Expression Of Estrogen Receptor α,-β Of Uterus And Ovary In Beagle Dog And Study Of Its Novel ERβ Isoforms

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 dogsParaffin 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 dogsImmunohistochemistry 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 proteinsERα 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 analysisCharacterisation 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 genesThe 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.