8]罗江霞.LH在IVF中应用的探讨[J]医学信息临床医学分册,2011,24(9):5750-5751.
[9]孙尚军.185例不孕症妇女血清生殖激素测分析[J].中国优生与遗传杂志,2004,12(1):80,90.
[10]周天胜. Beagle母犬生殖器官发育及其ERαmRNA、PRmRNA表达规律的研究[D].江苏:扬州大学,2007:38-40.
[11]吴献青,林秋华,张志胜.卵巢血管形成与卵巢生理[J].国外医学计划生育分册,2002,21(2):94-97.
[1]张洪海,马承宝,刘国世,等.犬科动物繁殖生物技术研究进展[J].中国畜牧杂志,2005,41(12):54-57.
[2] Green S, Wlater P, Kumar V, Krust A, Bornert J-M, Argos P&Chambon PHuman oestrogen receptor cDNA: sequence, expression and homology toV-erb-A[J]. Nature,1986,320:134-139.
[3] Mosselman S, Polman J, Dijkema R. ER beta: identification and characterizationof a novel human estrogen receptor[J]. FEBS Lett,1996,392(1):49-53.
[4] Byers M, Kuiper GGJM, Gustafsson J-A&Park-Sarge O-K Estrogenreceptor-beta mRNA expression in rat ovary: down regulation bygonadotropins[J]. Molecular Endocrinology,1997,11:172-182.
[5] Chu S, Fuller PJ. Identification of a splice variant of the rat estrogen receptor βgene [J]. Molecular and Cellular Endocrinology,1997,132:195–199.
[6] Walther N, Lioutas C, Tillmann G, et al. Cloning of bovine estrogen receptorbeta (ERβ): expression of novel deleted isoforms in reproductive tissues [J].Molecular and Cellular Endocrinology,1999,152:37–45.
[7] Camacho-Arroyoa I, lez-Arenasa AG, lez-Agu GG, et al. Changes in the contentof progesterone receptor isoforms and estrogen receptor alpha in the chick brainduring embryonic development [J]. Comparative Biochemistry and PhysiologyPart A,2003,136:447–452.
[8] Nagler JJ, Cavileer T, Sullivan J, et al. The complete nuclear estrogen receptorfamily in the rainbow trout: Discovery of the novel ERα2and both ERβ isoforms[J]. Gene,2007,392:164–173.
[9] Mhyre AJ, Dorsa DM. Estrogen activates rapid signaling in the brain: Role ofestrogen receptor α and estrogen receptor βin neurons and GLIA [J]. Neuro-science,2006,138:851-858.
[10] Imamov O, Shim GJ, Warner M, Gustafsson JA. Estrogen receptor beta in healthand disease [J]. Biol Reprod,2005,73:866-871.
[11] Bao B, Kumar N, Karp RM, et al. Estrogen receptor beta expression in relation tothe expression of luteinizing hormone receptor and cytochrome P450enzymes inrat ovarian follicles[J]. Biol.Reprod,2000,63:1747-1755.
[12] Shingo H, Torii, Kumagai D, et al. Expression of estrogen receptor α and βgenes in the mediobasal hypothalamus, pituitary and ovary during the canineestrous cycle[J]. Neuroscience Letters,2003,347:131-135.
[13] Varshney KC, Nair MG. An immunohistochemical study on the expression of sexsteroid receptors in canine mammary tumors [J]. ISRN Veterinary Science,2012:1-7.
[14] Takahashi N, Tonchev AB, Koike K, et al. Expression of estrogen receptor-βinthe postischemic monkey hippocampus [J]. Neuroscience Letters,2004,369:9–13.
[15] Yuri K, Morita N, et al. Differential expression of estrogen receptor mRNA andprotein in the female rat preoptic area [J].Neuroscience Letters,1997,239:81-84.
[16]李联祥,金东岭,高金生,等.人眼脸板腺和Zeis腺性激素受体定性定位的免疫组织化学研究[J].解剖学报,2006,37(1):82-86.
[17] Taylor AH, Alazzawi F. Immunolocalization of oestragen receptor beta in humantissues [J]. J Mol Endocrinol,2000,24(1):1452-1551.
[18] Hiroi H, Inoue S, Watanabe T, et al. Differential immunolocalization of estrogenreceptor alpha and beta in rat ovary and uterus [J]. J Mol Endocrinol,1999,22(1):372-441.
[19]石玉秀.组织学与胚胎学[M].北京:高等教育出版社,2007(2010重印):205-213.
[20]张东辉.实验小型猪组织学图谱[M].北京:科学出版社,2012,第一版:96-99.
[1]苗知春,罗志勇.雌激素受体信号转导的研究[J].生命的化学,2010,30(6):854-859.
[2] Klein-Hitpass L et a1．A13bp palindrome is a functional estrogen responsiveelement and interacts specifically with estrogen receptor．Nucleic Acids Res,1988,16:647-663.
[3] Koehler KF et a1．Reflections on the discovery and significance of estrogenreceptor beta[J]．Endocr Rev,2005,26:465-478.
[4] Korach KS et a1. Update on animal models developed for analyses of estrogenreceptor biological activity[J]．J Steroid Biochem Mol Bio1.2003,86:387-391.
[5] Kuiper GG, Carlsson B, Grandien K, et al. Comparison of the ligand bindingspecificity and t ranscript tissue distribution of estrogen receptors alpha and beta[J]. Endocrinology,1997,138:863–870.
[6] Loutradis D, Theofanakis Ch, Anagnostou E,et al. Genetic profile of SNP(s) andovulation I nduction. Curr Pharm Biotechnol,2012,13(3):417-25.
[7] Cinta Zapater, Fran ois Chauvigné, Beatriz Fernández-Gómez, et al. Alternativesplicing of the nuclear progestin receptor in a perciform teleost generates novelmechanisms of dominant-negative transcriptional regulation [J]. General andComparative Endocrinology,2013,182:24-40.
[8] Johnson JM, Castle J, Garrett-Engele P, et al.Genome-wide survey of humanalternative pre-mRNA splicing with exon junction microarrays. Science,2003,302:2141–2144.
[9] Ohshiro K, Mudvari P, Meng QC,et al. Identification of a Novel EstrogenReceptor-α Variant and Its Upstream Splicing Regulator [J]. Mol Endocrinol,2010,24(5):914–922.
[10] Poola I, Abraham J, Liu A. Estrogen receptor β splice variant mRNAs aredifferentially altered during breast carcinogenesis [J]. J Steroid Biochem Mol Bio,2002, l82:169-179.
[11] Poola I, Abraham J, Baldwin K, et al. Identification of ten exon deleted ERβmRNAs in human ovary, breast, uterus and bone tissues: alternate splicingpattern of estrogen receptor β mRNA is distinct from that of estrogen receptor α.FEBS Lett,2002,516:133-138.
[12] Figtree G, McDonald D, Watkins H, et al. Truncated estrogen receptorα46-kDaisoform in human endothelial cells [J]. Circulation,2003,107:120–126.
[13] Flouriot G, Griffin C, Kenealy M, et al. Differentially expressed messenger RNAisoforms of the human estrogen receptor-α gene are generated by alternativesplicing and promoter usage[J]. Mol Endocrinol,1998,12:1939–1954.
[14] Friend K, Ang L, Shupnik M. Estrogen regulates the expression of severaldifferent estrogen receptor mRNA isoforms in rat pituitary [J]. Proc Natl AcadSci USA,1995,93:5925–5930.
[15] Li L, Haynes MP, Bender JR. Plasma membrane localization and function of theestrogen receptor α variant (ER46) in human endothelial cells. Proc Natl AcadSci USA,2003,100:4807–4812.
[16] Vaillancourt KL, Dinsdale NL, Hurd PL. Estrogen receptor1promoterpolymorphism and digit ratio in men [J]. Am J Hum Biol,2012,24(5):682-689.
[17] Gunawan A, Kaewmala K, Uddin MJ, et al.Association study and expressionanalysis of porcine ESR1as a candidate gene for boar fertility and sperm quality[J]. Anim Reprod Sci,2011,128(1-4):11-21.
[18] Derecka K, Balkwill GD, Garner TP, et al. Occurrence of a quadruplex motif in aunique insert within exon C of the bovine estrogen receptor alpha gene (ESR1)[J]. Biochemistry,2010,49(35):7625-33.
[19] Maruyama NO, Lucas TF, Porto CS, et al. Estrogen receptor ESR1regulates thephospholipase C-inositol phosphate signaling in the hippocampus from rats inproestrous and estrous phases[J]. Steroids,2013,78(1):8-14.
[20] Griffin C, Flouriot G, Buck VS, et al. Identification of Novel Chicken EstrogenReceptor-α Messenger Ribonucleic Acid Isoforms Generated by AlternativeSplicing and Promoter Usage [J]. Endo,1998,139(11):4614-4625.
[21] Oliveira AG, Dornas RA, Mahecha GA, et al. Occurrence and cellulardistribution of estrogen receptors ERα and ERβ in the testis and epididymalregion of roosters [J]. Gen Comp Endocrinol,2011,170(3):597-603.
[22] Gamba L, Cubedo N, Ghysen A, et al.Estrogen receptor ESR1controls cellmigration by repressing chemokine receptor CXCR4in the zebrafish posteriorlateral line system[J].Proc Natl Acad Sci U S A,2010,107(14):6358-6363.
[23] Davies MPA, O’Neill PA, Innes H, et al. Correlation of mRNA for oestrogenreceptor beta splice variants ERβ1, ERβ2/ERβcx and ERβ5with outcome inendocrine-treated breast cancer [J]. Journal of Molecular Endocrinology,2004,33:773-782.
[24] Imamov O, Shim GJ, Warner M, et al. Estrogen Receptor beta in Health andDisease[J]. Biology of Reproduction,2005,73:866–871.
[25] Fitzgerald SD, Allred D, et al. Inhibition of estrogen receptor action by anaturally occurring variant in human breast tumors [J]. Cancer Res,1992,52:483–486.
[26] García Pedrero, Zuazua Pedro, Martínez-Campa Carlos, e al. The NaturallyOccurring Variant of Estrogen Receptor(ER) ER△E7Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ [J].Endocrinology,2003,144(7):2967–2976.
[1] Chu S, Fuller PJ. Identification of a splice variant of the rat estrogen receptor βgene [J]. Molecular and Cellular Endocrinology,1997,132:195-199.
[2] Couse JF, Curtis Hewitt S, Korach KS. Receptor null mice reveal contrasting rolesfor estrogen receptor alpha and beta in reproductive tissues [J]. J Steroid BiochemMol Biol,2000,74:287-296.
[3] Herynk MH, Fuqua SAW. Estrogen receptor mutations in human disease [J].Endocrine Reviews,2004,25(6):869-898.
[4] Lewandodowski S, Kalita K, Kaczmarek. Estrogen receptor β: potential functionalsignificance of a variety of mRNA isoforms [J]. FEBS Letters,2002,524:1-5.
[5] Petersen DN, Tkalcevic GT, Koza-Taylor PH, et al. Identification of estrogenreceptor2, a functional variant of estrogen receptor expressed in normal rattissues[J]. Endocrinology,1998,139:1082–1092.
[6] Maruyama K, Endoh H, Sasaki-Iwaoka H, et al. A novel isoform of rat estrogenreceptor with18amino acid insertion in the ligand binding domain as a putativedominant negative regular of estrogen action[J]. Biochem Biophys Res Commun,1998,246:142–147.
[7] Richard H, Price JR, Nancy Lorenzon, et al.2000Differential expression ofestrogen receptor beta splice variants in rat brain: identification andcharacterization of a novel variant missing exon4[J]. Molecular Brain Research,2000,80:260–268.
[1] Cushman RA, Hedgpeth VS, Echternkamp SE, et al. Evaluation of numbers ofmicroscopic and macroscopic follicles in cattle selected for twinning [J]. AnimSci,2000,78(6):1564-1567
[2] Lundy T, Smith PO, Connell A, et al. Populations of granulosa cells in smallfollicles of the sheep ovary[J]. Reprod Fertil,1999,115(2):251-262
[3] Miller PB, Charleston JS, Battaglia DE,et al. Morphometric analysis of primordialfollicle number in pigtailed monkey ovaries: symmetry and relationship with age[J]. Biol Reprod,1999,61(2):553-556
[4] McCoard SA, et al. Germ cell development in Meishan and White Composite gilts[J]. Anim Reprod Sci,2003,77(1-2):85-105
[5]刘玉堂等.东北虎雌性生殖系统的组织学[J].动物杂志,2002,37(2):38-41.
[6]俞诗源等.川金丝猴卵巢的组织学研究[J].兰州大学学报,2000,36(3):154-157.
[7]刘玉堂.水貂发情期卵巢动态结构研究[J].东北林业大学学报,1995,23(2):49-53.
[8]杨增明,孙青原,夏国良主编.生殖生物学[M].北京:科学出版社,2005,第一版:74-97.
[9] Hewitt DA. Effect of preovulatory endocrine events upon maturation of oocytes ofdomestic bitches [J].Reprod Fertil1997, Suppl51:83-91.
[10] Durrant BS, Pratt, Russ KD. Isolation and characterization of canine advancedpreantral and early antral follicles [J]. Theriogenology,1998,49:917-932
[11]杨秀平,肖向红主编.动物生理学[M].北京:高等教育出版社,2009,第二版:327-328.
[12]石玉修主编.组织学与胚胎学[M].北京:高等教育出版社,2007(2010重印):205-215.
[12]刘了,许秋香,刘娜,等.犬发情周期卵巢与子宫组织学观察[J].中国兽医杂志,2007,43(9):62-63.
[13]位兰,卢佳佳,薛帮群,等.八点黑獭兔不同发育时期子宫组织结构变化的研究[J].中国养兔,2009,9:8-13.
[14]段永霞.牦牛生殖周期中子宫的组织结构观察[D].兰州:甘肃农业大学,2010.
[15]施新猷主编.现代医学实验动物学[M].北京:人民军医出版社,2000,第一版:119-132.
[16]杨幼明,等. Beagle犬繁育性能及血液学生化学测定[J].上海实验动物科学，1994,14(3,4):144-147
[17]彭传贵,等. Beagle犬的饲养及繁殖[J].实验动物科学与管理,1994,11(4):22-24
[18]李厚达主编.实验动物学[M].北京:中国农业出版社,2003,第二版:263-265.
[19]张忠诚主编.家畜繁殖学[M].北京:中国农业出版社，2000，第三版：95-100.
[20] Chandra SA, Adler RR. Frequency of Different Estrous Stages in Purpose-bredBeagles: A Retrospective Study [J]. Toxicologic Pathology,2008,36(7):944-949.
[21]张洪海,马承宝,刘国世,等.犬科动物繁殖生物技术研究进展[J].中国畜牧杂志,2005,41(12):54-57
[22] Kutzler MA. Induction and synchronization of estrus in dogs [J]. Theriogenology,2005,64:766-775.
[23] Ithaca NY. Biology of gonadotrophin secretion in adult and female dog [J]. IntVeter inform Service,2001,9:102-104.
[24] Sung M, Armour AF, Wright PJ.The influence of exogenous progestin on theoccurrence of proestrous or estrous signs, plasma concentrations of luteinizinghormone and estradiol in deslorelin (GnRH agonist) treated anestrous bitches [J].Theriogenology,2006,66:1513–1517.
[25]余道伦,邢华,朱德建.犬辅助生殖技术研究进展[J].安徽农业科学,2006,34:1591-1592,1594.
[26]王禄华,朱丽丽,庞海涛,等. PMSG诱导犬发情的初步试验[J].黑龙江畜牧兽医,2008:98-99.
[27]黄家良.母犬诱导发情试验[J].黑龙江动物繁殖,2001,9:29-30.
[28]张玉西,卓炳德. PMSG对母犬诱发发情的作用及效果观察[J].中国兽医杂志,2007,43:62-63.
[29]谭建华,安铁沫. PGF2α、eCG及hCG3种生殖激素联用对母犬发情、卵泡发育及胚胎着床的影响[J].中国兽医学报,2002,22:394-396.
[30]孙兆增,曾林,洪宝庆,等.乙烯雌酚对间情期比格犬发情的诱导[J].中国比较医学杂志,2008,18:36-38,43.
[31]李小慧,杨利国.乏情母犬用溴隐亭处理后的发情表现与血清中孕酮和雌二醇水平变化[J].畜牧与兽医,2004,36:1-3.
[32]董佳涵,朱淑文.犬繁殖调控技术研究进展[J].中国比较医学杂志,2011,21(2):74-78.
[33] Rento JP, Boyd JS, Eckersll PD, et al. Ovulation and fertilization and earlyembryonic development in the bitch [J]. Journal of Reproduction and Fertility,1991,93:221-231.
[34] Kinney GM, Pennycook JW, Schriver MD, et al.Surgical collection and transferof canine embryo [J]. Biol Reprot,1979,20(S1):96A abst.
[35] Byeong CL, Min KK, Goo J, et al. Dogs cloned from adult stromatic cells [J].Nature,2005:436:641.
[1] Cheung E, Schwabish MA., Kraus WL. Chromatin exposes intrinsic differencesin the transcriptional activities of estrogen receptorsαandβ[J]. The EMBOJournal,2003,22(3):600-611.
[2] Green S,Walter P,Kumar V,et al. Human oestrogen receptor cDNA: sequence,expression and homology to v-erb-A.[J]. Nature,1986,320(6058):134-139.
[3] Greene GL, Gilna P, Waterfield M, et al. Sequence and expression of humanestrogen receptor complementary DNA [J].Science,1986,231:1150-1154.
[4] Kuiper GGJM, Enmark E, Pelto-Huikko M, et al. Cloning of a novel estrogenreceptor expressed in rat prostate and ovary [J]. Proc Natl Acad Sci USA,1996,93:5925–5930.
[5] Mosselman S, Polman J, Dijkema R. ERβ: identification and characterization ofa novel human estrogen receptor [J]. FEBS Lett,1996,392:49-53.
[6] Tremblay GB, Tremblay A, Copeland NG, et al. Cloning, chromosomallocalization, and functional analysis of the murine estrogen receptor β [J]. MolEndocrin,1997,11:353-365.
[7] Chu S, Fuller PJ. Identification of a splice variant of the rat estrogen receptor βgene [J]. Molecular and Cellular Endocrinology,1997,132:195-199.
[8] Bain DL, Heneghan AF, Connaghan-Jones KD, et al. Nuclear receptor structure:implications for function. Annu Rev Physiol2007;69:201–220.
[9] Aranda A, Pascual A. Nuclear hormone receptors and gene expression [J]. JPhysiological Reviews.2001,81(3):1269～1304.
[10] Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol andestrogen receptor signaling[J]. J Biol Chem,2001,276:36869–36872.
[11] Kuiper GG, Lemmen JG, Carlsson B, et al. Interaction of estrogenic chemicalsand phytoestrogens with estrogen receptorβ[J]. Endocrinology,1998,139:4252–4263.
[12] Katzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor transcription andtransactivation: Estrogen receptor alpha and estrogen receptor beta: regulation byselective estrogen receptor modulators and importance in breast cancer [J].Breast Cancer Res,2000,2:335–344.
[13] Nilsson S, Makela S, Treuter E, et al. Mechanisms of estrogen action[J]. PhysiolRev,2001,81:1535–1565.
[14] Klinge CM, Jernigan SC, Mattingly KA, et al. Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors alpha andbeta by coactivators and corepressors[J]. J Mol Endocrinol,2004,33:387–410.
[15] Shang Y, Brown M. Molecular determinants for the tissue specificity of SERMs[J]. Science,2002,295:2465–2468.
[16] Couse JF, Korach KS. Estrogen receptor null mice: what have we learned andwhere will they lead us? Endocr Rev,1999,20:358–417.
[17] McInerney EM, Weis KE, Sun J, et al. Transcription activation by the humanestrogen receptor subtype beta (ER beta) studied with ER beta and ER alphareceptor chimeras. Endocrinology,1998,139:4513–4522.
[18] Hall JM, McDonnell DP. The estrogen receptor beta isoform (ERbeta) of thehuman estrogen receptor modulates ERalpha transcriptional activity and is a keyregulator of the cellular response to estrogens and antiestrogens[J].Endocrinology,1999,140:5566–5578.
[19] Cheung E, Schwabish MA, Kraus WL. Chromatin exposes intrinsic differencesin the transcriptional activities of estrogen receptors alpha and beta[J]. EMBO J,2003,22:600–611.
[20] Yi P, Bhagat S, Hilf R, et al. Differences in the abilities of estrogen receptors tointegrate activation functions are critical for subtype-specific transcriptionalresponses [J]. Mol Endocrinol,2002,16:1810–1827.
[21] Mueller SO, Simon S, Chae K, et al. Phytoestrogens and their human metabolitesshow distinct agonistic and antagonistic properties on estrogen receptor alpha(ER alpha) and ER beta in human cells [J]. Toxicol Sci,2004,80:14–25.
[22] Harrington WR, Sheng S, Barnett DH, et al. Activities of estrogen receptoralpha-and beta-selective ligands at diverse estrogen responsive gene sitesmediating transactivation or transrepression [J]. Mol Cell Endocrinol,2003,206:13–22.
[23] Weatherman RV, Clegg NJ, Scanlan TS. Differential SERM activation of theestrogen receptors (ERalpha and ERbeta) at AP-1site [J]. Chem Biol,2001,8:427–436.
[24] Paech K, Webb P, Kuiper GG, et al. Differential ligand activation of estrogenreceptors ERalpha and ERbeta at AP1sites [J]. Science,1997,277:1508–1510.
[25] Waters KM, Safe S, Gaido KW. Differential gene expression in response tomethoxychlor and estradiol through ERalpha, ERbeta, and AR in reproductivetissues of female mice [J]. Toxicol Sci,2001,63:47–56.
[26] Frasor J, Barnett DH, Danes JM, et al. Response-specific and ligand dose-dependent modulation of estrogen receptor (ER)αactivity by ERβin the uterus[J]. Endocrinology,2003,144:3159–3166.
[27] Lindberg MK, Moverare S, Skrtic S, et al. Estrogen receptor (ER) β reducesERα-regulated gene transcription, supporting a “ying yang” relationshipbetween ERα and ERβ in mice [J]. Mol Endocrinol,2003,17:203–208.
[28] Lazennec G, Bresson D, Lucas A, et al. ERβ inhibits proliferation and invasionof breast cancer cells [J]. Endocrinology,2001,142:4120–4130.
[29] Frasor J, Chang EC, Komm B, et al. Gene expression preferentially regulated bytamoxifen in breast cancer cells and correlations with clinical outcome[J].Cancer Res,2006,66:7334–7340.
[30] Lin CY, Strom A, Li Kong S, et al. Inhibitory effects of estrogen receptor beta onspecific hormone-responsive gene expression and association with diseaseoutcome in primary breast cancer[J]. Breast Cancer Res,2007,9:R25.
[31] Williams C, Edvardsson K, Lewandowski SA, et al. A genome-wide study ofrepressive effects of estrogen receptor beta on estrogen receptor alpha signalingin breast cancer cells [J]. Oncogene,2007,1–14.
[32] Cowley SM, Hoare S, Mosselman S, et al. Estrogen Receptorsαandβformheterodimers on DNA[J]. J Biol Chem,1997,272:19858–19862.
[33] Pettersson K, Delaunay F, Gustafsson JA. Estrogen receptor β acts as adominant regulator of estrogen signaling [J]. Oncogene,2000,19:4970–4978.
[34] Liu MM, Albanese C, Anderson CM, et al. Opposing action of estrogen receptorsα and β on cyclin D1gene expression[J]. J Biol Chem,2002,2277:24353–24360.
[35] Matthews J, Wihlen B, Tujague M, et al. Estrogen receptor (ER)βmodulates ERα-mediated transcriptional activation by altering the recruitment of c-Fos andc-Jun to estrogen-responsive promoters[J]. Mol Endocrinol,2006,20:534–543.
[36] Chang EC, Frasor J, Komm B, et al. Impact of estrogen receptorβon genenetworks regulated by estrogen receptorαin breast cancer cells [J].Endocrinology,2006,147:4831–4842.
[37] Taylor A P, Osorio L, Craig R, et al. Tumor specific regulation of angiogenticgrowth factors and their receptors during recovery from cytotoxic therapy[J].Clinical Cancer Research,2002,8(4):1213-1222.
[38] Lannigan DA. Estrogen receptor phosphorylation [J]. Steroids,2003,68:1-9.
[39] Ueda S, Tsuda H, Sato K,et al. Alternative tyrosine phosphorylation of signalingkinases according to hormone receptor status in breast cancer overexpressing theinsulin like growth factor receptor type1[J]. Cancer Scence,2006,97(7):597-604.
[40] Shang YF, Hu X, James DR, et al. Cofactor dynamics and sufficiency in estrogenreceptor-regulated transcription [J]. Cell,2000,103:843-852.
[41] Hurtado A, Pinós T, Barbosa-Desongles A, et al. Estrogen receptor beta displayscell cycle-dependent expression and regulates the G1phase through anon-genomic mechanism in prostate carcinoma cells [J].Cell Oncol,2008,30(4):349-365.
[42] Koji H, Jeffrey RB. Vascular cell signaling by membrane estrogen receptors [J].Steroids,2005,70:382-387.
[43] Kousteni S, Bellido T, Plotkin LI, et al. Nongenotropic, sex-nonspecific signalingthrough the estrogen or androgen receptors: dissociation from transcriptionalactivity [J]. Cell,2001,104:719-730.
[44] Heldring N, Pike A, Andersson S, et al. Estrogen receptors: how do they signaland what are their targets [J]. Physiol Rev,2007,87:905–931.
[45] Acconcia F, Ascenzi P, Bocedi A, et al. Palmitoylation-dependent estrogenreceptor alpha membrane localization: regulation by17beta-estradiol [J]. MolBiol Cell,2005,16:231-237.
[46] Pedram A, Razandi M, Sainson RC, et al. A conserved mechanism for steroidreceptor translocation to the plasma membrane [J]. J Biol Chem,2007,282:22278-22288.
[47] Levin ER, Pietras RJ. Estrogen receptors outside the nucleus in breast cancer [J].Breast Cancer Res Treat,2008,108:351-361.
[48] Sotgia F, Rui H, Bonuccelli G, et al. Caveolin-1, mammary stem cells, andestrogen-dependent breast cancers [J]. Cancer Res,2006,66:10647-10651.
[49] Lin SL, Yan LY, Zhang XT, et al. ER-α36: a variant of ER-α, promotestamoxifen agonist action in endometrial cancer cells via the MAPK/ERK andPI3K/Akt pathways [J]. PLoS ONE|www.plosone.org,2010,5(2):1-9.
[50] Balfe PJ, McCann AH, Welch HM, et al. Estrogen receptor β and breast cancer[J]. EJSO,2004,30:1043-1050.
[51] Pearce ST, Jordan VC. The biological role of estrogen receptors α and β in cancer[J]. Critical Reviews in Oncology/Hematology,2004,50:3-22.
[52]吕颜枝.雌激素受体在山羊腺垂体生殖激素分泌细胞中的分布[D].陕西:西北农林科技大学,2011.
[53] Knapczyk K, Duda M, Szafranska B, et al. Immunolocalisation of oestrogenreceptors alpha (ERalpha) and beta (ERbeta) in porcine embryos and fetuses atdifferent stages of gestation [J]. Acta Vet Hung,2008,56(2):221-233.
[54] Slomczynska M, Wozniak J. Differential distribution of estrogen receptor-betaand estrogen receptor-alpha in the porcine ovary [J]. Exp Clin EndocrinolDiabetes,2001,109(4):238-244.
[55] Knapczyk K,Duda M,Durlej M,et al.Expression of estrogen receptor alpha(ERalpha) and estrogen receptor beta in the ovarian follicles and corpora lutea ofpregnant swine [J]. Domest Anim Endocrinol,2008,35(2):170-179.
[56] Couse JF, Curtis Hewitt S, Korach KS. Receptor null mice reveal contrastingroles for estrogen receptor alpha and beta in reproductive tissues [J]. J SteroidBiochem Mol Biol,2000,74:287-296.
[57] Lubahn DB, Moyer JS, Golding TS, et al. Alteration of reproductive function butnot prenatal sexual development after insertional disruption of the mouseestrogen receptor gene [J]. Proc Natl Acad Sci USA,1993,90:11162-11166.
[58] Krege JH, Hodgin JB, Couse JF, et al. Generation and reproductive phenotypesof mice lacking estrogen receptor β[J]. Pro Natl Acad Sci USA,1998,95:15677-15682.
[59] Ogawa S, Chester AE, Hewitt SC, et al. From the cover: abolition of male sexualbehaviors in mice lacking estrogen receptors alpha and beta (alpha beta ERKO)[J]. Proc Natl Acad Sci USA,2000,97:14737-14741.
[60] Hirata S, Shoda T, Kato J, et al. Isoform/variant mRNAs for sex steroid hormonereceptors in humans [J]. Trends in Endocrinology&Metabolism,2003,14(3):124-129.
[61] Johnson JM, Castle J, Garrett-Engele P, et al. Genome-wide survey of humanalternative pre-mRNA splicing with exon junction microarrays [J]. Science,2003,302:2141-2144.
[62] Estrogen receptorβ: Potential functional significance of a variety of mRNAisoforms [J].Lewandowski S, Kalita K, Kaczmarek L. FEBS Letters,2002,524:1-5.
[63] Ishii H, Kobayashi M, Munetomo A, et al. Novel splicing events and post-transcriptional regulation of human estrogen receptor α E isoforms [J]. TheJournal of Steroid Biochemistry and Molecular Biology,2013,133:120-128.
[64] Hirata S, Shoda T, Kato J, et al. Isoform/variant mRNAs for sex steroid hormonereceptors in humans [J]. Trends in Endocrinology&Metabolism,2003,14(3):124-129.