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1. Transposable elements acquire time- and sex-specific transcriptional and epigenetic signatures along mouse fetal gonad development

Author

Isabelle Stévant, Nitzan Gonen, and Francis Poulat

Subjects
transposable elements, sex determination, gonads, testis, ovary, gene expression regulation, Biology (General), QH301-705.5
Abstract

Gonadal sex determination in mice is a complex and dynamic process, which is crucial for the development of functional reproductive organs. The expression of genes involved in this process is regulated by a variety of genetic and epigenetic mechanisms. Recently, there has been increasing evidence that transposable elements (TEs), which are a class of mobile genetic elements, play a significant role in regulating gene expression during embryogenesis and organ development. In this study, we aimed to investigate the involvement of TEs in the regulation of gene expression during mouse embryonic gonadal development. Through bioinformatics analysis, we aimed to identify and characterize specific TEs that operate as regulatory elements for sex-specific genes, as well as their potential mechanisms of regulation. We identified TE loci expressed in a time- and sex-specific manner along fetal gonad development that correlate positively and negatively with nearby gene expression, suggesting that their expression is integrated to the gonadal regulatory network. Moreover, chromatin accessibility and histone post-transcriptional modification analyses in differentiating supporting cells revealed that TEs are acquiring a sex-specific signature for promoter-, enhancer-, and silencer-like elements, with some of them being proximal to critical sex-determining genes. Altogether, our study introduces TEs as the new potential players in the gene regulatory network that controls gonadal development in mammals.

Published
2024
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2. Deciphering the origins and fates of steroidogenic lineages in the mouse testis

Author

Herta Ademi, Cyril Djari, Chloé Mayère, Yasmine Neirijnck, Pauline Sararols, Chris M. Rands, Isabelle Stévant, Béatrice Conne, and Serge Nef

Subjects
CP: Developmental biology, Biology (General), QH301-705.5
Abstract

Summary: Leydig cells (LCs) are the major androgen-producing cells in the testis. They arise from steroidogenic progenitors (SPs), whose origins, maintenance, and differentiation dynamics remain largely unknown. Single-cell transcriptomics reveal that the mouse steroidogenic lineage is specified as early as embryonic day 12.5 (E12.5) and has a dual mesonephric and coelomic origin. SPs specifically express the Wnt5a gene and evolve rapidly. At E12.5 and E13.5, they give rise first to an intermediate population of pre-LCs, and finally to fetal LCs. At E16.5, SPs possess the characteristics of the dormant progenitors at the origin of adult LCs and are also transcriptionally closely related to peritubular myoid cells (PMCs). In agreement with our in silico analysis, in vivo lineage tracing indicates that Wnt5a-expressing cells are bona fide progenitors of PMCs as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis.

Published
2022
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3. Specific Transcriptomic Signatures and Dual Regulation of Steroidogenesis Between Fetal and Adult Mouse Leydig Cells

Author

Pauline Sararols, Isabelle Stévant, Yasmine Neirijnck, Diane Rebourcet, Annalucia Darbey, Michael K. Curley, Françoise Kühne, Emmanouil Dermitzakis, Lee B. Smith, and Serge Nef

Subjects
fetal Leydig cell, adult Leydig cell, androgen, testis, RNA sequencing, single cell RNA sequencing, Biology (General), QH301-705.5
Abstract

Leydig cells (LC) are the main testicular androgen-producing cells. In eutherian mammals, two types of LCs emerge successively during testicular development, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Both display significant differences in androgen production and regulation. Using bulk RNA sequencing, we compared the transcriptomes of both LC populations to characterize their specific transcriptional and functional features. Despite similar transcriptomic profiles, a quarter of the genes show significant variations in expression between FLCs and ALCs. Non-transcriptional events, such as alternative splicing was also observed, including a high rate of intron retention in FLCs compared to ALCs. The use of single-cell RNA sequencing data also allowed the identification of nine FLC-specific genes and 50 ALC-specific genes. Expression of the corticotropin-releasing hormone 1 (Crhr1) receptor and the ACTH receptor melanocortin type 2 receptor (Mc2r) specifically in FLCs suggests a dual regulation of steroidogenesis. The androstenedione synthesis by FLCs is stimulated by luteinizing hormone (LH), corticotrophin-releasing hormone (CRH), and adrenocorticotropic hormone (ACTH) whereas the testosterone synthesis by ALCs is dependent exclusively on LH. Overall, our study provides a useful database to explore LC development and functions.

Published
2021
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4. Deciphering Cell Lineage Specification during Male Sex Determination with Single-Cell RNA Sequencing

Author

Isabelle Stévant, Yasmine Neirijnck, Christelle Borel, Jessica Escoffier, Lee B. Smith, Stylianos E. Antonarakis, Emmanouil T. Dermitzakis, and Serge Nef

Subjects
single-cell RNA-seq, sex determination, testis, Sertoli cell, fetal Leydig cell, progenitors, Biology (General), QH301-705.5
Abstract

Summary: The gonad is a unique biological system for studying cell-fate decisions. However, major questions remain regarding the identity of somatic progenitor cells and the transcriptional events driving cell differentiation. Using time-series single-cell RNA sequencing on XY mouse gonads during sex determination, we identified a single population of somatic progenitor cells prior to sex determination. A subset of these progenitors differentiates into Sertoli cells, a process characterized by a highly dynamic genetic program consisting of sequential waves of gene expression. Another subset of multipotent cells maintains their progenitor state but undergoes significant transcriptional changes restricting their competence toward a steroidogenic fate required for the differentiation of fetal Leydig cells. Our findings confirm the presence of a unique multipotent progenitor population in the gonadal primordium that gives rise to both supporting and interstitial lineages. These also provide the most granular analysis of the transcriptional events occurring during testicular cell-fate commitment.

Published
2018
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5. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics

Author

Isabelle Stévant, Françoise Kühne, Andy Greenfield, Marie-Christine Chaboissier, Emmanouil T. Dermitzakis, and Serge Nef

Subjects
Biology (General), QH301-705.5
Abstract

Summary: Sex determination is a unique process that allows the study of multipotent progenitors and their acquisition of sex-specific fates during differentiation of the gonad into a testis or an ovary. Using time series single-cell RNA sequencing (scRNA-seq) on ovarian Nr5a1-GFP+ somatic cells during sex determination, we identified a single population of early progenitors giving rise to both pre-granulosa cells and potential steroidogenic precursor cells. By comparing time series single-cell RNA sequencing of XX and XY somatic cells, we provide evidence that gonadal supporting cells are specified from these early progenitors by a non-sex-specific transcriptomic program before pre-granulosa and Sertoli cells acquire their sex-specific identity. In XX and XY steroidogenic precursors, similar transcriptomic profiles underlie the acquisition of cell fate but with XX cells exhibiting a relative delay. Our data provide an important resource, at single-cell resolution, for further interrogation of the molecular and cellular basis of mammalian sex determination. : Using single-cell RNA sequencing of Nr5a1-expressing gonadal somatic cells during female and male sex determination, Stévant et al. deconvoluted the cell lineage specification process and sex-specific differentiation of both the supporting and the steroidogenic cell lineages at a transcriptomic level. Keywords: single-cell RNA-seq, sex determination, ovary, testis, granulosa cell, Sertoli cells, supporting cells, progenitors, differentiation, lineage specification, cell fate decision, gonad development, gene expression, transcriptomics

Published
2019
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6. Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling.

Author

Patrick Callier, Pierre Calvel, Armine Matevossian, Periklis Makrythanasis, Pascal Bernard, Hiroshi Kurosaka, Anne Vannier, Christel Thauvin-Robinet, Christelle Borel, Séverine Mazaud-Guittot, Antoine Rolland, Christèle Desdoits-Lethimonier, Michel Guipponi, Céline Zimmermann, Isabelle Stévant, Françoise Kuhne, Béatrice Conne, Federico Santoni, Sandy Lambert, Frederic Huet, Francine Mugneret, Jadwiga Jaruzelska, Laurence Faivre, Dagmar Wilhelm, Bernard Jégou, Paul A Trainor, Marilyn D Resh, Stylianos E Antonarakis, and Serge Nef

Subjects
Genetics, QH426-470
Abstract

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.

Published
2014
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7. Dual-topology of collagen XV and tenascin C acts in concert to guide and shape developing motor axons

Author

Laurie Nemoz-Billet, Martial Balland, Laurent Gilquin, Benjamin Gillet, Isabelle Stévant, Emilie Guillon, Sandrine Hughes, Gilles Carpentier, Elisabeth Vaganay, Mary-Julieth Gonzalez-Melo, Manuel Koch, Yad Ghavi-Helm, Florence Ruggiero, and Sandrine Bretaud

Abstract

During development, motor axons are guided towards their muscle target by various extrinsic cues including extracellular matrix (ECM) proteins those identities remain poorly documented. Using single-cell RNA-sequencing of differentiating slow muscle progenitors (SMP) in zebrafish, we charaterized the SMP as a major source of ECM proteins that were computationally predicted to form a basement membrane-like structure tailored for motor axon guidance. Multiplein vivoandin vitroapproaches further revealed that motor axon shape and growth relies on the timely expression of the attractive cue Collagen XV-B (ColXV-B) that locally provides motor axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Bioprinted micropatterns mimicking their unique topology provide compelling evidence that it represents a sufficient condition to elicit directional motor axon growth. Our study provides the first evidence that ECM topology and stiffness critically influence motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury.

Published
2023

8. Specific transcriptomic signatures and dual regulation of steroidogenesis between fetal and adult mouse Leydig cells

Author

Lee B. Smith, Diane Rebourcet, Isabelle Stévant, Emmanouil T. Dermitzakis, Annalucia Darbey, Yasmine Neirijnck, Françoise Kühne, Serge Nef, Pauline Sararols, and Michael Curley

Subjects
0301 basic medicine, endocrine system, medicine.drug_class, Alternative splicing, Intron, Biology, Androgen, Cell biology, Transcriptome, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, 030220 oncology & carcinogenesis, medicine, ACTH receptor, Receptor, Luteinizing hormone, Testosterone
Abstract

Leydig cells (LC) are the main testicular androgen-producing cells. In eutherian mammals, two types of LCs emerge successively during testicular development, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Both display significant differences in androgen production and regulation. Using bulk RNA sequencing, we compared the transcriptomes of both LC populations to characterise their specific transcriptional and functional features. Despite similar transcriptomic profiles, a quarter of the genes show significant variations in expression between FLCs and ALCs. Non-transcriptional events, such as alternative splicing was also observed, including a high rate of intron retention in FLCs compared to ALCs. The use of single-cell RNA sequencing data also allowed the identification of nine FLC-specific genes and 50 ALC-specific genes. Expression of the corticotropin-releasing hormone 1 (Crhr1) receptor and the ACTH receptor melanocortin type 2 receptor (Mc2r) specifically in FLCs suggests a dual regulation of steroidogenesis. The androstenedione synthesis by FLCs is stimulated by luteinizing hormone (LH), CRH and ACTH whereas the testosterone synthesis by ALCs is dependent exclusively on LH. Overall, our study provides a useful database to explore LC development and function.

Published
2021

9. Single‐cell transcriptomics reveal temporal dynamics of critical regulators of germ cell fate during mouse sex determination

Author

Anne-Amandine Chassot, Chloé Mayère, Isabelle Stévant, Emmanouil T. Dermitzakis, Pauline Sararols, Yasmine Neirijnck, Françoise Kühne, Chris M Rands, Marie-Christine Chaboissier, Serge Nef, University of Geneva [Switzerland], Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Université Côte d'Azur (UCA), Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Genève (UNIGE), Université de Genève = University of Geneva (UNIGE), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Chaboissier, Marie-Christine, and CHASSOT, Anne Amandine

Subjects
Male, 0301 basic medicine, endocrine system, Time Factors, X Chromosome, Somatic cell, [SDV]Life Sciences [q-bio], Gene regulatory network, Mice, Transgenic, Biology, Biochemistry, Mice, 03 medical and health sciences, 0302 clinical medicine, ddc:590, Downregulation and upregulation, Y Chromosome, Gene expression, Genetics, medicine, Animals, ddc:576.5, Molecular Biology, Gene, ComputingMilieux_MISCELLANEOUS, Gene Expression Profiling, Intron, Gene Expression Regulation, Developmental, Sex Determination Processes, Cell biology, [SDV] Life Sciences [q-bio], Germ Cells, 030104 developmental biology, medicine.anatomical_structure, Lineage differentiation, RNA splicing, Female, Single-Cell Analysis, Germ cell, 030217 neurology & neurosurgery, Biotechnology
Abstract

Despite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single-cell RNA sequencing of 14 914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the bone morphogenic protein (BMP) and nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex-specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex-specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data are available on a web platform and provide a molecular roadmap of GC sex determination at single-cell resolution, which will serve as a valuable resource for future studies of gonad development, function, and disease.

Published
2021

10. Two repeated motifs enriched within some enhancers and origins of replication are bound by SETMAR isoforms in human colon cells

Author

Yves Bigot, Thierry Lecomte, Ahmed Arnaoty, Isabelle Stévant, Peter Arensburger, Vincent Coustham, Aymeric Antoine-Lorquin, Martine Batailler, Laura Helou, Serge Guyétant, Nicolas Buisine, Bruno Pitard, Sassan Asgari, Linda Beauclair, Catherine Belleannée, Diversité, Génomes & Interactions Microorganismes - Insectes [Montpellier] (DGIMI), Université de Montpellier (UM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), California State Polytechnic University [Pomona] (CAL POLY POMONA), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), University of Queensland [Brisbane], Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Dynamics, Logics and Inference for biological Systems and Sequences (Dyliss), Inria Rennes – Bretagne Atlantique, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-GESTION DES DONNÉES ET DE LA CONNAISSANCE (IRISA-D7), Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-CentraleSupélec-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Physiologie moléculaire et adaptation (PhyMA), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Biologie des Oiseaux et Aviculture (BOA), Université de Tours (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Host-Pathogen Interactions in the Regulation of Immune Responses (CRCINA-ÉQUIPE 5), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Institut de Génomique Fonctionnelle de Lyon (IGFL), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Français du Cheval et de l'Equitation [Saumur] (IFCE)-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Université de Tours-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)

Subjects
0106 biological sciences, Gene isoform, DNA Repair, transposon, Colon, DNA repair, Regulatory Sequences, Nucleic Acid, Biology, DNA replication, Origin of replication, 01 natural sciences, 03 medical and health sciences, domestication, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, Humans, Protein Isoforms, expression enhancer, DNA binding, Enhancer, Gene, 030304 developmental biology, MITE, 0303 health sciences, Histone-Lysine N-Methyltransferase, Chromatin, Cell biology, Enhancer Elements, Genetic, CpG site, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, coevolution, 010606 plant biology & botany
Abstract

International audience; Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Published
2021

11. Origin, specification and differentiation of a rare supporting-like lineage in the developing mouse gonad

Author

Serge Nef, Violaine Regard, Pauline Sararols, Aitana Perea-Gomez, Andy Greenfield, Chloé Mayère, Cyril Djari, Isabelle Stévant, Dagmar Wilhelm, Diana Condrea, Michelle Simon, Richard Reeves, Corey Bunce, Françoise Kühne, Ivana Gantar, Marie-Christine Chaboissier, Laura Batti, Blanche Capel, Pam Siggers, Furong Tang, Simon Greenaway, Norbert B. Ghyselinck, Yasmine Neirijnck, University of Geneva [Switzerland], Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Duke University Medical Center, MRC Harwell Institute [UK], Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Melbourne, Institute of Genetics and Genomics in Geneva (iGE3), Université de Genève (UNIGE), Université Nice Sophia Antipolis (1965 - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)

Subjects
0303 health sciences, education.field_of_study, endocrine system, Gonad, Lineage (genetic), Gonadal ridge, Mesonephros, [SDV]Life Sciences [q-bio], Population, Biology, Coelomic epithelium, Cell biology, Rete ovarii, 03 medical and health sciences, 0302 clinical medicine, medicine.anatomical_structure, Rete testis, medicine, education, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLC) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially co-express Wnt4 and Sox9. SLCs become sexually dimorphic around E12.5, progressively acquire a Sertoli- or granulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Finally, we found that WNT4 is a crucial regulator of the SLC lineage and is required for the formation of the rete testis.TeaserDescription of an uncharacterized multipotent gonadal cell lineage involved in testis and ovary development

Published
2021

12. Pantoprazole, a proton‐pump inhibitor, impairs human sperm motility and capacitation in vitro

Author

Bastien Arnaud, Mayis Kaba, Pierre F. Ray, Isabelle Stévant, Jessica Escoffier, Guillaume Martinez, Christophe Arnoult, Emilie Le Blévec, Serge Nef, Jean-Pascal Hograindleur, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), and Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)

Subjects
Adult, Male, endocrine system, Urology, Endocrinology, Diabetes and Metabolism, [SDV]Life Sciences [q-bio], Lansoprazole, Motility, 2-Pyridinylmethylsulfinylbenzimidazoles, Membrane Potentials, Andrology, 03 medical and health sciences, Young Adult, 0302 clinical medicine, Endocrinology, Human fertilization, Capacitation, medicine, Humans, ddc:576.5, Phosphorylation, Pantoprazole, reproductive and urinary physiology, Sperm motility, ComputingMilieux_MISCELLANEOUS, Retrospective Studies, 030219 obstetrics & reproductive medicine, Hyperactivation, urogenital system, business.industry, Proton Pump Inhibitors, Middle Aged, Sperm, Spermatozoa, 3. Good health, Semen Analysis, Sperm Maturation, Reproductive Medicine, Fertilization, Rabeprazole, Sperm Motility, business, Sperm Capacitation, Omeprazole, medicine.drug
Abstract

Background The effects of PPIs on human sperm fertilizing capacity were poorly investigated although these drugs are widely over-used. Two publications retrospectively studied relationships between any PPI intake and sperm parameters from patients consulting at infertility clinics, but the conclusions of these reports were contradictory. Only two reports investigated the effects of lansoprazole and omeprazole on sperm motility and found lansoprazole to be deleterious and omeprazole to be neutral for sperm motility. The inconsistency of the PPI effect in the previous reports emphasizes the need for more basic research on human spermatozoa, taking into account the hypothesis that the different PPI drugs may have different effects on sperm physiology. Objectives Do PPIs, which are among the most widely sold drug in the word, impact negatively human sperm capacitation and sperm motility? Materials and methods The effects of PPIs on human sperm maturation and motility were analyzed by CASA, flow cytometry, and Western blot. Results We tested the impact of 6 different PPIs on human sperm motility and capacitation. We showed that pantoprazole, but not the other PPIs, decreased sperm progressive motility and capacitation-induced sperm hyperactivation. We therefore investigated further the effects of pantoprazole on sperm capacitation, and we observed that it had a significant deleterious effect on the capacitation-induced hyperpolarization of the membrane potential and capacitation-associated protein phosphorylation. Discussion and conclusion Our results indicate that exposure to pantoprazole has an adverse effect on the physiological competence of human spermatozoa. As the capacitation process takes place within the female tract, our results suggest that PPIs intake by the female partner may impair in vivo sperm maturation and possibly fertilization. Moreover, the absence of adverse effect by PPIs on mouse sperm emphasizes the need to develop reprotox assays using human material to better assess the effects of medication intake on sperm physiology.

Published
2020

13. Deciphering the origins and fates of steroidogenic lineages in the mouse testis

Author

Herta, Ademi, Cyril, Djari, Chloé, Mayère, Yasmine, Neirijnck, Pauline, Sararols, Chris M, Rands, Isabelle, Stévant, Béatrice, Conne, and Serge, Nef

Subjects
Male, Mice, Fetus, Testis, Androgens, Animals, Leydig Cells, Cell Differentiation
Abstract

Leydig cells (LCs) are the major androgen-producing cells in the testis. They arise from steroidogenic progenitors (SPs), whose origins, maintenance, and differentiation dynamics remain largely unknown. Single-cell transcriptomics reveal that the mouse steroidogenic lineage is specified as early as embryonic day 12.5 (E12.5) and has a dual mesonephric and coelomic origin. SPs specifically express the Wnt5a gene and evolve rapidly. At E12.5 and E13.5, they give rise first to an intermediate population of pre-LCs, and finally to fetal LCs. At E16.5, SPs possess the characteristics of the dormant progenitors at the origin of adult LCs and are also transcriptionally closely related to peritubular myoid cells (PMCs). In agreement with our in silico analysis, in vivo lineage tracing indicates that Wnt5a-expressing cells are bona fide progenitors of PMCs as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis.

Published
2020

14. Expression ofWnt5adefines the major progenitors of fetal and adult Leydig cells

Author

Isabelle Stévant, Serge Nef, Béatrice Conne, Chris M Rands, and Herta Ademi

Subjects
WNT5A, Fetus, In vivo, Fetal mouse, Lineage tracing, embryonic structures, Biology, Progenitor cell, Cell biology
Abstract

SummaryLeydig cells (LCs) are the major androgen-producing cells in the testes. They arise from steroidogenic progenitors, whose origins, maintenance and differentiation dynamics remain largely unknown. Here, we identifiedWnt5aas a specific marker of steroidogenic progenitors, whose expression begins at around E11.5-E12.5 in interstitial cells of the fetal mouse testis.In vivolineage tracing indicates thatWnt5a-expressing progenitors are initially present in large numbers in the fetal testis and then progressively decrease as development progresses. We provide evidence thatWnt5a-expressing cells arebona fideprogenitors of peritubular myoid cells as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis. Additionally, we show in the adult testis thatWnt5aexpression is restricted to a subset of LCs exhibiting a slow but noticeable clonal expansion, revealing hitherto unappreciated proliferation of fully differentiated LCs as a contribution to the adult LC pool.

Published
2020

15. Retinoic acid synthesis by ALDH1A proteins is dispensable for meiosis initiation in the mouse fetal ovary

Author

Morgane Le Rolle, Isabelle Stévant, Andreas Schedl, Marie-Christine Chaboissier, Norbert B. Ghyselinck, Eric Pailhoux, Fabio Da Silva, Geneviève Jolivet, Anne-Amandine Chassot, Jean-Marie Guigonis, Serge Nef, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Biologie de la Reproduction, Environnement, Epigénétique & Développement (BREED), École nationale vétérinaire d'Alfort (ENVA)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Genève (UNIGE), Université Côte d'Azur - Faculté de Médecine (UCA Faculté Médecine), Université Côte d'Azur (UCA), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-École nationale vétérinaire d'Alfort (ENVA)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Medical School of the University of Geneva, German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ), French National Research Agency (ANR)ANR-13-BSV2-0017-02 ARGONADSANR-11-LABX-0028-01ANR-18-CE14-0012 SexSpecsLa Ligue Nationale Contre le Cancer (Equipe Labelisée Ligue Nationale Contre le Cancer), ANR-13-BSV2-0017,ARGONADS,Acide rétinoique et devenir des cellules germinales(2013), ANR-18-CE14-0012,Sex-Specs,Homeostasie tissulaire selon le sex et son impact sur les maladies surrénaliennes(2018), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), UMR E4320 (TIRO-MATOs), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), Department of Genetic Medicine and Development [Geneva], Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Chaboissier, Marie-Christine, Blanc 2013 - Acide rétinoique et devenir des cellules germinales - - ARGONADS2013 - ANR-13-BSV2-0017 - Blanc 2013 - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Homeostasie tissulaire selon le sex et son impact sur les maladies surrénaliennes - - Sex-Specs2018 - ANR-18-CE14-0012 - AAPG2018 - VALID, Centres d'excellences - Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie - - SIGNALIFE2011 - ANR-11-LABX-0028 - LABX - VALID, Université Nice Sophia Antipolis (1965 - 2019) (UNS), École nationale vétérinaire - Alfort (ENVA)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), ANR-13-BSV2-0017-02 ARGONADSANR-11-LABX-0028-01ANR-18-CE14-0012 SexSpecsLa Ligue Nationale Contre le Cancer (Equipe Labelisée), ANR-11-LABX-0028,SIGNALIFE,Réseau d'Innovation sur les Voies de Signalisation en Sciences de la Vie(2011), and COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV]Life Sciences [q-bio], Retinoic acid, Endogeny, Oogenesis, chemistry.chemical_compound, 0302 clinical medicine, CYP26B1, PRIMORDIAL GERM-CELLS, BINDING, ddc:576.5, [SDV.BDD]Life Sciences [q-bio]/Development Biology, ComputingMilieux_MISCELLANEOUS, Research Articles, Mammals, 0303 health sciences, Multidisciplinary, integumentary system, SciAdv r-articles, CELL SEX DETERMINATION, LINEAGE, MEIOSIS, OOGENESIS, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, DIFFERENTIATION, Female, Research Article, EXPRESSION, FATE, Ovary, Tretinoin, Biology, ALDH1A2, 03 medical and health sciences, Meiosis, medicine, Animals, Gene, neoplasms, 030304 developmental biology, organic chemicals, Proteins, Cell Biology, GENE, biological factors, ALDH1A1, body regions, MICE, Germ Cells, chemistry, biology.protein, 030217 neurology & neurosurgery, Developmental Biology
Abstract

Retinoic acid synthesis is not essential for meiosis in fetal ovaries, challenging the dogma about the meiosis-inducing factors., In mammals, the timing of meiosis entry is regulated by signals from the gonadal environment. All-trans retinoic acid (ATRA) signaling is considered the key pathway that promotes Stra8 (stimulated by retinoic acid 8) expression and, in turn, meiosis entry. This model, however, is debated because it is based on analyzing the effects of exogenous ATRA on ex vivo gonadal cultures, which not accurately reflects the role of endogenous ATRA. Aldh1a1 and Aldh1a2, two retinaldehyde dehydrogenases synthesizing ATRA, are expressed in the mouse ovaries when meiosis initiates. Contrary to the present view, here, we demonstrate that ATRA-responsive cells are scarce in the ovary. Using three distinct gene deletion models for Aldh1a1;Aldh1a2;Aldh1a3, we show that Stra8 expression is independent of ATRA production by ALDH1A proteins and that germ cells progress through meiosis. Together, these data demonstrate that ATRA signaling is dispensable for instructing meiosis initiation in female germ cells.

Published
2020

16. A brief history of sex determination

Author

Serge Nef, Isabelle Stévant, and Marilena D. Papaioannou

Subjects
Male, 0301 basic medicine, Sex Characteristics, History, Ovary, Historical Article, Mythology, Sex Determination Processes, 030105 genetics & heredity, Biochemistry, Hormones, History, 17th Century, 03 medical and health sciences, 030104 developmental biology, Endocrinology, Aesthetics, Animals, Humans, ddc:576.5, Female, Hormone metabolism, Genes, sry, Molecular Biology, History, Ancient, Sex characteristics
Abstract

A fundamental biological question that has puzzled, but also fascinated mankind since antiquity is the one pertaining to the differences between sexes. Ancient cultures and mythologies poetically intended to explain the origin of the two sexes; philosophy offered insightful albeit occasionally paradoxical perceptions about men and women; and society as a whole put forward numerous intuitive observations about the traits that distinguish the two sexes. However, it was only through meticulous scientific research that began in the 16th century, and gradual technical improvements that followed over the next centuries, that the study of sex determination bore fruit. Here, we present a brief history of sex determination studies from ancient times until today, by selectively interviewing some of the milestones in the field. We complete our review by outlining some yet unanswered questions and proposing future experimental directions.

Published
2018

17. ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling

Author

Silvia Corrochano, Abigail Harris, Hans Clevers, Caroline Eozenou, Isabelle Stévant, Joelle Bignon-Topalovic, Nick Warr, Neila Belguith, Sara Wells, Bochra Ben Rhouma, Pam Siggers, Serge Nef, Daniel T. Grimes, Ken McElreavey, Rebecca D. Burdine, Feng Cong, Makoto Suzuki, Andy Greenfield, Danielle Sagar, Anu Bashamboo, Bon-Kyoung Koo, Raja Brauner, Medical Research Coucil Harwell [Oxford, UK] (MRC Harwell), MRC Harwell, Department of Molecular Biology [Princeton], Princeton University, Novartis Institutes for BioMedical Research (NIBR), Hubrecht Institute [Utrecht, Netherlands], University Medical Center [Utrecht]-Royal Netherlands Academy of Arts and Sciences (KNAW), Université de Genève = University of Geneva (UNIGE), Fondation Ophtalmologique Adolphe de Rothschild [Paris], Université Paris Descartes - Paris 5 (UPD5), Faculté de médecine - Faculty of Medicine [Sfax, Tunisie] (FMS), Université de Sfax - University of Sfax, Génétique du Développement humain - Human developmental genetics, Institut Pasteur [Paris] (IP), This work was supported by the Medical Research Council by core funding Grant MC_U142684167 (to A.G.) at the Harwell Institute, and the Agence Nationale de la Recherche Grant ANR-10-LABX-73 (to K.M.). S.N. acknowledges support from Swiss National Science Foundation Grant 31003A_173070. M.S. was a visiting scientist supported by the Strategic International Research Exchange Program between Princeton University and National Institutes of Natural Sciences, Japan. D.T.G. was supported by National Institute of Arthritis and Mucoskeletal and Skin Diseases Pathway to Independence Award 1K99AR070905. Work in the R.D.B. laboratory is supported by the National Institute of Child Health and Development Grant 2R01HD048584., We thank the husbandry team in Ward 5 of the Mary Lyon Centre at Harwell and the Frozen Embryo and Sperm Archive (FESA) and histology teams. We thank Dagmar Wilhelm for the kind gift of anti-FOXL2 antibody. We thank Phil Johnson for zebrafish husbandry. We acknowledge European Cooperation in Science & Technology (COST) Action BM1303 (DSDnet)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Université de Genève (UNIGE), Génétique du développement humain, Institut Pasteur [Paris], and Hubrecht Institute for Developmental Biology and Stem Cell Research

Subjects
0301 basic medicine, Male, Embryo, Nonmammalian, Sex Differentiation, Organogenesis, Wnt Proteins/antagonists & inhibitors, Disorders of Sex Development, DSD, MESH: Wnt Proteins / metabolism, MESH: Testis / metabolism, MESH: Disorders of Sex Development / genetics, MESH: Testis / pathology, MESH: Embryo, Nonmammalian / cytology, Mice, Embryo, Nonmammalian/cytology, MESH: Gene Expression Regulation, Developmental, Testis, Missense mutation, MESH: Animals, ddc:576.5, Developmental, Disorders of sex development, 10. No inequality, MESH: Ubiquitin-Protein Ligases / genetics, Exome sequencing, Cells, Cultured, beta Catenin, Zebrafish, Multidisciplinary, Cultured, MESH: Thrombospondins / genetics, MESH: SOX9 Transcription Factor / metabolism, Wnt signaling pathway, Gene Expression Regulation, Developmental, SOX9 Transcription Factor, beta Catenin/antagonists & inhibitors, MESH: beta Catenin / metabolism, Sex reversal, MESH: Gonads / pathology, Cell biology, MESH: Young Adult, Embryo, MESH: Gonads / metabolism, Female, MESH: Cells, Cultured, Adult, Adolescent, Ubiquitin-Protein Ligases, Cells, Thrombospondins/genetics, Mutation, Missense, MESH: beta Catenin / antagonists & inhibitors, SOX9, Biology, Gonads/metabolism, MESH: Embryo, Nonmammalian / metabolism, 03 medical and health sciences, Young Adult, MESH: Wnt Proteins / genetics, WNT signaling, medicine, Animals, Humans, Ubiquitin-Protein Ligases/genetics, MESH: SOX9 Transcription Factor / genetics, MESH: Zebrafish, RSPO1, Gonads, General, MESH: Mice, Nonmammalian/cytology, MESH: Disorders of Sex Development / pathology, MESH: Adolescent, MESH: Wnt Proteins / antagonists & inhibitors, MESH: Mutation, Missense, MESH: Humans, MESH: beta Catenin / genetics, ZNRF3, MESH: Adult, Sex determination, medicine.disease, MESH: Male, MESH: Ubiquitin-Protein Ligases / physiology, SOX9 Transcription Factor/genetics, Wnt Proteins, 030104 developmental biology, MESH: Thrombospondins / metabolism, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Gene Expression Regulation, Mutation, Testis/metabolism, Missense, Thrombospondins, MESH: Female, Function (biology), Disorders of Sex Development/genetics, MESH: Sex Differentiation
Abstract

International audience; Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-β-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/β-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Published
2018

18. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells

Author

Richard J. Griffeth, Isabelle Stévant, François P. Pralong, Jean-Luc Pitetti, Françoise Kühne, Meng-Chun Hu, Silvana A. Andric, Yasmine Neirijnck, Lee B. Smith, Serge Nef, Karen R. Kilcoyne, Pierre Calvel, Université de Genève (UNIGE), Génétique Animale et Biologie Intégrative (GABI), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Université Paris Saclay (COmUE), University of Edinburgh, University of Novi Sad, National Taiwan University, Department of Internal Medicine, University Hospital, School of Environmental and Life Sciences, and Newcastle University [Newcastle]

Subjects
0301 basic medicine, Male, steroidogenesis, medicine.medical_treatment, Cellular differentiation, [SDV]Life Sciences [q-bio], MOUSE, Biochemistry, testis development, Mice, 0302 clinical medicine, ddc:576.5, Testosterone, Receptor, Mice, Knockout, TRANSGENIC MICE, Leydig cell, TESTIS, Stem Cells, PROLIFERATION, Leydig Cells, Cell Differentiation, ADRENAL-GLAND, medicine.anatomical_structure, DIFFERENTIATION, Biotechnology, EXPRESSION, medicine.medical_specialty, endocrine system, Biology, MATURATION, 03 medical and health sciences, Growth factor receptor, progenitor Leydig cells, Internal medicine, GROWTH-FACTOR-I, Genetics, medicine, Animals, IGF, Molecular Biology, Insulin-like growth factor 1 receptor, adrenal gland, [SDV.GEN]Life Sciences [q-bio]/Genetics, Insulin, Receptors, Somatomedin, Receptor, Insulin, Insulin receptor, [SDV.GEN.GA]Life Sciences [q-bio]/Genetics/Animal genetics, 030104 developmental biology, Endocrinology, PROGENITOR CELLS, biology.protein, Adrenal Cortex, Corticosterone, 030217 neurology & neurosurgery
Abstract

The insulin family of growth factors (insulin, IGF1, and IGF2) are critical in sex determination, adrenal differentiation, and testicular function. Notably, the IGF system has been reported to mediate the proliferation of steroidogenic cells. However, the precise role and contribution of the membrane receptors mediating those effects, namely, insulin receptor (INSR) and type-I insulin-like growth factor receptor (IGF1R), have not, to our knowledge, been investigated. We show here that specific deletion of both Insr and Igf1r in steroidogenic cells in mice leads to severe alterations of adrenocortical and testicular development. Double-mutant mice display drastic size reduction of both adrenocortex and testes, with impaired corticosterone, testosterone, and sperm production. Detailed developmental analysis of the testes revealed that fetal Leydig cell (LC) function is normal, but there is a failure of adult LC maturation and steroidogenic function associated with accumulation of progenitor LCs (PLCs). Cell-lineage tracing revealed PLC enrichment is secondary to Insr and Igf1r deletion in differentiated adult LCs, suggesting a feedback mechanism between cells at different steps of differentiation. Taken together, these data reveal the cell-autonomous and nonautonomous roles of the IGF system for proper development and maintenance of steroidogenic lineages.-Neirijnck, Y., Calvel, P., Kilcoyne, K. R., Kühne, F., Stévant, I., Griffeth, R. J., Pitetti, J.-L., Andric, S. A., Hu, M.-C., Pralong, F., Smith, L. B., Nef, S. Insulin and IGF1 receptors are essential for the development and steroidogenic function of adult Leydig cells.

Published
2018

19. Protection Against XY Gonadal Sex Reversal by a Variant Region on Mouse Chromosome 13

Author

Madeleine Pope, Richard Reeves, Serge Nef, Isabelle Stévant, Nick Warr, Michelle Simon, Andy Greenfield, Ann-Marie Mallon, Sara Wells, and Catherine Livermore

Subjects
Male, Congenic, Disorders of Sex Development, Biology, testis, Y chromosome, ovotestis, 03 medical and health sciences, Mice, Genetics, Animals, Humans, ddc:576.5, Gonads, 030304 developmental biology, Chromosome 13, Gonadal Dysgenesis, 46,XY, 0303 health sciences, Genome, Ovotestis, Chromosomes, Human, Pair 13, 030305 genetics & heredity, Ovary, Chromosome, Gene Expression Regulation, Developmental, Nuclear Proteins, Sex reversal, Sex Determination Processes, Sex determination, Phenotype, DNA-Binding Proteins, Mice, Inbred C57BL, Genetics of Sex, Primary sex determination, Female, Transcription Factors
Abstract

XY C57BL/6J (B6) mice harboring a Mus musculus domesticus-type Y chromosome (YPOS), known as B6.YPOS mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.YPOS males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.YPOS colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.YPOS sex reversal. Here. we show that a B6.YPOS colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call Mod13, protects against B6.YPOS sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss Mod13’s influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.YPOS sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony.

Published
2019

20. Single cell transcriptomics reveal temporal dynamics of critical regulators of germ cell fate during mouse sex determination

Author

Chloé Mayère, Yasmine Neirijnck, Pauline Sararols, Chris M Rands, Isabelle Stévant, Françoise Kühne, Anne-Amandine Chassot, Marie-Christine Chaboissier, Emmanouil T. Dermitzakis, and Serge Nef

Subjects
endocrine system, medicine.anatomical_structure, Meiosis, Somatic cell, Gene expression, medicine, Gene regulatory network, Biology, Embryonic stem cell, Gene, Germ cell, Cell biology, Sexual reproduction
Abstract

SummaryDespite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single-cell RNA sequencing of 14,914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the Bone morphogenic protein (BMP) and Nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex-specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex-specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data is available on a web platform and provides a molecular roadmap of GC sex determination at single-cell resolution, which will serve as a valuable resource for future studies of gonad development, function and disease.

Published
2019

21. Dissecting Cell Lineage Specification and Sex Fate Determination in Gonadal Somatic Cells Using Single-Cell Transcriptomics

Author

Marie-Christine Chaboissier, Andy Greenfield, Isabelle Stévant, Françoise Kühne, Emmanouil T. Dermitzakis, Serge Nef, Institut de Biologie Valrose (IBV), Université Nice Sophia Antipolis (... - 2019) (UNS), and COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Male, Somatic cell, Granulosa cell, Sertoli cells, sex determination, gonad development, Lineage specification, Transcriptome, Mice, lineage specification, transcriptomics, 0302 clinical medicine, ddc:590, Testis, ddc:576.5, lcsh:QH301-705.5, [SDV.BDD]Life Sciences [q-bio]/Development Biology, education.field_of_study, single-cell RNA-seq, differentiation, Sertoli cell, granulosa cell, Cell biology, medicine.anatomical_structure, Differentiation, Female, supporting cells, Single-Cell Analysis, endocrine system, Gonad, Population, Progenitors, Mice, Transgenic, Cell fate determination, Biology, testis, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Supporting cells, medicine, Animals, Cell Lineage, Progenitor cell, Transcriptomics, education, Cell fate decision, Single-cell RNA-seq, progenitors, Granulosa Cells, Gonad development, Gene Expression Profiling, Ovary, Sex determination, Sex Determination Processes, 030104 developmental biology, lcsh:Biology (General), gene expression, cell fate decision, ovary, Gene expression, 030217 neurology & neurosurgery
Abstract

Summary: Sex determination is a unique process that allows the study of multipotent progenitors and their acquisition of sex-specific fates during differentiation of the gonad into a testis or an ovary. Using time series single-cell RNA sequencing (scRNA-seq) on ovarian Nr5a1-GFP+ somatic cells during sex determination, we identified a single population of early progenitors giving rise to both pre-granulosa cells and potential steroidogenic precursor cells. By comparing time series single-cell RNA sequencing of XX and XY somatic cells, we provide evidence that gonadal supporting cells are specified from these early progenitors by a non-sex-specific transcriptomic program before pre-granulosa and Sertoli cells acquire their sex-specific identity. In XX and XY steroidogenic precursors, similar transcriptomic profiles underlie the acquisition of cell fate but with XX cells exhibiting a relative delay. Our data provide an important resource, at single-cell resolution, for further interrogation of the molecular and cellular basis of mammalian sex determination. : Using single-cell RNA sequencing of Nr5a1-expressing gonadal somatic cells during female and male sex determination, Stévant et al. deconvoluted the cell lineage specification process and sex-specific differentiation of both the supporting and the steroidogenic cell lineages at a transcriptomic level. Keywords: single-cell RNA-seq, sex determination, ovary, testis, granulosa cell, Sertoli cells, supporting cells, progenitors, differentiation, lineage specification, cell fate decision, gonad development, gene expression, transcriptomics

Published
2019

22. The ReproGenomics Viewer: a multi-omics and cross-species resource compatible with single-cell studies for the reproductive science community

Author

Nathan Alary, Isabelle Stévant, Aurélie Lardenois, Thomas A Darde, Estelle Lecluze, Frank Tüttelmann, Frédéric Chalmel, Bernard Jégou, Antoine Rolland, Serge Nef, Olivier Collin, Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Genève = University of Geneva (UNIGE), Westfälische Wilhelms-Universität Münster = University of Münster (WWU), Plateforme bioinformatique GenOuest [Rennes], Université de Rennes (UR)-Plateforme Génomique Santé Biogenouest®-Inria Rennes – Bretagne Atlantique, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-GESTION DES DONNÉES ET DE LA CONNAISSANCE (IRISA-D7), Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Service Expérimentation et Développement (SED [Rennes]), Inria Rennes – Bretagne Atlantique, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), École des Hautes Études en Santé Publique [EHESP] (EHESP), n°18-CE14-0038-02, Agence Nationale de la Recherche, FRM n° DBI20131228558, Fondation pour la Recherche Médicale, SNF n° CRS115_171007, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, EST-17-256, French agency, CRU 326, Genes to Function, French National Institute of Health and Medical Research, French School of Public Health, ANR-16-CE14-0017,ARESSERC,DECOUVRIR LES VOIES NON-CANONIQUES DE SIGNALISATION RELAYEES PAR L'ACIDE RETINOÏQUE IN VIVO: LA CELLULE DE SERTOLI COMME MODELE D'ETUDE(2016), ANR-18-CE14-0038,MEIOSIL,Dynamique transcripionnelle lors de l'entrée en méiose(2018), Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Genève (UNIGE), University of Münster, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Plateforme Génomique Santé Biogenouest®-GESTION DES DONNÉES ET DE LA CONNAISSANCE (IRISA-D7), Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-CentraleSupélec-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), and Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1)

Subjects
Statistics and Probability, Source code, Databases, Factual, Computer science, media_common.quotation_subject, Genomics, JavaScript, Biochemistry, World Wide Web, Transcriptome, 03 medical and health sciences, 0302 clinical medicine, Software, ddc:576.5, Molecular Biology, 030304 developmental biology, computer.programming_language, media_common, 0303 health sciences, Cell studies, 030219 obstetrics & reproductive medicine, [INFO.INFO-DB]Computer Science [cs]/Databases [cs.DB], Sequence Analysis, RNA, business.industry, Computational Biology, Python (programming language), Computer Science Applications, Computational Mathematics, Computational Theory and Mathematics, Multi omics, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], business, computer
Abstract

MotivationRecent advances in transcriptomics have enabled unprecedented insight into gene expression analysis at a single-cell resolution. While it is anticipated that the number of publications based on such technologies will increase in the next decade, there is currently no public resource to centralize and enable scientists to explore single-cell datasets published in the field of reproductive biology.ResultsHere, we present a major update of the ReproGenomics Viewer, a cross-species and cross-technology web-based resource of manually-curated sequencing datasets related to reproduction. The redesign of the ReproGenomics Viewer's architecture is accompanied by significant growth of the database content including several landmark single-cell RNA-sequencing datasets. The implementation of additional tools enables users to visualize and browse the complex, high-dimensional data now being generated in the reproductive field.Availability and implementationThe ReproGenomics Viewer resource is freely accessible at http://rgv.genouest.org. The website is implemented in Python, JavaScript and MongoDB, and is compatible with all major browsers. Source codes can be downloaded from https://github.com/fchalmel/RGV.Supplementary informationSupplementary data are available at Bioinformatics online.

Published
2019

23. Characterizing the bipotential mammalian gonad

Author

Serge, Nef, Isabelle, Stévant, and Andy, Greenfield

Subjects
Male, endocrine system, Sex Differentiation, urogenital system, Organogenesis, Ovary, Sex Determination Processes, Testis, Animals, Humans, Cell Lineage, Female, ddc:576.5, Gonads
Abstract

Primary sex determination is the decision by which the bipotential embryonic gonad commits to either the testicular or ovarian fate. The developing gonad constitutes a unique paradigm for the study of lineage specification, cell fate commitment and the exploration of how distinct cell populations diverge from multipotent progenitors. After the separation of the adreno-gonadal primordium into two distinct primordia, somatic progenitor cells of the gonadal primordium undergo several cell fate decisions and sex-specific cell differentiation. The specification of the supporting and steroidogenic cell lineages into either Sertoli and Leydig cells in the testis, or granulosa and theca cells in the ovary is essential for germ cell development and endocrine function of the gonads. In this review, we focus on the early events leading to gonad formation, including the identity of gonadal progenitors, the genetic networks involved in cell lineage specification, the mutual antagonism of the pro-testis and pro-ovary networks and the importance of timing of developmental events orchestrating testis and ovary development. We discuss, and put into perspective, a number of experiments performed in mice or humans that have shed light on sex-determining mechanisms and, where possible, the clinical significance and limitations of such model organism data.

Published
2019

24. Characterizing the bipotential mammalian gonad

Author

Isabelle Stévant, Serge Nef, and Andy Greenfield

Subjects
endocrine system, 0303 health sciences, Gonad, urogenital system, Somatic cell, Cellular differentiation, Granulosa cell, Cell fate determination, Biology, Cell biology, Cell fate commitment, 03 medical and health sciences, medicine.anatomical_structure, medicine, Primary sex determination, Germ cell, 030304 developmental biology
Abstract

Primary sex determination is the decision by which the bipotential embryonic gonad commits to either the testicular or ovarian fate. The developing gonad constitutes a unique paradigm for the study of lineage specification, cell fate commitment and the exploration of how distinct cell populations diverge from multipotent progenitors. After the separation of the adreno-gonadal primordium into two distinct primordia, somatic progenitor cells of the gonadal primordium undergo several cell fate decisions and sex-specific cell differentiation. The specification of the supporting and steroidogenic cell lineages into either Sertoli and Leydig cells in the testis, or granulosa and theca cells in the ovary is essential for germ cell development and endocrine function of the gonads. In this review, we focus on the early events leading to gonad formation, including the identity of gonadal progenitors, the genetic networks involved in cell lineage specification, the mutual antagonism of the pro-testis and pro-ovary networks and the importance of timing of developmental events orchestrating testis and ovary development. We discuss, and put into perspective, a number of experiments performed in mice or humans that have shed light on sex-determining mechanisms and, where possible, the clinical significance and limitations of such model organism data.

Published
2019

25. Genetic Control of Gonadal Sex Determination and Development

Author

Isabelle Stévant and Serge Nef

Subjects
Male, endocrine system, Cellular differentiation, Organogenesis, Cell, Embryonic Development, Cell fate determination, Biology, Transcriptome, Cell fate commitment, 03 medical and health sciences, 0302 clinical medicine, Testis, Genetics, medicine, Animals, Humans, Cell Lineage, Epigenetics, Progenitor cell, Gonads, 030304 developmental biology, 0303 health sciences, urogenital system, Ovary, Gene Expression Regulation, Developmental, Cell Differentiation, Sex Determination Processes, Sertoli cell, medicine.anatomical_structure, Phenotype, Evolutionary biology, Female, 030217 neurology & neurosurgery
Abstract

Sex determination is the process by which the bipotential gonads develop as either testes or ovaries. With two distinct potential outcomes, the gonadal primordium offers a unique model for the study of cell fate specification and how distinct cell populations diverge from multipotent progenitors. This review focuses on recent advances in our understanding of the genetic programs and epigenetic mechanisms that regulate gonadal sex determination and the regulation of cell fate commitment in the bipotential gonads. We rely primarily on mouse data to illuminate the complex and dynamic genetic programs controlling cell fate decision and sex-specific cell differentiation during gonadal formation and gonadal sex determination.

Published
2018

26. Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex

Author

Isabelle Stévant, Julien Prados, Emmanouil T. Dermitzakis, Serge Nef, Subashika Govindan, Denis Jabaudon, Alexandre Dayer, and Ludovic Telley

Subjects
Male, 0301 basic medicine, Transcription, Genetic, Cerebral Ventricles/cytology/embryology, Neocortex, Bioinformatics, Green Fluorescent Proteins/genetics, Cerebral Ventricles, Transcriptome, ddc:616.89, Mice, Neurons/cytology, 0302 clinical medicine, Neural Stem Cells, Basic Helix-Loop-Helix Transcription Factors, Nerve Tissue Proteins/genetics, ddc:576.5, Neurons, Neuropeptides/genetics, Multidisciplinary, Neurogenesis, Neural stem cell, DNA-Binding Proteins, Corticogenesis, medicine.anatomical_structure, Excitatory postsynaptic potential, Female, Green Fluorescent Proteins, Nerve Tissue Proteins, GPI-Linked Proteins/genetics, Biology, GPI-Linked Proteins, 03 medical and health sciences, medicine, Animals, Progenitor cell, Progenitor, Basic Helix-Loop-Helix Transcription Factors/genetics, SOXB1 Transcription Factors, Neuropeptides, Neural Stem Cells/cytology, Neocortex/cytology/embryology, ddc:616.8, 030104 developmental biology, nervous system, SOXB1 Transcription Factors/genetics, Neurogenesis/genetics, T-Box Domain Proteins, Neuroscience, DNA-Binding Proteins/genetics, 030217 neurology & neurosurgery
Abstract

Tracking neuronal transcriptional programs Early in brain development, cortical neurons are born near the ventricles, then migrate to their functional destinations. Telley et al. used a fluorescent labeling technique to see what transcripts characterize these earliest stages of neural development. Waves of transcriptional programs are initiated, then passed by as the neuron progresses from proliferative to migratory and finally to connectivity phases. Science , this issue p. 1443

Published
2016

27. Single-cell transcriptomics of the mouse gonadal soma reveals the establishment of sexual dimorphism in distinct cell lineages

Author

Marie-Christine Chaboissier, Françoise Kühne, Isabelle Stévant, Emmanouil T. Dermitzakis, Andy Greenfield, and Serge Nef

Subjects
endocrine system, education.field_of_study, Gonad, Somatic cell, Population, Cell, Cell fate determination, Biology, Sertoli cell, Cell biology, Transcriptome, medicine.anatomical_structure, medicine, Progenitor cell, education
Abstract

SummarySex determination is a unique process that allows the study of multipotent progenitors and their acquisition of sex-specific fates during differentiation of the gonad into a testis or an ovary. Using time-series single-cell RNA sequencing (scRNA-seq) on ovarian Nr5a1-GFP+ somatic cells during sex determination, we identified a single population of early progenitors giving rise to both pre-granulosa cells and potential steroidogenic precursor cells. By comparing time-series scRNA-seq of XX and XY somatic cells, we demonstrate that the supporting cells emerge from the early progenitors with a non-sex-specific transcriptomic program, before pre-granulosa and Sertoli cells acquire their sex-specific identity. In XX and XY steroidogenic precursors similar transcriptomic profiles underlie the acquisition of cell fate, but with a delay in XX cells. Our data provide a novel framework, at single-cell resolution, for further interrogation of the molecular and cellular basis of mammalian sex determination.

Published
2018

28. Research Resource: The Dynamic Transcriptional Profile of Sertoli Cells During the Progression of Spermatogenesis

Author

Isabelle Stévant, Béatrice Conne, Pierre Calvel, Bernard Jégou, Céline Zimmermann, Henrik Kaessmann, Frédéric Chalmel, Jean-Luc Pitetti, Christelle Borel, Serge Nef, Department of Genetic Medicine and Development [Geneva], Université de Genève (UNIGE), Ecole Médicale, Département de Médecine Génétique et Développement, Center for Integrative Genomics - Institute of Bioinformatics, Génopode (CIG), Swiss Institute of Bioinformatics [Lausanne] (SIB), Université de Lausanne (UNIL)-Université de Lausanne (UNIL), École des Hautes Études en Santé Publique [EHESP] (EHESP), Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Groupe d'Etude de la Reproduction Chez l'Homme et les Mammiferes (GERHM), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Genève = University of Geneva (UNIGE), Université de Lausanne = University of Lausanne (UNIL)-Université de Lausanne = University of Lausanne (UNIL), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), and Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, Aryl hydrocarbon receptor nuclear translocator, Somatic cell, [SDV]Life Sciences [q-bio], Retinoic acid, Mice, Transgenic, Biology, Transcriptome, Mice, chemistry.chemical_compound, Endocrinology, Germ cell proliferation, medicine, Research Resource, Animals, ddc:576.5, Spermatogenesis, Molecular Biology, Transcription factor, Genetics, Spermatogenesis/genetics, Sertoli Cells, Gene Expression Profiling, Alternative splicing, General Medicine, Seminiferous Tubules, Sertoli cell, medicine.anatomical_structure, chemistry, Sertoli Cells/metabolism, Seminiferous Tubules/metabolism
Abstract

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

Published
2015

29. Single cell transcriptome sequencing: A new approach for the study of mammalian sex determination

Author

Isabelle Stévant and Serge Nef

Subjects
0301 basic medicine, Mammals, Sequence Analysis, RNA, Context (language use), Computational biology, Biology, Sex Determination Processes, Biochemistry, Transcriptome, 03 medical and health sciences, 030104 developmental biology, Endocrinology, Single-cell analysis, Cellular heterogeneity, Single cell transcriptome, Sex Determination Process, Animals, Humans, ddc:576.5, Identification (biology), Experimental methods, Single-Cell Analysis, Gonads, Molecular Biology
Abstract

Mammalian sex determination is a highly complex developmental process that is particularly difficult to study due to the limited number of gonadal cells present at the bipotential stage, the large cellular heterogeneity in both testis and ovaries and the rapid sex-dependent differentiation processes. Single-cell RNA-sequencing (scRNA-seq) circumvents the averaging artifacts associated with methods traditionally used to profile bulk populations of cells. It is a powerful tool that allows the identification and classification of cell populations in a comprehensive and unbiased manner. In particular, scRNA-seq enables the tracing of cells along developmental trajectories and characterization of the transcriptional dynamics controlling their differentiation. In this review, we describe the current state-of-the-art experimental methods used for scRNA-seq and discuss their strengths and limitations. Additionally, we summarize the multiple key insights that scRNA-seq has provided to the understanding of mammalian sex determination. Finally, we briefly discuss the future of this technology, as well as complementary applications in single cell -omics in the context of mammalian sex determination.

Published
2017

30. First landscape of binding to chromosomes for a domesticated mariner transposase in the human genome: diversity of genomic targets of SETMAR isoforms in two colorectal cell lines

Author

Serge Guyetant, Catherine Belleannée, Aymeric Antoine-Lorquin, Martine Batailler, Alban Girault, Thierry Lecomte, Sassan Asgari, Vincent Coustham, Solenne Bire, Isabelle Stévant, Benoît Piégu, Nicolas Buisine, Bruno Pitard, Linda Beauclair, Yves Bigot, Ahmed Arnaoty, Dynamics, Logics and Inference for biological Systems and Sequences (Dyliss), Inria Rennes – Bretagne Atlantique, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-GESTION DES DONNÉES ET DE LA CONNAISSANCE (IRISA-D7), Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Rennes (UR)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Institut de Recherche en Informatique et Systèmes Aléatoires (IRISA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT), Université de Rennes (UR), Génétique, immunothérapie, chimie et cancer (GICC), UMR 7292 CNRS [2012-2017] (GICC UMR 7292 CNRS), Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), University of Queensland [Brisbane], Laboratoire de Physiologie de la Reproduction, Institut National de la Recherche Agronomique (INRA), LBTM, Institut de Biotechnologie, Ecole Polytechnique Fédérale de Lausanne (EPFL), Evolution des régulations endocriniennes (ERE), Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Unité de recherche de l'institut du thorax (ITX-lab), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-Institut National de Recherche en Informatique et en Automatique (Inria)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Rennes 1 (UR1), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Bretagne Sud (UBS)-École normale supérieure - Rennes (ENS Rennes)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Université de Rennes (UNIV-RENNES), Université de Tours-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National de Recherche en Informatique et en Automatique (Inria)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-CentraleSupélec-IMT Atlantique Bretagne-Pays de la Loire (IMT Atlantique), Institut Mines-Télécom [Paris] (IMT)-Institut Mines-Télécom [Paris] (IMT)-Université de Bretagne Sud (UBS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), and Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1)

Subjects
Gene isoform, Genetics, 0303 health sciences, Genomics, Biology, ENCODE, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, chemistry, Cell culture, 030220 oncology & carcinogenesis, Human genome, MADE1, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Hsmar1, Gene, DNA, Transposase, 030304 developmental biology
Abstract

Setmar is a 3-exons gene coding a SET domain fused to a Hsmar1 transposase. Its different transcripts theoretically encode 8 isoforms with SET moieties differently spliced. In vitro, the largest isoform binds specifically to Hsmar1 DNA ends and with no specificity to DNA when it is associated with hPso4. In colon cell lines, we found they bind specifically to two chromosomal targets depending probably on the isoform, Hsmar1 ends and sites with no conserved motifs. We also discovered that the isoforms profile was different between cell lines and patient tissues, suggesting the isoforms encoded by this gene in healthy cells and their functions are currently not investigated.

Published
2017

31. Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements

Author

Mojca Tajnik, Alejandro Reyes, Isabelle Stévant, Inigo Martincorena, Sebastian Eustermann, Kathi Zarnack, Jernej Ule, Julian König, Simon Anders, and Nicholas M. Luscombe

Subjects
Alu element, Biology, General Biochemistry, Genetics and Molecular Biology, Evolution, Molecular, Transcriptome, 03 medical and health sciences, Splicing factor, Exon, 0302 clinical medicine, Alu Elements, Splicing Factor U2AF, Humans, Immunoprecipitation, 030304 developmental biology, Genetics, 0303 health sciences, Sequence Analysis, RNA, Biochemistry, Genetics and Molecular Biology(all), Gene Expression Profiling, Heterogeneous-Nuclear Ribonucleoprotein Group C, High-Throughput Nucleotide Sequencing, Nuclear Proteins, Exons, Ribonucleoproteins, Gene Knockdown Techniques, Human genome, RNA Splice Sites, ICLIP, 030217 neurology & neurosurgery, HeLa Cells, Minigene
Abstract

SummaryThere are ∼650,000 Alu elements in transcribed regions of the human genome. These elements contain cryptic splice sites, so they are in constant danger of aberrant incorporation into mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about the molecular mechanisms preventing their inclusion. Here, we present a mechanism for protecting the human transcriptome from the aberrant exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites. Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt transcript function. Minigene experiments explain disease-associated mutations in Alu elements that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a critical role as a genome-wide sentinel protecting the transcriptome. The findings have important implications for human evolution and disease.

Published
2013

32. Loss of function mutation in the palmitoyl-transferase HHAT leads to syndromic 46,XY disorder of sex development by impeding Hedgehog protein palmitoylation and signaling

Author

Stylianos E. Antonarakis, Christel Thauvin-Robinet, Marilyn D. Resh, Laurence Faivre, Sandy Lambert, Isabelle Stévant, Christèle Desdoits-Lethimonier, Hiroshi Kurosaka, Paul A. Trainor, Federico Santoni, Christelle Borel, Bernard Jégou, Armine Matevossian, Antoine Rolland, Serge Nef, Frédéric Huet, Pierre Calvel, Françoise Kühne, Jadwiga Jaruzelska, Béatrice Conne, Patrick Callier, Séverine Mazaud-Guittot, Periklis Makrythanasis, Pascal Bernard, Céline Zimmermann, Michel Guipponi, Anne Vannier, Dagmar Wilhelm, Francine Mugneret, Laboratoire de cytogénétique (CHU de Dijon), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Génétique des Anomalies du Développement (GAD), IFR100 - Structure fédérative de recherche Santé-STIC-Université de Bourgogne (UB), Department of Genetic Medicine and Development [Geneva], Université de Genève (UNIGE), Monash University [Clayton], Department of Experimental Cardiology, Academic Medical Center - Academisch Medisch Centrum [Amsterdam] (AMC), University of Amsterdam [Amsterdam] (UvA)-University of Amsterdam [Amsterdam] (UvA)-Heart Failure Research Center (HFRC), Centre de génétique - Centre de référence des maladies rares, anomalies du développement et syndromes malformatifs (CHU de Dijon), Environnement viral et chimique & reproduction, Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Service de Biologie Moléculaire, EHESP-ARENES (EHESP-ARENES), École des Hautes Études en Santé Publique [EHESP] (EHESP), Groupe d'Etude de la Reproduction Chez l'Homme et les Mammiferes (GERHM), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand ( CHU Dijon ), Génétique des Anomalies du Développement ( GAD ), IFR100 - Structure fédérative de recherche Santé-STIC-Université de Bourgogne ( UB ), Department of Genetic Medicine and Development, University of Geneva Medical School, Academic Medical Center [Amsterdam] ( AMC ), University of Amsterdam [Amsterdam] ( UvA ) -University of Amsterdam [Amsterdam] ( UvA ) -Heart Failure Research Center (HFRC), Université de Genève ( UNIGE ), Institut de recherche, santé, environnement et travail ( Irset ), Université d'Angers ( UA ) -Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -École des Hautes Études en Santé Publique [EHESP] ( EHESP ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ) -Université des Antilles ( UA ) -Université d'Angers ( UA ) -Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -École des Hautes Études en Santé Publique [EHESP] ( EHESP ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ) -Université des Antilles ( UA ), Virus, Environnement et Reproduction, Groupe d'Etude de la Reproduction Chez l'Homme et les Mammiferes ( GERHM ), Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -IFR140-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -IFR140-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Department of Genetic Medicine and Development, University of Geneva Medical School-University of Geneva Medical School, Université de Bourgogne (UB)-IFR100 - Structure fédérative de recherche Santé-STIC, Université de Genève = University of Geneva (UNIGE), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), and Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, Cancer Research, [SDV]Life Sciences [q-bio], medicine.disease_cause, Cell Fate Determination, Mice, Testis, Morphogenesis, Missense mutation, ddc:576.5, Genetics (clinical), Mutation, Homozygote, Cell Differentiation, Hedgehog signaling pathway, Pedigree, Cell biology, Female, Signal transduction, Signal Transduction, Research Article, medicine.medical_specialty, lcsh:QH426-470, Lipoylation, Molecular Sequence Data, Mutation, Missense, Biology, Palmitoylation, HHAT, Internal medicine, Genetics, medicine, Animals, Humans, Hedgehog Proteins, Amino Acid Sequence, Molecular Biology, Hedgehog, Ecology, Evolution, Behavior and Systematics, Disorder of Sex Development, 46,XY, [ SDV ] Life Sciences [q-bio], Sequence Homology, Amino Acid, Biology and Life Sciences, Sex Determination, lcsh:Genetics, Endocrinology, 46, XY Disorders of Sex Development/*genetics, Acyltransferases/chemistry/*genetics/metabolism, Hedgehog Proteins/*metabolism, Lipoylation/*genetics, Signal Transduction/*genetics, Testis/embryology, Lipid modification, Acyltransferases, Developmental Biology
Abstract

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development., Author Summary Disorders of gonadal development represent a clinically and genetically heterogeneous class of DSD caused by defects in gonadal development and/or a failure of testis/ovarian differentiation. Unfortunately, in many cases the genetic aetiology of DSD is unknown, indicating that our knowledge of the factors mediating sex determination is limited. Using exome sequencing on a case of autosomal recessive syndromic 46,XY DSD with testicular dysgenesis and chondrodysplasia, we found a homozygous missense mutation (G287V) within the coding sequence of the O-acetyl-transferase HHAT gene. The HHAT gene encodes an enzyme required for the attachment of palmitoyl residues that are critical for multimerization and long range signaling potency of hedgehog secreted proteins. We found that HHAT is widely expressed in human organs during fetal development, including testes and ovaries around the time of sex determination. In vitro assays show that G287V mutation impairs HHAT palmitoyl-transferase activity and mice lacking functional Hhat exhibit testicular dysgenesis as well as other skeletal, neuronal and growth defects that recapitulate most aspects of the syndromic 46,XY DSD patient. These data provide the first clinical evidence of the essential role played by lipid modification of Hedgehog proteins in human testicular organogenesis and embryonic development.

Published
2014

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