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Depletion of the P43 Mitochondrial T3 Receptor Increases Sertoli Cell

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Depletion of the P43 Mitochondrial T3 Receptor Increases Sertoli Cell Depletion of the p43 mitochondrial T3 receptor increases Sertoli cell proliferation in mice Betty Fumel, Stéphanie Roy, Sophie Fouchécourt, Gabriel Livera, Anne-Simone Parent, Francois Casas, Florian Jean Louis Guillou To cite this version: Betty Fumel, Stéphanie Roy, Sophie Fouchécourt, Gabriel Livera, Anne-Simone Parent, et al.. De- pletion of the p43 mitochondrial T3 receptor increases Sertoli cell proliferation in mice. PLoS ONE, Public Library of Science, 2013, 8 (9), pp.1-15. 10.1371/journal.pone.0074015. hal-01129770 HAL Id: hal-01129770 https://hal.archives-ouvertes.fr/hal-01129770 Submitted on 28 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. Depletion of the p43 Mitochondrial T3 Receptor Increases Sertoli Cell Proliferation in Mice Betty Fumel1,2,3,4, Ste´phanie Roy1, Sophie Fouche´court1, Gabriel Livera5, Anne-Simone Parent6, Franc¸ois Casas7,8, Florian Guillou1* 1 INRA, UMR85 Physiologie de la Reproduction et des Comportements, Nouzilly, France, 2 CNRS, UMR7247 Physiologie de la Reproduction et des Comportements, Nouzilly, France, 3 Universite´ Franc¸ois Rabelais de Tours, Tours, France, 4 IFCE, Nouzilly, France, 5 Universite´ Paris Diderot, Sorbonne Paris Cite´, INSERM U967, CEA/DSV/ iRCM/SCSR Laboratoire de De´veloppement des Gonades, Fontenay-Aux-Roses, France, 6 Developmental Neuroendocrinology Unit, GIGA Neurosciences, University of Lie`ge, CHU Sart Tilman, Lie`ge, Belgium, 7 INRA, UMR 866 Dynamique Musculaire et me´tabolisme, Montpellier, France, 8 Universite´ de Montpellier 1 et 2, Montpellier, France Abstract Among T3 receptors, TRa1 is ubiquitous and its deletion or a specific expression of a dominant-negative TRa1 isoform in Sertoli cell leads to an increase in testis weight and sperm production. The identification of a 43-kDa truncated form of the nuclear receptor TRa1 (p43) in the mitochondrial matrix led us to test the hypothesis that this mitochondrial transcription factor could regulate Sertoli cell proliferation. Here we report that p43 depletion in mice increases testis weight and sperm reserve. In addition, we found that p43 deletion increases Sertoli cell proliferation in postnatal testis at 3 days of development. Electron microscopy studies evidence an alteration of mitochondrial morphology observed specifically in Sertoli cells of p432/2 mice. Moreover, gene expression studies indicate that the lack of p43 in testis induced an alteration of the mitochondrial-nuclear cross-talk. In particular, the up-regulation of Cdk4 and c-myc pathway in p432/2 probably explain the extended proliferation recorded in Sertoli cells of these mice. Our finding suggests that T3 limits post-natal Sertoli cell proliferation mainly through its mitochondrial T3 receptor p43. Citation: Fumel B, Roy S, Fouche´court S, Livera G, Parent A-S, et al. (2013) Depletion of the p43 Mitochondrial T3 Receptor Increases Sertoli Cell Proliferation in Mice. PLoS ONE 8(9): e74015. doi:10.1371/journal.pone.0074015 Editor: Joe¨l R. Drevet, Clermont-Ferrand Univ., France Received April 30, 2013; Accepted July 26, 2013; Published September 9, 2013 Copyright: ß 2013 Fumel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was received from the Agence Nationale pour la Recherche; grant ‘‘Biodiversite´,e´volution des e´cosystemes, e´cosystemes productifs, agronomie’’ (SF, FG), Institut national de la recherche agronomique (FG, SF, FC) and a fellowship Re´gion Centre (BF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction TRa1 transcript. The p43 is a mitochondrial T3 receptor ubiquitously expressed that stimulates mitochondrial transcription Sertoli cells (SC) are essential in spermatogenesis. It is well and protein synthesis in the presence of T3 [13]. The physiological established that the number of SC conditions the efficiency of importance of p43 was recently revealed by the use of mice spermatogenesis as one SC can only support a limited number of overexpressing or lacking this mitochondrial receptor. In partic- germ cells [1]. SC number is defined during the foetal and pre- ular, these works establish that p43 receptor strongly affects muscle pubertal periods. The SC proliferation arrest mainly depends on mass and the metabolic and contractile features of myofibers [14– thyroid hormone T3. In rodents, during the post natal period, the 16]. In addition, p43 was found to regulate insulin secretion and arrest of SC proliferation and entry into differentiation is glucose homeostasis [17]. correlated with a peak of T3 blood level [2]. Neonatal In this work, we focused our study on testis because the presence hypothyroidism in mice and rats leads to an increase in the of high-affinity binding sites for T3 has been reported in rat Sertoli weight of adult testis and epididymis due to an increase in the daily cell mitochondria [18]. Here we report that p43 specific depletion sperm production [3–8]. in mice increases testis weight and sperm reserve. In addition, we For a while, it was assumed that thyroid hormone acts found that p43 deletion increases Sertoli cell proliferation in exclusively through nuclear receptors: TRa1, TRa2, TRb1, postnatal testis at 3 days of development. Moreover, gene TRb2 encoded by the THRA and THRB genes [9]. Among T3 expression studies provided some molecular mechanisms under- receptors, TRa1 is ubiquitous and it was previously described that lying the ability of T3 to arrest Sertoli cell proliferation. In its total deletion in mice leads to an increase in testis weight and particular, c-myc and Cdk4 could be the central targets of the sperm production [10]. Moreover, a specific expression of mitochondrial-nuclear crosstalk, controlling the proliferation of dominant-negative TRa1 isoform in SC exhibited similar pheno- Sertoli cells. In summary, we demonstrate that the T3 limits post- typic features to the TRa1 knock-out mice: heavier testicular weight and higher sperm reserve [11]. In 1995, a 43-kDa natal Sertoli cell proliferation in part through its mitochondrial T3 truncated form of the nuclear receptor TRa1 was identified in receptor p43. the mitochondrial matrix (p43) [12]. This protein is synthesized by the use of internal initiation site of translation occuring in the PLOS ONE | www.plosone.org 1 September 2013 | Volume 8 | Issue 9 | e74015 p43 Receptor Controls Sertoli Cell Proliferation Materials and Methods Serum FSH Levels Serum FSH levels were determined in a volume of 100 ml using Animals a double Ab method and a RIA kit (rFSH RIA), kindly supplied by All animal experiments were performed according to European the National Institutes of Health (Dr. A. F. Parlow, National directives (86/609/CEE) and approved by the Comite´ d’Ethique Hormone and Peptide Program, Torrance, CA). Rat FSH antigen en matie`re d’Expe´rimentation Animale : Re´gion Languedoc- (NIDDK-rFSH-I) was labeled with 125I by the chloramine-T Rousillon. Our animal studies were conducted in accordance with method and the hormone concentration was expressed using the guidelines for the care and use of laboratory animals issued by the rat FSH reference preparation (NIDDK-rFSH-RP-2) as standard. french Ministry of Agriculture. Intraassay and interassay coefficients were less than 7 and 10%, Mice were fed a standard laboratory diet and tap water ad respectively. The sensitivity of the assay was 0.125 ng/100 ml. libitum. The p432/2 mice lacking specifically the mitochondrial T3 receptor were generated by Franc¸ois Casas as described Testis Histology previously [17]. To only inhibit p43 translation from TR1 mRNA, Testis histology (seminiferous tubule organization) was analyzed we deleted the internal translation start in exon 3 using site after fixing in Bouin’s fluid and embedding in paraffin. Sections directed PCR mutagenesis to change the methionine codon at (4 mm) were stained with haematoxylin for microscopic observa- position 109 to a leucine codon. Targeting construct was tion. electroporated into 129SV embryonic stem (ES) cells. We generated chimeras by blastocyst injection of ES cells and Determination of SC Proliferation Index in Vivo obtained germline transmissionof the mutated allele. We mated p432/2 and WT mice at post-natal day 3 (P3) and 10 (P10) them with C57BL/6 females. Offspring inheriting the p43- were injected with 50 mg/g (of body weight) of 5-bromo-2- mutated allele were intercrossed to generate the p432/2 line. deoxyuridine (BrdU) (Sigma) 3 hours before sacrifice and BrdU Homozygote p432/2 mice born from the 3 independent ES cell was incorporated in proliferating cells. Testis were fixed in Bouin’s clones were viable, fertile, and appeared normal. All the mice used fluid, embedded in paraffin and sectioned (4 mm). After antigen in these studies were back-crossed at least 10 times into the retrieval in a boiling sodium citrate buffer (pH 6.0), endogenous C57BL/6 background. peroxidase activity was quenched with H2O2. BrdU immunode- The colony was generated by crossing p432/2 mice with wild- tection was performed using a mouse IgG monoclonal antibody type (WT) C57BL/6 breeders and generated future generation of (Roche; 1/200), and revealed using a peroxidase labelled polymer WT controls. conjugated to goat anti-mouse IgG (Dako) and 3 39diaminoben- zidine chromogen (DAB) (Dako).
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