135 NEW INSIGHTS ON THE ROLE OF 17β-ESTRADIOL IN CORPUS LUTEUM LIFESPAN OF NON-PREGNANT BITCHES

Abstract

Canine corpus luteum (CL) is a transient endocrine gland responsible for the synthesis of 17β-oestradiol (E2) during diestrus and acts in an autocrine and/or paracrine manner within this structure. The mechanism of action of E2 depends on the expression ratio of its receptors ERa and ERβ. Binding to ERa has a proliferative effect and to ERβ an antiproliferative effect. The aim of this study was to better understand the possible signalling pathways mediated by ESR1 and ESR2 in the formation and regression of canine corpus luteum. The CL were collected via ovariosalpingohysterectomy from nonpregnant bitches (n = 30) on Days 10, 20, 30, 40, 50, and 60 (n = 5/group) post-ovulation. Eighteen CL (n = 3/group) were subjected to RNA sequencing (RNA-Seq) to identify differentially expressed (DE) genes during diestrus because 3 replicates per group is the minimum number required to obtain good results in the sequencing. The DE genes were submitted to oPOSSUM 3 software (http://opossum.cisreg.ca/oPOSSUM3/) for detection of over-represented conserved transcription binding sites (TFBS) related to ESR1 and ESR2 (coding genes for ERa and ERβ, respectively). We validated the expression from 10 of these genes by RT-PCR using GAPDH as the reference gene. Protein expression of the IGF-related genes was also evaluated by immunohistochemistry. The RNAseq results were analysed by Cufflinks. Data were tested for homogeneity and normality using the Kolmogorov-Smirnov test. The RT-PCR data were compared by the one-way ANOVA test. The correlations between the RNAseq and RT-PCR results were verified by Pearson correlation. The difference was considered significant when P < 0.05. All statistical analyses were performed with GraphPadPrism 5 (GraphPad Software Inc., San Diego, CA, USA). Differential gene expression analysis among groups during the luteal phase showed the presence of 5116 DE genes in at least one comparison, and 1106 genes that have not been recorded to the canine genome yet. Among all DE genes, we found 295 genes showing TFBS related to ESR1 and ESR2. Of these genes, 4 that had TFBS in common with ESR2 (LEF-1, PAPPA, NDGR2, and ATP1A1) and 1 with ESR1 (CAV1) were selected for validation, and the other 5 genes were chosen because they control cell proliferation (CTNNB1, CCND1, IGFBP3, IGFBP4, and IGFBP5). The selected genes belonged to IGF system-related genes (PAPPA, IGFBP3, IGFBP4, and IGFBP5), Wnt/betacatenin signalling (CTNNB1, LEF-1, and CCND1), and genes regulated by oestrogen hormones (NDRG2 and ATP1A1) and plasma membrane ER (CAV1). The results suggest that during the first half of diestrus, E2 signalling appears to be mediated by ERa via interaction with caveolin-1 (non-genomic pathways): IGF system and Wnt/β-catenin signalling were identified as one of the cascades activated by this interaction, with a major role in the proliferative process. During the second half of diestrus, ERβ appears to regulate NDGR2 and ATP1A1 gene expression, contributing to the regression of the CL. Therefore, the results suggest that E2 might activate both luteotrophic and regression-related factors in canine CL.