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Differentially Expressed Genes and Gene Networks Involved in Pig Differentially expressed genes and gene networks involved in pig ovarian follicular atresia Elena Mormede, Stéphane Fabre, Agnes Bonnet, Danielle Monniaux, Christele Robert-Granie, Magali San Cristobal, Julien Sarry, Florence Vignoles, Florence Gondret, Philippe Monget, et al. To cite this version: Elena Mormede, Stéphane Fabre, Agnes Bonnet, Danielle Monniaux, Christele Robert-Granie, et al.. Differentially expressed genes and gene networks involved in pig ovarian follicular atresia. Phys- iological Genomics, American Physiological Society, 2017, 49 (2), pp.67-80. 10.1152/physiolge- nomics.00069.2016. hal-01529483 HAL Id: hal-01529483 https://hal.archives-ouvertes.fr/hal-01529483 Submitted on 27 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Physiol Genomics 49: 67–80, 2017. First published December 9, 2016; doi:10.1152/physiolgenomics.00069.2016. RESEARCH ARTICLE Systems Biology and Polygenic Traits Differentially expressed genes and gene networks involved in pig ovarian follicular atresia Elena Terenina,1 Stephane Fabre,1 Agnès Bonnet,1 Danielle Monniaux,2 Christèle Robert-Granié,1 Magali SanCristobal,1 Julien Sarry,1 Florence Vignoles,1 Florence Gondret,3,4 Philippe Monget,2 and X Gwenola Tosser-Klopp1 1GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Castanet Tolosan, France; 2INRA UMR 0085, CNRS UMR 7247, Université Francois Rabelais de Tours, IFCE, Physiologie de la Reproduction et des Comportements, Nouzilly, France; 3INRA, UMR1348 Pegase, Saint᎑Gilles, France; and 4AgroCampus-Ouest, UMR1348 Pegase, Saint᎑Gilles, France Submitted 15 June 2016; accepted in final form 2 December 2016 Terenina E, Fabre S, Bonnet A, Monniaux D, Robert-Granié C, atresia. This process may be considered as physiologically SanCristobal M, Sarry J, Vignoles F, Gondret F, Monget P, normal, allowing the ovary to generate the ovulatory quota Tosser-Klopp G. Differentially expressed genes and gene networks characteristic of the species, at each sexual cycle. involved in pig ovarian follicular atresia. Physiol Genomics 49: A number of studies in antral growing follicles have indi- 67–80, 2017. First published December 9, 2016; doi:10.1152/physi- cated that follicular atresia is initiated and caused by apoptosis olgenomics.00069.2016.—Ovarian folliculogenesis corresponds to the development of follicles leading to either ovulation or degenera- (programmed cell death) of granulosa cells (53, 56, 71, 107). tion, this latter process being called atresia. Even if atresia involves Two general pathways have been suggested to be involved in apoptosis, its mechanism is not well understood. The objective of this the physiological induction of apoptosis: negative induction by study was to analyze global gene expression in pig granulosa cells of survival factor withdrawal and positive induction by the bind- ovarian follicles during atresia. The transcriptome analysis was per- ing of a specific ligand, such as tumor necrosis factor-alpha or formed on a 9,216 cDNA microarray to identify gene networks and Fas ligand, to its membrane receptor (41). In follicles, apopto- candidate genes involved in pig ovarian follicular atresia. We found sis is triggered when endocrine and/or intra-follicular concen- 1,684 significantly regulated genes to be differentially regulated trations of survival factors, particularly gonadotropins and between small healthy follicles and small atretic follicles. Among some growth factors, are inadequate (93). It is now recognized them, 287 genes had a fold-change higher than two between the two follicle groups. Eleven genes (DKK3, GADD45A, CAMTA2, that a diverse spectrum of pro- and antiapoptosis susceptibility CCDC80, DAPK2, ECSIT, MSMB, NUPR1, RUNX2, SAMD4A, and genes are thus expressed in germ cells and/or somatic cells of ZNF628) having a fold-change higher than five between groups could the ovary, including members of the BCL2 and caspase gene likely serve as markers of follicular atresia. Moreover, automatic families (108). Many of these genes are regulated by gonado- confrontation of deregulated genes with literature data highlighted 93 tropins and growth factors, and changes in the temporal pattern genes as regulatory candidates of pig granulosa cell atresia. Among of cell death gene expression suggest that an intimate associ- these genes known to be inhibitors of apoptosis, stimulators of ation exists between the products of these genes and the apoptosis, or tumor suppressors INHBB, HNF4, CLU, different inter- activation of apoptosis. leukins (IL5, IL24), TNF-associated receptor (TNFR1), and cyto- The biochemical characteristics of apoptosis have been in- chrome-c oxidase (COX) were suggested as playing an important role in porcine atresia. The present study also enlists key upstream regu- vestigated to discover causative factors and markers identify- lators in follicle atresia based on our results and on a literature review. ing follicles at the early stages of atresia. Results from studies The novel gene candidates and gene networks identified in the current of ovarian gene expression in vivo and in vitro have indicated study lead to a better understanding of the molecular regulation of that ovarian products, such as steroid hormones, transforming ovarian follicular atresia. growth factor family members, insulin-like growth factor (IGF) 1, IGF-binding proteins (IGFBPs), and inhibins could pig ovary; folliculogenesis; atresia; gene expression; cDNA microar- ray; biomarkers; upstream regulators; functional pathways play a role in atretic process (2, 42). However, most of this information has been acquired based on candidate-gene ap- proaches. OVARIAN FOLLICULOGENESIS REFERS to the development of folli- In the present study, we propose a high-throughput tran- cles leading to either ovulation or degeneration. This degener- scriptomic approach to get a better understanding of the mo- ation process is called atresia. In mammals, it has long been lecular actors, their regulatory genes, and physiological recognized that the majority of follicles present at birth become networks involved in pig follicle atresia. A specific microar- atretic, so that only Ͻ1% of follicles ovulate during ovarian ray has been developed to identify genes expressed in follicular growth and development (53, 107). Thus, the most granulosa cells along the terminal ovarian follicle growth in highly probable fate for a follicle is to degenerate by entering the pig (17). This represents a useful tool to study porcine ovarian follicular atresia. The aims of this study were 1)to analyze global gene expression changes in pig granulosa Address for reprint requests and other correspondence: E. Terenina; GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Castanet Tolosan, France (e-mail: cells of ovarian follicles during atresia and 2) to identify the [email protected]). gene networks and propose new candidates genes that can 1094-8341/17 Copyright © 2017 the American Physiological Society 67 Downloaded from www.physiology.org/journal/physiolgenomics by ${individualUser.givenNames} ${individualUser.surname} (194.167.077.053) on July 30, 2018. Copyright © 2017 American Physiological Society. All rights reserved. 68 TARGET GENES OF PIG OVARIAN FOLLICULAR ATRESIA further serve as targets to characterize and modulate atresia Microarray data analysis. Raw data and normalized data were in granulosa cells. submitted to the GEO database as GSM596969 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?accϭGSM596969). MATERIAL AND METHODS Data exploration and statistical analysis were performed using R software (http://www.r-project.org). The data coming from complex Collection of ovaries. Estrous cycles of 10 sows were synchronized probes hybridization were analyzed on a natural logarithmic scale. As by oral administration of 20 mg/day altrenogest (Regumate; Hoechst- a first step, negative spots with Ͼ7 intensity values were checked on Roussel, Paris, France) for 18 days. Ovaries were removed by lapa- images and replaced by the median of negative spots in case of rotomy 24 or 96 h after the last altrenogest feeding. All procedures obvious overshining effect or hybridization stain. Then, luciferase- were approved by the Agricultural and Scientific Research agencies positive controls were removed, and the correlations of the gene and conducted at an INRA experimental farm (animal experimenta- expressions from each membrane were examined within condition. tion authorization C37-175-2) in accordance with the guidelines for Hybridizations with a correlation coefficient Ͻ0.80 were discarded. Care and Use of Agricultural Animals in Agricultural Research and Spots with low signal value (below the average of empty spots ϩ 3 Teaching. standard deviations) were considered as unexpressed and were ex- Granulosa cell isolation and RNA extraction. Once ovaries were cluded from the analysis. Finally, the negative control spots were collected, all visible (Ͼ1 mm in diameter) antral follicles were
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