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The Oviductal Transcriptome Is Influenced by a Local Ovarian Effect in the Sow Rebeca López-Úbeda1,2, Marta Muñoz3, Luis Vieira1,2, Ronald H

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The Oviductal Transcriptome Is Influenced by a Local Ovarian Effect in the Sow Rebeca López-Úbeda1,2, Marta Muñoz3, Luis Vieira1,2, Ronald H López-Úbeda et al. Journal of Ovarian Research (2016) 9:44 DOI 10.1186/s13048-016-0252-9 RESEARCH Open Access The oviductal transcriptome is influenced by a local ovarian effect in the sow Rebeca López-Úbeda1,2, Marta Muñoz3, Luis Vieira1,2, Ronald H. F. Hunter4, Pilar Coy1,2,5* and Sebastian Canovas1,2,5* Abstract Background: Oviducts participate in fertilization and early embryo development, and they are influenced by systemic and local circulation. Local functional interplay between ovary, oviduct and uterus is important, as deduced from the previously observed differences in hormone concentrations, presence of sperm, or patterns of motility in the oviduct after unilateral ovariectomy (UO). However, the consequences of unilateral ovariectomy on the oviductal transcriptome remain unexplored. In this study, we have investigated the consequences of UO in a higher animal model as the pig. Methods: The influence of UO was analyzed on the number of ovulations on the contra ovary, which was increased, and on the ipsilateral oviductal transcriptome. Microarray analysis was performed and the results were validated by PCR. Differentially expressed genes (DEGs) with a fold change ≥ 2andafalsediscoveryrateof10%were analyzed by Ingenuity Pathway Analysis (IPA) to identify the main biofunctions affected by UO. Results: Data revealed two principal effects in the ipsilateral oviduct after UO: i) down-regulation of genes involved in the survival of sperm in the oviduct and early embryonic development, and ii) up-regulation of genes involved in others functions as protection against external agents and tumors. Conclusions: Results showed that unilateral ovariectomy results in an increased number of ovulation points on the contra ovary and changes in the transcriptome of the ipsilateral oviduct with consequences on key biological process that could affect fertility output. Keywords: Pig, Oviductal transcriptome, Microarray, Ovariectomy Background livestock reproduction and also improve in vitro culture Among all structures involved in the fertilization process, conditions used in assisted reproductive technologies it is in the oviduct where the most decisive episodes for (ART) [9]. fertilization take place [1, 2]. Recently, the oviduct tran- Oviducts are influenced by the systemic circulation, scriptome has aroused the interest of different groups, that changes throughout the reproductive cycle [1], pri- including ourselves; these have studied changes in hu- marily due to the action of ovarian steroid hormones man [3, 4] and animal oviductal transcriptome profiles [10, 11] and prostaglandins [12–14]. In addition, local under different conditions [5–8], trying to elucidate functional interplay between ovary, oviduct and uterus are the consequences for fertilization and early embryo also important [15, 16]. The existence of a local regulatory development. mechanism has been documented in women and animal In contrast to extensive studies focused on interactions species from anatomical studies and functional evaluation between ovary and endometrium, interactions with the of blood distribution (Additional file 1: Table S1), and it is oviduct appear to have been overlooked. A better under- already known that small changes in concentrations of standing of the local regulatory interactions between progesterone can affect the oviduct secretions such as ovary and oviduct could help us to make progress in amino acids and ions [17, 18], so it could be expected that unilateral ovariectomy (UO) would have conse- * Correspondence: [email protected]; [email protected] quences in the oviduct, but this question remains unex- 1 Department of Physiology, Veterinary Faculty, University of Murcia, Campus plored until now. de Espinardo, 30100 Murcia, Spain Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. López-Úbeda et al. Journal of Ovarian Research (2016) 9:44 Page 2 of 10 The objective of this study was to investigate differ- Pathways, networks and biofunctions from ences in oviductal transcriptome caused by the pres- Ingenuity Analysis ence or absence of the local effect of the ovary IPA software generated a single functional network able following UO, in a specific area of the oviduct (ampul- to integrate 17 of the 18 selected genes (12 up and 5 lary-isthmic junction) where fertilization and zygote down-regulated genes; marked red and green, respect- formation occurs [19]. ively) as shown in Fig. 1, with a very high score (51 score We employed an in vivo model in which no hormonal value). The gene SAL1 is the only gene not included in treatment (animals were in natural oestrus) or previous sur- the network because the Ingenuity database does not gery were performed. The principal tool used to achieve recognize it. Associated top network functions were: cel- our objective was the Porcine Gene Expression Microarray lular growth and proliferation, cellular development, (ID 026440 Agilent Technologies, Madrid, Spain). cell-to-cell signalling interaction, tissue development and lipid metabolism. Interestingly, UBC, TGFB1, ERK1/2, TNF and INSULIN are candidates to be involved in the Results cited functions as inferred from the gene network. Al- Compensatory ovulation mechanism though these genes were not found to be differentially The number of points of ovulation and ovarian weights expressed in the microarray analysis, they are targeted at the first and second surgery were compared (Table 1). by several DEGs. Unilateral ovariectomy had a significant effect on the The molecular and cellular functions related to DEGs number of ovulation points, which were 3 times higher (Additional file 3: Table S3) were cell death and survival in the ovaries obtained in the second surgery (7.6 ± 1.6 (n = 9), cell morphology (n = 7), small molecule bio- vs. 22.00 ± 2.4; p < 0.017). In contrast, removal of one chemistry (n = 7), amino acid metabolism (n = 4) and ovary had no significant effect on the weight of the carbohydrate metabolism (n = 4). Acute Phase Response remaining ovary except for sow number 5 where ovary Signaling, cAMP-mediated signaling, Complement Sys- weight increased after ovariectomy. No other morpho- tem, Hematopoiesis from Pluripotent Stem Cells and logical changes were noted in the ovary or connective CNTF Signaling were the most significant canonical tissue. pathways affected (Additional file 3: Table S3). Genes differentially expressed between ovariectomized Predicted upstream regulators and control oviducts We interrogated IPA for upstream microRNA (miRNA) Bioinformatics analysis of the microarray data showed regulators of down-regulated genes in this study. Results 18 DEGs (with a fold change > 2 and a false discovery showed 9 predicted miRNAs that could act on CFH, rate of 10 %) in ipsilateral oviduct (respect to ovariec- SEPP1, RPL37A or RBBP6. Additionally, one miRNAs tomy) vs. contraleral oviduct. Twelve genes were up- (miR-680-5p) was identified as regulator of RBBP6 and regulated (MSC, IGHA1, MIS18BP1, SEMA3B, AKAP5, RPL37A (Fig. 2). Moreover, we also obtained a prediction LZTS3, MTOR, TMEM184B, ALOX12, RPP40, PRAP1 of other (not miRNAs) upstream regulators that could and PTH1R; Table 2). In contrast, six genes were down- control genes that were identified as down and up- regulated (RPL37A, SEPP1, ITIH2, CFH, RBBP6, SAL1; regulated genes in UO gilts. Results showed 15 factors Table 2). Quantitative RT-PCR results for 3 selected that could act as upstream regulators (Fig. 3). Some fac- genes (ALOX12, CFH and SAL1) confirmed microarray tors could act simultaneously as a regulator of several results (up- or down-regulation; Table 3), but differ- genes, for example HOXA9and TP63 are transcription ences in the fold change were detected, as typically factors which actually activate transcription. They were have been described in other reports. found as potential upstream regulators for the RBBP6, Table 1 Animal data sheet First surgery Second surgery Sow No. of previous farrowings No. Ovulateda follicles Ovary weight (g) No. Ovulateda follicles Ovary weight (g) 1 3 8 11.97 25 10.98 2 2 10 7.79 23 5.06 3 4 2 8.90 25 11.02 4 3 11 13.89 Not ovulated 11.72 5 2 7 5.88 15 48.07 aPaired-samples tTestanalysis for mean ± s.e.m. comparisons showed significant differences (p < 0.01) for the No. of ovulated follicles betweenthefirst (7.60 ± 1.6) and secondsurgery (22.00 ± 2.4) López-Úbeda et al. Journal of Ovarian Research (2016) 9:44 Page 3 of 10 Table 2 Genes up- or down-regulated by ovariectomy Gene bank accession Symbol Entrez gene name Fold change AK350490 MSC musculin 2.88 AB194101 IGHA1 immunoglobulin heavy constant alpha 1 2.75 XM_003353476 MIS18BP1 MIS18 binding protein 1 2.61 XM_001927855 SEMA3B semaphorin 3B 2.58 XM_001924844 AKAP5 A-kinase anchor protein 5-like 2.55 AK392511 LZTS3 leucine zipper, putative tumor suppressor family member 3 2.27 XP_003127632 MTOR mechanistic target of rapamycin (serine/threonine kinase) 2.19 XM_001924228 TMEM184B
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