1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 ReproSciences

2015

3 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Cette réunion n’aurait pu être organisée sans le soutien d’un certain nombre d’organismes et d’Instituts. Nous les remercions très sincèrement.

GDR 3606 Repro. Université de Rennes 1. Rennes Métropole. Institut National de la Recherche Agronomique. Institut de Recherche en Santé Environnement et Travail (IRSET, U1085) Structure fédérative de recherche Biosit. Université Européenne de Bretagne. Centre National de la Recherche Scientifique.

Institut de recherche sur la santé l’environnement et le travail

4 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril 2015 Bienvenue à Rennes Au nom du comité scientifique et du comité local d’organisation, c’est avec grand plaisir que nous vous accueillons à Rennes à l’occasion des premières journées du GDR 3606 REPRO organisées sur le campus de Beaulieu de l’Université de Rennes 1. ReproSciences 2015 est la première édition d’un événement appelé à être réorganisé tous les deux ans

dans d’autres villes françaises. L’objectif est de rassembler une communauté française parfois trop 2015 Rennes du 13 au 15 avril 15 au 13 du Rennes dispersée avec pour ambition de stimuler les discussions et les interactions entre les différents acteurs de la recherche dans le domaine de la reproduction. Avec l’aide d’un comité scientifique national extrêmement large, nous nous sommes efforcés d’établir un programme aussi riche que possible et reflétant les différentes facettes de la fonction physiologique qui nous rassemble. Le domaine est particulièrement riche et nous voudrions ici remercier tous ceux qui nous ont aidé dans cette tâche. Nous remercions également tous ceux qui ont accepté de prendre la parole et dire à ceux qui n’auront pas le privilège de nous présenter leur travail qu’il y aura d’autres occasions. Compte tenu de la situation financière des organismes, nous avons choisi d’organiser la réunion avec le souci d’épargner au maximum les crédits du GDR, de manière à pouvoir développer d’autres actions. Nous avons fait le choix de privilégier la participation des jeunes chercheurs en leur offrant l’inscription gratuite et des bourses de voyage. Par ailleurs, une session “jeunes chercheurs” avec des présentations flash a été incorporée au programme qui, d’autre part, laisse de larges espaces de discussions devant les posters. L’organisation de ces journées n’aurait pas été possible sans le soutien financier de l’Université de Rennes 1, de Rennes Métropole, du CNRS, de l’INRA, de l’INRIA, de l’Université Européenne de Bretagne, de l’IRSET, de la SFR Biosit et du GDR Repro. Qu’ils en soient très sincèrement remerciés. Notre gratitude va également à tous ceux qui, à des titres divers, se sont impliqués dans l’organisation de cette réunion. Le comité d’organisation 5 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

6 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril 2015 avril SOMMAIRE

2015 Rennes du 13 au 15 au 13 du Rennes

Présentation ------Page 4

Les comités ------Page 7

Quelques Informations Générales ------Page 10

Programme détaillé ------Page 22

Résumés des communications. ------Page 28

Liste des auteurs ------Page 87

7 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril 2015

Comité Scientifique National

Isabelle Allemand Gabriel Livera Julien Bobe Micheline Misrahi Xavier Bonnefont Danielle Monniaux

2015 François Brion Eric Pailhoux Rennes du 13 au 15 avril 15 au 13 du Rennes Thierry Charlier Catherine Patrat Frédérique Clément Vincent Prévot Joëlle Cohen-Tannoudji Celia Ravel Yves Combarnous Eric Reiter Corinne Cotinot Sophie Rousseaux Nathalie Dejucq-Rainsford Olivier Sandra Nicolas de Roux Valérie Simonneaux Nathalie di Clemente Hervé Tostivint Sylvie Dufour Daniel Vaiman Anne Duittoz François Vialard Joëlle Dupont Catherine Viguié Stéphane Fabre Patricia Fauque Pascal Favrel Florian Guillou René Habert Hélène Jammes Bernard Jégou Olivier Kah Catherine Labbé Emmanuel Lemazurier 8 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015 Comité Local d’Organisation

Milissia Ben Maamar (Graphisme) Anne Moreau (Gestion) Blandine Blanchard (Gestion) Catherine Nouyrigat (Gestion) Julien Bobe Jean Louis Novo (Services techniques)

Joel Cano-Nicolau Elisabeth Pellegrini 2015 Frédéric Chalmel Patricia Pilot Rousseau (Secrétariat Rennes du 13 au 15 avril 15 au 13 du Rennes

Thierry Charlier Marylène Sauvée (Gestion des amphis) Pascal Coumailleau Colette Vaillant Nathalie Dejucq-Rainsford Véronique Villalon (Gestion) Jean-Pierre Dussol (Site paiement) Pierre Gaudriault Marie-Madeleine Gueguen Yann Guiguen Bernard Jégou Olivier Kah Catherine Labbé Jean-Jacques Lareyre Florence Le Gac M. Legavre et toute son équipe (Restauration) Laurianne Lesné Christèle Lethimonier-Desdoits Séverine Mazaud-Guittot Rémy Morand (Gestion des amphis)

9 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

10 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril 2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes Informations Générales

11 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril 2015

2015 ReproSciences 2015 se tient à l'université de Rennes 1, sur le Campus de Beaulieu, 263 avenue du Général Rennes du 13 au 15 avril 15 au 13 du Rennes

Leclerc à Rennes les 13, 14 et 15 avril 2015.

Contact: Olivier Kah Research Institute in Health, Environment and Occupation (INSERM U1085) Team NEED, Case 1302. Université de Rennes 1 [email protected]

Patricia Pilot Rousseau (Secrétariat) [email protected]

Enregistrement: Lundi 13 avril 2015 de 11 heures 30 à 13 heures 30 Au club des professeurs (à gauche après le hall de l’entrée principale de l’Université).

12 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Comment venir sur le Campus de Beaulieu ? Rennes du 13 au 15 avril

2015

Le Campus de Beaulieu est situé au 263 avenue du Général Leclerc à Rennes . Arrêt de bus Tournebride. • Coordonnées GPS : 48.115554 ; -1.636807

Le Campus est très facile d’accès :

▪ En bus (réseau STAR) : Environ 10-12 minutes du centre ville: 2015

A partir des arrêts « Place de Bretagne », “République” où “Musée des Beaux Arts”, (Voir plan ci-

Rennes du 13 au 15 avril 15 au 13 du Rennes après) Bus ligne n° 6 , Bus ligne n°C4 et Bus ligne 40 (express)

L’arrêt “Tournebride” est situé juste en face de l’entrée principale du campus de Beaulieu.

Le bâtiment N°2 est juste en face de l’arrêt “Tournebride” à environ 100 mètres de l’autre côté de la rue (Voir plan ci-après)

▪ En voiture: Il y a un grand parking gratuit devant le bâtiment principal. • En vélo: Le Vélo STAR est le système de vélo en libre service proposé par Rennes Métropole : • 900 vélos disponibles 24h/24 et 7j/7 dans 83 stations, positionnées en grande majorité près d’une station de métro, d’un arrêt de bus, de car ou de la gare. • A pied: Si vous voulez vous dégourdir les jambes, vous pouvez marcher et longer la rivière (environ 40 minutes du centre en marchant bien). Très agréable. • Les locaux sont accessibles aux personnes handicapées. • Espace non fumeur. • Accès wifi (Réseau EDUROAM disponible)

13 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Auberge de jeunesse

Campus de Beaulieu, Bât. 2

Centre ville: Zone 100m où se trouvent les hôtels Tournebride

Place de Musée des Bretagne Beaux Arts

République

400m

Gare

Bon à savoir Itinéraire des bus 6 et C4 • Le C4 passe toutes les 8 minutes Arrêts des bus C4 et 6 • Le 40 est un express qui s’arrête à République et Musée des Beaux Arts puis va direct à Tournebride • De la Gare à République il y a un métro. Vous pouvez prendre le bus avec le même ticket

14 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

15 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

16 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

17 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

18 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

19 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

20 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril 2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

21 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 2015

avril

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

22 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

Programme

23 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

LUN DI 13 AVRIL 2015 Après midi

11h30-13h30 Enregistrement et café d’accueil 13h30-13h50 Introduction générale Olivier Kah (Rennes).

Session 1: Modérateurs Corinne Cotinot et François Vialard

13h50-14h20 Julien Bobe (Rennes) “Mécanismes moléculaires impliqués dans la qualité des oeufs de poissons”. 14h20-14h40 Véronique Duranthon (Jouy-en-Josas) “Effets de l’environnement sur l’embryon de Mammifères, conséquences pour le phénotype adulte”. 14h40-15h00 Virginie Maillard (Nouzilly) “Métabolisme lipidique et reproduction femelle: rôle au niveau ovarien”. 15h00-15h20 Samir Hamamah (Montpellier) ‘’The regulation of the human cumulus-oocyte complex: The MicroRNAs ‘’

15h20-16h50 CAFÉ ET POSTERS.

Session 2: Modérateurs Daniel Vaiman et Charlotte Moretti

16h50-17h10 Thierry Fournier (Paris) “Développement du placenta humain: Rôle du récepteur nucléaire PPARg et des gènes cibles”. 17h10-17h30 Olivier Sandra (Jouy en Josas) “De l’existence d’un senseur endométrial chez les mammifères”. 17h30-17h50 Marie-Noëlle Dieudonné (Montigny le Bretonneux) “Rôles du facteur pré-implantatoire (PIF) à l’interface fœto-maternelle”

17h50-18h45 Conférence plénière. Modérateur Hervé Tostivint Pierre-Henri Gouyon (Paris) “Sexe, Conflits et Coopérations” 18h45-21h00 COCKTAIL DÎNATOIRE (Hall)

24 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

MARDI 14 AVRIL 2015 Matin

Session 3: Modérateurs Valérie Simonneaux et Pascal Coumailleau

09h00-09h30 Nicolas de Roux (Paris) “Apport de la génétique dans la compréhension de l’activation neuroendocrine de l’axe gonadotrope de la vie foeœtale à la puberté”. 09h30-09h50 Hervé Tostivint (Paris) “Impact des tétraploïdisations sur le répertoire des hormones reproductrices des vertébrés”. 09h50-10h10 Paolo Giacobini (Lille) “Shaping the reproductive system: lessons from semaphorins” 10h10- 10h30 Paul Klosen (Strasbourg) “Mélatonine et TSH dans le contrôle saisonnier de la reproduction chez les rongeurs”

10h30-11h00 PAUSE CAFÉ

Session 4: Modérateurs Joëlle Cohen-Tannoudji et Antoine D. Rolland

11h00-11h20 Matthieu Keller (Nouzilly) “Contrôle olfactif de la fonction de reproduction.” 11h20-11h40 Denis Tagu (Rennes) “Changer de mode de reproduction: Le cas des pucerons pour l’étude de l’équilibre entre génétique et environnement”. 11h40-12h00 Frédéric Chalmel (Rennes) “Génomique intégrative de la spermatogenèse chez les mammifères. 12h00-12h20 Aurélien Capitan (Paris). “Apports du modèle bovin et des données de génotypage et de séquençage à haut-débit dans la connaissance du contrôle génétique de la fertilité”.

12h20-14h00 BUFFET ET POSTERS.

25 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

MARDI 14 AVRIL 2015 Après midi

Session 5: Modérateurs Danielle Monniaux et Céline Guigon

14h00-14h30 Nathalie di Clemente (Paris) “Hormone anti-Müllérienne et fonction ovarienne normale et pathologique” 14h30-14h50 Gabriel Livera (Fontenay aux Roses) “Contrôle de la transition mitose/méiose” 14h50-15h10 Norbert Ghyselink (Strasbourg) “Role of retinoic acid receptor (RAR) signalling in post-natal male germ cell differentiation” 15h10-15h30 Philippe Michel (Lyon) “Modélisation mathématique du développement folliculaire basal” 15h30-15h50 Philippe Touraine ( Paris) “Génétique des insuffisances ovariennes primitives”

15h50-17h20 CAFÉ ET POSTERS.

Session 6: Modérateurs Nicolas de Roux et Yves Tillet

17h20-19h00 SESSION JEUNES CHERCHEURS

20h30-22h00 Conférence Grand Public, Professeur Jean-Pierre Bourguignon, Endocrinologie Pédiatrique – Liège “La puberté: le grand chambardement de la tête aux pieds?” Espace des Sciences, les Champs libres, 10 Cours des alliés, 35000 Rennes

26 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

MERCREDI 15 AVRIL 2015 Matin

Session 7: Modérateurs François Brion et Catherine Viguié

09h00-09h30 Sakina Mhaouty-Kodja (Paris). “Effets et mécanismes d’action du bisphenol A dans les réponses neuroendocrines et comportementales liées à la reproduction chez la souris”. 09h30-09h50 Nathalie Hinfray (Verneuil-en-Halatte) “Effets des fongicides azolés sur le système endocrinien et la reproduction.” 09h50-10h10 Marie Postel (Paris) “Modélisation multi-échelle de la folliculogenèse terminale” 10h10-10h30 Louis Bujan (Toulouse) “Projet GAMATOX: Effets des traitements du cancer sur le gamète mâle humain”.

10h30-11h00 PAUSE CAFÉ

Session 8: Modérateurs Célia Ravel et Guillaume Halet

11h00-11h20 Arnaud Reignier (Nantes). “Time lapse monitoring: a tool fot clinical research and embryo assessment”. 11h20- 11h50 Saadi Khochbin (Grenoble) “Bases moléculaires de la programmation post-méiotique du génome mâle”.

11h50-12h30 PRIX ET CLÔTURE

27 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

28 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

R ÉSUMÉS

29 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

30 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Présentations Orales

Classement par ordre alphabétique Pa

31 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

What makes a good egg ? Molecular mechanisms defining GAMATOX Project: impact of cancer treatments on semen 2015 egg developmental competence in teleost fish. characteristics, sperm DNA fragmentation and sperm aneuploïdy: a multicenter prospective study from the Julien Bobe1, Aurélien Bouleau1,2, Caroline Cheung1, Ozlem Yilmaz1, CECOS Network Thaovi Nguyen1, Amine Bouchareb1, Amélie Juanchich1, Daniel Zarski1, Louis Bujan1 and Marie Walschaerts1, Nathalie Rives2, Sylvianne Hennebicq3, Iratxe Rojo1, Stéphanie Gay1, Aurélie Lecam1, Jérôme Montfort1, Hélène Guillaume Martinez3, Véronique Duchesne2, Jacqueline Saias4, Florence Rime1, Christian Fauvel2, Violette Thermes1 Brugnon5, Jacques Auger6, Isabelle Berthaut7, Ethel Szerman8, Nathalie Moinard1, Myriam Daudin1 1 Equipe différenciation Sexuelle et Ovogenèse, INRA LPGP, F-35000 1 Université de Toulouse; UPS; Groupe de Recherche en Fertilité Humaine (EA Rennes 2 IFREMER, LALR, F-34250 Palavas Les Flots 3694, Human Fertility Research Group) and CECOS, Toulouse, France; and [email protected] following CECOS centers and research team associated : 2Rouen, 3Grenoble, avril 15 au 13 du Rennes 4Marseille, 5Clermont-Ferrand, 6Paris Tenon, 7 Paris Cochin, 8Caen. [email protected] Egg quality (i.e. the egg’s ability to be fertilized and subsequently develop into a normal embryo) is highly variable in the wild or Testicular Germ Cell Tumor (TGCT) is the most common cancer in young men and under aquaculture conditions. Yet, the egg components – and TGCT incidence has increased in several countries over the past 50 years. Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) affect also young men who wish to associated molecular processes – responsible for its procreate. Prognosis of these cancers has improved very markedly over the last developmental competence remain poorly understood. In teleost decades due to the treatment mainly based on chemotherapy and radiotherapy. fish, in which a high fecundity can be observed in comparison to Several late adverse effects of chemotherapy or radiotherapy have been described but mainly in retrospective studies and very few studies, with discrepant results, have other vertebrate models, it is possible to sample individual egg explored sperm DNA fragmentation and sperm aneuploïdy following such treatments. clutches in which both developmental success assessment and In this context, we performed the national multicenter prospective research project analytical studies can be performed in parallel. Several types of “GAmete MAle TOXicity” (GAMATOX I) which enrolled patients with testicular germ cell tumors (n= 129), patients with Hodgkin Lymphoma or no Hodgkin lymphoma approaches have been conducted to draw the molecular portrait of (n=75). Patients performed semen samples before cancer treatment and 3, 6, 12, 24 a developmentally competent fish egg by studying its composition months after the treatment ending. Routine semen analyses were performed according in terms of maternal mRNAs, miRNAs, and proteins. Correlative to the WHO recommendations in each center while specific analyses for the sperm DNA fragmentation and the sperm aneuploïdy were centralized in Toulouse, Grenoble studies have shown a link between the abundance of specific and Rouen centers. All samples were registered with the GERMETHEQUE biobank mRNAs and/or proteins in the eggs and the overall developmental (France). Results were explored according to each cancer type and each treatment success of the corresponding egg clutches. In rainbow trout regimen. Predictive factors for sperm recovery following treatment were studied by multivariate analyses. (Oncorhynchus mykiss), a correlation between developmental Treatments have drastic effects on spermatogenesis and the capacity and the time success and the abundance of nucleoplasmin (npm2) mRNA in the needed to recover were dependant of the type of treatment and of pretreatment sperm egg was previously established. In zebrafish (Danio rerio), a model characteristics. Compared to control group of normal men pretreatment alterations existed in certain cancer groups. Sperm DNA fragmentation and sperm aneuploïdy species with transparent eggs and rapid development, a knock- were increased following treatment but were also increased before treatment in down (KD) approach showed that maternally-inherited npm2 lymphoma group. mRNA was crucial to allow developmental success beyond zygotic The results of the GAMATOX I project were relevant for the counseling of cancer patients, before and after treatment, about the risks for the male gamete and the genome activation (ZGA). Similar approaches at the proteome and progeny. However, the new genome and epigenome technology explorations will be miRNA repertoire levels have yielded interesting results that are applied in the next project: GAMATOX II, in order to define more precisely the currently being further analyzed. Taking advantage of the wide alterations induced by such treatment and to evaluate the period safety after the end of treatment. diversity of fish models (over 30000 species), future studies will be This work was supported by a grant from the French Ministry of Health, PHRC designed to identify key molecular mechanisms that could be N° 20030222. Regulatory and ethical submissions were performed by the shared by evolutionary distant species. University Hospital of Toulouse.

32 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Genetic tools to improve reproduction traits in dairy cattle Integrative genomics and mammalian spermatogenesis 2015

Capitan A.1,2*, Michot P.1,2, Baur A.1,2, Saintilan R. 1,2, Hozé C. 1,2, Frédéric Chalmel1,*, Antoine Rolland1,*, Bertrand Evrard1, Sophie Valour D. 1,4, Guillaume F.3, Boichon, D.5, Barbat A. 2, Boichard D.2, Chocu1, Nolwen Hernio1, Emmanuelle Com1, Charles Pineau1, Michael Schibler L.1 and Fritz S.1,2 Primig1, Nathalie Rioux-Leclercq, Nathalie Dejucq-Rainsford2 & Bernard Jégou1 1UNCEIA, 149 rue de Bercy, 75012 Paris, France 2INRA, UMR1313 Génétique Animale et Biologie Intégrative, Domaine de 1 Inserm U1085-Irset, Rennes, France Vilvert, 78352 Jouy-en-Josas, France 2 CHU Pontchailloux, 2, rue Henri Le Guillou, 35033 Rennes cedex 9, 3EVOLUTION, 69 rue de la Motte Brûlon, 35706 Rennes, France France. 4INRA, UMR 1198 Biologie du Développement et Reproduction, Domaine [email protected] de Vilvert, 78352 Jouy-en-Josas, France avril 15 au 13 du Rennes 5MIDATEST, Les Nauzes, 81580 Soual, France Spermatogenesis is a complex and tightly regulated process [email protected] leading to the continuous production of male gametes, the spermatozoa. Within the testes, male germ cells first proliferate Fertility is a major concern in dairy cattle industry and has been the to amplify their number, next shuffle and reduce their genome subject of numerous studies over the last twenty years. through two consecutive meiotic divisions, and finally Surprisingly, most of them focused on rough female phenotypes differentiate dramatically into cells specialized for mobility and and despite their important role in reproductive success, male and fecundation. This developmental process requires the sequential embryo related traits have been poorly studied. In recent years, and coordinated expression of thousands of genes, including the rapid and important evolution of technologies in genetic many that are testis-specific. The molecular networks underlying research led to the development of genomic selection. In a chain normal and pathological spermatogenesis have been widely reaction, the generalization of this method combined with the investigated in recent decades, and many high-throughput extreme achievement of the artificial insemination industry have expression studies have studied genes and proteins important led to the constitution of large data bases of genotyping and for male fertility. During this presentation, I will focus on studies sequencing data as well as refined phenotypes and pedigree that have attempted to link the transcriptome and proteome in records. These resources offer unprecedented opportunities in spermatogenesis or have combined transcriptomic and term of fundamental and applied research. Here we present five proteomic data to gain insight into testicular functions and germ examples of them with a focus on reproduction related traits i.e. cell biology. the detection of QTL for male fertility and semen quality traits (i), and, for refined phenotypes associated with female fertility (ii); the Supported by the Institut national de la santé et de la recherche médicale identification of recessive embryonic lethal mutations by depletion (Inserm), the Université de Rennes 1, the Agence nationale de sécurité of homozygous haplotypes (iii) or by mining whole genome sanitaire de l'alimentation, de l'environnement et du travail [ANSES sequencing data (iv); and finally the contributions of HD SNP n°EST-13-081 to F.C.], the Fondation pour la recherche médicale [FRM chips, whole genome sequencing and imputation to the increase of n°DBI20131228558 to F.C.], and the European Union [FEDER to F.C]. the power of QTL detection methods and to the identification of their causal variants (v).

33 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Apport of Human genetics in the understanding of the neuroendocrine control of the gonadotropic axis Anti-Müllerian hormone and ovarian function 2015

2015 Nathalie di Clemente1 Nicolas de Roux 1. Univ Paris Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et Inserm U1141. Université Paris Diderot. Labaratoire de Biochimie Adaptative (BFA), F-75013 Paris, France; CNRS UMR 8251, F-75013 Paris, Hormonale. Hopital Robert Debré. 48 Bld Sérurier. 75019 Paris. France; Physiologie de l'axe gonadotrope INSERM U1133, F-75013 Paris, [email protected] France. [email protected] Anti-Müllerian hormone (AMH) is a 140 kDa glycoprotein belonging to the The development of the neuroendocrine control of the TGF-β family. Its existence was postulated by Pr Alfred Jost in the early gonadotropic axis is complex. It starts during the fetal life, fifties to explain the regression in male fetuses of Müllerian ducts, the inhibited at the end of gestation, reactivated after birth for few anlagen of uterus and tubes in females. AMH must be cleaved to allow its C- weeks, this axis is then inhibited during childhood until a second terminal fragment to bind AMH specific type II receptor, AMHR-II. Then, avril 15 au 13 du Rennes reactivation around 10 years which marks the start of the puberty. avril 15 au 13 du Rennes AMH activates the same signalling pathway than Bone Morphogenetic Proteins (BMPs): the type I receptors Alk 2, 3 and 6 and the Smad1,5, 8 This sequence of activation-inhibition is fundamental to develop a proteins. In males, AMH expression starts when Sertoli cells begin to normal reproduction function, but the mechanisms remain poorly differentiate, decreases at puberty mainly under the influence of androgens, understood. but stays detectable in adults. The only pathology due to a defect of AMH Human genetics of rare disorders of gonadotropic axis activation synthesis or sensitivity is the persistent Müllerian duct syndrome, a rare has led to major advances to understand the functional plasticity case of male pseudohermaphroditism characterized by the presence of of this axis. Initially focused on isolated or syndromic uterus and tubes in otherwise virilized males. In addition, serum AMH is a gonadotropin deficiency, novel perspectives have recently valuable marker for the diagnostic of sexually ambiguous babies, for the follow-up of boys puberty or the treatment of men with hypogonadotropic emerged with the description of genetic defects causing central hypogonadism. In the eighties, AMH was shown to be also synthesized in precocious puberty. In addition to the description of novel females by granulosa cells of growing follicles of the ovary where AMH neuropeptides, current studies try to characterize the molecular represses both primordial follicle recruitment and FSH-dependent follicle mechanisms controlling the plasticity of the GnRH neuronal maturation. The discovery in years 2000 that serum AMH was a marker of network. The most recent results on human genetics of pubertal ovarian reserve brought light to ovarian AMH. Since that time, many groups disorders will be presented. have extended this result and showed that serum AMH was also a prognostic marker of ovarian stimulation, making serum AMH a useful tool in assisted reproductive technology. Because despite this growing interest in ovarian AMH, its regulation and mechanism of action were still unclear, these last years, our group has made use of numerous and complementary tools to fill this lack of data. We have shown that AMH expression is stimulated by FSH and BMPs, and regulated differentially by estradiol depending on estrogen receptors. We have also demonstrated that AMH signals through Alk3 type I receptor and Smad 1 and 5 proteins in granulosa cells and identified a new AMH target gene, Inhibitor of differentiation/Deoxyribonucleic Acid-Binding 3. In addition, we have studied how AMH could be involved in the polycystic ovary syndrome (PCOS), the main cause of women infertility, which is characterized by high serum AMH levels. We have shown that both AMH and AMHR-II are overexpressed in granulosa cells from PCOS women and that this is partly due to a dysregulation of these genes by LH.

34 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

PIF, a major actor for pregnancy Effects of the environment on early mammalian embryo. 2015 2015 Consequences for adult phenotype. Hadia MOINDJIE1, Esther DOS SANTOS1,2, Florence BOITRELLE1,2, Valérie SERAZIN1,2, Nathalie MELAINE3, Eytan BARNEA4, François Véronique Duranthon. VIALARD1,2 , Marie Noëlle DIEUDONNE1, INRA, UMR1198 Biologie du Développement et Reproduction, F-78350 1 : GIG EA2493, UFR des sciences de la santé Simone Veil, UVSQ, Jouy-en-Josas, France Montigny le bretonneux, France2 : Medical biology laboratory, CHI de [email protected] Poissy, Poissy, France. 3: Biogenouest, Rennes, France 4: BioIncept, LLC, Cherry Hill NJ, USA. In Mammals, fetal environment is known to affect adult health, giving rise to [email protected] the DOHaD concept (Developmental Origin of Health and Disease). More recently, this concept of sensitivity to the environment with long term The preimplantation factor (PIF) is a 15aa peptide secreted very early avril 15 au 13 du Rennes consequences has been extended to the periconceptional period. Especially, avril 15 au 13 du Rennes by viable mammalian embryo. First identified in 1995, its impact has preimplantation period of development has been shown to be very sensitive been clearly shown since 2010. Afterwards, many results have been to environmental conditions. This was quite unexpected since in most obtained, showing its pleiotropic effects and implications in the mammalian species, preimplantation development has been obtained in immune and inflammatory processes. Concerning the reproductive vitro and is compatible with full term development after transfer to a recipient biology, more elements indicate a major role of PIF during pregnancy. mother. One of the most specific examples of such long term effect has PIF, embryo secreted, has been shown to have an autocrine positive been developed in the mouse model where females were fed a low protein effect on embryo development. Recently, using mass spectrometry, a diet for 3.5 days from fertilization onwards. This maternal diet skewed cell low PIF level in IVF embryo culture media has been detected. If a allocation to the first embryonic lineages at the blastocyst stage and induced correlation between PIF level and IVF success was established, PIF a compensatory fetal and perinatal growth positively correlated to could be considered as a new non invasive biomarker for IVF. cardivascular, metabolic and behavioural abnormalities (1). In vitro development of mammalian embryo has also been reported to have long PIF has been also detected in maternal blood circulation. Recently, term effects, which is particularly worrisome within the framework of using bovine model, it was shown that PIF detection in maternal Assisted Reproductive Technologies. To analyze the effect of different circulation was correlated with live birth in early pregnancy. environments on epigenetics modifications and on gene expression during Furthermore, it has been reported that PIF could modulate disrupting the preimplantation period of development, we used the rabbit embryo as a immune and apoptosis pathways, cells proliferation and adhesion in model of early mammalian embryo with a delayed onset of embryonic human endometrial stromal cells. Moreover, an intense PIF genome activation, which is the case in most mammals (including human) immunostaining was observed in human trophoblastic cells from first except the mouse. In the rabbit, in vivo developed embryos can be easily trimester placentas. A decrease of PIF labeling was observed at term. recovered at each stage of development. We showed that the kinetics of Finally, we recently showed that PIF promotes invasion in human embryonic genome de-methylation during the preimplantation development primary extravillous trophoblasts (EVT), confirming its pro-invasive varies with embryo culture conditions and differs from in vivo development effect initially described in the cell line HTR-8/SVneo in 2010. The pro- (2). We also modified the embryo environment in vivo by feeding rabbit invasive regulatory effect of PIF in EVT was associated with a females with an hyperlipidic/hypercholesterolemic diet. We showed that modulation of metalloproteinase activity and mRNA integrin gene expression and trophoblast function were altered as soon as the expressions mediating by multiple signaling pathways. Further blastocyst stage in such females (3,4). 1. Sun C, 2014 Development. 141(5):1140-50. analyses are currently in progress in our laboratory to precise the 2. Reis e Silva AR, 2012. Epigenetics. 7(5):440-6. molecular mechanisms implicated in the PIF effects on human 3. Picone O, 2011 Theriogenology. 75(2):287-99. placenta and endometrium. In conclusion; PIF appears to be a key 4. Tarrade A, 2013. PLoS One. 8(12):e83458. factor of foeto-maternal interface. Supported by: Agence de la Biomédecine, INRA-PHASE, Labex Revive

35 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Role of PPAR-gamma and of its target genes in placental Role of Retinoic Acid Receptor (RAR) signalling in post-natal 2015 2015 development. male germ cell differentiation

Thierry Fournier Norbert B. Ghyselinck1, Aurore Gely-Pernot1, Mathilde Raverdeau1, UMR-S1139, Inserm-Paris Descartes, Faculté de Pharmacie, Paris, France Nadège Vernet1, Betty Féret1, Muriel Klopfenstein1, Christine Dennefeld1, Fondation PremUp “Grossesse et Prématurité”, Paris, France Marius Teletin1,2, Manuel Mark1,2 DHU “Risques et Grossesse”, Maternité Port Royal, Paris, France 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, Département [email protected] de Génétique Fonctionnelle et Cancer, CNRS UMR7104, INSERM U964, The peroxisome proliferator-activated receptor-γ (PPAR γ) is a member of Université de Strasbourg, Illkirch, France the nuclear receptor superfamily that controls in a ligand-dependent 2Hopitaux Universitaires de Strasbourg, France manner the expression of a large array of genes involved in the control of [email protected] energy homeostasis, cell differentiation, proliferation, apoptosis, and the avril 15 au 13 du Rennes avril 15 au 13 du Rennes inflammatory process. Unexpectedly, genetic studies performed in mice All-trans retinoic acid (ATRA), the active metabolite of vitamin A, is established that PPAR γ is essential for placental development. During synthesised by dedicated enzymes called retinaldehyde dehydrogenases pregnancy, the placenta ensures multiple functions, which are directly (ALDH1A1 to A3). Then it acts either through activating nuclear receptor involved in the initiation, outcome of gestation and foetal growth. In the heterodimers made of ATRA receptors (RARA, RARB, RARG) and rexinoid human placenta, PPAR γ is highly and specifically expressed in the two receptors (RXRA, RXRB and RXRG), or through non genomic effects. It is trophoblast subtypes i.e. the villous trophoblast that represents the known for decades that ATRA is instrumental to male germ cell endocrine and exchange tissue and the invasive extravillous differentiation, but its origin and its mechanism of action in the seminiferous cytotrophoblasts (EVCT) involved in implantation, immune-tolerance and epithelium remained elusive. To address these questions, we have analysed uterine artery remodelling. Activation of PPAR γ induces accumulation of the phenotypes of mice lacking either ATRA-synthesizing activities in Sertoli lipids, villous trophoblast differentiation and inhibits EVCT invasiveness. cells (SC), the supporting cells of the germ cell lineage, or retinoid receptors Oxidized LDLs that contain potential PPAR γ ligands, but not native LDLs, (RAR and RXR) in spermatogonia (SG). We demonstrate that (i) ALDH1A- induce PPAR γ transcriptional activity and inhibit trophoblast invasion in dependent synthesis of ATRA by SC is indispensable during the first vitro. Recently, human cytomegalovirus (HCMV) was shown to activate spermatogenic cycle to initiate differentiation of SG; (ii) RARA in SC trophoblastic PPAR γ for its own replication and consequently inhibits mediates the effects of ATRA, notably through activating expression of MAFB transcription factor, whose Drosophila homologue is mandatory to invasiveness of infected cytotrophoblasts. Analysis of PPAR γ target genes revealed trophoblastic factors described to control trophoblast invasiveness germ cell differentiation; (iii) ablation of RXR in SG recapitulates the set of and surprisingly chorionic gonadotropin hormone (hCG), known to be defects observed both upon ablation of RAR in SG and upon vitamin A mainly produced by the endocrine villous trophoblast. Analysis of hCG deficiency; (iv) ATRA enhances expression of the SALL4A transcription factor in SG. This effect depends on activation of RARG and RXRA bound gene expression revealed opposite regulation by PPAR γ in the two to a conserved regulatory region located in the Sall4 gene. This indicates trophoblast subtypes. Finally, a hyperglycosylated form of hCG (hCG-H) that RAR/RXR heterodimers are the functional units in spermatogonia only produced by invasive EVCT was shown to promote trophoblast driving the ATRA-induced transition from the undifferentiated to the invasion and angiogenesis through a TGFß signalling pathway and differentiating state. Moreover, they cast light on the long-searched independently to its binding to the LH-hCG receptor. Together, these data mechanism through which ATRA cell-autonomously controls expression of underscore the major role of PPAR γ and its target genes, such as hCG the KIT tyrosine kinase receptor to trigger this transition. Our data also and hCG-H, in the control of human trophoblast differentiation and invasion, establish for the first time that the effects of ATRA on SG differentiation in and suggest that over-activation of this nuclear receptor following HCMV the seminiferous epithelium are indirect, via SC. infection or by excess of ligands at the maternal–foetal interface could Supported by the RAPSSODI, MOLMECHMEIOSIS and ARGONADS ANR impair implantation and placentation and therefore embryonic development. projects

36 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Shaping the reproductive system: lessons from semaphorins Le sexe : conflits et coopération" 2015 2015

Paolo Giacobini 1 Pierre-Henri Gouyon, Professeur au Muséum National d’Histoire Naturelle, à 1 Inserm, Development and Plasticity of the Neuroendocrine Brain, Jean- l’ENS, à l’AgroParisTech et à Sciences Po. Pierre Aubert Research Center, U1172, Lille, France [email protected] [email protected] La sexualité, c'est l'échange. L'échange de matériel génétique Reproductive competence in mammals depends on the projection entre deux organismes qui en produisent un nouveau procédant of gonadotropin-releasing hormone (GnRH) neurons to the des deux. Dans ce sens large, la sexualité se trouve dans tous les hypothalamic median eminence (ME) and the timely release of groupes d'organismes vivants, bactéries, archées, eucaryotes

GnRH into the hypothalamic—pituitary–gonadal axis. In adult avril 15 au 13 du Rennes (plantes, animaux, champignons...). Pourquoi les êtres vivants ont- avril 15 au 13 du Rennes rodents, GnRH neurons and the specialized glial cells named ils adopté une caractéristique si compliquée? Comment procèdent- tanycytes, periodically undergo cytoskeletal plasticity. During the ils? Les modes de sexualité observés dans la nature sont d'une ovarian cycle, under conditions of low gonadotropin output, diversité incroyable. Pourquoi des mâles et des femelles, ou des GnRH-secreting axon terminals are distant from the pericapillary hermaphrodites? Certaines espèces ont abandonné le sexe. Les space of the ME, thus impairing the access of the neurohormone femelles (parthénogénétiques) se débrouillent seules. Pourquoi to the pituitary portal circulation, but they undergo extensive font-elles ça? Et pourquoi pas les autres? En effet, il semble bien axonal growth toward the vascular wall at the onset of the que les femelles aient tout à gagner à abandonner le sexe ; de ce preovulatory surge, when massive GnRH release has to occur to point de vue, le fait qu’elles le pratiquent s’apparente à de trigger ovulation. However, the mechanisms that regulate this l’altruisme. Elles favorisent la diversité de la population au plasticity are still largely unknown. This talk summarizes recent détriment de leur propre reproduction. Une telle caractéristique studies analysing the contribution of specific guidance molecules semble alors sélectionnée à l’échelle de la lignée évolutive et non named semaphorins in the development and adult function of pas à l’échelle individuelle. Chez les humains par exemple, le sexe gonadotropin-releasing hormone (GnRH) neurons. pourrait-il disparaître? Le sexe est au centre d’un réseau ce These studies started to shed light on the molecular mechanisms situations de conflits et de coopération qui montrent toute la responsible for the progression of the estrous cycle in rodents complexité de nous pose une multitude de questions tant and suggest that this phenomenon relies on the antagonistic biologiques que sociologiques. Comme on le dit pour l'amour (et effects of two semaphorins whose expression in the median les maths), on ne peut pas le faire en public, mais on peut en eminence is periodically influenced by circulating sex hormones parler...

Supported by the ANR-14-CE12-0015-01 RoSes and GnRH

37 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

The regulation of the human cumulus-oocyte complex: Azole fungicides in zebrafish: new effects for old molecules 2015 2015 The MicroRNAs Nathalie Hinfray1, Rüdiger W. Schulz2, Yann Guiguen3, François Brion1 S Hamamah 1INERIS, DRC/VIVA, Ecotoxicology unit, Verneuil-en-Halatte, France.2Utrecht University, Reproductive Biology Group, Utrecht, The INSERM U 1203 ‘Human early embryo development and pluripotency’ Arnaud de Villeneuve hospital, Montpellier, France Netherlands.3INRA, LPGP, Sexual differentiation and oogenesis unit, Rennes, France [email protected] [email protected] Azole is a class of diverse compounds discovered several decades ago and

essentially used as antifungals in agriculture and medicine. Their primary An enormous amount of knowledge about the human oocyte and mode of action is to inhibit the fungal enzyme 14α-demethylase, which CCs have been generated over the last years, due in part to the produces ergosterol, an important component of the cell membranes of fungi.

recent advances in gene expression technologies using avril 15 au 13 du Rennes Despite this specific mode of action, azoles are also characterized by their avril 15 au 13 du Rennes microarray, CGH array and high-fidelity RNA amplification. capacity to disrupt the endocrine system of vertebrate through multiple Numerous small endogenous non-coding transcripts, termed mechanisms notably by altering steroidogenesis, a key physiological microRNAs (miRNAs), have been found to execute key functions process responsible for the biosynthesis of steroidal hormones. For instance, in silencing expression of specific target genes in plant, animal azole compounds affect both expression and enzymatic activities of several steroidogenic enzymes in vertebrate models, including fish, leading to and human systems. reproductive disorders. Because of their uses, their presence in the aquatic environment (surface waters of rivers, lakes and estuaries; sewage sludge) Changes in miRNA expression profiles have been linked to has been recently reported in different industrialized countries raising the pathologies such as cancer and infertility: female mice with global need to assess hazard and risk posed to aquatic organisms. In this context, miRNA deficiency are sterile from several causes, including several experiments have been performed to explore the effects of the defects in oocyte function. In addition, the messenger RNA pharmaceutical azole, clotrimazole, on the endocrine system in the zebrafish. (mRNA) expression in mice and bovine during oogenesis shows In males, we found that clotrimazole was able to affect the testicular physiology by affecting steroidogenesis, androgen release and that a large proportion of maternal genes are under the control of spermatogenesis (Hinfray et al., 2011, Baudiffier et al. 2012, 2013). miRNAs. Thus, miRNA profiling offers an effective means of However, the most striking effect was observed in females. Indeed, we acquiring novel and valuable information regarding the regulation found that exposure of adult female zebrafish to clotrimazole led to a of transcripts involved in human reproduction. dramatic masculinisation as revealed by the complete sex-reversal of the phenotypic sex. Remarkably, this sex-reversal occurred rapidly leading to The miRNAs study of oocyte-cumulus complex offers a well-differentiated testicular tissue after 42 days of exposure. By using promising opportunity, by a non-invasive method, to evaluate cyp19a1a-GFP transgenic zebrafish, we further demonstrated that clotrimazole led to a time-dependent inhibition of GFP expression in ovary ovarian failure and pregnancy outcome which preceded the histological differentiation of testis demonstrating the crucial role played by aromatase in the process of masculinisation. Altogether, our study demonstrates that clotrimazole significantly affect the gonad endocrinology and physiology of fish revealing new and striking effects on its ability to reverse the phenotypic sex of adult female. Based on our data, it is clear that further studies are needed to address the issue raised by the presence of azoles in the aquatic environment as regards to their potential impact on wild population of fish. Supported by the 190 program of the French ministry of environment and the post-grenelle program NEMO.

38 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Olfactory control of reproductive function Molecular basis of post-meiotic male genome programing 2015 2015

Matthieu Keller1, Chantal Moussu1, Didier Chesneau1, Laura Szymanski1, Emilie Montellier1, Hitoshi Shiota1, Sophie Barral1, Thierry Buchou1, Mélanie Jouhanneau1 & Pablo Chamero1 Afsaneh Goudarzi1, Fayçal Boussouar1, Jonathan Gaucher1, Matthieu 1Physiologie de la Reproduction & des Comportements, UMR 7247 Gérard2, Yingming Zhao3, Sophie Rousseaux1, Saadi Khochbin1 INRA/CNRS/Université de Tours/IFCE, Nouzilly, France 1 - INSERM, U823; Université Joseph Fourier - Grenoble 1; Institut Albert [email protected] Bonniot, Grenoble, F-38700 France In many vertebrate species, olfactory informations exchanged during 2 - Laboratoire d'Etude du Métabolisme des Médicaments, , DSV / iBiTec-S social interactions have profound consequences on both reproductive / SPI, CEA Saclay, 91191 Gif sur Yvette, Cedex, France physiology and behavior. Indeed, olfactory cues can virtually affect all 3 - Ben May Department of Cancer Research, The University of Chicago, the steps of the reproductive cycle. These olfactory informations are Chicago, IL 60637, USA. [email protected] processed by various olfactory sub-systems and especially by the avril 15 au 13 du Rennes avril 15 au 13 du Rennes main and the accessory (or vomeronasal) olfactory systems. In In mammals, post-meiotic male genome reorganization and rodents, it is quite well established that the chemosignals affecting compaction can be considered as conceptually related to sporulation in reproductive physiology and behavior are mainly dependent on lower eukaryotes or pollen formation in plants, since all these olfactory cues processed through the accessory olfactory pathway processes consist in preparing the genome to confront the hostile which is closely connected to the hypothalamus, thereby controlling external environment. All involve post-meiotic genome compaction reproductive function. To illustrate the role of male olfactory mechanisms of unclear nature. In mammals, the current knowledge chemosignals on female mice, we will present here data on the control implies a post-meiotic stepwise replacement of histones by transition of puberty onset and sexual behavior. Indeed, we have shown that proteins and protamines, which finally pack the genome into the mature male soiled bedding contains various androgen-dependent spermatozoid. chemosignals such as (1R, 5S, 7S)-3,4-dehydro-exo-brevicomin, 6- Our recent investigations on the molecular basis of post-meiotic male hydroxy-6-methyl-3-heptanone or (S)-2-sec-butyl-4,5-dihydrothiazole genome programming have demonstrated that not only that advance vaginal opening and enhance uterus weight in hyperacetylation-dependent histone replacement but also the meiotic prepubertal females. By using surgical approaches and the use of and post-meiotic gene transcription programs are largely controlled by mice with conditional cell-specific ablation of the vomeronasal G a single member of the BET double bromodomain family, Brdt. Our protein Gαo, we have shown that the olfactory compounds contained parallel investigations of histone variants show that in post-meiotic cells, in male bedding are processed by the vomeronasal olfactory system histone hyperacetylation and Brdt’s action are not sufficient for the and especially the vomeronasal type 2 receptors (V2Rs) which are replacement of histones and that a prior global incorporation of testis- located in the basal layer of the vomeronasal epithelium. Then, using specific H2A and H2B histone variants is required. Finally, we also c-Fos as a marker of cellular activation, we have delineated the neural demonstrate that the whole male germ cell expression program is network involved in the processing of male chemosignals. We show a directed by new and yet uncharacterized histone post-translational significant effect of odor on c-Fos-expression in areas mainly modifications which shape the male genome and drive the meiotic and receiving olfactory information from the vomeronasal system, thus post-meiotic male-specific gene expression program. We have showing that these areas may be responsible for communicating odor therefore discovered unique and essential regulators of male germ cell information that drives puberty acceleration. Finally, we provide differentiation, which, in a developmentally controlled manner, first evidence that the peripubertal exposure to male odors has also long- drive a specific spermatogenic gene expression program and later term behavioral consequences as it promotes an early preference for control the tight packaging of the male genome. male odors in adulthood.

Supported by the ANR JC PHEROSEX. 39 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Melatonin and TSH in the seasonal control of reproduction in Control of the mitotic/meiotic switch 2015 2015 rodents Marie-Justine Guerquin, Virginie Rouiller-Fabre, René Habert, Benoit Paul Klosen1, Marion Ciancia1, Sébastien Milési1, Marie-Emilie Sébert1, Souquet, Emilie Abby, Ronan Le Bouffant, Sébastien Messiaen, Sophie Kamontip Rasri1,2, Marie-Pierre Laran-Chich1, Valérie Simonneaux1 Tourpin, Delphine Moison, Clotilde Duquenne, Gabriel Livera Laboratory of development of the gonads, UMR967 1INCI, CNRS UPR 3212, Strasbourg INSERM/CEA/University Paris Diderot, Sorbonne Paris Cité & University 2 Fac Medecine, Thammasat University, Bangkok, Thailand Paris XI, 92265 Fontenay aux Roses, France [email protected] [email protected]

Many animal species synchronize their reproductive activity with the The timing of meiotic entry is a crucial event in the life of all seasons in order to have their offspring being born at a favourable moment avril 15 au 13 du Rennes sexually reproducing organisms. In Mammals, embryonic germ avril 15 au 13 du Rennes of the seasonal cycle. The nocturnal secretion of the pineal hormone cells follow a sexually dichotomic fate with female germ cells melatonin is known to be the key synchronizing cue for seasonal entering meiosis and male ones escaping this differentiation physiology. Melatonin controls the production and secretion of the thyroid stimulating hormone (TSH) by the pars tuberalis of the adenohypophysis. process. In the mouse fetal ovary all germ cells enter rapidly into In 2008, the tanycytes, highly specialized glial cells of the hypothalamus, meiosis between 13.5 and 15.5 days post-conception. During the have been recognized as the main target of the pars tuberalis TSH for the same period, in the testis germ cells progressively stop seasonal control of reproduction. TSH stimulates the production of proliferating and enter into quiescence. Over the recent years, Deiodinase 2 (Dio2) by the tanycytes. This enzyme activates many intrinsic regulators of these events have progressively been tetraiodothyronine T4 to its active form triiodothyronine T3. The current identified while the upstream regulators are still a matter of consensus is that this local T3 production then controls the gonadotropic debates. axis through a neuroendocrine pathway that remains to be uncovered. We have shown that a chronic intracerebroventricular (icv) infusion of TSH Based on original organ cultures, co-cultures and cell sorting is able to fully reactivate the photoperiodically inhibited gonadotropic axis of experiments we evidenced that the timing of meiosis in fetal germ hamsters exposed to a short photoperiod. This reactivation coincides with cells does not depend on their chromosomal constitution but rather the restauration of a long day pattern in the expression of the RFamides from signals providing from the surrounding somatic cells. Those kisspeptin and RFRP, both potent regulators of GnRH neuron activity. This experiments allowed defining various activities based on secreted suggests that TSH acts through these neurons to control the gonadotropic testicular substance some inhibiting meiosis and other slowing axis. However, currently no precise signalling pathway between tanycytes down proliferation. The role of retinoic acid, that has been and RFamide neurons has been described. During the photoperiodic reactivation of the hamster gonadotropic axis by a proposed as a key factor for governing meiotic entry, was then long day photoperiod, we noticed that the secretion of LH, and thus of investigated and surprisingly it appears to have a modest impact GnRH, was increased before the expression of RFamides started to rise, on the mitotic/meiotic switch in both rodents and human ovaries. an observation that questions the initial hypothesis. Furthermore, acute icv Even more strikingly, we propose that part of the meiotic program infusion of TSH in sexually active Djungarian hamsters induces an increase is likely retinoic acid-independent based on the identification of in circulating testosterone without notably affecting RFamide new regulators of the meiotic entry. It thus appears that the abrupt immunostaining. switch from mitosis to meiosis requires a more complex interplay These observations suggest the existence of a signalling pathway independent of RFamides through which TSH is able to control the than anticipated relying on both retinoic acid-dependant and gonadotropic axis. This pathway might also explain the circadian rhythm in retinoic acid independent signalling. circulating sex steroids observed in various species.

40 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Lipid metabolism and female reproduction: role at the ovary Effects of oral exposure to bisphenol A on neuroendocrine 2015 2015 level. and behavioral responses related to reproduction in male and Virginie Maillard1, Sébastien Elis1, Sandrine Fréret1, Valérie Labas1,2, female mice Ana-Paula Teixeira-Gomes2,3, Véronique Cadoret1,4, Philippe Monget1 and Svetlana Uzbekova1,2. Sakhina Mhaouty-Kodja 1 Team BINGO, PRC, UMR0085-7247, INRA-CNRS-Université de Tours- Neuroscience Paris Seine, Team “Neuroplasticity of Reproductive Behaviors” IFCE, Nouzilly, France 2 Laboratoire de Spectrométrie de masse, PAIB, Université Pierre et Marie Curie, INSERM U 1130, CNRS UMR 8246; INRA PRC, Nouzilly, France3 ISP, INRA UMR1282, Nouzilly, France F75005, Paris, France 4 CHRU de Tours, LBR, Tours France. [email protected] [email protected] There are human reproduction concerns associated with extensive use Besides their role of energy sources, intracellular lipids and their derivatives are avril 15 au 13 du Rennes of bisphenol A (BPA)-containing plastic, and in particular, the leaching avril 15 au 13 du Rennes well-known to be essential components of biological membranes, cell-to-cell interaction, and in regulation of different cellular processes as proliferation, of BPA into food and beverages. In this context, we investigated apoptosis, hormone synthesis... In mammals, oocytes develop inside the ovarian whether and how exposure to oral BPA at reference doses interferes follicles; this process is strongly supported by the surrounding follicular cells with sex steroids in the developmental organization and adult activation (cumulus, granulosa and theca cells) and follicular fluid. Folliculogenesis and of neural structures underlying the expression of sexual behavior and final oocyte maturation are regulated at the endocrine and paracrine levels and regulation of the hypothalamus-pituitary-gonad axis in mice. Indeed, are strongly influenced by dietary fat supplementation and lipid metabolism. testosterone and its neural metabolite estradiol play a key role in the The BINGO team investigates the roles of lipid metabolism and dietary n-3 permanent masculinization and defeminization of these neural areas in polyunsaturated fatty acid (FA) supplementation at ovarian level and the molecular factors involved in these processes. males during the perinatal period. During this period, the ovaries are Firstly, our data showed in bovine that several genes of lipid metabolism inactive and the female brain is protected from the potential (lipolysis, lipogenesis, FA transport and oxidation) are upregulated in cumulus masculinizing effects of estradiol. In adulthood, testosterone and cells at different times of in vitro maturation relating to stages of oocyte meiosis estradiol are important in the activation of male and female responses. progression. We showed that inhibition of FA oxidation in cumulus cells strongly Developmental exposure of mice to BPA at the no-observed-adverse- influences meiosis progression and survival of enclosed oocytes. Moreover in effect-level (NOAEL, 5 mg/kg body weight.day) and tolerable daily bovine granulosa cells, FA synthesis and oxidation were found to regulate cell proliferation and steroidogenesis. Currently several components of lipid intake (TDI, 50 µg/kg body weight.day) doses induced sex-dependent metabolism in follicular cells are analysed during basal follicular growth thanks to effects. In exposed males, testosterone levels, sexual behavior and the a model of ovine cultured follicles in vitro. neuroanatomical organization of brain areas underlying these Secondly, using mass spectrometry imaging and transcript analysis, we responses were unchanged. In female mice, BPA at TDI dose observed differences in spatial distribution of lipids and in expression of several increased sexual behavior, kisspeptin cell number in the preoptic area lipid metabolism genes between the compartments of the porcine ovary follicles, and estradiol levels. Adult exposure of male mice to BPA at TD, but not emphasizing the potential lipogenic and lipolytic activity of oocyte and theca cells, NOAEL dose, reduced sexual behavior without affecting circulating respectively. Thirdly, we showed in dairy cows that early post-partum application of n-3 marine levels of testosterone or olfactory preference. polyunsaturated FA enriched diet tended to improve fertility compared to control Analyses of the potential mechanisms underlying BPA effects suggest diet. This effect of FA supplementation suggests that either oocyte competence that this compound exacerbates effects of estradiol in the female to develop or uterine and oviductal compartments could be affected. Our present postnatal/prepubertal brain, whereas it acts as an anti-androgenic project aims to explore the effects of n-3 marine FA enriched diet on oocyte compound in the adult male brain. These findings will be discussed in competence and/or embryo quality in cattle, to identify ovarian tract cell targets of the context of current knowledge of the roles of neural androgen and these n-3 FAs (oocytes and follicular cells) and finally to understand the involved mechanisms. estrogen receptors and potential mechanisms of BPA effects in male Supported by INRA, Apis-gène and Région Val de Loire. and female reproduction.

41 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

MULTISCALE MODEL-BASED INSIGHT ON OVARIAN A Multiscale model for the terminal development of ovarian 2015 2015 FOLLICULAR DEVELOPMENT follicles

F. Clement1, P. Michel2, D. Monniaux3 et T. Stiehl4 Benjamin Aymard1,2, Frédérique Clément2, Danielle Monniaux3, Marie 1 INRIA Paris-Rocquencourt Research Centre, Domaine de Voluceau Postel1,2 Rocquencourt, 78153 Le Chesnay, France. 1 UPMC - Paris 06, Laboratoire Jacques-Louis Lions 2 Université de Lyon, CNRS, Ecole Centrale de Lyon, Institut Camille

2 INRIA Paris-Rocquencourt. 3 INRA, UMR85 Physiologie de la 13au 15avril Jordan, 69134 Ecully Cedex, France. Reproduction et des Comportements, Nouzilly. [email protected] 3 INRA, UMR85 Physiologie de la Reproduction et des Comportements, F- 37380 Nouzilly, France; CNRS, UMR7247, F-37380 Nouzilly, France; This talk will present a mathematical model of the selection process in Université François Rabelais de Tours, F-37041 Tours, France; IFCE, F- ovarian follicles, which determines the number of ovulations occurring during 37380 Nouzilly, France avril 15 au 13 du Rennes each ovarian cycle, together with a numerical method dedicated to the du Rennes 4 Interdisciplinary Center for Scientific Computing (IWR), Heidelberg quantitative calibration of its main parameters. The ovulatory follicle(s) is University, 69120 Heidel- berg, Germany. (are) selected within a cohort of growing follicles which compete with each [email protected] other for FSH resource. The purpose of the study is a better understanding of the selection process and the identification of mechanisms that can We present a stochastic individual-based model describing the promote multiple ovulations. first stages of follicular development (the initiation of follicular The follicles recruited for the latest stages of development start from a quite development from the pool of resting follicles), where the somatic homogenous state (comparable number of granulosa cells and maturity), cell population is structured with respect to age (progression and their trajectories progressively diverge according to their differential within the cell cycle) and space (radial distance from the oocyte). response to FSH in terms of cell proliferation and final differentiation. In turn, FSH secretion is modulated on the pituitary level by the secretion of ovarian The model accounts for the molecular dialogue existing between hormones cumulating the contribution of all follicles, weighted by their the oocyte and granulosa cells. The model accounts for the individual maturity. The endpoint of the selection process occurs when molecular dialogue existing between the oocyte and granulosa estradiol levels reach a threshold and trigger the hypothalamic surge of cells, as well as the three-dimensional morphogenesis of follicles : GnRH, followed by the LH surge and subsequent ovulation of the selected (i) detailed spatial distribution of individual granulosa cells, (ii) follicles organization as concentric layers or functional cell clones, and (iii) The mathematical model has multiscale features: on the microscopic scale, increase in the follicle size. The model can help to explain a system of coupled transport equations, whose unknowns are the cell densities, describes the evolution in space and time of the distribution of pathological situations of imbalance between oocyte growth and cells within each follicle, according to their age (progression in or exit from follicular cell proliferation. the cell cycle) and maturity. The functional domain is divided into zones corresponding to different cell states (proliferation, differentiation, sensitivity This work is part of the Inria Large Scale Initiative REGATE (Regulation of to apoptosis) and cell cycle phases. On the mesoscopic scale, the follicle the GonAdoTropE axis) individual maturity and cell number are obtained by integrating the cell density to obtain different aggregated quantities. Finally, on the macroscopic scale, the ovarian maturity is obtained by summing the individual maturities. The dynamic feedback-loop between the hypothalamo-pituitary axis and the ovaries is accounted for through interactions between variables defined on the different scales.

42 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Time lapse monitoring: a tool for clinical research and Talking about an endometrial biosensor in mammals 2015 embryo assessment 2015 Olivier Sandra Arnaud Reignier1,2, Jenna Lammers1,2, Carole Splingart1,2, Aurore Catteau1, Laurent David2, Thomas Fréour1,2 INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en-

au 15 avril Josas, France 1 Service de médecine et de biologie de la reproduction, CHU de Nantes, [email protected] France 2 UMR 1064, INSERM, Nantes, France In mammals, the birth of a viable and healthy progeny involves a [email protected] continuum of complex biological processes and several

Rennes du 13 13 du Rennes checkpoints (or hurdles) that have to be passed successfully. For a Among all the strategies available in order to improve success

long time, successful pregnancy has been thought to be restricted avril 15 au 13 du Rennes rates in IVF cycles, a lot of work has been done on embryo to embryo quality. Nevertheless, recent data have shown that culture conditions and embryo quality evaluation. Most IVF endometrium (the tissue layer covering the internal part of the centres use conventional incubators and select embryo according uterus) can elicit a tailored biological response to embryos to punctual morphological evaluation, but this strategy has presenting distinct post-implantation fates. Indeed biological several limitations. Recently developed commercial devices functions (e. g. metabolism and immune function), molecular associating more stable culture conditions and time lapse pathways (e.g. oxidative phosphorylation) and individual genes are observation of embryo development provide new insights into affected in endometrium facing various types of embryos early embryo development in IVF cycles. One of the main benefits (produced by artificial insemination, in vitro-fertilization or somatic of these systems resides in the use of different models or cell nuclear transfer) and may affect the issue of pregnancy. These algorithms known to improve clinical outcomes by predicting findings have led to the concept that endometrium is an early embryo viability even if more studies (random prospective trials) biosensor of embryo developmental potential, useful for the have confirm its specificity in selection of embryos with high prediction of pregnancy issues. This biological property first reproductive potential evidenced in cattle has been recently applied to human species Moreover, mammalian preimplantation embryo development is a then has been extended to selection of embryos by the complex process in which knowing the exact timing and sequence endometrium. Hence mammalian endometrium appears as a of events can be a source of many useful information in the field dynamic and reactive tissue whose physiology can be negatively of research, notably in the study of exogenous factors on embryo affected by environmental factors or types of embryos. This development or in the evaluation of some of the cofactors of compromised endometrial quality can affect embryo development infertility. during implantation with consequences on pregnancy outcome and

long-term health of the offspring.

43 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Changing of reproductive mode, a balanced affair between Impact of tetraploidization events in the repertoire of the genetics and environment. Exemple taken for aphids reproductive hormones in vertebrates 2015 2015

TAGU, D.1; LEGEAI, F.1 ; JAQUIERY, J.1; MIEUZET, L.1; MAHEO, F1. ; Hervé Tostivint1, LETERME, N.1; BONHOMME, J.1 ; NOUHAUD, P.1; RISPE, C.1; 1 Evolution des Régulations Endocriniennes. CNRS UMR 7221. Muséum LAROSE, C. ;1 GAGGIOTTI, O.2; STOECKEL, S.1 ; SIMON, J-C1 . National d’Histoire Naturelle. Paris 1 INRA, UMR1349 IGEPP, F-35653 Le Rheu, France [email protected] 2 LECA UMR CNRS 5553, Université Joseph Fourier BP 53 38041 Grenoble, France It is now well established that two rounds of whole genome [email protected] duplication (2R) took place in the vertebrate lineage after its

One key issue for the success of pest management is the understanding of separation from invertebrate chordates, about 500 million years

mechanisms involved in pest adaptations to environmental pressures. avril 15 au 13 du Rennes ago. This means that vertebrates initially possessed four copies of avril 15 au 13 du Rennes Aphids are among the main insect pests in countries of temperate and each gene inherited from their chordate ancestor. Even though a continental climates. They feed from phloem sap and provoke damage on large part of these copies were subsequently lost during evolution, plants. The success of aphids as pests is related to their peculiar life a number of them were preserved and are still present in living history traits, in particular their reproductive mode alternating asexual vertebrate species. parthenogenesis and sexual reproduction. An additional complexity is the The aim of our presentation will be to show the impact of 2R in the presence of lines or populations that have become entirely asexual. This work aims at integrating population genomics, quantitative genetics and repertoire of the reproductive hormones, namely kisspeptins (Kiss), transcriptomics to get comprehensive insights into these variations of gonadotropin-releasing hormones (GnRH) and glycoprotein reproductive mode in aphids, by linking phenotypic plasticity (molecular hormones (LH and FSH), and their receptors. An important bases of clonal and sexual phases within a given genotype) and conclusion of our talk will be that, when compared with other polymorphism (co-existence of sexual and asexual lines within a given vertebrates such as fish for example, mammals including human species). are far from possessing the richest repertoire of these molecules. We first identified loci of the pea aphid genome involved in differences in reproductive mode by genome scanning of multiple sexual and asexual populations. Since the variation of reproductive mode is shaped by climate factors, we sampled sexual populations in regions that have a cold winter and asexual populations in regions that have a mild winter. To identify the genomic regions linked to reproductive phenotypes, we genotyped 124 individuals at 378 microsatellite markers chosen to cover different scaffolds of the referenced annotated genome. We detected 5 genomic regions under divergent selection loci between asexual and sexual populations. The second step was to identify quantitative trait loci (QTLs) for the reproductive mode in the pea aphid. We i) generated F1 and F2 individuals from F0 that present contrasted phenotype for the reproductive mode, ii) genotyped these individuals, iii) assessed the phenotype (i.e. reproductive mode), and iv) constructed a genetic linkage map. Interestingly, the major QTL corresponds to the locus identified from the genome scan approach. This opens hypthesis on the function underlying this locus. Supported by ANR GW_Aphid and ANR miRNAdapt projects

44 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Genetics of Premature Ovarian Insufficiency 2015

Anne Bachelot 1,2 and Philippe Touraine1,2

1 Service d’Endocrinologie et Médecine de la reproduction, IE3M, Hôpitaux Universitaires Pitié-Salpêtrière Charles Foix; Centre des Maladies Rares de la Croissance; Centre des pathologies Gynécologiques Rares 2 Université Pierre et Marie Curie, Paris 6 [email protected]

Premature ovarian insufficiency (POI) is a disorder affecting approximately 1% of women under 40 years of age. POI avril 15 au 13 du Rennes encompasses a heterogeneous spectrum of conditions, through two major mechanisms: follicle dysfunction and follicle depletion. Although causes such as autoimmunity, monosomy X and environmental factors play a role in POI, the aetiology in most cases remains unknown. These last 10 years, genetic studies have been set up top better understand the role of either chromosome X or genes located on autosomal genes. The well- described association between Xfra permutation and POI leads to the current practice for searching such anomaly in any POI patient. Emphasis has also been put to search for gene candidates based on animal models leading to POI; therefore multiples genes have been identified as potentially involved in POI. However, clinical presentations of POI are heterogeneous and not systematically similar to the phenotypes observed in certain animal models. Since these mutations are most frequently described in clinical case reports, the opportunity which is now discussed is to get new approaches including GWS or exomic studies. All these aspects will be discussed.

45 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

46 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

2015 Rennes du 13 au 15 avril 15 au 13 du Rennes

Posters

Classement par ordre alphabétique 1er auteur

47 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

A role for estrogen in the development of KNDY neurons? SETting the stage for GnRH signaling: evidence for a 2015 2015

functionnal interplay between GnRH receptor and its Caroline Alfaïa, Mélanie Faure, Vincent Robert and Isabelle regulatory partner SET Franceschini Charlotte Avet, Ghislaine Garrel, Chantal Denoyelle, Joëlle Cohen- UMR Physiologie de la reproduction et des comportements. 37380 Tannoudji, Violaine Simon Nouzilly [email protected] Univ Paris Diderot, Sorbonne Paris Cité, Unité Biologie Fonctionnelle et Adaptative (BFA), CNRS UMR 8251; Equipe: Physiologie de l'axe 13au 15avril Kndy neurons express the Kiss1 gene encoding kisspeptin (Kp), a potent gonadotrope INSERM U1133, F-75013 Paris, France. neuropeptide secretagogue of GnRH that plays a fundamental role in [email protected] sexual differentiation and regulation of reproductive life cycles. Considering Reproductive function is under the control of the hypothalamic that prenatal exposure to estrogenic compounds can produce adverse neurohormone Gonadotropin-Releasing Hormone (GnRH), which Rennes du 13 au 15 avril 15 au 13 du Rennes effects on sexual differentiation and reproductive function and lead to activates a G-protein coupled receptor (GnRHR) expressed in pituitary du Rennes altered patterns of Kiss1 expression postnatally, we hypothesize that gonadotrope cells. Mechanisms regulating GnRHR coupling to its developing Kndy neurons could represent an early target of estrogens signaling pathways are still elusive. Recently, we identified the first during fetal life. The purpose of this study is to understand how Kndy interacting partner of GnRHR, the proto-oncogene SET (1), which was neurons are set up during fetal development and if estrogens could initially known as an inhibitor of protein phosphatase 2A and a interfere with this development. We took advantage of a knock-in mouse regulator of gene expression. We demonstrated that SET induces a expressing GFP under control of the Kiss-1 promoter. The anatomical distribution and antigenic phenotype of GFP-immunoreactive (ir) cells was signaling switch of GnRHR from calcium towards cAMP pathway and studied by immunohistochemistry from embryonic day E12.5 to E16.5 with also showed using siRNA and cell permeable peptides in aT3-1 a variety of antibody markers. GFP-ir cells were first detected at E13.5 in gonadotrope cells that GnRHR couples to the cAMP pathway through the mantle layer of the tuberal hypothalamus. At E14.5 GFP-ir cells were interaction of SET with the first intracellular domain (ICL1) of the clustered on either side of the infundibular recess and extended posteriorly receptor. We demonstrated, using GST pull down assays, that both N- along the ventral midline up to the mammillary recess; at E16.5 they were and C-terminal domains of SET directly interact with ICL1. less widespread along the postero-anterior axis, accumulating around the Interestingly, GnRH agonist (GnRHa) treatment rapidly decreases infundibulum. Moreover, the number of GFP-ir cells tripled between E13.5 SET expression in aT3-1 gonadotrope cells as early as 30 minutes and 16.5. Spatiotemporal differences in the antigenic phenotype of GFP-ir and until 24 hours, highlighting for the first time a role of GnRH in cells were further noted: Erα-ir was first detected at E14.5 in over half GFP- regulating SET protein. This regulation was not accompanied by any ir cells. At this stage, most Erα-ir cells in the hypothalamus expressed GFP. change in SET mRNA content as evidenced by real time PCR. Our The posterior GFP-ir cell population displayed a more immature profile than results highlight two mechanisms driving SET downregulation by the anterior one, as suggested by sox-2-ir and Erα-ir. In addition, Kp-ir GnRHa: a post-traductionnal regulation involving the proteasomal increased between E14.5 and E16.5 when it labeled nearly all GFP-ir cells. pathway and a post–transcriptionnal regulation targeting mRNA SET At E16.5, proximity between Kp-ir fibers and GnRH neurons and between GnRH-ir fibers and GFP/kp-ir neurons was noted. Preliminary data also into the RISC complex. Our data suggest that GnRH not only suggest the onset of sex differences in the antigenic phenotype of GFP-ir regulates SET expression but also modulates its phosphorylation cells after E13.5. Taken together, these results are consistent with the state thereby influencing its activity notably by regulating its interaction hypothesis that developing Kndy neurons may undergo a sex-specific with other proteins and its subcellular localization. Altogether, our differentiation during fetal life and represent one of the earliest cellular work shows that GnRH may regulate its own signaling by acting on targets of estrogens in the developing hypothalamus. SET level and phosphorylation and suggests that a regulatory loop between GnRHR and SET fine-tunes GnRHR coupling to the cAMP pathway in gonadotrope cells. (1) Avet et al. J. Biol. Chem., 2013, 288(4): 2641-54.

48 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Effect of season and steroid on RFRP3 expression in ewe: a Spermatogonial Stem Cells: the Gdnf-Gfra1 pathway 2015 2015 three dimensions analysis of neurons distribution and regulation is spermatogenetic dependent in trout, and differs neurotransmitter markers. from that in mouse.

Julien Bartzen-Sprauer, Hugues Dardente, Karine Anger, Vincent Johanna Bellaiche, A.Sophie Goupil, Elisabeth Sambroni, J.Jacques Robert, Caroline Decourt, Massimiliano Beltramo Lareyre, Florence Le Gac UMR Physiologie de la Reproduction et des Comportements (INRA, Fish Physiology and Genomics INRA, BIOSIT, Biogenouest, Campus de UMR85; CNRS, UMR7247; Université François Rabelais Tours; IFCE) Beaulieu, Rennes. [email protected] F-37380 Nouzilly, France. [email protected] We recently characterized putative spermatogonial stem cells (SSCs) in trout spermatozoa [Bellaiche et al 2014 a and 2014b]. What makes these cells self-

A neuropeptide of the RF-amides family, RFRP3 (RF-amide related avril 15 au 13 du Rennes renew or differentiate to produce spermatozoa is barely understood, in particular avril 15 au 13 du Rennes peptides-3), has been implicated in the central control of reproduction in in non-mammalian species. Our research explores possible regulations of the mammals. Initially identified by homology to GnIH, that inhibit LH secretion spermatogonial stem cell niche in teleost, locally by paracrine factors and in bird, its physiological functions in mammals appear more complex and peripherally by hormonal regulation. In the present study, we focused on the variable. Gdnf/Gfra1 pathway, known to play a major role in SSC self-renewal in rodents. It has been reported that RFRP3 expression is influenced by season and Using qPCR measurements in purified testicular cell populations, the gdnfb was sexual hormones. For example in ewe RFRP3 neurons are less abundant found expressed in testicular somatic cells and in spermatogonia. In contrast, the during the breeding season. We investigated the effect of a combination of transcript of the gdnf receptor, gfra1a, was specifically expressed in a population progesterone analog (flugestone acetate, FA) treatment and season on of undifferentiated-spermatogonia (und-Spg) purified by centrifugal elutriation. RFRP3 gene expression by in situ hybridization (ISH) on Ile de France Transplantation studies demonstrated that this particular cell population had a ewes (n=5 per group). Different rostrocaudal levels of the hypothalamus high “stemness” potential in terms of gonadal colonization and production of fertile spermatozoa [Bellaiche et al 2014a]. It also preferentially expressed were analyzed and neurons labeled by ISH counted on microscope images nanos2, a putative SSC marker in trout. Furthermore, by flow cytometry and using a Mercator Software. Labeled neurons were present in the dorso- immunohistochemistry we find that only a sub-fraction of the und-Spg (12%-20%) medial hypothalamus (DMH) and more scattered neurons observed in the expressed gfra1a and nanos2. nearby hypothalamic regions. Under FA treatment neurons expressing In trout, spermatogenesis develops along a strict annual cycle. We show that RFRP3 in the DMH were slightly less abundant during breeding season gdnfb and its receptor were expressed in a spermatogenetic activity dependent compared to non-breeding season. To define if there is a subpopulation of manner. Interestingly, a dramatic increase of the gdnfb transcript towards the neurons that is most affected by the reproductive status and FA treatment end of the reproductive cycle coincided with the progressive cessation of we perform a tridimensional analysis of neurons distribution. A grid was differentiated spermatogonia proliferation. These results suggest that, in trout, applied on photomicrographs and RFRP-3 neurons were counted in each Gdnfb is involved in the repression of und-A-Spg differentiation. In rodents, Fsh case. Our analysis showed a subpopulation in DMH core that account for was found to up regulate Gdnf. We demonstrate that in trout, in vitro Fsh the seasonal difference observed and is possibly less affected by FA treatment stimulated the expression of the receptor gfra1a1, but not of its ligand, treatment. gdnfb. Fsh treatment also stimulated the proliferation of und-Spg co-cultured with At present it is unknown which other neurotransmitters are present in testicular somatic cells. [Bellaiche et al 2014b] Based on those results we propose that the Gfra1 positive cells correspond to RFRP3 neurons. To assess possible coexpression we performed double the putative SSCs in rainbow trout and that the balance between SSC self- ISH using glutamate and GABA neuron markers (vglut2 and gad65). renewal and differentiation during the trout spermatogenetic cycle is possibly Preliminary results suggest that GAD65 and Vglut2 are not expressed in under paracrine regulation by Gdnfb and under peripheral regulation by Fsh via RFRP-3 neurons. the control of gfra1 expression. Further studies are in progress to establish if RFRP3 neurons contain other Bellaiche J., Lareyre J.J., Cauty C., Yano A., Allemand I., Le Gac F. 2014a. Biol Reprod, 90. 79. neurotransmitters and/or progesterone and corticosteroid receptors. Bellaïche J., Goupil AS., Sambroni E., Lareyre J.J., Le Gac F. 2014b. Biol Reprod, 91. 94. Funding : ANR Repramide / Bourse region Centre Supported by EU LIFECYCLE project and CRB-Anim (Infrastructure ANR)

49 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

No evidence of LH secretion inhibition by RFRP treatment in Ibuprofen targets several cell types in the human fetal testis 2015 ewe in vitro

Beltramo Massimiliano, Karine Anger, Robert Vincent, Julien-Bartzen- Millissia Ben Maamar1,2, Laurianne Lesné1,2, Antoine Rolland1,2, Isabelle Sprauer, Lomet Didier, Caroline Decourt Coiffec1,2, Christèle Desdoits-Lethimonier1,2, David M. Kristensen3, UMR Physiologie de la Reproduction et des Comportements (INRA, Vincent Lavoué4, Nathalie Dejucq-Rainsford1,2, Séverine Mazaud- UMR85; CNRS, UMR7247; Université François Rabelais Tours; IFCE) F- Guittot1,2 & Bernard Jégou1,2,5 37380 Nouzilly, France. [email protected] 1Inserm (Institut national de la santé et de la recherche médicale), IRSET, The discovery of the mammalian orthologue (RFRP) of the avian U1085, SFR Biosit, Campus de Beaulieu, Rennes CEDEX, France gonadotropin inhibiting factor (GnIH) created the expectation that this was 2Université de Rennes I, Campus de Beaulieu, Rennes CEDEX, France the long sought-after inhibitory system controlling GnRH release. However, avril 15 au 13 du Rennes 3Department of Biomedical Sciences, Faculty of Health Sciences, University data obtained in rodent showed that pending on species, sex, and of Copenhagen, Copenhagen, Denmark. 4CHU Rennes, Service reproductive status (breeding vs non-breeding) administration of RFRP Gynécologie et Obstétrique, Rennes, France. 5EHESP - School of Public could decrease, increase or have no effect on LH secretion. Contrasting Health, Avenue du Professeur Léon Bernard, Rennes, France data were also reported in ewe with either an inhibitory (Clarke 2008 & [email protected] 2011) or no effects observed (Caraty et al 2013). In order to clarify this contentious finding we performed additional experiments in ewe (Ile de Many drugs are prohibited during pregnancy because birth defects issues France breed). go along with their consumption. Epidemiological studies have shown an To assess effect on basal and stimulated LH level in anoestrus season we association between exposure of the human fetus during pregnancy to infused intravenously, for 4 hours, either RFRP3 at different concentrations paracetamol and/or other mild analgesics and an increased risk of (250, 500 and 1000 µg•hour-1) or saline (n=6 per group). Two hours after cryptorchidism, but none of them have singled out ibuprofen. The aim of this perfusion initiation LH secretion was stimulated by an intravenous bolus study is to determine whether ibuprofen can disrupt the endocrine balance injection of kisspeptin 10 (5 nmoles/ewe). No difference was observed of the human fetal testis through an in vitro system based on the culture of human fetal testes exposed or not to different concentrations of ibuprofen between RFRP3 treated animals and control on either basal or stimulated -7 -4 LH level. (from 10 to 10 M). Morphology, hormonal production and cell markers by Considering that in non-breeding season a high inhibitory tone could have qPCR were assessed. No morphological changes were observed on the masked the inhibitory effect of RFRP3 we decided to test RFRP3 effect on Leydig and Sertoli cells, unlike the germ cells which seem to diminish with preovulatory LH surge in the oestrus season. To this aim 12 ewes were the highest doses of ibuprofen. A decrease in the production of testosterone, ovariectomized and implanted with 2 cm estradiol implant. To simulate an AMH and the prostaglandin PGE2 was shown in the youngest fetuses (8- artificial luteal phase a vaginal sponge containing flugestone acetate was 9.86 developmental weeks). This was confirmed by qPCR with repression of inserted for 14 days. To induce an LH surge 50 µg of estradiol benzoate Sertoli cell markers like SOX9, AMH and DHH but not KRT18; Leydig cell were injected 24 hours after sponge withdrawal. RFRP3 (500 µg•hour-1) or markers like CYP17A1 but not DLK1; and germ cell markers like LIN28A but not POU5F1. Ibuprofen, like other mild analgesics and at concentrations saline perfusion started 10 hours after estradiol benzoate injection and -4 lasted for 24 hours. No significant difference was observed either on the close to the human peak plasma concentration (around 10 M) cause maximal LH level achieved during the surge or on the timing of LH surge endocrine disturbances in the human fetal testis. We suggest here that between RFRP3-treated and saline-treated ewes. ibuprofen targets several cell types in the human fetal testis with a critical Our results lend further support to the hypothesis that in ewe RFRP3 does age window of sensitivity. Unlike other mild analgesics tested in the not play a significant role in controlling LH secretion but do not exclude laboratory (paracetamol, indomethacin and aspirin), ibuprofen is the first over possible role for example in food intake and/or stress and anxiety drug showing an effect on germ cells. modulation. Funded by INSERM, Rennes 1 University. ANSM (AAP-2012-037) Supported by ANR (Repramide grant).

50 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Role of Gata6 during mouse preimplantation development Hypogonadism associated with Cyp19a1 (Aromatase) post- 2015 transcriptional upregulation in Celf1-KO mice Sylvain Bessonnard, Pierre Pouchin, Claire Chazaud. GReD, UMR CNRS 6293/INSERM 1103/Université Clermont-Auvergne, Gaella Boulanger a,b , Marie Cibois a,b, Justine Viet a,b, Alexis Fostier c, Clermont-Ferrand, France Stéphane Deschamps a,b, Sylvain Pastezeur a,b, Catherine Massart d, [email protected] Bernhard Gschloessl a,b, Luc Paillard a,b and Carole Gautier-Courteille a,b a Université de Rennes 1; b CNRS UMR 6290, IFR 140, Rennes, France c INRA, UR1037, LPGP IFR 140, Rennes, France d Centre d'Investigation Clinique Inserm 0203, CHU Rennes At E3.5, the mouse embryo is composed of a monolayer of [email protected] trophectoderm surrounding the inner cell mass (ICM). The ICM is Rennes du 13 au 15 avril 15 au 13 du Rennes heterogeneous, composed of Epiblast (Epi) and Primitive CELF1 is a multifunctional RNA-binding protein that controls Endoderm (PrE) precursor cells. The heterogeneity is visualized several aspects of RNA fate. The targeted disruption of the Celf1 by the exclusive expression of Nanog, an Epi marker, and Gata6, gene in mice causes male infertility due to impaired a PrE marker in a “salt and pepper” pattern. Our aim is to spermiogenesis, the post-meiotic differentiation of male gametes. understand the mechanisms regulating the differentiation Here, we investigated the molecular reasons that underlie this between Epi and PrE within the ICM. testicular phenotype. The transcription factor Gata6 is suggested to have an important By measuring sex hormone levels, we detected low concentrations role in PrE formation. Indeed, Gata6 is required for the induction of testosterone in Celf1-null mice. We investigated the effect of of PrE in ES cells embryoid bodies and E4.5 mutant embryos are Celf1 disruption on the expression levels of steroidogenic enzyme lacking the PrE epithelium. Thus, is Gata6 sufficient for PrE genes. We observed that Cyp19a1 was upregulated, whereas the determination and differentiation? In addition how does it relate to expression of other genes was not modified. Nanog and the RTK pathway activities? Cyp19a1 encodes aromatase, which transforms testosterone into estradiol. Administration of testosterone or the aromatase inhibitor Letrozole partly rescued the spermiogenesis defects, indicating that a lack of testosterone associated with excessive aromatase contributes to the testicular phenotype. In vivo and in vitro interaction assays demonstrated that CELF1 binds to Cyp19a1 mRNA, and reporter assays supported the conclusion that CELF1 directly downregulates Cyp19a1. We conclude that CELF1 represses Cyp19a1/Aromatase post- transcriptionally to achieve high concentrations of testosterone compatible with spermiogenesis completion.

51 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Estrogen receptors and effects of 17-b estradiol on human Impact of Bisphenol A on spermatogenesis’s establishment in 2015 spermatozoa motility. rats

Vanessa Brouard1, Emeline Bovet-Courtois2, Ethel Szerman-Poisson2, Vanessa Brouard 1,2, Hélène Bouraima-Lelong 1,2, Christelle Delalande 1,2 Hélène Bouraima-Lelong1 et Christelle Delalande1. 1 EA2608, Laboratoire OEstrogènes, Reproduction, Cancer; Université de 1 : EA2608 USC INRA, OeReCa, University of Caen Basse-Normandie, Caen Basse Normandie, CS 14032, Caen, France ; 2 INRA USC, CS 14032, esplanade de la paix, CS14032, 14032 Caen cedex 5, France. 2 : CECOS, Caen, France. [email protected] CHU de Caen, 14033 Caen cedex 9, France [email protected] Several chemical compounds present in our environment are endocrine disruptors. Among these compounds, Bisphenol A (BPA) is able to bind It is now clearly established that estrogens are implicated in male estrogen receptors and can activate signaling pathways. Estrogens play reproduction. Their production and the expression of estrogen receptors by avril 15 au 13 du Rennes roles in reproductive male functions as spermatogenesis and final testicular cells allow them to act locally. The estrogen receptors ESR1 and maturations of sperm. Rats’ exposure to BPA at low dose and at period of ESR2 were also retrieved on spermatozoa in different species and recently establishment of first spermatogenesis will allow estimating the impact of a new transmembrane estrogen receptor (GPER) was described in murine BPA on events regulated by estrogens. germ cells. This receptor would be implicated in non genomic actions of Prepuberal S.D. rats of 15 days post-partum (dpp) were randomized in two estrogens by the activation of signaling pathways. Many studies suggest groups: Bisphenol A group (BPA: 50µg/kg/day) and control group (DMSO) that estrogens could be implicated in spermatozoa’s quality; in fact, 17b- (8 animals/group). A phytoestrogens free food (SDS-Dietex) was used form estradiol and xenoestrogens stimulated the capacitation and acrosomic the weaning. Treatments were administered by intra-peritoneal injection for reaction of pig, mouse and human spermatozoa whereas they decreased 14 days. At the end of the treatment, animals were anesthetized, the blood stallion spermatozoa’s motility. was collected and testes and epididymis were isolated and weighed. Our objective was to determine if human spermatozoa expressed GPER by Tissues were then fixed in 3% PFA for histological analysis or frozen for western blot, to localize it and ESR1 and ESR2 by confocal microscopy, to genes expression analysis. quantify the expression of ESR1 and ESR2 by cytometry and to study the The relative testes weights, the percent of seminiferous tubules (ST) with effects of 17b-estradiol on spermatozoa’s motility by computer assisted lumen and with acrosomal vesicles show an increase for the BPA group. No sperm analysis (CASA).Sperm samples are obtained at the CECOS (CHU, modification of estradiol and testosterone levels is observed. Number of Caen) after patient consent. apoptotic cells per ST decreases for BPA group but number of ST stained In addition to ESR1 and ESR2, some spermatozoa samples expressed isn’t different between BPA and control group. Expression of specific genes GPER. Two forms of 38 kDa and 42 kDa were observed, the last form of germ cells (C-Kit), Sertoli cells (SCF) and of specific Blood-Testis-Barrier could correspond to the N-glycosylated protein. GPER is localized at the (BTB) proteins: Occludine and β-catenine, is decreased in BPA group. base of the head whereas ESR1 and ESR2 are localized on the flagellum. Testes of prepubertal rats exposed to BPA present an increase of ST with For ESR1, sometimes, the signal is more important on the mid piece of the lumen and with acrosomal vesicles; this can reflect an advanced flagellum. The percentage of positive spermatozoa for ESR1 was higher spermatogenesis. Decreases of genes expression of specific cells (germ than that expressing ESR2. cells and Sertoli cells) and of BTB can led to a disruption of BTB and -9 17b-estradiol at 10 M added during 5 minutes induced an elevation of the spermatogenesis. These results are in accordance with Li et al. (2009) who rapid and/or progressive motility in normal samples. This result seemed not describe a decrease of gene expression of BTB proteins in adults rats to be related to the number of positive spermatozoa for ESR1 and ESR2 exposed to BPA. This disruption doesn’t seem to depend of hypothalamus- between normosperm and asthenozoosperm. Therefore, estrogens and hypophysis axis because we don’t observe a modification of hormonal levels. estrogen receptors could be implicated in spermatozoa’s quality So BPA exposure can affect the local regulation of spermatogenesis in parameters like the motility; the use of agonists and antagonists of the prepubertal rats. different estrogen receptors will allow us to determine the nature of receptors implicated.

52 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Characterization of the adult hypothalamic neurogenic niche Brain derived neurotrophic factor in the ovary of zebrafish 2015 in sheep and influence of an environmental factor: the photoperiod Pietro Cacialli1,2, Livia D’angelo1, Paolo De Girolamo1, Carla Lucini1, Elisabeth Pellegrini2, Olivier Kah2 , Luciana Castaldo1 Lucile Butruille1, Martine Batailler1, Martine Migaud1 1 INRA, UMR PRC, Nouzilly, France 1Department of Veterinary Medicine and Animal Productions, University of [email protected] Naples Federico II, via Veterinaria, 1, 80137 Napoli, Italy In mammals recent studies have demonstrated the presence of an adult 2Team NEED, IRSET, IFR 140, Rennes, France neurogenic niche in the hypothalamus, a key region that controls [email protected] physiological functions, such as reproduction. In sheep, a long lived mammalian model, the existence of a neurogenic niche has also been Brain derived neurotrophic factor (BDNF) is a member of the Rennes du 13 au 15 avril 15 au 13 du Rennes shown in the hypothalamus and DCX-positive cells were found in the neurotrophin family, whose other components are nerve growth vicinity of this hypothalamic neurogenic niche, indicating the presence of factor (NGF), neurotrophin (NT) 3, NT 4/5 and NT 6/7. BDNF has numerous adult-born neurons in this structure (Batailler et al, 2014). In this seasonal model, reproduction is characterized by alternation of two been highly conserved molecule during the vertebrate evolution. It periods: a period of reproduction during short days and a period of sexual has been demonstrated that the DNA-deduced amino-acid rest during long days. We have recently reported seasonal increases in sequence of the processed mature BDNF of the teleost both proliferation rates and DCX’s expression in the hypothalamus during fish Xiphophorus maculatum shows 90% identity with the mouse short days (Migaud et al, 2011, Batailler et al., submitted). sequence. Also, the primary amino acid sequences of zebrafish This study aims at evaluating (i) whether the markers linked to the various (Danio rerio) and human BDNF are 91% identical. It is largely niche cell types (neural stem cells, oligodendrocytes, microglia, etc.) are known that BDNF in the nervous system promotes neuronal also sensitive to photoperiod by comparing their expression between short and long days (ii) the migratory potential of the sheep hypothalamic growth, differentiation, survival and synaptogenesis. However, neuroblasts. BDNF, similar to other neurotrophins, acts on several peripheral Through an immunohistochemical approach, we assessed the variation in organs. In the ovary, BDNF is involved in mammalian oocyte the expression of the niches’ components by estimating the expression of development, early embryo cleavage and blastocyst formation. various markers in the short and the long photoperiods. We showed a However, to date, there are no data concerning BDNF in teleost variation in the density of labeling for neural stem cells and basal lamina fish ovary. Thus, this study aims to investigate the presence and markers according to photoperiod. However, there is no change in the distribution of BDNF in the ovary of zebrafish, a teleost fish widely expression of markers of oligodendrocytes and microglia. This data suggest that photoperiod drives cytoarchitectural rearrangements within the used as vertebrate model. The identification of the different stages sheep hypothalamic neurogenic niche. Electron microscopy technique will of oocytes was carried out by morphological basis and BDNF was be used to determine more specifically to which extend the cytoarchitecture investigated by immunohistochemistry, in situ hybridization and of the cells lining the third ventricle is affected by photoperiod. qPCR. Our results showed BDNF expression in follicle cell layer in Next, a neuroimaging approach using micron-sized iron oxide particles later stage. In conclusion, these preliminary findings demonstrated (MPIOs) injection was developed to explore the migratory potential of the that BDNF is synthesized and stored in the ovary of zebrafish, sheep hypothalamic neuroblats (DCX+ cells). In a pilot experiment we suggesting an involvement of this neurotrophin in oocyte showed that MPIOs are incorporated by cells lining the third ventricle by development. endocytosis and can be detected by Magnetic-Resonance Imaging (MRI), indicating that the use of MPIOs and RMI for the detection of a possible migration route in the hypothalamus is feasible in sheep. Once the hypothalamic migratory path determined we will identify the phenotype of the newly born neurons by immunohistochemistry approach. 53 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Development of preantral follicles in culture: Cellular Introducing the International Network of Young Researchers 2015 characterization and gene expression in Male Fertility

Véronique Cadoret1,2,3,4, Cynthia Frapsauce1,2,3,4, Peggy Jarrier1,2,3, Pierre Calvel1, Yoni Baert2, Ida Björkgren3, Jennifer Borgmann4, Judit Virginie Maillard1,2,3, Agnès Bonnet5, Alain Roulet6, Philippe Monget1,2,3, Castillo5, Chiara Chianese6, Dorte Louise Egeberg7, Anne Jørgensen7, Danielle Monniaux1,2,3, Dominique Royère1,2,3,4, Yann Locatelli1,2,3,7, Michelle Welsh8, Alexandra Amaral9 Fabrice Guerif1,2,3,4 1Department of Developmental and Stem Cell Biology, Institut Pasteur, 1 INRA, UMR85; 2 CNRS, UMR7247; PRC, Nouzilly, France. 3 Université Paris, France; 2Embryology and Genetics (EMGE), Vrije Universiteit Brussel, François Rabelais, Tours, France. 4 CHRU de Tours, LBR, Tours, France Brussels, Belgium; 3University of Turku, Turku, Finland; 4Center of 5 INRA, UMR1388 GenPhySE, 6 GeT-PlaGe; Castanet-Tolosan, France Reproductive Medicine and Andrology, Münster, Germany; 5Lead Pharma 7 MNHN, Réserve de la Haute Touche, Obterre, France. Medicine B. V., Nijmegen, The Netherlands; 6Department of Experimental [email protected] avril 15 au 13 du Rennes and Clinical Biomedical Sciences, University of Florence, Florence, Italy; 7University Department of Growth and Reproduction, Rigshospitalet, In the context of fertility preservation, ovarian tissue cryopreservation Copenhagen, Denmark; 8College of Medical, Veterinary and Life Sciences, followed by culture of isolated follicles is a promising approach. It is then University of Glasgow, Glasgow, UK; 9Max Planck Institute for Molecular crucial to develop a culture system preserving the integrity of the oocyte- Genetics, Berlin, Germany. [email protected] granulosa-theca interactions while allowing an optimal follicular maturation in order to produce in vitro an oocyte of good quality for subsequent The International Network for Young Researchers in Male Fertility fertilization. Therefore, appropriate parameters must be determined to (INYRMF http://www.youngresearch.eu/) is a non-profitable, evaluate the best conditions for follicular in vitro development. European association that aims at promoting interactions and The animal model chosen was the prepubertal lamb. After isolation from ovarian cortex strips, preantral follicles were cultured individually in collaborations between young scientists in the field of male microdrops under oil in αMEM+ medium supplemented with insulin. Then, reproduction and fertility. Through our website and social networks, we compared follicles cultured for 1, 6, 12 and 18 days to follicles of similar we provide an easy-to-use platform where our members can sizes developed in vivo. communicate, exchange advices and discuss about projects and In this culture system, the follicles developed from the preantral stage (180- methodologies. We also inform our members about scientific 240µm) until the antral stage (up to 800µm) over an 18 days period. events in our research field, job opportunities and other relevant Follicular development involved oocyte growth (x1.3), follicular cell news, both through the webpage and by Newsletters. We organize proliferation (x20), and appearance and development of the antrum (from day 6). However, through comparison with in vivo development, annual meetings where members can meet, present and discuss quantitative differences in growth parameters were found. Particularly, their work and get constructive feedbacks from experienced oocyte growth and antrum development were significantly reduced, but cell scientists in a very friendly and informal environment. For the year proliferation was enhanced in cultured follicles (p<0.001). These 2015, the annual INYRMF meeting will be held in Florence, Italy. differences in follicular morphogenesis were accompanied by a lower Also of importance for the French community of researchers in expression of genes encoding factors of the AMH, BMP and KIT systems, reproductive science is that both the annual INYRMF meeting and but a higher expression of differentiation gene markers such as the European Testis Workshop will be held in France in 2016. steroidogenic enzymes, FSH, LH and estradiol receptors. In conclusion, despite a harmonious follicle development in culture, the These events will be a great occasion to present and promote the observed patterns of gene expression suggested an early maturation of the contribution of French research to our field. Therefore we cultured follicles. encourage any young French scientist in the field to visit our Supported by INRA and Région Centre. webpage or meet us during the Reprosciences meeting in Rennes Contribution of the Toulouse Genomic Platform to make a free registration to our network. 54 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Estrogenic effects of two progestins, Levonorgestrel and Correlation between chromosomes localization and DNA 2015 2015 Norethindrone, in zebrafish oxidative damage in sperm nucleus.

Joel Cano-Nicolau1, François Brion2, Olivier Kah1 Alexandre Champroux1, Joëlle Henry-Berger1, Rachel Guiton1, Fabrice 1 Team NEED, IRSET, IFR 140, Rennes, France Saez1, Joël Drevet1, Ayhan Kocer1. 2 Unité d’Ecotoxicologie in vitro et in vivo, INERIS, Verneuil-en-Halatte, 1GReD laboratory, CNRS UMR6293-INSERM U1103-Clermont University, France. [email protected] team “Mechanisms of mammalian post-testicular infertility” 63171 BP80006, Aubière cedex, France. du 13 au 15avril Brain aromatase (cyp19a1b) is a gene that is expressed in radial glial cells [email protected] of adult zebrafish and that is very sensitive to estrogens; thus, cyp19a1b is commonly used as an endpoint to monitor the potentially estrogenic effects Sperm DNA oxidative damage (SDOD) is one of the causes Rennes avril 15 au 13 du Rennes of endocrine disruptors (EDs) found in the environment. The estrogen- frequently associated with reproductive failures in natural or dependent cyp19a1b up-regulation requires the presence of functional artificial conception. We generated earlier transgenic mice models estrogen receptors (ERs) and, interestingly, occurs in glial cell context only. showing oxidative stress in the epididymis compartment and Previous studies have shown that, while progesterone has no effects on cyp19a1b expression in the brain of zebrafish some progestins, notably spermatozoa with high level of DNA oxidation that were Levonorgestrel and Norethindrone, stimulate cyp19a1b expression in the associated with reproductive failures. Using such spermatozoa brain of adult zebrafish, an effect blocked by the anti-estrogen inhibitor ICI we demonstrated that SDOD is localized in discrete regions of the 182-780. In order to decipher the mechanisms underlying those effects, i.e. sperm nucleus (peripheral and basal nuclear domains; Noblanc et direct binding of progestins to ERs or their metabolization into estrogenic al., 2013). To characterize more precisely the sperm nuclear compounds, we an in vitro glial cell-based assay using cyp19a1b as the regions concerned by oxidation we developed a DNA target gene (Le Page et al. 2006). To this end, the ER-negative glial cell immunoprecipitation strategy using an anti-7,8-dihydro-8-oxo-2’- line U251-MG was transfected with the three zebrafish ER subtypes (zfER- α, -β1, and -β2) and the cyp19a1b promoter linked to luciferase reporter déoxyguanine (8-OHdG) antibody coupled with genome wide gene. Using estradiol (E2) as a positive control, we analyzed the dose- sequencing. We show that all chromosomes are not affected response (10-9 to 10-6M) effects of natural progesterone (P4) and two homogenously by oxidation and that smaller chromosomes (such synthetic progestins: Levonorgestrel and Norethindrone. As expected, P4 as chromosomes 19 and Y) show higher susceptibility to oxidative has no effects on the activation of the cyp19a1b promoter at any damage. Chromosome localization within the sperm nucleus concentration However, Norethindrone and to a lesser extent using Fluorescence In Situ Hybridization (FISH) and immuno- Levonorgestrel caused a dose-dependent expression of luciferase. All FISH assays partly explain that chromosome susceptibility to these effects were suppressed by ICI 182-780. Using ER binding assays, we demonstrated that the affinity of oxidative damage is correlated with their position in the sperm Levonorgestrel and Norethindrone for the three zfERs was very weak, only nucleus. at doses superior to 10 -6M. This suggests that the activity of progestin in Supported by the Region Auvergne vivo and in vitro is probably due to metabolization through 5α-reductase activity. However, we failed to block the luciferase fold induction of progestins using the 5α-reductase inhibitor finasteride. All together, these data provide evidences that Levonorgestrel and Norethistrone have estrogenic activity in vivo and in vitro. However, the underlying mechanisms ate still not completely clear. Supported by the PROOF project.

55 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Epigenetic transgenerational actions of the widely used Novel Role for Anti-Müllerian Hormone in the Regulation of 2015 herbicide Atrazine. GnRH Neuron Excitability and Hormone Secretion

Chunxiang Hao1*, Aurore Gely-Pernot1*, Emmanuelle Becker1, Christine Irene Cimino1,2, Filippo Casoni1,2, Xinhuai Liu3, Andrea Messina1,2, Jyoti Kervarrec1, Bernard Jégou1,2 and Fatima Smagulova1# Parkash1,2, Soazik P Jamin4, Sophie Catteau-Jonard1,2,5, Francis 1Inserm U1085 IRSET, 263 Avenue du Général Leclerc, 35042, Rennes, Collier2,5, Marc Baroncini 1,2, Didier Dewailly1,2,5, Allan E Herbison3, France. 2EHESP, Avenue du Professeur Léon-Bernard, 35043, Rennes, Vincent Prevot1,2, Paolo Giacobini1,2 France . * These authors made an equal contribution 1 Inserm, Jean-Pierre Aubert Research Center, U1172, Development and [email protected] Plasticity of the Postnatal Brain, Lille, France 2 Université de Lille 2, Institut de Medecine Predictive et de Recherche The environmental toxicants have recently been shown to promote an Therapeutique (IMPRT-IFR114), Lille, France Rennes du 13 au 15 avril 15 au 13 du Rennes epigenetic transgenerational inheritance of disease states including 3 Centre for Neuroendocrinology and Department of Physiology, University testis diseases and male infertility. We hypothesize that pesticides of Otago School of Medical Sciences, Dunedin 9054, New Zealand. 4 change the DNA methylation and histone modifications in germ line Inserm U1085-IRSET, Université de Rennes 1, Rennes, France. 5 Service and these changes can be transferred to several generations via de Gynécologie Endocrinienne et Médecine de la Reproduction, Hôpital Jeanne de Flandre, CHU de Lille, Lille, France germline cells. To test our hypothesis outbred (CD1) and inbred [email protected] (C57BL/6J) strains of mice were treated by atrazine during gestation period from embryonic day E5.5 until E15.5. The male progeny of Polycystic ovary syndrome (PCOS) is the most common cause of treated mice were crossed with untreated females for a several generations. Mice from F3 generation were sacrified, reproductive female infertility affecting up to 10% of all women worldwide. The organs and some other tissue were dissected and saved for analysis. reproductive dysfunction involves persistently rapid gonadotropin- The effects of atrazine were evaluated by analyzing the cells releasing hormone (GnRH) pulsatility, which favors the pituitary morphology and physiology (H&E staining, FACS analysis, germ cells synthesis of luteinizing hormone (LH) over follicle-stimulating count, TUNEL assay…) of testis tissue. To analyze a genome wide hormone (FSH), indicating that abnormal GnRH release is an distribution of pivotal marks of actively transcribed genes and meiosis important pathophysiological feature in many cases of PCOS, we performed high throughput sequencing of H3K4me3 histone marks. although the origin of this dysregulation remains unknown. Another To analysis the effect on genes expression we performed high hallmark of PCOS is elevated levels of circulating anti-Müllerian throughput RNA-sequencing using Illumina technology. hormone (AMH). Besides the ovaries, AMH and its receptors are Our preliminary results showed that atrazine doesn’t affect the weight expressed in multiple areas of the murine brain; however, the of reproductive organs and testicular morphology. The RNA-seq possible extra-ovarian effects of AMH on the hypothalamic- analysis showed that 58 transcripts are differentially expressed in pituitary-gonadal axis have never been investigated. testis of F3 males progeny of treated females. Out of differential Here, we show that AMH is an important regulator of GnRH transcripts 35 are genes with known functions and the roles of rest 23 neuronal function, stimulating the firing activity of GnRH neurons transcripts are not elucidated yet. Further work will address the as well as the secretion of GnRH and LH, highlighting how question if gene expressions are affected in other organs including brain and liver. High throughput sequencing of H3K4me3 histone aberrant AMH signaling in GnRH neurons might culminate in marks showed that at least 1000 genomic loci have changed PCOS. H3K4me3 marks in testis of F3 generation of mice. Supported by ANR-2010-JCJC-1404-01 project Our preliminary data demonstrates that atrazine can cause the epigenetic transgenerational phenotype in mouse. 56 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Reference preparations and bioassays for gonadotropin Isolation and molecular characterization of Sertoli and 2015 2015 preparations: why do pharmacopeas ignore scientific data ? undifferentiated spermatogonial cell populations using GFP- expressing transgenic zebrafish lines. Yves Combarnous, Julie Mariot, Camille Jouanny, Sandrine Rafert, Danièle Klett & Claire Cahoreau Edouard Curran, Aude Gautier, Florence Le Gac, Jean-Jacques Lareyre INRA UPR1037 (Fish Physiology and Genomics), BIOSIT, Ouest- INRA-CNRS Physiologie de la Reproduction et des Comportements 37380 Genopole, Campus de Beaulieu, Rennes, France. Nouzilly (France) [email protected] [email protected] The preservation of the genetic resources (mitochondrial and nuclear The classical bioassay for natural pituitary FSHs is the ovarian weight genomes) and the regeneration of individuals are important economic stimulation in immature rats in the presence of a low and constant dose of and ecological issues for the sustainability of the aquaculture sector Rennes du 13 au 15 avril 15 au 13 du Rennes avril 15 au 13 du Rennes hCG (LH activity). Given the short half-life of natural FSHs, 6 injections are and conservation of the biodiversity, respectively. In fish, the performed over three days in order to get a sustained FSH stimulation of cryopreservation of the ovocytes and embryos is impossible but the ovaries (Steelman & Pohley, 1953) The equine chorionic gonadotropin (eCG; PMSG) like hCG possesses a transplantation of cryopreserved diploid spermatogonial stem cells carboxy-terminal peptide (CTP) and therefore exhibits a prolonged half-life. (SSC) in pre-hatching recipient embryos is a promising approach Moreover it exhibits both LH and FSH activity. In consequence of these two fulfilling the requirements. However, the rareness, and our incapacity properties, no additional hCG is required for the measurement of its to efficiently purify and amplify in vitro the SSCs limit the use and the ovarian weight stimulation and due to its long half-life, one single injection dissemination of this procedure. In order to unravel mechanisms is sufficient to exert its full activity over several days (Cole & Erway, 1941). involved in the control of SSC self-renewal, we have undertaken the This assay has been successfully used by all researchers in the field but identification of paracrine factors that are released from the Sertoli for a completely unknown reason the european pharmacopea states that cells within the germinal niche and could act on their cognate for the assay of PMSG, 5-6 injections must be performed as it is the case membrane bound receptors expressed on SSC. In the present study, for FSHs that exhibit short half-lives. we have produced transgenic zebrafish lines that express GFP This procedure is particularly risky as PMSG preparations with degraded specifically in the Sertoli cells (Dr_Gsdf:eGFP) or in spermatogonia carbohydrate moieties and therefore with shorten half-lives will appear as (Dr_Vasa:eGFP) using promoter fragments of cell-specific genes active as intact preparations. (gsdf and vasa respectively). The expression pattern of the transgene The bFSH fused with hCG CTP exhibits only FSH activity (like natural was validated using whole mount RNA in situ hybridization and FSHs) but has a long half-life (like natural eCG). Therefore the FSH assay of Steelman & Pohley was modified as to take into account the expected immunocytochemistry techniques. Note that transgene expression long half-life of bFSH fused with CTP (Combarnous et al, 2012 unpubl.). was not observed in the primordial germ cells (PGCs) of the Therefore, only one single injection was performed instead of 6 but still in developing Dr_vasa:eGFP embryos but was mainly detected in the presence of a low and constant dose of hCG (LH activity). isolated or doublets of undifferentiated spermatogonial cells in the This procedure is strongly recommended as the presence of CTP testis of pubertal males. The fluorescent cell populations were isolated dramatically increases the in vivo bioactivity of FSH through the from adult testes using a differential plating approach followed by a augmentation of its half-life. This effect is moderately observed when fluorescent assisted cell sorting. The enrichment of the candidate cell multiple injections are carried out. types was determined using real time quantitative PCR and a panel of These exemples clearly illustrate the fact that the number of injections must 15 genes representative of different testicular cell types and germ cell be reduced to only one when the effect of prolonged half-life has to be stemness. We demonstrate that the transgenic zebrafish lines are taken in consideration in in vivo gonadotropins bioassays. valuable models to purify highly enriched populations of Sertoli cells or germ cells expressing high levels of certain but not all germ stem cell markers previously proposed in mammals and in fish. 57 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Maternal factors: their expression, control and role in oocyte-

015 The rabbit as a model for the study of gene pathways 2 cumulus dialogue, oocyte maturation and early embryo involved in gonad differentiation in foetal development 2015 development Nathalie Daniel-Carlier, Erwana Harscoët, Dominique Thépot, Aurélie Rozenn Dalbies-Tran1-3, Svetlana Uzbekova1-3, Véronique Cadoret1-4, Auguste, Eric Pailhoux, Geneviève Jolivet. Fabrice Guérif1-4 UMR INRA-ENVA Biologie du Développement et Reproduction, 78350 1 INRA, UMR85 Phsiologie de la Reproduction et des Comportements, Jouy-en-Josas, France 37380 Nouzilly. 2 CNRS, UMR7247, 37380 Nouzilly. 3 Université François [email protected] Rabelais de Tours, 37041 Tours. 4 Service de Médecine et Biologie de la Reprodction, CHRU de Tours, 37044 Tours In mammals, gonad differentiation initiates early in foetal life. The [email protected] molecular mechanisms that underlie gonad differentiation through Rennes du 13 au 15 avril 15 au 13 du Rennes foetal and neonatal life until puberty have already been studied Rennes du 13 au 15 avril 15 au 13 du Rennes The ultimate function of the ovary is to produce oocytes that can be intensively in mouse species. A cascade of genes has thus been fetilized and generate viable embryos. This requires prior optimal described as the main actors, which regulate specific steps. It is now evolution of the oocyte within the follicle. Oocyte development is accepted that early events, which occur during foetal life profoundly refected in and influenced by its surrounding cumulus, as a close and determine the sequence of further gonad development. However, essential dialog exists between the two compartments. Our studies im foetal life is extremely short in mice when compared with other at describing the cellular and molecular events that accompany mammalian species (especially livestock species such as cows, goats, optimal differentiation of the oocyte-cumulus complex, and how they sheep and pigs) and humans. Even though the same events re altered by physiological or environmental factors that can affect punctuate overall differentiation, the different milestones are not ubsequent embryo development. reached at the same time and probably do not have the same effect in We have developed and applied microgenomics technologies each species. It is therefore difficult to rely on data obtained in the (microarray, RNAseq, microfluidic qPCR, proteomics) and revealed mouse to further explain the origin of aberrations in gonad gene expression profiles in the bovine and human oocyte and/or development that may be encountered in other species, especially in cumulus in various physiological situations, according to meiotic stage, humans. oocyte potential to develop into a blastocyst, age or metabolic The aim of the present study was to report a first insight into gonad status… as the basis to unravel underlying regulations. In particular, in differentiation in the rabbit that is an attractive species because of its the fully grown oocyte, expression relies mostly on post-transcriptional 31-day long gestation, the timing of female meiosis around birth and regulation of maternal RNA, including selective cytoplasmic the 15-day delay between gonadal switch and the onset of meiosis in polyadenylation, under control of sequence elements in the 3’ the female. The study was achieved by measuring the expression of untranslated region; it may also involve long and short non coding genes already characterized as being important actors in sex RNA. determination and gonad differentiation in mouse species such as the In parallel, known and novel (Breast Cancer Anti-Estrogen Resistance SRY, Aromatase, STRA8, DMC1 and NANOS2 genes. At the same 4, Stem-Loop Binding Protein 2…) mammalian oocyte-specific genes time, gonads were analysed histologically throughout the have been isolated from a library. Functional genomics experiments developmental period, alongside immunohistological localizations of are underway to investigate their role in oocyte-cumulus dialogue, proteins when this was possible. maturation and/or embryo development. BCAR4 was indeed Interestingly, our data highlighted some rabbit-specific findings with characterized as a novel maternal-effect gene in bovine. Mutation or respect to the gonad differentiation process. deregulation of such genes may underlie certain cases of so far unexplained infertility in women. 58 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Induction of synchronized fertile ovulation by a new selective Molecular and cellular characterization of bovine extra- 2015 agonist of kisspeptin receptor embryonic primary cultured cells: an in vitro model to study trophoblast, endoderm and mesoderm crosstalks before Decourt Caroline1, Robert Vincent1, Lomet Didier1, Anger Karine1, implantation Galibert Mathieu2, Madinier Jean-Baptiste2, Marceau Philippe2, Delmas Agnès2, Caraty Alain1, Aucagne Vincent 2, Beltramo Massimiliano1. Séverine A. Degrelle1-4, Thierry Fournier 2-3, Christophe Richard1,5, Pierre 1UMR Physiologie de la Reproduction et des Comportements (INRA, Adenot1,6, Danièle Evain-Brion2-4, Isabelle Hue1 UMR85; CNRS, UMR7247; Université François Rabelais Tours; IFCE) F- 1 INRA UMR1198 BDR, Jouy-en-Josas, France 37380 Nouzilly, France; 2Centre de Biophysique Moléculaire (CNRS UPR 2 INSERM UMR S1139, U767, FSPB, Paris, France 4301) F-45071 Orléans cedex 2, France. [email protected] or 3 UPD, SPC, Paris, France [email protected] 4 PremUP, Paris, France The neuropeptide kisspeptin (Kp) is the most potent stimulator of avril 15 au 13 du Rennes 5 INRA, UE331 UCEA, Leudeville, France GnRH secretion and therefore an interesting target to develop 6 INRA, MIMA2, Jouy-en-Josas, France treatments to manage reproduction and heal related pathologies. [email protected] Endogenous Kp isoforms (54, 14, 13 and 10 amino acids in length) have short in vivo half-life and continuous administration is required to In mammals, extra-embryonic tissues are essential to support obtain the wanted effects. Single injection, however, is preferable to embryo patterning but also embryo survival, especially in late continuous administration. To meet this need we designed analogs of implanting species. These tissues are composed of 3 cell types: the 10 amino acid isoform (Kp10) with improved pharmacokinetics trophoblast, endoderm and mesoderm, each one being affected by and pharmacodynamics. somatic cell nuclear transfer (SCNT). At present it is unclear how Kp10 scaffold was customized by combining modifications improving these cells interact. In this study, we have established primary cell resistance to degradation and reducing renal clearance. Combination of various modifications leads to analogs having equal efficacy, similar cultures of extra-embryonic tissues from bovine embryos collected or better potency, and a prolonged half-life in blood serum compared at day-18 after artificial insemination. After trypsic digestions and to Kp10. Compounds were active by intramuscular injection at very percoll gradients, these extra-embryonic cells were cultured for up low doses (5 to 15 nmoles/ewe). Nine hours after analog injection, LH to a week on a plastic dish (endoderm, mesoderm) or level were still three-fold higher than basal. When injected in oestrus matrigel/collagenIV coated dish (trophoblast). We validated each ewes pretreated with flugestone acetate (FA) for 14 days, the best Kp culture by comparing it to the corresponding cells from in vivo analog produced a superior synchronization of LH surge, and micro-dissected embryos (microarrays, immunofluorescence). consequently of ovulation, compared to available treatment (injection These primary cell cultures were a powerful tool to start studying of pregnant mare serum gonadotrophin). In presence of a ram treated their cellular properties (CYTOOTM chip) and will further allow in ewes showed all behavioral signs of oestrus. Ovulations triggered by vitro studies on cellular interactions among extra-embryonic Kp analog were fertile as indicated by the rate of pregnancy (7 out of tissues (among control cells or control vs. SCNT derived cells), 10) obtained after servicing. and potentially between extra-embryonic vs. embryonic tissues. In conclusion we generated Kp analogs with improved pharmacokinetics and pharmacodynamics capable, after a single PremUp foundation (S.A.D. post-doctoral fellowship), INRA intramuscular injection, to induce synchronized fertile ovulation in ewe pretreated with FA. These molecules hold a strong potential to funding: ACI PHASE 2008, 2009, AIP AGROBI to I. Hue & EMBIC improve management of livestock reproduction and to treat human European network of excellence headed at INRA by O. Sandra reproductive disorders. Supported by Région Centre (Reprokiss grant). 59 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Ibuprofen disrupts hormonal profiles within the human adult VITRIFICATION OVOCYTAIRE EN DON D’OVOCYTES: 2015 2015 testis Constitution d’une banque d’ovocytes. Deville M 2, Lopes M1, Griveau JF1, Jouve G1, Veau S1, Morcel Christèle Desdoits-Lethimonier 1,2, Laurianne Lesné 1,2, Séverine K2,Leveque J2, Kazdar N1, Viard P1, Ravel C1 Mazaud-Guittot1,2, Millissia Ben Maamar 1,2, David M. Kristensen3, 1 Service de Biologie de la reproduction-CECOS, CHU de Rennes, 16 Bd Karim Bensalah4, Christian Platel4, Nathalie Dejucq-Rainsford1,2& de Bulgarie, 35 000 Rennes Bernard Jégou1,2,5 2 Service de Gynécologie,CHU de Rennes, 16 Bd de Bulgarie, 35 000 1Inserm(Institut National de la Santé et de la Recherche Médicale), IRSET Rennes U1085, SFR Biosit, Campus de Beaulieu, Rennes, France ; [email protected] 2Université de Rennes I, Campus de Beaulieu, Rennes CEDEX, France ; Contexte: Grâce à la mise en place de la technique de vitrification 3Department of Biomedical Sciences, Faculty of Health Sciences, (technique de congélation ultra-rapide des ovocytes) dans les centres University of Copenhagen, Denmark ; 4Centre Hospitalier universitaire avril 15 au 13 du Rennes d’AMP, l’organisation des traitements inhérents au don d’ovocytes a avril 15 au 13 du Rennes Pontchaillou, 2 rue Henri Le Guilloux, Rennes, France. 5EHESP, School été bouleversée, offrant désormais un meilleur confort aux of public health, av. du Pr Léon Bernard, 35043 Rennes CEDEX. receveuses puisque le transfert embryonnaire peut à présent être [email protected] programmé, selon les mêmes principes que ceux du transfert d’un embryon congelé. Ibuprofen is one of the most used Non Steroidal Anti-Inflammatory Matériel et Méthodes: Dans notre laboratoire, au CECOS de Rennes, Drugs (NSAIDs) worldwide. It is used for its analgesic, anti-pyretic and la vitrification a été introduite dès 2012 et, depuis janvier 2014, tous anti-inflammatory properties. Recent study from our laboratory has les ovocytes destinés au don sont vitrifiés. Nous utilisons un système demonstrated that paracetamol and two NSAIDs - aspirin and de vitrification fermé, ce qui garantit une sécurité sanitaire optimale. indomethacin - can exert endocrine disrupting effects within the Le système utilisé est le RAPID-I™ VITRIFICATION SYSTÈME human adult testis ex vivo (Albert et al., 2013). The present study (VITROLIFE) associé au milieu de cryoconservation VitKit®- aimed at investigating the possible endocrine disruptive capabilities of Freeze/Thaw (Irvine Scientific) selon les recommandations du ibuprofen in the human testis. Using the Testis Explant Assay fabricant. Deux ovocytes intacts sont attribués à chaque don sur 3 previously described (Desdoits-Lethimonier et al., 2012), we showed dons ce qui correspond à 6 ovocytes intacts au total par receveuse. that after 24 or 48h of exposure, ibuprofen at concentrations lower or Résultats: Sur 779 ovocytes vitrifiés, le taux de survie des ovocytes equivalent to its peak plasma concentration significantly decreased réchauffés est de 78%, le taux de fécondation des ovocytes ayant testosterone production, whereas insulin like factor-3 (INSL3) survécu au réchauffement est de 72.5% et le taux de clivage des production was not affected. Ibuprofen also displayed anti- ovocytes fécondés est de 98%. Le taux de grossesse clinique en don prostaglandinic properties (PGD2 and PGE2) after 24h of exposure d’ovocytes par transfert d’embryons issus d’ovocyte vitrifié est de 22% which can be compared to other NSAIDs as follows: indomethacin > alors que le taux de grossesse clinique en don d’ovocytes par ibuprofen > aspirin > paracetamol. Furthermore, ibuprofen also transfert d’embryons issus d’ovocytes frais est de 22.8%. affected Sertoli cell function. Thus, it inhibited both Anti-Mullerian Conclusion: Grâce à la vitrification, une véritable banque d’ovocytes a Hormone (AMH) and inhibin B levels. In conclusion, ibuprofen can be pu être constituée, sur le même principe que la banque de sperme. considered as an endocrine disruptor with multiple targets in the Nous pouvons proposer aujourd’hui une prise en charge optimale à human adult testis ex vivo. Further experiments are needed to nos patientes, en conservant les mêmes taux de grossesse avec des decipher the mechanisms underlying these endocrine disruptive ovocytes vitrifiés qu’avec des ovocytes frais. La vitrification ovocytaire properties in the adult human testis. permet ainsi une meilleure gestion de la liste d’attente, les délais étant Supported by Inserm, University of Rennes 1, and grants from the Agence particulièrement longs en France. Nationale de Sécurité du Médicament et des Produits de Santé (Ansm; Financements : Agence de BioMédecine AAP-2012–037). 60 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Expression of Adipokines and several Lipid Metabolism Expression and effect of NAMPT (visfatin) on progesterone 2015 Actors in Adipose Tissue and Granulosa Cells during secretion in hen granulosa cells lactation in dairy cows. Mélodie Diot1,2,3,4, Maxime Reverchon1,2,3,4, Christelle Ramé1,2,3,4, Diot Mélodie1,2,3,4, Ramé Christelle1,2,3,4, Touzé Jean-Luc1,2,3,4, Briant Yannick Baumard5, Joëlle Dupont1,2,3,4 Eric5, Dupont Mickaël5, Dupont Joëlle1,2,3,4 . 1INRA, UMR85 Physiologie de la Reproduction et des Comportements, F- 1INRA, UMR85 Physiologie de la Reproduction et des Comportements, F- 37380 Nouzilly, France. 2CNRS, UMR7247, F-37380 Nouzilly, France 37380 Nouzilly, France . 2CNRS, UMR7247, F-37380 Nouzilly, France. 3Université François Rabelais de Tours, F-37000 Tours, France 3Université François Rabelais de Tours, F-37000 Tours, France. 4IFCE, F- 4IFCE, F-37380 Nouzilly, France. 5INRA, UE 1295, Unité Expérimentale 37380 Nouzilly, France . 5INRA, UEPAO 1297, Nouzilly France. Pôle d’Expérimentation Avicole de Tours, F-37380 Nouzilly, France. [email protected] [email protected] avril 15 au 13 du Rennes In dairy cows, the energetic cost of milk production during early lactation is In mammals, NAMPT is an adipokine mainly produced by adipose greater than energy consumed resulting in a prolonged period of negative tissue and exists in intracellular and extracellular compartments. The energy balance (NEB) and consequent mobilization of body tissue reserves. intracellular form of NAMPT is a nicotinamide The amount of body tissue mobilized negatively affects reproductive phosphoribosyltransferase whereas the extracellular form is considered performance. Some evidence indicates that molecules produced by as an adipokine. In human, NAMPT regulates metabolism but also adipose tissue named adipokines are not only expressed in adipose tissue reproduction and more precisely ovarian functions. In hens, no study but also in the reproductive tract and more precisely in ovary. Our has yet identified and investigated the role of NAMPT in ovary. Here, laboratory has previously shown that these adipokines can regulate ovarian we investigated the presence of NAMPT in hen ovarian follicles and its functions including steroidogenesis. In this study we have investigated the role in granulosa cells. By RT-PCR, Western-Blot and expression of adipokines and their receptors and some lipid metabolism actors in subcutaneous adipose tissue and granulosa cells in nine dairy immunocytochemistry, we found mRNA transcript and protein of cows at different times of body fat mobilization (1, 2, 5 months and 305 NAMPT in theca and granulosa cells from preovulatory follicles. By RT- days after calving). Biopsies of adipose tissue were performed at the qPCR, we observed that mRNA NAMPT levels were higher in dewlap. Granulosa cells from small follicles purified on a percoll gradient granulosa than in theca cells and they decreased during follicle were obtained after ovum pick up. As expected, in adipose tissue, we have development in theca cells where it remained unchanged in granulosa showed by RT-qPCR that expression of several actors involved in cells. NAMPT protein quantities were significantly higher in theca as lipogenesis were decreased after one or two months post-partum and compared to granulosa cells and were unchanged during follicular increased after 5 months post-partum or during the dry period. Opposite development. Plasma NAMPT levels, determined by ELISA and results were observed for some actors involved in the lipolysis. Concerning immunoblot, were significantly reduced in adult as compared to young the adipokines, adiponectin, visfatin, leptin and apelin mRNA expression hen. In vitro, treatment with human recombinant NAMPT (100 ng/ml, increased in adipose tissue during the lactation whereas resistin and 48h) halved basal and IGF1-induced progesterone secretion and this chemerin increased until 2 months postpartum and then decreased at 5 was associated with a reduction in STAR and HSD3B protein levels months postpartum and at the dry period. Interestingly, we have observed and MAPK3/1 phosphorylation levels by granulosa cells. These effects similar variation of messengers of these adipokines and most of actors of were abolished by the addition of FK866, a specific inhibitor of NAMPT the lipid metabolism in granulosa cells except for leptin where the expression was unchanged.Taken together, our data suggest that negative enzymatic activity. Moreover, NAMPT had no effects on granulosa cell energy balance is associated not only to variation of adipokine and lipid proliferation. In conclusion, NAMPT is present in chicken ovarian cells metabolism actor expression in adipose tissue but also in granulosa and and inhibits progesterone production in granulosa cells. consequently could affect steroidogenesis and fertility. Supported by the Région Centre Adipofertikines project Supported by the Région Centre Adipofertikines project

61 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Cardiovascular Alterations in Preeclampsia Adiponectin in human villous trophoblast cells: opposite 2015 2015 effects on energetic metabolism and mitochondrial function Aurélien Ducat1, Ludivine Doridot1, Rosamaria Calicchio1, Céline Méhats1, Jean-Luc Vilotte2, Johann Castille2, Sébastien Jacques1, Franck Fabien Duval1, Esther Dos Santos1,2, François Vialard1,3, Philippe de Letourneur1, Christophe Buffat, Paul Laissue4, Francisco Miralles1, Mazancourt1,4, Marie-Noëlle Dieudonné1 Daniel Vaiman1. 1 : GIG-EA 2493, Université de Versailles-St Quentin, UFR des Sciences 1 INSERM U1016, Institut Cochin, Paris, France. 2 INRA UMR1313, de la Santé, 78180 Montigny le Bretonneux, France. Génétique Animale et Biologie Intégrative, Jouy-en-Josas, France. 2 : Service de Biologie Médicale, CHI de Poissy-Saint-Germain, 78300 3 Laboratoire de biologie moléculaire, Genetique Oncologique et Poissy, France. 3 : Département de Biologie de la Reproduction, Endocrinienne, Hôpital de la Conception - AP-HMINRA, Marseille. Cytogénétique, Gynécologie et Obstétrique, CHI de Poissy-Saint-Germain, 4 Universidad del Rosario, Genetics/GENIUROS Group, Colombia 78300 Poissy, France. 4 : Service de Biochimie et Génétique Moléculaire, [email protected] avril 15 au 13 du Rennes Hôpital A. Paré, 92100 Boulogne, France. avril 15 au 13 du Rennes [email protected] Preeclampsia (PE) is a complication of human pregnancy characterized by a gestational hypertension associated with proteinuria occurring from mid- The placenta provides nutrient exchanges between the mother gestation. Worldwide, this syndrome affects ~5% of pregnant women, and and the fetus and presents an important metabolic activity. is a leading cause of maternal mortality. Endothelial and cardiac dysfunctions are well-described characteristics of preeclampsia, but up to Adiponectin is a cytokine produced principally by the adipose now, studying the molecular effects of preeclampsia on the maternal tissue which controls glucose and lipid metabolisms. It is well cardiovascular system has not been possible, owing to the obvious known that maternal serum adiponectin is inversely related to difficulty of analysing maternal tissue. birth weight, suggesting a negative effect of adiponectin on fetal We recently developed a mouse model of preeclampsia by transgenesis of growth. These effects seem to be associated with a control of the transcription factor STOX1, discovered in 2005 as a protein involved in nutrient transporters in human placenta. However, the molecular the pathophysiology of preeclampsia. Crossing transgenic males with WT mechanisms are presently not clearly elucidated. females induces a severe preeclamptic phenotype in the females, with gestational hypertension, proteinuria, increased plasma levels of sENG and In this work, we studied the direct effect of adiponectin on human sFLT1, and kidney histological anomalies reminiscent of those visible in primary cytotrophoblasts from first trimester placenta. We have human patients. shown that in these cells, adiponectin i) inhibits the expression of We focused on the question of cardiovascular alterations, using purified the two principal glucose transporters (GLUT1 and GLUT12) ii) endothelial cells from the limb muscles of mice carrying either WT or enhances glucose consumption and total ATP production but transgenic embryos, and carried out for a first time an RNAseq analysis of decreases lactate production iii) down-regulates the expression of these cells. We discovered very significant alterations of gene networks genes involved in mitochondrial biogenesis (PGC1α, ERRγ, involved in inflammation, cell cycle and cardiac hypertrophy. Studying the SIRT1, NRF1) and the number of functional mitochondria. heart of mice, we could observe an abnormal cardiac hypertrophy accompanied by histological anomalies. In addition, we performed human Adiponectin appears to regulate human placental functions by experiments by exposing endothelial cells (HUVEC) to plasma from either promoting aerobic glycolysis and by inhibiting mitochondrial normotensive or preeclamptic pregnant women. Bioinformatics analysis biogenesis. These opposite effects suggest a pro-apoptotic role of revealed very significant similarities between the in vivo alterations of the adiponectin as described in other cell types. These findings may murine endothelium and the gene expression profile of the HUVEC. These help us to better understand the role of adiponectin in placental data pave the way to understanding the molecular effects of preeclampsia physiopathology like intrauterine growth restriction. in the cardiovascular system of women.

62 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Dmxl2 is involved in follicle formation in the mouse. BPS and BPF are not safe alternatives to BPA 2015 2015

Maëva Elzaiat1, Clara Gobé1, Bruno Passet2, Nicolas Meunier3, Soria Eladak1, Tiphany Grisin1, Delphine Moison1, Marie-Justine Marjolaine André1, Luc Jouneau1, Patrice Congar3, Aurélie Allais-Bonnet1, Guerquin1, Eric Pailhoux1 and Maëlle Pannetier1. Stéphanie Pozzi-Gaudin2, Alexandra Benachi2, Gabriel Livera1, Virginie Rouiller-Fabre1, and René Habert1. 1INRA, UMR 1198, Biologie du Développement et Reproduction, F-78350 1: Laboratoire de Développement des Gonades, Unité de Stabilité Jouy-en-Josas, France. 2INRA, UMR1313 Génétique Animale et Biologie génétique, cellules souches et radiations. INSERM U 967 - CEA - Intégrative, F-78350 Jouy-en-Josas, France. 3INRA, UR1197 Université Paris Diderot - 92265 Fontenay-aux-Roses, France. 2: Service Neurobiologie de l’Olfaction, F-78350 Jouy-en-Josas, France. de Gynécologie-Obstétrique et Médecine de la Reproduction, Hôpital A. [email protected] Béclère, Université Paris Sud - 92140 Clamart, France. rené[email protected] avril 15 au 13 du Rennes Bisphenol A (BPA) is a widely studied typical endocrine-disrupting avril 15 au 13 du Rennes We identified DMXL2 from RNA-sequencing data as a gene chemical, and one of the major new issues is the safe replacement of this commonly used compound. Bisphenol S (BPS) and bisphenol F preferentially expressed in differentiating-ovaries in the goat (BPF) are already or are planned to be used as BPA alternatives. species. In order to determine its role during ovarian Recently, using the culture system that we historically developed and morphogenesis, we investigated functional studies in mice. named Fetal Testis Assay (FeTA), we brought the first experimental Dmxl2 invalidation (KO) is lethal within the few hours following evidence showing that 10 nmol/L BPA reduces basal testosterone birth. The analysis of its expression in several tissues let us show secreted by human fetal testis explants and that the susceptibility to that the heart, olfactory mucosa and brain expressed Dmxl2 BPA is at least 100-fold lower in rat and mouse species [1]. during development. Our results further suggest that the lethality Here, using again the FeTA system without LH, i.e. the experimental may be due to an alteration of the synaptic transmission. conditions in which mouse and human fetal testes are most sensitive Besides, both male and female gonads express Dmxl2. Notably in to BPA [2], we found that, as for BPA, 10 nmol/L BPS or BPF are the ovary, its expression increases few days before follicle sufficient to decrease basal testosterone secretion by human fetal formation. We show that DMXL2 protein is dynamically expressed testes. The three bisphenols exhibited often non-monotonic dose- in germ cells and granulosa cells; DMXL2 is strongly detected in response curves. With fetal mouse testes, we observed that the the oocytes whereas its expression in the granulosa cells minimum effective concentrations were 1,000 nmol/L for BPA and fluctuates depending on the stage of folliculogenesis. To study BPF and 100 nmol/L for BPS. Finally, 10,000 nmol/L BPA, BPS or Dmxl2 implication during follicle formation, and to bypass the BPF reduced Insl3 expression in cultured mouse fetal testes. This is the first report describing BPS and BPF adverse effects on one issue of lethality at birth, we performed allograft of KO ovaries on physiological function in humans and rodents [2]. nude recipient mice. KO grafts show an alteration in follicle Supported by University Paris Diderot, INSERM, CEA, MEDDE formation, followed by ovarian dysgenesis. (Antiopes), ANSES (PNR-EST). To conclude, our work shows that Dmxl2 is involved in follicle References formation in mice, and makes it a new candidate to explain some 1. ’Tumba-Byn T, Moison D, Lacroix M, Lecureuil C, Lesage L, Prud’homme cases of premature ovarian failure. The conditional knock-out of SM, Pozzi-Godin S, Frydman R, Benachi A, Livera G, Rouiller-Fabre V, Habert R. Differential effects of bisphenol A and diethylstilbestrol on human, rat and mouse fetal Dmxl2 in germ cells or in granulosa cells is in progress in the Leydig cell function. PLoS One 2012;7: e51579. laboratory (please refer to Clara Gobé’s poster) and will permit to 2. Eladak S, Grisin T, Moison D, Guerquin MJ, N'Tumba-Byn T, Pozzi-Gaudin precise its specific role in each cell types. S, Benachi A, Livera G, Rouiller-Fabre V, Habert R. A new chapter in the bisphenol A Supported by ANR and FRM story: bisphenol S and bisphenol F are not safe alternatives to this compound. Fertil Steril. 2015; 103:11-21. 63 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Study of SLC26A8 in HUMAN ASTHENOZOOSPERMIA Inflammatory response of testicular cells is modified by 2015 Elma EL KHOURI, Thassadite DIRAMI, Baptiste RODE, Nathalie DA metformin 2015 SILVA, Patrick LORES, Jean-Philippe WOLF, Gérard GACON, Thierry BIENVENU, Emmanuel DULIOUST and Aminata TOURE. Faure Mélanie1, Alves Sabine1, Guibert Edith1, Michailidis Georgios2, Institut Cochin, Paris. INSERM U1016, CNRS UMR 8105, Université Paris Anastasiadou Maria2, Brillard Jean-Pierre1, Froment Pascal1 Descartes. 1 INRA, UMR85 Physiologie de la Reproduction et du Comportement, F- Ion fluxes play an essential role in the control of sperm motility and 37380 NOUZILLY capacitation (a maturation event occurring in the female genital tract and 2 Laboratory of Physiology of Reproduction of Farm Animals, Department required for fertilization); in particular, calcium, chloride and bicarbonate of Animal Production, School of Agriculture, Aristotle University of are essential for both processes by activating cAMP-PKA-dependent Thessaloniki, Greece phosphorylation pathways. We previously described SLC26A8, also known [email protected] Rennes du 13 au 15 avril 15 au 13 du Rennes as Testis Anion Transporter 1 (SLC26A8), as a male germ cell-specific member of a large family of anion transporters known as SLC26 (Solute avril 15 au 13 du Rennes It is well established that infections of the male reproductive tract contribute Linked Carrier 26) proteins. Most SLC26 proteins display transport activity to alteration of fertility. The testis is considered as an immune-privileged towards monovalent and/or divalent anions (including sulfate, chloride, site, and testicular Sertoli cells have been identified as key players for bicarbonate, iodide, oxalate) and in humans, genetic abnormalities of conferring this immune privilege. These cells present immune properties SLC26A2, SLC26A3, SLC26A4, and SLC26A, have been causally such as immune factors secretion, phagocytosis and constitute also the associated with diastrophic dysplasia, chloride diarrhoea, Pendred blood testicular barrier which limit damage of germ cells. Metformin is an syndrome and deafness respectively. We showed that homozygous insulin-sensitizer which presents anti-inflammatory properties and improved deletion of Slc26A8 in the mouse leads to male sterility due to the total lack the oxidative status. Our purpose was to better understand the of sperm motility, impairment of sperm capacitation and severe structural inflammatory response induced by metformin in Sertoli cells. We have defects of the flagellum (midpiece disorganization, hairpin-like bending of shown its activity on chicken testicular cells and more particularly on the the flagellum and atrophy of the annulus). Consistent with this phenotype, protective cells of germ cells, called Sertoli cells. SLC26A8 in mature sperm localizes at the equatorial segment of the head First results have shown that metformin increased phagocytosis induced by and at the annulus, a ring shape structure at the junction between the dead germ cells in Sertoli cells, measured by oil-O-red staining. In addition, midpiece and the principal piece of the flagellum. we have analysed the Sertoli cells stimulation in presence of a kinetic of In various epithelia, members of the SLC26 family (SLC26A3, A4, A5, A6 stimulation by LPS (lipopolysaccharide used to mimic a bacterial infection), and A9) complex with the Cystic Fibrosis Transmembrane conductance conducted using 0, 6, 12, 24, 48 hours. The expression measured by Regulator, the chloride/bicarbonate channel mutated in cystic fibrosis, and rtqPCR of some interleukins (secreted when the immune system is are able to regulate CFTR transport activity. Interestingly, the CFTR activated) were increased (IL-1β, IL-6, IL-8) after LPS or after metformin channel has been shown to localize at the equatorial segment of the head stimulation. As known, LPS activates the NFkappaB pathway and cells pre- and at the flagellum (midpiece) of mature sperm, and to be required for treated by metformin presented a synergic effect on NFkappaB activation sperm motility and capacitation. induced by LPS and measured by reporter gene expressing luciferase. We will report our findings about SLC26A8 and CFTR functional Despite metformin stimulated several receptors of the immune system cooperation in the sperm and the identification of novel SLC26A8 called Tolls like Receptors (TLR), it did not modify expression of the TLR4. mutations associated with human asthenozoospermia. TLR4 is the receptor bound by LPS and which stimulate NFkappaB Acknowledgments pathway. This work was supported by the Ministère de l’Education Nationale et de la Recherche We can conclude that chicken Sertoli cells possess the set of immune Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre National system, but their response following a bacterial infection was slower than de la Recherche Scientifique, Université Paris Descartes, Agence Nationale de la Recherche (ANR-07-JCJC-0099; ANR-12-BSV1-0011-01 MUCOFERTIL) and the circulating immune cells. Metformin influence the response of Sertoli Association Vaincre la Mucoviscidose (RF20110600465 cells in presence of bacterial infection.

64 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Study of the reproduction for the development of biomarkers Ontogenesis of estradiol synthesis and its regulation by 2015 in ecotoxicology in the fresh water zebra mussel Dreissena gonadotropins in prepubertal female mice 2015 polymorpha François CM1, Magre S1, Giton F2, Petit F1, Cohen-Tannoudji J1, Guigon

CJ1 Alban Franco1, Gabrielle Magniez1, Jean-Marc Porcher2, Sandrine 1 Equipe PAG, Université Paris 7, unité BFA, CNRS UMR 8251, Inserm Joachim2, Laurence Delahaut1, Isabelle Bonnard1, Alain Geffard1, Marc U1133, Paris. 2 Inserm U955, Hôpital Henri Mondor, Créteil Bonnard1 [email protected] 1 UMR I-02 SEBIO, Université de Reims Champagne Ardennes 2 UMR I-02 SEBIO, Verneuil en Halatte [email protected] In mice, the follicles that start to grow after birth constitute the first follicular waves. They develop rapidly to the antral stage and then undergo a avril 15 au 13 du Rennes massive depletion after 14 days post-natal (dpn), so that few of them

During the last two decades, pharmaceuticals compounds have been avril 15 au 13 du Rennes ovulate at puberty (30 dpn). These follicles may ensure the endocrine detected in several aquatic ecosystems and their potential impacts on the ovary capacity during the prepubertal period, including estradiol (E2) reproduction of non-target organisms have to be evaluated. Zebra mussels production. However, little is known about the ontogenesis of E2 synthesis D. polymorpha, commonly distributed in Europe is frequently used for the in the prepubertal female mouse and its possible regulation by the biomonitoring of freshwater systems. In view to define relevant biomarkers gonadotropins FSH and LH. The aim of this study is to analyze the we considered three important steps of the reproductive process: ontogenesis of E2 synthesis and to determine whether it could be regulated - Spermatogenesis. The aim was to obtain a rapid measure of by gonadotropins, as in cycling females. spermatogenesis disturbance using flow cytometry. Quantification in germ Circulating E2 was detectable by HPLC/MS as early as 7 dpn, and reached cells of the DNA content by staining with propidium iodide and analysis by maximal concentrations around 14 dpn. Analyses of steroidogenic flow cytometry was performed. The percentage of cells in Q, G0G1, S and enzymes involved in E2 synthesis (cyp11a1, cyp17a1, cyp19a1, hsd3b) G2M measured, was used to calculate an index of maturity for each and of FSH (fshr) and LH receptors (lhcgr) revealed that their relative samples. A correlation was found between the stage determinated by expression levels increased progressively in the ovary until 14 dpn to histological observation and this value. This tool was used to evaluate the decrease thereafter, except for those of hsd3b and fshr that further impact of the exposure at carbamazepine (0.05, 0.5, 5µg/L) during six increased. Plasma LH and FSH levels were high as early as 7 dpn, peaked months in mesocosms. After a three month exposure (July) and 6 months at 12-14 dpn, and then dropped after 17 dpn. The fact that the levels of (September), a significant delayed maturity was measured at the highest circulating E2 and ovarian steroidogenesis markers became maximal at 12- concentration. 14 dpn when plasma LH and FSH levels were high, strongly suggest that - Spermatozoa quality. Different responses were investigated as DNA gonadotropins are already involved in E2 synthesis in this period. integrity (Comet assay), viability, acrosome integrity, fertility. Concerning Preliminary studies on organotypic cultures of 12 dpn ovaries supported carbamazepine, three times (1h, 3h and 6h) and 3 concentrations (0.1, 1 this hypothesis, as co-treatment by LH and FSH stimulated steroidogenic and 10µg/L) were tested ex vivo. No effect was observed on viability and enzymes expression. Noteworthy, in situ hybridization studies showed that acrosome integrity but DNA integrity tested showed an increase of DNA up to 14 dpn, early antral follicles displayed characteristics of preovulatory damage after 1h, 3h and 6h at each concentration suggesting a possible follicles, as shown by the expression of lhcgr in granulosa cells, usually effect on the genetic information transfer. In vivo exposure of mussels in observed at this stage in thecal cells in cycling females. mesocosms revealed genotoxic effect of carbamazepine on spermatozoa In conclusion, this study reveals that the ovarian endocrine capacity after 3 months. develops rapidly after birth to become significant around 12-14 dpn, when - Larval development. In a transgenerational point of view, it’s necessary to circulating gonadotropins are high. We propose that the unique endocrine control the embryo-larval development of progeny. Optimal conditions to features of the initial follicular waves may contribute to such a rapid ovarian obtain larval development in the labs are in the process of validation. maturation. Supported by the TRECOPOLEM and DOREMIPHARM project

65 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

The transferrin expression by Sertoli cell was critical to the A newly cyp11c1-GFP transgenic zebrafish model to study 2015 corticosteroidogenesis and its perturbation by endocrine

early morphological events of testis cord formation. 2015 active substances at early developmental stages Betty Fumel, Stéphanie Roy, Amina Sow, Isabelle Fontaine, Florian Guillou INRA-CNRS-IFCE-Université de Tours Joint Research Unit for Clémentine Garoche1, Marie Picot1, Nathalie Hinfray1, Jean-Marc au 15 avril Reproductive and Behavioural Physiology, Tours Research Centre, France. Porcher1, Olivier Kah2, François Brion1 [email protected] 1INERIS, DRC/VIVA, Ecotoxicology unit, Verneuil-en-Halatte, France 2IRSET, Team NEED, Rennes, France [email protected] Sertoli cells play a central role in early testicular differentiation

and male sex determination as well as in germ cell development In fish, 11β-hydroxylase (11βH) is the enzyme responsible for the Rennes du 13 du Rennes in adult life. Sertoli cells are somatic cells located in the biosynthesis of cortisol, the main corticosteroid in fish, which is implicated Rennes du 13 au 15 avril 15 au 13 du Rennes seminiferous epithelium of the testis and without their physical in the regulation of multiple physiological processes including stress and metabolic support germ-cell differentiation, meiosis and response, energy metabolism, hydromineral balance, reproduction and the transformation into spermatozoa could not occur. A failure in immune system. The gene coding for 11βH, namely cyp11c1, is expressed Sertoli cells malfunction or poor development causes disorders in early in interrenal cells during the development participating to the spermatogenesis and leads to impaired semen quality and emergence of the corticosteroid axis. To further investigate the expression of cyp11c1 and its potential deregulation by stressors and pharmaceutical possibly testis cancer. Although mammalian spermatogenesis is ligands, we established a transgenic zebrafish line that expresses Green absolutely dependent of the cord formation and survival of Sertoli Fluorescent Protein (GFP) under the control of the zebrafish cyp11c1 cells, little is known about the mechanisms by which drive the promoter. Using in vivo fluorescence imaging techniques on whole larvae, cord testis formation and survive of seminiferous tubules through we showed an expression of the reporter gene from 4 to 7 days post- adult life. Transferrin (Trf), a serum glycoprotein, plays an fertilization (dpf) in an anatomical region where interrenal cells are known essential role in the transport of iron from sites of absorption and to be present. Tissue sections and immunostaining with specific 11βH storage to iron-requiring cells. It is synthesized primarily in the antibodies revealed that GFP is co-localized with the endogenous cyp11c1 protein in interrenal cells. This well agrees with the expression and liver and at low levels in other organs, such as the brain, localization of other steroidogenic interrenal marker genes such as star and mammary glands, and testes. Trf is one of the most abundant cyp11a1 in wild-type zebrafish at this stage of development (To et al. 2007). proteins secreted by Sertoli cells. Trf receptors are present at the To further characterize the cyp11c1-GFP zebrafish model, GFP was cell surface of germ cells, particularly in spermatogonia and quantified in 6-dpf old zebrafish after exposure to glucocorticoids receptor spermatocytes and in lower quantities in round spermatids. The (GR) agonists (Dexamethasone, DEX) and antagonists (Mifepristone, MIF). Trf role in spermatogenesis and controls of the production of germ Exposure to DEX (0.1µM, 1µM and 10µM from 4 to 6 dpf) increased GFP cells remains still unknown. In this issue we have investigate if intensity in a concentration dependent manner, suggesting that GR is down expression of Trf by Sertoli cells decrease the efficiency of involved in the transcriptional regulation of cyp11c1 during development. This effect was abolished when zebrafish larvae were co-exposed with MIF. mouse spermatogenesis. We have generated transgenic mice Interestingly, exposure to DEX induced whole-body cortisol in a expressing a specific Trf shRNA in Sertoli cells. We shown that concentration-dependent manner which is consistent with the effect seen Trf down expression in Sertoli cells bring about the misformation on 11βH. Altogether, these data suggest that the newly developed of testis cord. This result suggested that the Trf level expressed transgenic cyp11c1-GFP zebrafish is an interesting model to study the by the Sertoli cells was critical to the early morphological events expression of 11β-hydroxylase gene in interrenal cells and its perturbation of testis cord formation. by endocrine active compounds acting on the HPI axis, which is critical for cortisol homeostasis.

66 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Acknowledgments: This research was supported by the ANR PROO Materno-fetal toxicokinetics of bisphenol A and its main 2015 2015 metabolite, BPA-glucuronide Study of the dimerization of GPR147, the RfRP3 receptor; Influence on his cell signalling Glenn Gauderat, Nicole Picard-Hagen, Pierre-Louis Toutain, Marlène Lacroix, Catherine Viguié, Sylvie Puel, Véronique Gayrard. Gassem Rami, Foulon-Gauze Florence, Noly Alicia, Xavier CAYLA UMR 1331 TOXALIM - E3 GPE : Gestation Perturbateurs Endocriniens PRC, UMR6175, INRA centre Val de Loire, Nouzilly, FRANCE Ecole Nationale Vétérinaire de Toulouse, 23, chemin des Capelles - BP [email protected] [email protected]. 87614. F-31076 TOULOUSE cedex 3, France The GPR147, also known as Neuropeptide FF1 Receptor (Bonini et al., [email protected] 2000), is a seven-transmembrane G protein-coupled receptor that binds the Gonadotropin inhibitory hormone (GnIH) or the RFamide-related Previous studies in experimental animals have shown that maternal peptide-3 (RfRP3) peptide (Smith & Clarke, 2010). Ligand binding on this avril 15 au 13 du Rennes exposure to bisphenol A (BPA) during late pregnancy leads to high plasma avril 15 au 13 du Rennes receptor signals through Gαi and inhibits cAMP production (Ubuka & concentrations of glucuronoconjugated BPA (BPAG) in fetus compared to Tsutsui, 2014). GPR147 is present in GnRH hypothalamic neurons in mother due to its inabilty to cross the placental barrier. In this context, our which he could meet GnRH receptor as well as with GPR54, the kisspetine project aimed at addressing the issue of the possible reemergence of receptor, then participating altogether to the regulation of the hypothalamic- bisphenol A by deglucuronidation reactions, at the level of the feto- pituitary-gonadal axes (HPG). Today, in addition to the known neuronal placental unit through an in vivo toxicokinetic approach in fetal sheep. crosstalk of GnRH, Kisspetin (KnDy) and RfRP3 neurons. In mammalian, Eight 12-mo old Lacaune pregnant ewes were used. One fetus per ewe the effect of RfRP3 on GnRH seems depend of the species, sex and was chronically catheterized between 107 and 118 days of gestation with season and is still a matter of debate. Furthermore, we don’t clearly know if catheters placed in one carotid artery and one jugular vein. During the first these receptors physically interact in a cell and what could be the part of the experiment, arterial fetal blood samples were collected at consequences of these interactions. regular intervals after fetal intravenous administrations of the same molar This research project, dedicated to the study of the influence GPR147 dose of 21.9 µmol/kg BPA or BPAG (fetal body weight around 2.5kg), 3 dimerization on his action, is indeed part of my current masters-II training days apart to monitor the temporal decay of fetal BPA and BPAG plasma and which implies cellular imagery studies. My first goal is to demonstrate concentrations. In parallel, maternal blood samples were simultaneously the functionality of the fluorescent chimeric receptors produced in the collected and total urine was collected every 3-4-hours for 36 h after BPA laboratory (with full or partial fluorescent protein), then I will study the and BPAG administration to the fetus. During the second part of the consequences of receptors dimerization by following their signaling in living experiment that took place at least 15 days after cesarean, the ewes cells with the help of signaling sensor. I will perform ratiometric FRET received intravenous administrations of BPA or BPAG (at 21.9 µmol/kg and (Förster Resonance Energy Transfer) experiments with reporters able to 8.8 µmol/kg respectively) 3 days apart in order to determine maternal evaluate kinases (PKA, PKC and ERK with AKAR, CKAR, EKAR sensors toxicokinetic parameters. respectively) and second messenger (cAMP with ICUE sensor) levels in Our results suggest that about 30% of the BPAG dose administrated to living cells. fetus was transferred to maternal circulation over 72h. This fraction of First results indicate that the chimeric receptors are functional and BPAG was of the same order than the estimated value of the percentage of furthermore with the help of Bimolecular Fluorescence Complementation BPAG converted to BPA by the fetus during the same period, calculated (BiFC) (Kerpolla, 2008) we are able to detect some homo- and hetero- from the area under the fetal plasma concentration time curve of BPA, receptor dimerizations. obtained after BPAG administration. While BPA administered to the mother References: was rapidly eliminated in urine as BPAG, the inability of BPAG produced by Bonini et al., J. Biol. Chem 2000; 275: 39324-39331 the fetus to cross the placenta associated to the BPA-BPAG conjugation- Kerppola TK; Ann Rev Biophys 2008; 37: 465-487 deconjugation futile cycle at the level of the fetal unit could act as a Smith JT. & Clarke IJ. Trends Endocrinol Metab.2010; 21(4) : 255–260 mechanism prolonging the fetal exposure to the active form of BPA. Ubuka T. & Tsutsui K. Gen Comp Endocrinol. 2014; 209: 148–161 Supported by the ANR (13-CESA0007-1, Modelexpo) and Région Midi- Supported by ANR Repramide project Pyrénées 67 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Estradiol decreases the motility of ejaculated stallion Effects of caffeine and its metabolites on the human fetal 2015 spermatozoa via a GPER- mediating pathway.

testis ex-vivo. 2015

Camille Gautier1, Isabelle Barrier-Battut2, Christelle Delalande1, Hélène Gaudriault Pierre1,2, Mazaud-Guittot Séverine1,2, Lavoué Vincent3, Bouraïma-Lelong1 Coiffec Isabelle1,2, Jégou Bernard1,2,4

1 EA2608-USC INRA2006 OeReCa, Université de Caen Basse-Normandie, au 15 avril 1Irset-Inserm UMR1085, SFR Biosit, , Rennes, France Caen, France. 2 IFCE, site du Haras du Pin, La Jumenterie du Pin, 61310 2Université de Rennes 1, Campus de Beaulieu F- 35042, Rennes CEDEX, Exmes, France . [email protected] France 3CHU Rennes, Service Gynécologie et Obstétrique, F-35000 Rennes, France. 4EHESP-School of public health, Avenue du Professeur The stallion is the mammal producing the largest amount of testicular Léon Bernard, F-35043 Rennes, France. [email protected] estrogens (Raeside, 1932). These steroid hormones are synthetized

13 du Rennes Caffeine is a natural member of the alkaloid family, found in more than 63 plant mainly by the Leydig cells in the testis, but also by the epididymis. Rennes du 13 au 15 avril 15 au 13 du Rennes species (coffee, cocoa beans, kola nuts, tea leaves…). It is the most consumed Recently, we showed the presence of the three estrogen receptors psychoactive compound in the world, and more than 85% of the world’s (ERα, ERβ and GPER) on ejaculated stallion spermatozoa, identifying population use it daily1, including pregnant women2. There are experimental them as a putative target for estradiol. Indeed, during this course in evidences suggesting that caffeine and its metabolites may be able to interfere male and female genital tracts, spermatozoa are exposed to estradiol. with normal Leydig cell development and testosterone production in rat fetal Estradiol could be one of the factors which regulate the different steps testis3. In the present study, the possible impact of caffeine and of 2 of its metabolites, of spermatozoa maturations undertaken to acquire the fertilizing theophylline which have been used to pharmaceuticals drug and theobromine ability: survival, motility, capacitation and acrosomic reaction. In order which is naturally found in chocolate4, was investigated on the human fetal to determine if estradiol could regulate some stallion spermatozoa’s testosterone production and human fetal testis morphology. maturations, we have tested the effects of estradiol on motility and Our first series of experiments evidence that caffeine at doses between 10-9 and -5 -5 -7 capacitation of ejaculated spermatozoa. The samples were obtained 10 M, and theophylline at doses between 10 and 10 M, decrease fetal from stallions from Jumenterie du Pin (IFCE) during breeding season. testosterone production ex vivo in a dose-dependent manner after 72h of exposure. Chemicals were dissolved in the DMSO which is use control during the Spermatozoa were incubated in Tyrode buffer supplemented or not culture. These effects were found in the absence of any gross morphology with NaHCO3 and BSA with and without E2 to evaluate the effect of changes in the testis. estradiol on capacitation. The induction of capacitation was evaluated In conclusion, we demonstrate that under our experimental conditions, caffeine by flow cytometry, with the measure of tyrosine phosphorylation, with and at least one of its metabolite exert anti-androgenic effects. Further monoclonal antibody 4G10. To evaluate the potential effect on motility, experiments will investigate effects of paraxanthine, a third metabolite of caffeine spermatozoa were incubated in Tyrode’s buffer supplemented or not and attempt to identify the main pathways involved in caffeine anti-androgenic with E2 10-7M or fulvestrant 10-7 M (ERα and ERβ specific antagonist) effects. 1: Harris M., The buzz on caffeine. Veg Times., 2004. during 10, 20, and 30 minutes and then to determine the receptor 2: Stanto CK., Effects of caffeine consumption on delayed conception. Am J implicated, spermatozoa were incubated with the agonist of ERα (PPT -7 -7 -6 Epidemiol, 1995. 10 M), ERβ (DPN, 10 M) and GPER (G1, 10 M) during 20 min. 3: Pollard I., Influence of caffeine administered during pregnancy on the early Motility was evaluated with computer assist sperm analysis (Ivos, differentiation of fetal rat ovaries and testes. J Dev Physiol., 1990 4 Hamilton-Thorne). We showed that capacitation is not regulated by : Winston J Craig., Caffeine and theobromine levels in cocoa and carob products. estradiol in opposite to the effect described in porcine spermatozoa Journal of Food Science, 1984 (Ded et al., 2012). Some parameters of motility of freshly ejaculated

Supported by Fondation pour la Recherche Médicale, Inserm, Université de spermatozoa were significantly decreased after 20 minutes incubation Rennes 1, EHESP-School of public health and by the Agence nationale de with estradiol. Incubation with the specific agonists of the estrogen sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES receptors showed that this effect is exclusively mediated by GPER.

68 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Analysis of molecular events characteristics of Characterization of the spermatogonial stem cells in the 2015 2015 spermatogenesis initiation in stallion catshark Scyliorhinus canicula: new insights into the evolution of the niche? Camille Gautier1, Isabelle Guenon1, Christelle Delalande1, Hélène Bouraïma-Lelong1 Aude Gautier, Adrien Bosseboeuf and Pascal Sourdaine 1 Université de Caen Basse-Normandie, EA2608-USC INRA2006 OeReCa, UMR BOREA, UCBN, IBFA, Caen, France Campus 1, esplanade de la Paix CS14032, 14032 Caen [email protected] [email protected] Spermatogonial stem cells (SSCs) are crucial for the maintenance of a Initiation of spermatogenesis requires an active steroidogenesis and the continuous spermatogenesis. The self-renewal of SSC is controlled in a close establishment of blood-testis barrier (BTB). Recently the importance of microenvironment called “niche”. Spermatogonia resulting from SSCs divisions, estrogens in regulating testicular functions has been established. The avril 15 au 13 du Rennes leave the niche and gradually differentiate while proliferating. The phylogenetic avril 15 au 13 du Rennes stallion puberty happens from month 12th to 18th but the specific position of the catshark among Chondrichthyes at the root of Vertebrates makes chronology of molecular events and BTB formation remains to be it a valuable model for comparative studies. In addition, the anatomical structure described. To determine if there is a link between estrogen synthesis and of its testis allow an easy access to the germinative area. Located between the tunica albuginea and the main testicular blood vessel, this area contains single, implementation of BTB at puberty, we have examined the gene expression paired and clusters of undifferentiated spermatogonia surrounded by Sertoli cell of steroidogenic enzymes (Steroidogenic acute regulatory protein, STAR; precursors. Upon leaving this zone, both spermatogonia and Sertoli cell 3β-hydroxysteroid dehydrogenase, 3βHSD ; Aromatase encoding by cyp19, precursors proliferate and progressively form a cyst composed of spermatoblasts steroid receptors (Androgen receptor, AR ; estrogen receptors, ESR1, in which one Sertoli cell is initially associated with one spermatogonium. This ESR2), BTB genes (βcatenin ; Connexin 43, Cx43 ; tight junction protein1, cyst formation coincides with the end of Sertoli cell divisions and further four Tjp1) and cell maturation markers (c-kit ; anti-Mullerian hormone, AMH) in mitosis of differentiated spermatogonia lead to preleptotene spermatocytes. Our prepubertal, peripubertal, postpubertal and adult equine testis. aim was to characterize molecular markers to differentiate the spermatogonial Testes were collected by routine castration (La Clinique de Livet, La subpopulations, including SCCs, in the catshark. Furthermore, an in vitro model Jumenterie du Pin and l’INRA de Jouy en Josas), from a total of 33 horses. of the testicular germinative area was established. cDNA sequences were Animals were divided into 4 groups: prepubertal (9-11 month), peripubertal identified for GDNF Family Receptor alpha1 (GFRα1), Promyelocytic Leukemia (12-18 month), postpubertal (19 month-3 years) and adult (4-14 years). Zinc Finger protein (PLZF), Pou2, high mobility group box protein 3 (HMGB3) Testes were snap frozen and stored at -80°C before mRNA isolation and and mini-chromosome maintenance protein 6 (MCM6). On the basis of the quantitative real time PCR analysis. transcript expression analyses of those factors by RT-PCR and in situ hybridization, four subpopulations were defined: (i) pou2+/gfrα1+/plzf+/hmgb3- STAR, 3βHSD and CYP19 gene expression reach their maximum after 3 /mcm6- single and paired spermatogonia; (ii) years, suggesting that steroidogenesis is maximal after 3 years. ESR1 and pou2+/gfrα1+/plzf+/hmgb3+/mcm6+ undifferentiated spermatogonia; (iii) pou2- ESR2 gene expression show large intragroup variations, the latter could be /gfrα1+/plzf+/hmgb3+/mcm6+ differentiating spermatogonia and (iv) pou2-/gfrα1- due to an effect independent of age. AR gene expression increases with /plzf+/hmgb3+/mcm6+ differentiated spermatogonia. The primary co-culture of age. βcatenin and Tjp1 mRNA levels increase with age and are higher after undifferentiated spermatogonia and somatic cells from the germinative area has 3 years. Cx43 gene expression increases from 18 month and is higher after been successful for long term maintenance of spermatogonia. Addition of GDNF 3 years. Expression of AMH gene is reduced after 18 months, has promoted the development of clones of spermatogonia expressing stem-cell corresponding to differentiation of Sertoli cells and activation of markers. In conclusion, chondrichthyan SSCs seem to share common molecular steroidogenesis. C-kit mRNA level increases from 18 months, suggesting markers and regulation pathways with other vertebrates. Transplantation of that the germ line becomes functional at this age. potential SSCs in the catshark is now a challenge to take up to further High CYP19 mRNA level are found at the same period of establishment of characterize those cells. BTB, thus estrogen synthesis could be correlated with events linked to spermatogenesis development in the equine testis. Supported by the PEPTISAN project (CRBN and FEDER)

69 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

The widely used herbicide atrazine affects epigenetic Invalidation of Dmxl2 in female germ cells and fertility 2015 processes during meiosis in male mice trouble. 2015

Aurore Gely-Pernot1*, Chunxiang Hao1*, Emmanuelle Becker1, Igor Clara Gobé, Maëva Elzaiat, Marjolaine André, Aurélie Allais-Bonnet, Eric Stuparevic1,3, Christine Kervarrec1, Frédéric Chalmel1, Michael Primig1, Pailhoux, Maëlle Pannetier Bernard Jégou1,2 and Fatima Smagulova1# INRA, UMR 1198, Biology of Development and Reproduction, F-78350 1Inserm U1085 IRSET, 263 Avenue du Général Leclerc, 35042, Rennes, Jouy-en-Josas, France. France. 2EHESP, Avenue du Professeur Léon-Bernard, 35043, Rennes, [email protected] France, 3Present address: University of Zagreb, Faculty of Food Technology and Biotechnology, 10000, Zagreb, Croatia. * These authors Recent researches in our laboratory showed the implication of made an equal contribution. [email protected] Dmxl2 gene in follicle formation in mouse ovaries (refer to Maëva Atrazine is a widely used agricultural herbicide. It is the most common avril 15 au 13 du Rennes

contaminant present in groundwater in many countries. Many studies using avril 15 au 13 du Rennes Elzaiat’s poster). In the differentiating ovary Dmxl2 is expressed rodent models showed that the administration of atrazine affects both by germ cells and somatic cells. Then, when folliculogenesis steroidogenesis and gonad function in both sexes. Atrazine demasculinizes starts, the protein is detected in oocyte cytoplasm at every folliclar male gonads and induces a partial or complete feminization in fish, stages; but also in granulosa cells, faintly in primordial or primary amphibians and reptiles. In mammals, this compound induces reductions in follicles and strongly in follicle of later stages. Dmxl2 encodes for androgen level and induction of estrogen synthesis that lead to a a scaffold protein presenting several protein/protein interaction reproductive toxicity. However, the effects of atrazine on meiosis, a key step of reproduction, are largely unknown. We hypothesize that atrazine domains, which appear to be involved in cell trafficking. In the can affect meiosis via interfering histone modifying enzymes and chromatin female gonad, this protein could play a specific role in each cell remodelling events. To test this hypothesis, we treated C57black6J mice types together with being involved in oocyte and granulosa cells with atrazine at 25mg/kg/day during 3 weeks and two weeks without communication. To determine the role of Dmxl2 in germ cells and pesticide introduction. Mice were sacrified, effects of atrazine were in supporting cells, conditional invalidation of this gene is realized evaluated by analyzing cells morphology and physiology (H&E staining, thanks to 2 transgenic lines of mice: one expressing the Cre FACS analysis, Cell count, TUNEL assay…) and by molecular biology recombinase under the control of the Vasa gene promoter in techniques using Affymetrix microarrays, Immunohistochemistry of chromosomal spreads and testis sections. To understand if atrazine has an germ cells (Vasa-Cre); the other under the control of the AMH impact on histones H3k4me3 marks distribution we performed Genome receptor II promoter in female supporting cells (Amhr2-Cre). wide sequencing of H3K4me3 histone marks. We found a meiotic delay in Up to now, only mice invalidated for Dmxl2 in germ cells have treated mice, associated with an increased number of cells having been obtained. Regarding to our first results, females (Vasa-Cre; persistent meiotic DNA double strand breaks (DSBs) and histone Dmxl2flox/flox) seem to be able to reproduce. However, ongoing modifications marks associated with the presence of the breaks. Genome fertility tests tend to show a lower fertility, (decreasing with age). wide analysis of H3K4me3 marks revealed altered patterns in 823 genomic Histological analyses of these mice ovaries at 7 weeks showed loci. H3K4me3 marks are changed in genes involved in a wide range of cellular functions including GTPase activity, apoptosis, ubiquitin pathways important mass cells and a few antrum follicles. Analyses at older and regulation of steroid hormone function. We found that H3K4me3 marks stages are pursued. To conclude, invalidation of Dmxl2 in the are enriched in regions where meiotic recombination is initiated via the germ cell line has no effect on initial follicle formation, but seems formation of DSBs and depleted within the pseudoautosomal region of the to be involved in ovarian function maintenance at adult stages. X chromosome. Our data demonstrate that atrazine exposure results in impaired Supported by FRM chromosome synapsis and disruption of meiotic DSB repair progression leading to delayed meiosis and reduced numbers of spermatozoa. 70 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

1alpha,25-dihydroxyvitamin D3 effects on 30-day-old rat Sex- and photoperiod-dependant variations in the Syrian 2015 2015 Sertoli cells: gamma-glutamyl transpeptidase activity and hamster RFRP system glucose uptake Jo B Henningsen1, Vincent-Joseph Poirel1, Jens D Mikkelsen2, Francois Renata Gonçalves 1,2, Ana Paula Zanatta1,2, Leila Zanatta3, Christelle Gauer1 and Valérie Simonneaux1 Delalande2, Fátima Regina Mena Barreto Silva1, Hélène Bouraïma- 1Institut des Neurosciences Cellulaires et Intégratives, CNRS, University of Lelong2. Strasbourg, Strasbourg, France 2Neurobiology Research Unit, 1Universidade Federal de Santa Catarina, Florianópolis, Brasil Rigshospitalet, Copenhagen University Hospital, Denmark 2Université de Caen Basse-Normandie, Caen, France [email protected] 3Universidade Comunitária da Região de Chapecó, Chapecó, Brasil [email protected] Photoperiodic signals are transduced into the rhythmic release of the pineal Rennes du 13 au 15 avril 15 au 13 du Rennes avril 15 au 13 du Rennes hormone melatonin, which synchronizes reproduction with season. RF- Sertoli cells have an important role in the male reproductive system, related peptide (RFRP)-3 is considered to play a role in the seasonal maintaining the microenvironment for germ cells development by, for regulation of reproduction and its expression is strongly down-regulated by example, secreting nutrients as lactate, the main energetic substrate for melatonin in short photoperiod (SP). RFRP-3 regulates reproductive germ cells. The gamma-glutamyl transpeptidase (GGTP) is a membrane- activity unclear. In this study we investigated the photoperiod-dependent bound enzyme considered a Sertoli cell marker, having an important role in variations in the RFRP system in female and male Syrian hamster and reproduction since animals deficient on GGTP are infertile. 1alpha,25- furthermore evaluated the effects of chronic intracerebroventricular RFRP- dihydroxyvitamin D3 (1,25D3) the active form of Vitamin D, is critical for the 3 administration in female Syrian hamster. maintenance of normal reproduction since reduced fertility is observed in We found no differences in the neuroanatomical distribution of RFRP- male rats deficient on vitamin D. The aim of this work was to study the immunoreactivity and GPR147 (RFRP-3 receptor) mRNA between female effect of 1,25D3 on GGTP activity, glucose uptake and lactate secretion in and male Syrian hamster. Both GPR147 and RFRP3 nerve terminals are 30-day-old rat Sertoli cells. Sertoli cells were obtained from 30-day-old found in similar areas: mainly in hypothalamic (POA/OVLT, MPN/AVPV, Wistar rats by sequential enzymatic digestion. For the glucose uptake PVN, AH, VMH and ARC) and thalamic areas (BST, Hb and PVT). experiments, cells were incubated with 14C-DG (0.1 microCi/ml) in the However, RFRP-neuronal expression and GPR147 mRNA levels are -9 presence or absence of 1,25D3 for 1 h (10 M). For the GGTP assay and significantly higher in female than in male. Furthermore the SP inhibition of lactate determination, cells were incubated for 1, 3, 6, 24, 48 or 72 h in the GPR147 mRNA levels as well as RFRP-neuronal expression is a stronger -9 presence or absence of 1,25D3 (10 M). Inhibitors, when used, were added in female as compared to male. In females as well, RFRP-fiber density in 30 min before the hormone. After incubation time, the cells were the MPN/AVPV is increased in SP as compared to long photoperiod (LP). homogenized in cold 0.1 M Tris buffer, pH 8.5 for the enzyme assay and Intracerebroventricular RFRP-3 administration in sexually active LP female the medium was collected for lactate measurement. 1,25D3 was able to hamsters decreased gonadal weight, whereas in SP-adapted females increase the GGTP activity after 6 h (132%) and 72 h (113%) of hormone RFRP-3 significantly stimulates gonadal size despite photoinhibitory exposure. H-89 and KT-5720 were able to inhibit this effect (42% and 45%, conditions respectively) while Stearoilcarnitine and Ro31-0432 did not modify the RFRP and its receptor GPR147 are regulated by photoperiod in both male hormone stimuli. 1,25D3 also stimulated glucose uptake (124%) and lactate and female Syrian hamster. Our results suggest a more sensitive RFRP secretion after 3 h (118%), 6 h (115%) and 72 h (113%). These findings system in females, possibly playing a role in modulating the pre-ovulatory demonstrate that the 1,25D3 increase testicular GGTP activity via PKA, and LH surge. The effects of RFRP-3 depend on the reproductive state of the stimulates glucose uptake and lactate secretion in 30-day-old rat Sertoli female and interestingly RFRP-3 potently stimulates reproductive activity cells, suggesting that the hormone may be involved in male reproductive despite photoinhibitory conditions functions. Supported by CNPq, CAPES, FINEP, PPGBQA-UFSC. Supported by the Université de Strasbourg (Idex) and ANR Repramide.

71 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

The conserved 3’-5’ exoribonuclease Exosc10 is essential for Morphokinetic study of early embryo development using 2015 2015 embryogenesis and spermatogenesis in the mouse time-lapse microscopy.

Soazik P. Jamin, Fabrice G. Petit, Christine Kervarrec, Michael Primig Nadia Kazdar1, Patricia Viard1, Célia Ravel1 Inserm U1085-IRSET, Université de Rennes 1, Rennes, France 1CHU de Rennes, Service de Biologie de la Reproduction, Rennes, France. [email protected] [email protected]

One particular challenge of Assisted Reproductive Technologies (ART) is Exosc10 encodes a conserved 3'-5' exoribonuclease important for the selection of in vitro-fertilized embryos with the highest success rate of RNA processing and degradation. The yeast and fly orthologs implantation. Recently, kinetic analysis of early embryo development has been proposed as a novel evaluation tool for the classification of embryos (Rrp6) are essential for normal growth and development. avril 15 au 13 du Rennes with the best potential for implantation. However, the reliability of the avril 15 au 13 du Rennes However, little is known about the role of Exosc10 in mammals, criteria defined thus far is still controversial. Here, we examined new kinetic apart from siRNA interference data generated in vitro, indicating and morphologic parameters which aim to best predict in vitro-fertilized that the gene might not be absolutely required for cell division. To embryos implantation. In particular we investigated the potential relevance investigate the function of Exosc10 in vivo, we generated a of the morphology of the zona pellucida (ZP).The study was conducted on mouse gene deletion model. Interestingly, Exosc10-/- mice 66 patients aged 21-40, with blood levels of AMH > 1ng/ml, and who display an early embryonic lethal phenotype, which shows that benefited from one mono-embryo transfer. The successive cleavages of embryos fertilized by Intracytoplasmic Sperm Injection (ICSI) was followed the protein is important for the onset of cell division during initial by time-lapse video-microscopy. We have examined the duration of the stages of embryogenesis. Therefore, we have generated transitions between the 2-3 cells (CC2) and the 3-4 cells (S2) stages, conditional mutant mice using the Cre-lox system to delete together with the time required for the embryos to achieve the 5-cells stage Exosc10 specifically in mitotically growing spermatogonia. Initial in vitro (t5; expressed in hours post-ICSI: hpi). The thickness of the ZP was results reveal that both testis and epididymis are smaller and calculated as an average of 10 measures.We have found that the thickness germ cell development is affected in these mutants. of the ZP can vary between patients from 14 to 21µm. A global analysis performed on all the embryos included in the study, show that a thinner ZP (16,5 ± 0,70 µm; n=22 versus 18,5 ± 3,32 µm; n=44) was correlated with a higher implantation rate, which is in agreement with the assisted hatching strategy frequently used in ART laboratories. The implantation rate of embryo who developed with an early t5 (< 48.8hpi), and therefore classified as C+ according to Meseguer et al (Hum Reprod 2011; 26, 2658-71), was slightly higher than for embryos classified A+, ie with an expected t5 between 48.8 and 56.6 hpi. As expected, embryos classified as A+ exhibited a better implantation rate with a thinner ZP (50% n= 6 for zp≤15.5µm vs 20% n=10 for zp>15.5µm). Surprisingly, C+ embryos were equally successful in implanting with a zp≤15.5µm ( 44% n=9) or >15.5µm (39% n=14). This preliminary result suggests that, unexpectedly, embryos classified on the basis of their developmental kinetics only as C+ have, in fact, the best potential for implantation. Therefore, we propose that the thickness of the ZP is a pertinent criteria for the selection of the embryos for transfer, in order to offer the best chances of a successful pregnancy.

72 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Intact Cell MALDI Mass Spectrometry and Top Down Targeting MT1 and MT2 melatonin receptors with fluorescent 2015 proteomic approaches to identify potential biomarkers of ligands: assay on cell cultures and brain tissue. 2015 oocyte maturation and quality in surrounding cumulus cells C. Lagaraine1, V. Bozon1, M. Beltramo1, P. Delagrange2, G. Guillaumet3, in human and bovine. F. Suzenet3, L. Dufourny1

15avril 1PRC, INRA-CNRS-Univ. Tours-IFCE, Centre INRA Val de Loire, Nouzilly, Valérie Labas2, Véronique Cadoret1,4, Ana-Paula Teixeira-Gomes2,3, France. 2Institut de Recherches SERVIER, Croissy sur Seine, France Audrey Gargaros2, Fabrice Guerif 1,4, and Svetlana Uzbekova1,2. 3ICOA, CNRS, Université d’Orléans, Orléans, France 1 Team BINGO (Biologie Integrative de l’Ovaire), INRA PRC (Physiologie [email protected] de la Reproduction et des Comportements), UMR7247, Nouzilly, France Melatonin is the main synchronizer of circadian and seasonal 2 Laboratoire de la Spectrométrie de Masse, plate-forme PAIB2, INRA, functions and is also involved in numerous others physiological Nouzilly processes. In mammals, melatonin binds to 2 high affinity G-protein avril 15 au 13 du Rennes 3 INRA, Infectiologie et Santé Publique, UMR1282, Nouzilly au 13 du Rennes coupled receptors, MT1 and MT2. These 2 receptors have been 4 Service de la Reproduction, CHRU, Tours cloned in several mammals. The paucity of mRNA for these receptors [email protected] [email protected] did not allow determining the location and identity of MT1 and MT2 Reproducible fingerprints in mass range < 20 kDa could be obtained by expressing cells using in situ hybridization. Furthermore, many Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) on different attempts to develop primary antibodies against these receptors failed. microorganisms and eukaryotic cells. We adapted ICM-MS on few cells The study of melatonin receptors is complicated by the lack of biopsies of cumulus cells (CC) surrounding bovine and human oocytes selective pharmacological molecule to determine the specific before in vitro fertilization (IVF). CC gathered from individual immature or involvement of each subtype in cellular and physiological functions. mature oocytes were analysed by ICM-MS with sinapic acid matrix using Therefore the aim of the present study was to develop new MALDI-TOF mass spectrometer (Waters) operating in positive linear mode. fluorescent agonists to localize MT1 and MT2 receptors in cells and Spectral profiles were collected in the mass range 2000-20000 m/z and tissue. For this purpose, melatonin was linked to fluorescent analysed with MassLynx software. Alignment, peak intensity quantification molecules or transformed into a fluorescent molecule. These 2 and statistics were done using Progenesis-MALDI (Nonlinear Dynamics). strategies lead to the synthesis of more than 50 different ligands that More than 130 peaks were detected in CC in both species, and several of were first pharmacologically characterized on membrane preparations. them varied between different physiological conditions (p<0.01) in relation Those having Ki in nanomolar range for MT1 and/or MT2 receptors with either oocyte maturation (immature vs mature) or oocyte competence were subsequently tested on different cell cultures expressing MT1 or to develop after IVF into viable embryo (blastocyst vs arrested embryo). In order to identify m/z peaks, Top Down proteomic approach consisting in MT2 receptor subtype. Tests were also performed after melatonin direct fragmentation of intact molecular species was carried out. preincubation to check for staining specificity. Finally, the most Protein/peptide extracts from human and bovine CC were enriched for promising fluorescent ligands were tested on brain tissue sections small molecular species and analysed by tandem MS using LTQ Velos from ewes killed in short photoperiod to check for the presence of Orbitrap. 26 and 11 major peaks were formerly identified using ProSight PC fluorescence at the level of the pars tuberalis, the region having the in bovine and human CC, respectively. Among them different histones, most intense concentration of melatonin receptors, and of the ubiquitin, vimentin, heat shock and thymosin family proteins and others mediobasal hypothalamus. Among the fluorescent ligands tested, were identified. several provided good pharmacogical data but none gave specific In conclusion, m/z peaks detected in CC by differential and quantitative labelling on cells. The most suitable for microscopic observations ICM-MS profiling could be identified using top down proteomic approach were linked to cyanin and Bodipy. Future studies will focus on more and might be used as potential biomarkers of oocyte competence in discriminative ligands in order to map these receptors on tissue. assisted reproduction technology protocols. Supported by Région Centre. Supported by INRA and Val-de-Loire Region. 73 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

A microRNA-132/212 pathway mediates GnRH activation of Overexpression of hyaluronidase-1 oppositely alters cell 2015 FSH expression growth and cAMP-stimulated estrogen synthesis in human 2015 granulosa KGN cells.

Jérôme Lannes, David L’hôte, Ghislaine Garrel, Jean-Noël Laverrière, Jérémy Le Masson1,2,, Marion Vanneste 1,2, Jérôme Levallet 1,2,3, Joëlle Cohen-Tannoudji, Bruno Quérat. Isabelle Guénon1,2,3, Pierre-Jacques Bonnamy 1,2,3 Université Paris-Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et 1 ComUE Normandie Univ . 2 UNICAEN, OeReCa, F-14032Caen, France Adaptative (BFA), F-75013 Paris, France; Centre National pour la 3 INRA, USC 1377, F-14032Caen, France.. Recherche Scientifique (CNRS) UMR 8251, Paris, France; Institut National [email protected] de la Santé et de la Recherche Médicale (INSERM) U1133 Physiologie de

l’axe gonadotrope, Paris, France Hyaluronan is a linear, high molecular weight, and non-sulfated [email protected] glycosaminoglycan and is widely distributed in the extracellular matrix. Despite avril 15 au 13 du Rennes

avril 15 au 13 du Rennes the well-defined role of hyaluronan in the expension of cumulus-oocyte complex Gonadotropin-releasing hormone (GnRH) plays a key role in the vertebrate at the time of ovulation, both expression and biological functions of hyaluronan- reproductive system by stimulating biosynthesis and secretion of pituitary catabolizing enzymes (namely hyaluronidases 1 and 2) in ovarian tissue are still gonadotropins. Although a wealth of knowledge has accumulated on largely unknown. To explore potential effects of HA-catabolizing activity in transcriptional control of gonadotropin subunit genes, the potential involvement of ovarian functions, we have induced a stable overexpression of hyaluronidase1 microRNAs (miRNAs) has still to be explored. During the last decade, miRNAs (HYAL1) in human granulosa KGN cells. As compared with wild-type (WT) and emerged as critical regulators of gene expression by modulating target mRNA control plasmid-transfected (CTRL) cells, HYAL1 overexpressing cells (OVER) availability (degradation or inhibition of translation) through their capture into the cultured for 96h in MEM medium supplemented with 5% charcoal-stripped fetal RNA-induced silencing complex (RISC). In this study, we investigated the role of calf serum displayed a two-fold increase in cell growth associated with the two miRNAS that target the same transcripts, miRNA-132 and miRNA-212 on the acquisition of a more epitheloid morphology. Increase in cell growth appeared as GnRH-induced Follicle-Stimulating Hormone (FSH) expression. Both miRNAs the consequence of increasing expression of CCND1 (cyclin D1) as determined are encoded by the same intronic sequence and are activated by GnRH. We first by real-time PCR. HYAL-1 overexpression was also associated a 2-fold showed in rat pituitary cells that blocking miR-132/212 action by locked nucleic expression of CD44 and some genes encoding HA-metabolizing enzymes such acid overexpression reduced the activation of FSH secretion by GnRH. It also as HYAL2 and HAS2. Overexpression also markedly inhibited cAMP-induced abolished the GnRH stimulation of Fshb mRNA steady state level. The estradiol synthesis (-60%) through transcriptional repression of CYP19A1. mechanism of this mediation was then explored in mouse gonadotrope LβT2 Addition of dextran sulfate (DxS), an inhibitor of hyaluronidase activity, not only cells. The GnRH stimulation of Fshb mRNA was reproduced in these cells by abolished the repression of cAMP-induced CYP19A1 expression in OVER cells overexpressing one or both miRNAs and was prevented by blocking both but also markedly increased that expression in all cell types, suggesting the miRNAs together. Sirt1 deacetylase mRNA is a potential target of miR-132/212. involvement of endogenous hyaluronidase in the repression of aromatase We showed that GnRH treatment induced a capture into RISC of miR-132 and expression. Endogenous hyaluronidase was also involved in KGN cell growth as Sirt1 mRNA, resulting in a lowered level of SIRT1 deacetylase and a concomitant demonstrated by the DxS-induced exit of cell cycle in all cell types. The use of increase in the acetylated form of Forkhead Box O1 (FOXO1), a transcriptional different chemical inhibitors of intracellular signalling pathways revealed that the repressor of Fshb. Blocking miR132/212 prevented GnRH-induced FOXO1 repression of PI3K (Phosphatidylinositol-3-Kinase) and JNK (c-Jun N-terminal acetylation. Over-expression of an acetylated-mimicking mutant of FOXO1 kinase) pathways mimicked the DxS effects. All these data suggest that induced an increase in Fshb mRNA expression, likely via its observed exit from endogenously activated JNK and PI3K pathways in KGN cells promote cell the nucleus and consequently, the release of the inhibitory action on Fshb growth and repress cAMP-stimulated CYP19A1 expression that are further promoter. Overall, we show that GnRH increases miR-132/212 that target Sirt1 amplified by hyaluronidase overexpression. In conclusion, HYAL-1 could play a mRNA into the RISC complex. The lower level of SIRT1 deacetylase results in an key role in folliculogenesis (early terminal growth, phenotypic differentiation increase in the acetylated form of FOXO1 and a decrease in its transcriptional between cumulus and antral granulosa cells) by oppositely regulating granulosa inhibitory action on Fshb subunit gene. This is the first demonstration of an cell growth and steroidogenic response to FSH. obligatory microRNA pathway in the GnRH-regulated expression of a gonadotropin gene. Lannes J. is a recipient of a PhD grant from Université Paris-Diderot. 74 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Has Uranium an endocrine disruptor-like activity after Gametogenesis of triploid Pacific Oyster Crassostrea gigas 2015 chronic exposure ? A focus on the rat male reproductive 2015 function Christophe Lelong1, Anne Sophie Martinez1, Beatrice Adeline1, Guillaume Rivière1, Nolwenn Dheilly1, Aude Jouaux1, Pascal Favrel1, Pierre Boudry2, Audrey Legendre1, Margaux Tymen1, Philippe Lestaevel1, Stéphane Clothilde Heude Berthelin1, Kristell Kellner1 Grison1, Maâmar Souidi1, Karine Tack1 1- UMR BOREA. UCBN, CNRS-7208 –UPMC, MNHN, IRD-207, 14032 1 Institut de radioprotection et de sûreté nucléaire (IRSN), PRP-HOM, Caen cedex. SRBE, LRTOX, Fontenay aux Roses, France. [email protected] 2- Unité PFOM, UMR 6539 LEMAR, Ifremer, Brest Some environmental contaminants interact with hormones and may exert [email protected] adverse effects which could explain the increased risk of fertility problems and reproductive dysfunctions observed since several years. Uranium is a heavy metal naturally found in the environment and its many uses in civil or Triploidy can occur in many animal species but is often lethal. In avril 15 au 13 du Rennes military technologies give cause of concern about human health risks, avril 15 au 13 du Rennes invertebrates, amphibians and fishes, triploids are viable but they are often particularly for their reproductive function. In complement to reproductive sterile, or unfertile. Although triploid oysters Crassostrea gigas have studies, a recent experimental study has suggested that uranium will be an generally been considered to be sterile, gametogenesis events are endocrine-disrupting chemical, causing estrogen receptor-dependent sometimes recorded. A classification of gametogenesis stages has been responses in female mice. The objective of our project is to identify and established allowing the description of gametogenesis in triploid oysters but evaluate endocrine activity and reproductive effects of low dose of natural also comparison with diploid animals (Jouaux et al., 2010): The α-pattern uranium on male reproductive parameters. We compare general corresponds to animals displaying numerous proliferating gonia (PCNA parameters (body and reproductive organ weights) and RTQPCR analysis labelling at stage I), resulting in abundant gametes at stage III whereas the in two models of chronic contamination by drinking water (40 mg/l, supra- β-pattern is associated with locked gametogenesis (only few mature environmental and non toxic concentration) after in utero and postnatal gametes at sexual maturity) with accumulation of abnormal gonia from exposure of male Sprague-Dawley rats. We show that uranium increased stage I to III. The granular aspect of TUNEL labelling in the perinuclear significantly testis weights after in utero exposure although no effect was area of stage Iα animals suggests the occurrence of apoptotic events. observed in postnatal exposed animals. RTQPCR analysis evidenced a Reproductive effort was evaluated by quantitative approaches as well. We significant differences in genetic expressions of cyp11a1 (- 36%; ø), also used a microarray analysis to compare female and male gonad cyp19a1 (- 11%; ø), HSD17B3 (- 21%; ø), HSD3B1(- 47%; + 75%) and AR transcriptomes of 2n, 3nβ and 3nα during their gametogenesis (Dheilly et (- 43%; + 60%) RNAs for in utero and postnatal models, respectively. al, 2014). In comparison to 2n, we observed a difference of gene These preliminary results suggest a modulation of androgen and estrogen expression related to DNA repair, apoptosis, cell division. A misregulation balance in two groups of exposed animals to low dose of uranium, which of the cell cycle checkpoint during mitosis may be involved in the could be more deleterious for in utero exposed animals. Focus on successful, but delayed development of gonad in 3nα individuals. reproductive function markers, which are hormonally regulated, confirms Moreover, the sterility of 3nβ individuals is associated with the disruption of that mRNA expression of some spermatogenesis and BTB integrity sex differentiation mechanisms. Indeed, 3nβ females express male-specific markers are modulated. Further analysis with (immuno)histological genes and 3nβ males express female-specific genes. Some of these genes observations, hormonal assays are necessary to complete these first have previously been characterized in C gigas as sex-determining genes in results and study if adult animals exposed during critical windows have 2n oysters, based in particular on their early gonadic cell expression. In 3n, reproductive disorders. Together, these data will provide a first study of some of them also exhibit a peak of expression in stage III, the time- endocrine-disruptor activity of low doses of natural uranium on male window for sex determination already defined in 2n oysters (Santerre et al., reproductive function after chronic contamination. Indeed, identify 2014). endocrine disruptor properties of low dose of uranium is a real challenge for This study in triploid Pacific oysters provides evidence that a disruption of risk assessment and could suggest this involvement in the increase of male sex differentiation and mitosis may be responsible for the impaired reproductive dysfunctions. gametogenesis. 75 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Elements of the germinal niche in an alternative Role of Melatonin receptors in the synthesis of melatonin in 2015 hermaphrodite: the mollusc Crassostrea gigas the pineal gland. 2015

Christophe Lelong1, Sébastien Chong1, Romain Travers1, Béatrice Julie Lépinay1, Dominique Gennetay1, Didier Chesneau1, Philippe Adeline1, Didier Goux2, Kristell Kellner1 and Clothilde Heude Berthelin1. Delagrange2, Catherine Taragnat1, Véronique Bozon1 1- UMR BOREA. UCBN, CNRS-7208 –UPMC, MNHN, IRD-207, 14032 1-UMR7247, Physiologie de la Reproduction et des Comportements, Caen cedex. Nouzilly, France. 2-Institut de Recherche Servier, Croissy sur Seine, 2- CMABio, SF ICORE, UCBN, 14032 Caen cedex France [email protected] [email protected]

Pineal gland is the main site of melatonin synthesis (MLT), neurohormone Rennes du 13 au 15 avril 15 au 13 du Rennes The lophotrochozoan mollusc, Crassostrea gigas, is an alternative involved in mammalian reproduction. Night duration is the major factor Rennes du 13 au 15 avril 15 au 13 du Rennes hermaphrodite. Germinal niche in this species can lead to the controlling MLT synthesis. MLT binds two GPCR with high affinity, MT1 reversible development of male or female line with every annual and MT2. Cogé and al. showed that the ovine pineal gland expressed mRNA encoding both MT receptors. The presence of the ligand and its reproductive cycle. We have studied the ultrastructure of the receptors in the same organ suggests the regulation of MLT synthesis by gonad at early stages of gametogenesis, especially the early MT receptors. The aim of our work is to study functional properties of MT niche. It is defined by an organization with early germ cells that receptors and their role in the MLT synthesis in ovine pineal gland. show specific stem cell characteristics (as chromatoid body or low Primary cultures of pinealocytes were realized from sheep’s pineal glands nucleoplasmic ratio) and surrounded by somatic cells. Germinal aged less than 6 months. To decrease the endogenous MLT in culture stem cells markers have been identified from recent supernatant, two different treatments were used: the first was to wash the transcriptomic analysis of C. gigas, and the expression by RT- cells before pharmacological stimulations, and the second was to inhibit tryptophan hydroxylase, enzyme involved in MLT synthesis, with 4-chloro- qPCR and in situ hybridization of Vasa and Piwi homologs was L-phenylalanine (PCPA) and to empty the vesicles of 5-HT with reserpine. strictly localized on the early niche. Functional tools are currently The efficiency of inhibition is determined by incubating the cells with the developed in order to explore the functioning of the germinal isoproterenol (ISO, adrenergic receptor agonist) and measuring the amount niche. Strategies of cell enrichments led to obtain a population of MLT secreted by RIA. The activation of the MAP kinase Erk 1/2 pathway with both germ and somatic cells, and specific populations with and the Gi-protein were studied by incubating the cells with specific either early germ cells or somatic cells. These different cell pharmacological agents of α1, β adrenergic receptors, MT receptors fractions were qualified by molecular expression of specific (luzindole, inverse agonist) and with 0.1 µM MLT +/- 5 ng/ml of PTX (Gi- protein inhibitor). The expression of MT receptors on the surface of cells, markers. A first cell sorting of putative germ stem cells was the binding affinity and the internalization mechanism were studied with the developed on the basis of aldehyde dehydrogenase activity in the radioligand, 2-[125I] –iodomelatonin, at various concentrations and with germinal population. We aim now to study the fine molecular and different incubation times. cellular regulation on these germ and somatic cells during the MT receptors are functional in pineal gland and are able to transduce an early reinitiation of gametogenesis, but also the mechanisms intracellular signal through the activation of Gi-protein and MAP kinase Erk involved in the maintenance of the undifferentiated state of stem 1/2 pathway activation. More, MT receptors would appear to regulate cells in this molluscan species . negatively the MLT synthesis and finally, their number expressed on the membrane would depend of the breeding or anoestrus period. During short days, the binding affinity of MT receptors is higher than in long days, and the number of sites is decreased Supported by Institut de Recherche Servier

76 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

FOXL2 is a new progesterone-regulated gene in the Topaz1: a new actor of the spermatogenesis 2015 2015 endometrium Alix Luangpraseuth-Prosper1, Fanny Husson1, Elodie Lesueur1, Corinne Audrey Lesage-Padilla1*, Caroline Eozenou1*, Gareth Healey2, Takashi Cotinot1, Béatrice Mandon-Pépin1§ Shimizu3, Jean-François Oudin1, Daniel Vaiman4, Akio Myamoto3, I 1INRA, UMR 1198 Biologie du Développement et Reproduction, F-78350 Martin Sheldon2, Pierrette Reinaud1, Gilles Charpigny1, Maëlle Pannetier1, Jouy en Josas, France Olivier Sandra1 [email protected] [email protected] 1 INRA, UMR1198 Biologie du Développement et Reproduction, Jouy-en- Josas, France Our laboratory have pointed out and characterized a new gene 2 Centre for Reproductive Immunology, Swansea University, Swansea, UK which was called TOPAZ1 (Testis and Ovary-specific PAZ 3 Obihiro University of Agriculture and Veterinary Medicine, Hokkaido,

Rennes du 13 au 15 avril 15 au 13 du Rennes domain gene 1) (Baillet el al, 2011). Because this gene is highly avril 15 au 13 du Rennes Japan conserved in Vertebrates and is specifically expressed in germ 4 Institut Cochin, INSERM U1016, Paris, France. co-first authors; [email protected] cells, the role of TOPAZ1 has been studied during gametogenesis. In mammals, mutual actions of estrogens and progesterone on their uterine For this, the mouse Topaz1 gene has been disrupted via a knock- receptors are essential for endometrium receptivity and conceptus in model. implantation. In cattle we showed that FOXL2 -a key gene for ovarian Whereas the fertility of invalidated female mice is not disturbed, differentiation and maintenance- is expressed and regulated in homozygous Topaz1-/- male are sterile. They have a reduced endometrium during oestrous cycle, a finding confirmed in murine and testis size compared to wild type testis and histological analysis human endometrium. The present study aims to determine if FOXL2 is a progesterone-target gene in the bovine endometrium. Using various highlighted an absence of spermatids (round and elongated) and experimental models in cattle, our results indicated (i) a negative spermatozoa in Topaz1-/- testis. These analyses also revealed correlation between FOXL2 gene expression and progesterone (P4) blood that the perturbation of spermatogenesis takes place between 15 levels (ii) a significant reduction of FOXL2 transcript level in ovariectomized and 20 dpp. The lumen of their epididym is also completely cows supplemented with P4 for 6 days (2.2-fold vs. control ovariectomized devoid of spermatozoa. Thus, meiosis seems to stop before cows, P < 0.05) (iii) a significant decrease in FOXL2 mRNA level in bovine -5 haploid germ cells formation. Moreover, we showed that neither endometrial explants incubated with P4 (10 M) for 48h (2.4-fold vs. control retrotransposon repression in germ cells nor chromosome pairing explants, P < 0.05). No impact of oestradiol on FOXL2 gene expression was detected in these conditions. In order to confirm the regulation of during prophase I of meiosis are disturbed in Topaz1-/- testis FOXL2 promoter by P4, COS7 cells were transfected with a caprine (Luangpraseuth-Prosper et al., in preparation). FOXL2 reporter gene and progesterone receptor (PR) A or B expression Transcriptomic analysis have been realized from two vectors. In the presence of PRA and PRB, P4 (10-7 M) stimulated the developmental stages (15 and 20 dpp) of controls and Topaz1-/- activity of FOXL2 promoter (2.8-fold). Mutation of the P4 Response testis and showed gene expression variations. The validation of Element (PRE) in the caprine FOXL2 promoter abrogated the activity of the differential expressed genes by quantitative PCR and the this promoter in P4-treated COS7 cells overexpressing PRA/PRB. characterization of the biological pathways disturbed in absence Collectively, our data show that reduced FOXL2 expression in the of Topaz1 are on the way in the laboratory. endometrium during the luteal phase results from the down-regulation of PRA/B known to occur in the presence of P4. Determining the biological All of these analyzes should point out a better understanding of actions of FOXL2 will be mandatory to define the contribution of this Topaz1 function in mammals spermatogenesis. transcription factor in the regulation of sensor and driver properties of the endometrium. Supported by ANR-08-GENM-037, INRA PHASE division and MESR

77 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

HIV-1 interacts with human testicular germ cells Analysis of mutations involved in Disorders of Sexual 2015 Development using a new model of mouse supporting cells 2015 Dominique Mahé1, 2, 3, Giulia Matusali1, 2, 3*, Claire Deleage1, 2, 3*, culture. Raquel Alvarenga4, Anne-Pascale Satie1, 2, 3, Karim Bensalah5, Laurence Guézenec6, Bernard Jégou1, 2,3 , Nathalie Dejucq-Rainsford1, Namya Mellouk1, Pierre Calvel1, Anu Bashamboo1, Ken McElreavey1 2,3 1Human Developmental Genetics Unit, Department of Developmental and 1 INSERM U1085-IRSET, Campus de Beaulieu, Rennes CEDEX F-35042, Stem Cell Biology, Institut Pasteur, Paris, France France ; 2 Université de Rennes I, Rennes CEDEX F-35042, France ;3 [email protected] SFR Biosit, Rennes, France ;4 Laboratoire de Biologie Cellulaire, ICB- UFMG, Brésil ;5 Centre Hospitalier Universitaire de Pontchaillou, Service Disorders of sexual development (DSDs) are rare and Urologie, 2 rue Henri Le Guilloux, Rennes CEDEX 9 35033, France ;6 heterogeneous disorders characterized by a complete or partial Rennes du 13 au 15 avril 15 au 13 du Rennes Centre Hospitalier Universitaire de Pontchaillou, Centre de coordination

Rennes du 13 au 15 avril 15 au 13 du Rennes mismatch between genetic and phenotypic sex. In particular, 46, des prélèvements, 2 rue Henri Le Guilloux, Rennes CEDEX 9 35033, XY DSD with complete gonadal dysgenesis and 46, XX testicular France ; * equal contribution. [email protected] Recent reports of the endogenisation of SIV in primates demonstrate that DSD are genetic disorders in which the development and lentiviruses can infect the germinal lineage. Testicular germ cells (TGC) of both differentiation of the embryonic testes or ovaries, respectively, are infected men and macaques have been shown to harbor HIV/SIV nucleic acids in affected. The development of high throughput genomic tools such situ by several teams including ours (Le Tortorec A, PloS ONE 2008). Although as CGH arrays or exome sequencing is beginning to provide HIV binds but cannot enter human spermatozoa (Ceballos A, J Exp Med, 2009), deeper insights into the genetic architecture associated with viral DNA has been detected in a few spermatozoa from HIV-1 infected men, suggesting a clonal infection of their progenitors, the TGC. In this context, we human DSD. Nevertheless, their aetiology still remains unknown aimed at investigating the ability of human TGC to interact with HIV-1. TGC were in almost 50% of the cases, mainly because of the lack of broad, isolated from normal human testes obtained at autopsy or following orchidectomy. rapid and reproducible functional assays to validate the candidate TGC preparations, composed of haploid spermatids, tetraploid spermatocytes pathogenic variants issued from genomic analyses. and diploid spermatogonia and spermatocytes cells, were on average 94% pure and contained less than 4% of contaminating testicular somatic cells and 2% of To tackle this issue, we recently developed a model of mouse CD45+ leukocytes, as revealed by flow cytometry. As expected, TGC were supporting cells culture to study pathogenic variants associated to devoid of CD4. However, they expressed at their membrane alternate HIV-1 DSD. From mice bearing SF1prom-GFP transgene, we were able receptors (heparan sulfate proteoglycans, the mannose receptor and to dissect, dissociate and grow total cells from male or female galactocerebroside), as well as CCR3. HIV-1 binding on untreated or pronase- treated TGC, was evaluated by p24 ELISA quantification and show HIV-1 embryonic gonads and transfect those with wild type or mutant attachment of both HIV-1 R5 (SF162) and X4 (IIIB) strains in a dose dependent versions of DSD associated genes fused with a C-term Cherry. manner, involving .cellular heparin sulfate and to a lesser extent, the mannose This protocol enable us to specifically purify GFP+/Cherry+ cells receptor. The viral envelope gp120 was partly involved in this binding. (i.e. supporting, transfected cells) by FACS, in order to monitor The seminoma T-cam2 cell line, which displays the receptor described above, the effects of the candidate genes on male and female genetic was used to assess whether the male germ line can support viral entry and further steps of viral cycle. . HIV-1 entry and reverse transcription could be programs by qPCR. The data collected so far by testing human detected in infected T-cam2 as revealed by confocal imaging for the viral protein variants of steroidogenic factor 1 (SF1) tend to show that primary p24 detection and qPCR for HIV DNA. In addition, the use of VSV-G culture of mouse supporting cells is a viable technique to pseudotyped virus, which increases the efficiency of viral entry, indicates that T- functionally validate the pathogenicity of DSD-associated genetic cam2 can also support further steps of viral cycle, e.g. integration of viral DNA into host DNA. The development of primary culture of spermatogonia and their variants infection is underway to determine whether HIV-1 can enter these cells. Acknowledgements : This work was funded by Inserm, ANRS and Sidaction. .

78 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

PreImplantation Factor (PIF) Promotes Human Trophoblast Exploring the function of spindlin genes during mouse 2015 2015 Invasion spermiogenesis.

Hadia Moindjie1, Esther Dos Santos1,2, Laurence Loeuillet3, Philippe de Charlotte Moretti1, Maria-Elisabetta Serrentino1, Julie Cocquet1 Mazancourt1,5, Eytan R. Barnea6, François Vialard1,4 and Marie-Noëlle 1INSERM U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, Dieudonne1. France; Université Paris Descartes, Sorbonne Paris Cité, Faculté de 1. GIG-EA 2493, Université de Versailles-St-Quentin, U.F.R Sciences de la Médecine, Paris, France. [email protected] Santé, France. 2. Service de Biologie Médicale, Centre Hospitalier de Poissy-Saint Germain, France. 3. Service d’Anatomo-pathologie, Centre In the mouse, deletions of the long arm of the Y chromosome (MSYq-) are Hospitalier de Poissy-Saint Germain, France. 4. Département de Biologie responsible for the deregulation of hundreds of genes, many of which are de la Reproduction, Cytogénétique, Gynécologie et Obstétrique, Centre encoded by the sex chromosomes; this deregulation leads to spermiogenesis Hospitalier de Poissy-Saint Germain, France. 5. Service de Biochimie et avril 15 au 13 du Rennes defects and male infertility. Sly gene, a member of the Sycp3 superfamily, is one avril 15 au 13 du Rennes Génétique Moléculaire, Hôpital Ambroise Paré, Boulogne, France. of the five multicopy genes present on MSYq and Sly deficiency has been shown 6. BioIncept, LLC, Cherry Hill NJ, USA. PIF* Proprietary to be at the basis of the gene deregulation and sperm defects observed in MSYq- males via changes at the chromatin level. Another member of the Sycp3 [email protected] family and found to be up-regulated in Sly-deficient mice is Slx, an X-linked multicopy gene. Intriguingly, the absence of SLX leads to a down-regulation of Successful human embryo implantation depends on a deep and highly spermiogenic XY genes and male infertility. SLX and SLY are therefore thought controlled invasion of extravillous trophoblast (EVT) in the maternal to be cornerstones in the regulation of XY genes during mouse spermiogenesis. endometrium. Invasion process is regulated, in part, by matrix However, the phenotype observed in Sly-deficient mice is less severe than in metalloproteinase (MMP) activity and integrin expression. MSYq-, paving the way for the implication of others regulators. Our favorite PreImplantation Factor (PIF) is a peptide secreted by viable mammalian candidate is Ssty, another multicopy gene bore by the MSYq segment which embryos. Moreover, it is detected in placenta and maternal blood encodes a spermatid-specific protein and appears to interact with SLY/SLX circulation. Recently, it was shown that PIF promotes invasion in proteins. SSTY protein shares ~70% homology with Spin1, an ubiquitous trophoblast cell lines. nucleolar protein shown to control gene expression at the level of chromatin and The present study was undertaken to assess the presence of PIF in human to interact with the trimethylated histone H3K4. SSTY and Spin1 are part of the placenta during pregnancy and to characterize its effects on primary Spindlin family which encompasses three other members on the X chromosome (Spin2c, Spin2d and Spin4). As of today, nothing is known about these other human trophoblast invasion. members. At the fetomaternal interface, intense PIF labelling was detected during The aim of the present study is to investigate the role of Ssty and other Spindlin early gestation in trophoblastic cells. However, a decrease of PIF labelling members in spermiogenesis and in the underlying chromatin regulation was observed at term. Furthermore, PIF significantly promoted invasion of processes. We found that Ssty and Spin2d are specifically expressed in round human EVT. This pro-invasive effect of PIF in EVT was associated with (i) spermatids, and that SSTY protein co-localizes with the X or Y chromatin. We increased matrix metalloproteinase MMP-9 activity and (ii) reduced tissue are currently developing antibodies, mouse and cellular models to further inhibitor of metalloproteinase-1 (TIMP-1) mRNA expressions. PIF also investigate their localization and molecular mechanism. Specifically, we are regulated αv and α1 integrin mRNA expressions. Using a genome setting up experiments to establish if they are all able to interact with histone microarray, we have shown that p53 signaling pathway is activated by PIF post-translational modifications and with SLY/SLX proteins. We are also in EVT and could be implicated in this proinvasive effect. developing coimmunoprecipitations followed by mass spectrometry analyses with In summary, this work describes the direct positive effect of PIF on the the intent to discover new partners. Knowing that several Spindlin members are control of human trophoblastic invasion by modulating MMP/TIMP balance highly conserved in rodents and humans, this project could be particularly and integrin expressions. Moreover, these results provide insight into the relevant for the study of human male infertility. Funded by ANRJC EPICHROMXY possible role of PIF in pathological pregnancy characterized by insufficient or excessive trophoblast invasion like pre-eclampsia or intrauterine growth restriction. 79 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Genome-wide analyses of the X and Y chromosomes during Characterization of a de novo complex chromosomal 2015 2015 mouse spermiogenesis rearrangement with five breakpoints associated with azoospermia Charlotte Moretti1, Frederic Tores2, Maria-Elisabetta Serrentino1, Julie Cocquet1 A. Mouka 1, 2, V. Izard 3, S. Brisset 1, 2, L. Drévillon 1, G. Tachdjian 1, 2, 1INSERM U1016, Institut Cochin, Paris, France; CNRS, UMR8104, Paris, L. Tosca 1, 2 France; Université Paris Descartes, Sorbonne Paris Cité, Faculté de 1AP-HP, Service d’Histologie, Embryologie et Cytogénétique, Hôpitaux Médecine, Paris, France. 2Plateforme de Bioinformatique, Université Paris Universitaires Paris-Sud, Hôpital Antoine Béclère, 92140, Clamart, France. Descartes - Sorbonne Paris Cité, Institut Imagine, Paris, France. 2Université Paris-Sud, 94276 Le Kremlin-Bicêtre cedex, France [email protected] 3AP-HP, Service de Gynécologie-Obstétrique et Médecine de la In mammals, the X and Y chromosomes are transcriptionally silenced by Reproduction, Hôpitaux Universitaires Paris-Sud, Hôpital Antoine Béclère, an epigenetic process named MSCI for meiotic sex chromosome avril 15 au 13 du Rennes 92140, Clamart, France. [email protected] avril 15 au 13 du Rennes inactivation. It is initiated at pachytene stage by phosphorylation of histone H2AX by ATR and MDC1-mediated spreading of this signal over the sex Complex chromosomal rearrangements (CCRs) are balanced or chromosomes, and followed by recruitment of repressive histone marks unbalanced structural aberrations involving three or more chromosomal such as trimethylated lysine 9 of histone H3 (H3K9me3) and breakpoints with exchange of genetic material between two or more heterochromatin proteins. Because some of these repressive marks and chromosomes. CCRs are rather rare event in the general population and proteins are still visible on the sex chromosomes after meiosis, it was carriers display various phenotypes (normal phenotype, infertility, initially thought that the X and Y chromosomes remain mostly silenced in malformations, mental retardation and/or congenital abnormalities). Male spermatids and therefore do not carry genes important for spermiogenesis. carriers are at risk of reproductive failure as a result of spermatogenesis This dogma has been challenged by studies showing a significant number disruption. Indeed, CCRs can be associated with abnormal segregation of of X genes (re)activated after meiosis, coinciding with accumulation of derivative chromosomes and production of chromosomally abnormal sperm. active chromatin marks on the X and Y chromosomes, such as histone In this report, we describe a rare and de novo CCR associated with lysine crotonylation (Kcr) and trimethylated lysine 4 of histone H3 azoospermia in a 36 years-old man. The CCR was characterized by (H3K4me3). karyotype, FISH (fluorescence in situ hybridization) and array-CGH To conclude on the expression and regulation of XY genes during mouse (microarray Comparative Genomic Hybridization) Agilent 1M 2.1Kb spermiogenesis, we have re-analyzed expression datasets (RNA- resolution assays. Successive analyses by FISH allowed to estimate the sequencing) from purified mouse germ cells at different stages of location of breakpoints and thus a gene cartography of these regions. spermatogenesis, using the last version of the mouse genome mm10. We Results showed that the rearrangement was more complex than initially show that over a 1000 of XY genes are highly expressed in round and assumed with a two-step CCR and five breakpoints were identified. The elongating spermatids, many of which with relevant putative roles in sperm first event, insertion of a part of an inversed chromosome 12 into the short differentiation. arm of a chromosome 7, involved three breakpoints located on We then sought to better characterize the chromatin composition of the X 12p11.1q11/12q13.11, 12q21.2/12q21.33 and 7p21.2-21.3. The second and Y in spermatids via chromatin immunoprecipitation-sequencing event, pericentric inversion of chromosome 12, involved two others analyses for 10 different chromatin marks (histone variants and histone breakpoints located on 12p13.31-12p13.2 and 12q21.33/qter. The post translational modifications). With this approach, we show that the cartography associated with these breakpoints showed several genes of coverage of several active chromatin marks does not differ between the potential interest involved in the reproductive function such as ARL4, sex chromosomes and autosomes, contrary to conclusions drawn from FOXJ2, NEDD1, SYCP3 and NFYB. Array-CGH analysis did not immunofluorescence analyses. There are however significant differences in highlighted DNA copy number variation on estimated breakpoints or other marks which could be at the basis of the co-regulation of XY genes elsewhere. This case report underlined the potential of conventional and during sperm differentiation. This is particularly pertinent since deregulation molecular technics to precise mechanism of CCRs formation and to identify (up- or down-regulation) of XY genes after meiosis leads to male infertility. breakpoint DNA sequence Supported by ANRJC EPICHROMXY 80 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Sperm storage in female reproductive tract: study of Anti-Müllerian hormone expression is differentially regulated 2015 molecules involved

by oestradiol and dehydrotestosterone in control women and 2015

dysovulatory women with the polycystic ovarian syndrome Cindy RIOU1,2, Audrey GARGAROS1, Grégoire HARICHAUX1, Aurélien BRIONNE3, Joël GAUTRON3, Xavier Druart1, Valérie LABAS1, Nadine Chrystèle Racine1, Joëlle Taieb1,2, Michaël Grynberg1,2, Hady El GERARD1 Hachem1,2, Renato Fanchin1,2, Joëlle Cohen-Tannoudji1, Nathalie di 1 UMR7247 Physiologie de la Reproduction et des Comportements, INRA, Clemente1, Alice Pierre1 Nouzilly. 2 ALLICE Elevage Innovation Service, Paris. 3 Unité de 1. Univ Paris Diderot, Sorbonne Paris Cité, Biologie Fonctionnelle et Recherches Avicoles, INRA, Nouzilly [email protected] Adaptative (BFA), F-75013 Paris, France; CNRS UMR 8251, F-75013 Paris, France; Physiologie de l'axe gonadotrope INSERM U1133, F-75013 Paris, Because of prolonged sperm storage in their oviduct, domestic France. 2. AP-HP, Service de Biochimie Hormonologie Gynecologie avril 15 au 13 du Rennes hens can produce fertile eggs for up to 3 weeks following a single Obstetrique, Hopital Antoine Beclere, Clamart F-92140, France. avril 15 au 13 du Rennes [email protected] artificial insemination (AI). The oviduct secretions may have an impact on sperm survival but its composition during fertilization is Contex: Anti-Müllerian hormone (AMH) is a member of the transforming unknown. In the present study, we compared the proteomic growth factor family which exerts a repressive role on folliculogenesis. AMH content of uterine fluid collected from two distinct lines of hens. and its specific receptor AMHR-II are expressed by granulosa cells of The first displays a shorter period of sperm storage (10 days, line growing follicles. These last years, serum AMH has been recognized as a DPF-) whereas the second displays a longer period of sperm reliable marker of the ovarian follicular status. In particular, serum AMH is storage (21 days, DPF+). The aim was to identify proteins or elevated in women with the polycystic ovarian syndrome (PCOS), the most common cause of female infertility, which is characterized by an increased peptides that may be involved in spermatozoa survival. Uterine number of small follicles. We had previously shown that AMH and AMHR-II fluid was collected 10h after oviposition either before and 24h expression are up-regulated in granulosa cells from PCOS women after AI. Bottom up approach using SDS-PAGE and nano LC- undergoing in vitro fertilization treatment and that oestradiol repressed MS/MS was performed. Data were matched against NCBInr AMH expression in granulosa cells from control women. database (2014) using Mascot and identifications were validated Objectives: In this work, using real-time RT-PCR experiments, we by the peptide and protein Prophet algorithm using Scaffold compared the regulation by oestradiol (E2) and dehydrotestosterone (DHT) software. To determine the differences in protein expression, of AMH and AMHR-II expression in primary culture of granulosa cells (GCs) from control and dysovulatory PCOS women and we studied the spectral counting and XIC quantitative methods were employed expression level of estrogen and androgen receptors. using Scaffold Q+ (p<0.05, ratio >2). Two proteins were up- Results: We confirmed that E2 inhibited AMH mRNA in GCs from control regulated and one was down-regulated in oviductal secretion of women. We showed that in GCs from PCOS women, DHT up-regulated both lines in response to the presence of sperm in SST. However, AMH mRNAs (+68,8 % p=0.0488, n=10) and that E2 had no effect. ER this response implies a panel of 9 proteins which abundance was alpha was overexpressed in these cells (+ 99%, p=0.0068) compared to either more or less, or specific, in DPF+ line than in DPF-. In those from control women. AMHR-II expression was not modified by either conclusion, the presence of sperm in genital tract induced DHT or E2 treatments in both groups of patients. Conclusion: Our results demonstrate that DHT and E2 regulation of AMH quantitative differences of the protein content of the uterine fluid, expression is altered in PCOS GCs, in a way which promotes AMH in DPF- and in DPF+ hen lines. These differences imply proteins overexpression. Because we had previously shown that the effect of E2 on which are known as male proteins (sperm, seminal plasma, testis). AMH transcription is stimulatory via ER alpha and an inhibitory through ER Analysis of sperm protein modifications after storage will help us beta, overexpression of ER alpha in GCs from PCOS women could explain to understand the functional implication of these candidates. why E2 did not repress AMH expression in these cells. Supported by the OVISPERM regional project 81 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Dynamic landscape of chromatin accessibility during rat The Prostaglandin D2 signaling has a tumor suppressor role 2015 spermatogenesis in the male germline in mouse 2015

Moïra Rossitto, Joelle Simony-Lafontaine*, Florence Bernex*, Francis Antoine Rolland1,*, Bertrand Evrard1,*, Aurélie Lardenois1, Ando Poulat and Brigitte Boizet-Bonhoure Randriamanantena1, Bernard Jégou1, Fréderic Chalmel1 Genetic and Development department, Institute of Human Genetics CNRS 1 Inserm U1085-Irset, Université de Rennes 1, F-35042 Rennes, France UPR1142, Montpellier, France. * RHEM Histology platform Montpellier, * These authors contributed equally to this work : France. [email protected] [email protected]

During mouse testicular development, prostaglandin D2 (PGD2) is Spermatogenesis is a complex and tightly regulated process leading produced by two enzymes: the lipocalin-type prostaglandin D2 to the continuous production of male gametes, the spermatozoa. synthase (L-Pgds), an enzyme that is male-specifically expressed at avril 15 au 13 du Rennes Within the testes, male germ cells first proliferate to amplify their avril 15 au 13 du Rennes E(embryonic stage) 12.5 by Sertoli cells and by differentiating germ number, next shuffle and reduce their genome through two cells, and the hematopoietic Pgds (H-Pgds), which is expressed in consecutive meiotic divisions, and finally differentiate dramatically into both sexes (1). In the fetal testis, PGD2 acts during somatic cells specialized for mobility and fecundation. This developmental differentiation to help maintaining Sox9 gene expression (2) and process requires the sequential and coordinated expression of contributes to the embryonic germline differentiation (3). In absence of thousands of genes which are in part regulated by epigenetic -/- PGD2, the embryonic L/H-Pgds germ cells show a Carcinoma in situ modifications such as DNA methylation, histone post-translational (CIS) like phenotype, abnormally proliferating and expressing the modifications and chromatin remodelling. In this study we combined pluripotent markers Oct4, Sox2 and Nanog after E15.5 at a time they FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and should be differentiated and arrested into the G0/G1 phase of the cell high-throughput next generation sequencing (FAIRE-seq) to identify cycle (Moniot et al., 2014). nucleosome-free regions and potential active regulatory elements In order to study further the role of PGD2 in the germline biology, the from chromatin in 7 somatic and germ cell types of rat testis. Following double L/H-Pgds mutation has been transferred from the C57BL/6J read mapping, peak calling and statistical filtrations, we identified genetic background to the 129svJ background (ten backcrosses); this around 70’000 genomic regions that display significant accessibility unstable genetic background spontaneously develop testicular tumors variations across cellular samples. These included regions showing a (incidence 2% in wild animals). Analysis of L-/-/H-/-, L+/-/H-/- or L+/-/H+/-- preferential decondensation in each testicular somatic cell type, i.e. in Pgds testes (F9-F10) revealed a germline hyperplasia, increased Leydig cells, peritubular cells or Sertoli cells, as well as in proliferative proliferation of the spermatogonia cell population and increased spermatogonia, meiotic spermatocytes or post-meiotic spermatids. apoptosis of adult testicular germ cells. Also 28% of the animals Surprisingly we also evidenced an unexpected large number of open developed a testicular tumor (Teratoma) and close to 80% of the adult chromatin regions in spermatozoa; given the high nuclear 8 weeks testis showed polynuclear germ cells or/and Carcinoma in condensation of these transcriptionally silent cells, such regions could situ cells, within the seminiferous tubules and the epididymis. These correspond to loci important for the early zygote development. Finally, abnormal cells are expressing the CIS marker Lin28. by combining these results to RNA-seq data we identified a subset of These data identified the roles of the PGD2 signaling in the control of regions in the vicinity of genes whose expression displays the same the balance proliferation/differentiation of the adult male germ cells dynamics during spermatogenesis. These open chromatin regions and identified PGD2 as a new tumor suppressor pathway of the therefore represent promising candidates for the identification of germline. regulatory elements driving the testicular/germ cell gene expression 1- Rossitto et al., 2015 Reproduction, 149, R49-58. program.Supported by the University of Rennes 1 2- Moniot et al. 2009 Development, 136, 1813-1821. 3- Moniot et al. 2014 Development, 141, 3561-71 82 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

A new BMP4 binding protein synthetised by pituitary cells: SLY controls XY gene expression via multiple chromatin 2015 marks and affects global chromatin remodeling in

Thrombospondin-1 acts as BMP4 antagonist 2015

spermatids. Céline Sallon1, Ida Boulay1, Joël Fontaine1, Delphine Logeart- Avramoglou2, Xavier Cayla1, Grégoire Harichaux3, Valérie Labas3, Sylvie Serrentino ME, Moretti C, Ialy-Radio C. and Cocquet J. Canépa3, Catherine Taragnat1,3 INSERM, U1016, Institut Cochin, Paris, France. CNRS, UMR8104, Paris, 1 INRA, CNRS, Université François Rabelais de Tours, IFCE, UMR PRC, France .Université Paris Descartes, Sorbonne Paris Cité, Faculté de 37380 Nouzilly, France. 2 CNRS, Univ Paris Diderot, B2OA, UMR 7052, Médecine, Paris, France. [email protected] 75010 Paris. 3 INRA, Plateforme d’Analyse Intégrative des Biomolécules, 37380 Nouzilly, France [email protected] The chromatin of male germ cells is extensively remodeled and compacted

At the pituitary level, the bone morphogenetic proteins (BMPs), members of during spermatogenesis to produce functional spermatozoa. This process avril 15 au 13 du Rennes

the TGFß superfamily play roles in different differentiated cell types. For avril 15 au 13 du Rennes is characterized by progressive changes in the chromatin structure, such instance, in gonadotrope cells, the regulation of FSH synthesis is affected as hyper-acetylation of histones and culminates with the replacement of by BMPs. Several BMP ligand mRNAs, as well as BMP receptors, are histones by transition proteins and then by protamines. Our laboratory has present in sheep pituitary suggesting that BMPs can exert previously shown that Sly, a spermatid-specific gene encoded by the paracrine/autocrine actions on FSH synthesis (Faure et al. 2005; Sallon et mouse Y chromosome, is essential for normal sperm differentiation as its al. 2010). The first aim of the study was to determine whether ovine absence induces a spectrum of sperm anomalies including reduced sperm pituitary cells produced BMPs. The potential presence of BMPs in motility, deformed sperm heads, abnormal chromatin remodeling and conditioned medium (CM) from ovine pituitary cells cultured for 48h, as well increased DNA damage, that lead to male infertility. Interestingly, Sly- as in ovine serum, was investigated by using a bioactivity test based on deficient round spermatids display a deregulation of over 400 genes, many embryonic mesenchymal cells (C3H10T1/2 cells) transfected with a BMP- of which are encoded by the sex chromosomes. The molecular mechanism responsive element fused to firefly luciferase reporter gene. The results remained unknown. showed that pituitary CM, in contrast to ovine serum, did not exhibit BMP Here we show how SLY controls spermatid differentiation at the molecular activity whatever the treatment (GnRH, estradio or activin) or the incubation level. By chromatin immunoprecipitation (ChIP)-sequencing we period (6h-48h) applied to the pituitary cells. Interestingly, this assay characterize the genome-wide localization of SLY in round spermatids, demonstrated that pituitary CM contained factor(s) able to inhibit BMP-4 showing that SLY associates with the 5’-UTR of the genes whose action. Moreover, GnRH increased this inhibitory activity. In the second part expression is altered in its absence. SLY localization is not restricted to XY of the work, we conducted the identification of the putative factor(s) genes as it also binds to the 5’-UTR of autosomal genes, many of which responsible of the inhibition for BMP action. To explore the hypothesis that have key function in chromatin remodeling. the factor(s) can be BMP-4 binding protein, BMP-4 was immobilized on a At the chromatin level, absence of SLY in round spermatids leads to BIACORE sensorchip and pituitary CM were injected on the chip for multiple changes in histone post translational modifications at the promoter analysis by surface plasmon resonance. The result demonstrated that of expressed genes: increase in the trimethylated lysine 4 of histone H3 pituitary CM contain factor(s) able to bind BMP-4. The bound factor was (H3K4me3) and histone crotonylation (KCr), and decrease of dimethylated then recovered from the chip and analysed by high resolution mass lysine 79 of histone H3 (H3K79me2) and trimethylated lysine 9 of histone spectrometry allowing for identification of one molecule, the H3 (H3K9me3), in round spermatids. thrombospondin-1 (TSP-1). Subsequent analyses confirm that TSP-1 is Moreover, the events orchestrated by SLY in round spermatids have a produced by pituitary cells and is capable to bind BMP-4 and to antagonize direct consequence on chromatin remodeling in elongating spermatids, as its effect as evaluated with rh TSP-1 or with TSP-1 enriched CM in the we also observe a decrease in histone acetylation. bioassay. In conclusion, we identified a new inhibitor of BMP-4 Altogether our data shed light into the molecular mechanisms by which produced by ovine pituitary cells. We hypothesize that this factor could SLY coordinates the chromatin remodeling steps crucial for sperm regulate the bioavailability of BMPs reaching the pituitary by the blood way. differentiation. Funded by ANRJC EPICHROMXY Supported by the EU LIFECYCLE project 83 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Postnatal astrogenesis and female sexual maturation in Ram seminal plasma proteome and impact on sperm liquid 2015 rodents preservation 2015

C. Soleilhavoup1, G. Tsikis1, V. Labas1,2, G. Harichaux1,2, P. L. Ariane Sharif1*, Cécile Allet1, Giuliana Pellegrino1, Danièle Leroy1, Aude Kohnke1, J.L. Dacheux1, Y. Guérin1, J.L. Gatti1, S. P. de Graaf3 and Caillet1, Anne Loyens1, Juergen Siepmann2, Gabriel Corfas3, Sergio R. X.Druart1 Ojeda4 & Vincent Prevot1 1) INRA, UMR 85 Physiologie de la Reproduction et des Comportements, 1Inserm, U1172, Jean-Pierre Aubert Research Center, Development and 13au 15avril F-37380 Nouzilly, France plasticity of the neuroendocrine brain, Lille cedex, France and Université de 2) INRA, Plate-forme d’Analyse Intégrative des Biomarqueurs, Laboratoire Lille 2, IMPRT, Lille, France.. 2Inserm U1008, College of Pharmacy, Univ. de Spectrométrie de Masse, F-37380 Nouzilly, France Lille Nord de France, Lille, France and College of Pharmacy, Freie 3) Faculty of Veterinary Science, The University of Sydney NSW 2006,

Universitaet Berlin, Berlin, Germany. 3Division of Neuroscience, Children's du Rennes Australia.

Hospital and Harvard Medical School, Boston Massachusetts 02115, USA. avril 15 au 13 du Rennes [email protected] 4Division of Neuroscience, Oregon National Primate Research Center-

Oregon Health & Science University, Beaverton, OR 97006, USA. [email protected] Seminal plasma is composed of secretions from the epididymis and the accessory sex glands The initiation of mammalian puberty requires an increased pulsatile and plays a critical role in fertilizing ability of spermatozoa. In secretion of GnRH from specialized neurons of the hypothalamus. rams, analysis of seminal plasma by GeLC MS/MS has allowed This increase is brought about by coordinated changes in the identification of more than 700 proteins, including a high transsynaptic and glial-neuronal communication. Here, we show that abundance of Binder of Sperm family proteins (BSP1, BSP5, pubertal activation of GnRH secretion in female rodents involves the SPADH1, SPADH2), the spermadhesins family (bodhesin2), participation of astrocytes born during the infantile period in the lactoferrin and newly identified proteins like UPF0762 (C6orf58 hypothalamus. Our results suggest that a significant fraction of GnRH gene). When spermatogenesis was stopped by scrotal insulation, neurons recruit and retain in their immediate vicinity newly generated changes in the proteome profile revealed the sperm origin of 40 cells born on postnatal day 8. These newborn cells differentiate into seminal proteins, such as glycolysis pathway enzymes, the astrocytes and stay morphologically associated with GnRH neuron cell chaperonin containing TCP1 (CCT) complex and the 26S bodies throughout sexual development and in sexually mature animals. proteasome complex. Sperm mobility after liquid preservation Local inhibition of cell proliferation in the surroundings of GnRH (24h in milk at 15°C) is male dependent and can be correlated to neuron cell bodies during the infantile period by stereotaxic injection of beads releasing the anti-mitotic paclitaxel caused delayed puberty and differences in the seminal plasma proteome, detected by spectral impaired adult ovarian cycle in female rats. Experiments carried out in counting. The negative association of zinc alpha-2 glycoprotein mice deficient in both erbB1 and erbB4 signaling in astrocytes, which (ZAG) with semen preservation was confirmed by the use of exhibit impaired sexual development and mature reproductive function, recombinant hZAG, which induced an increase in mobility of fresh showed that erbB signalling was required for the long-term sperm, but then decreased sperm mobility after 24h of incubation. maintenance of the association between astrocytes born during the Several sperm proteins interacting with the cytoskeleton, infantile period and GnRH neurons. Altogether, our results raise the glycolysis enzymes and sperm-associated proteins involved in exciting possibility that the birth of new astrocytes morphologically and capacitation correlated with better liquid storage and can be functionally connected to GnRH neurons is a key maturational event considered as seminal biomarkers of sperm preservation. required to initiate GnRH secretion in female rodents. Keywords: seminal plasma; spermatozoa; zinc alpha Supported by the FRM and the ANR glycoprotein; preservation; proteome; spectral counting 84 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

New proteomic tools for reproductive phenotyping: the male Early development of bovine embryos is influenced by the 2015 2015 fowl fertility example origin of feeders during in vitro culture. Anaïs Vitorino Carvalho1, Luc Jouneau1, Catherine Archilla1, Ludivine Laura Soler Vasco1,2,3,4, Aurore Thélie1,2,3,4, Valérie Labas1, Ana-Paula Laffont1, Sylvie Ruffini1, Emilie Corbin2, Pascal Mermillod2, Véronique Teixeira-Gomes5,6,7, Isabelle Grasseau1, Grégoire Harichaux1, Elisabeth Duranthon1 Blesbois1,2,3,4 . [email protected] 1INRA, UMR1198 Biologie du Développement et Reproduction, F-78350 1INRA, UMR85 Physiologie de la Reproduction et des Comportements, F- Jouy-en-Josas, France 37380 Nouzilly, France; 2CNRS, UMR7247, F-37380 Nouzilly, France 2INRA, UR85 Physiologie de la Reproduction et des Comportements, 3Université François Rabelais de Tours, F-37000 Tours, France. 4IFCE, Nouzilly, France. [email protected] Institut Français du Cheval et de l'Equitation, F-37380 Nouzilly, France. 5INRA, Plate-forme d'Analyse Intégrative des Biomolécules, Laboratoire de The early development of embryo is clearly impacted by its environment Spectrométrie de Masse, F-37380 Nouzilly, France. 6INRA, UMR 1282 avril 15 au 13 du Rennes and more specifically by the oviduct secretions in vivo. In cattle, an in vitro avril 15 au 13 du Rennes Infectiologie et Santé Publique, F-37380 Nouzilly, France. 7Université model of co-culture of bovine oviduct epithelial cells (BOEC) with the François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F- embryo has been developed to mimic the in vivo oviduct/embryo crosstalk. 37000 Tours, France On the other hand, several other co-culture systems were previously developed including co-culture with monkey VERO fibroblasts to improve Accurate reproductive phenotyping is essential for optimizing genetic resources bovine embryo quality. Nevertheless, to the best of our knowledge, no preservation, and for improving farm animal production indexes especially in comparison of co-culture systems using BOEC and other cells has been intensively bred animal as chicken. Fast and reliable tools to define the done yet to analyze if there is a specific impact of BOEC on bovine embryo. reproductive ability of male chicken are therefore largely needed. Fertility can be To answer to this question, we compared the influence of BOEC as well as accurately evaluated trough in vivo egg fertilization rate, which is cumbersome VERO cells on bovine early development. Because co-culture with BOEC and expensive. A simpler alternative is the use of in vitro sperm quality tests, cells require culture at 20%O2, we included two control conditions: embryos although they are often highly variable and poorly correlated with fertility. The cultured at 20%O and embryos cultured at 5% O in SOF medium + 5% aim of this project is to develop a new fast, reliable and simple tool for fertility 2 2 SVF. No difference of cleavage rates were observed. No difference in the screening. A preliminary study of our laboratory has recently suggested the efficiency of intact-cells MALDI-TOF mass spectrometry (ICM-MS) for high- timing and rate of 16 cell stage development were observed despite this throughput male fertility evaluation (Labas et al., 2014; 2015). Our aim is now to stage just follows EGA which is very sensitive to environmental conditions. methodologically improve and clinically validate this tool in a bigger chicken Blastocyst rates are currently analyzed. To have new insight on the embryo population (n=72), including two different genetic lines (broiler and layer). The quality at the 16-cell stage, a high throughput transcriptomic analysis was fertility of 36 broiler and 36 layer roosters was defined through different in vivo developed. Therefore, a new bovine microarray was designed including and in vitro tests. Fresh spermatozoa (3 ejaculates/male) were subjected to an more than 26 700 transcripts and 250 retroviral EST. Considering an automated ICM-MS method, and MS spectra were processed and analyzed with adjusted p value < 0.05, only one gene was differentially expressed specific software. Fertility-predictive mathematic models were then constructed between 5% O2 and 20%O2 conditions. Thus, 16-cell embryos seem to not and validated. In parallel, the molecular composition of the spectra was respond to the induced oxidative stress. The direct comparison of the two characterized through high-throughput top-down protein identification by co-culture conditions revealed only 19 differential expressed genes. upgrading our former protocol. We were able to obtain predictive models for Nevertheless, the presence of VERO or BOEC induced differential each genetic line with high recognition and validation rates. Animals were expression of 125 and 1162 genes respectively when compared to 5% O classified by those models with good sensitivity and specificity. Furthermore, we 2 and 1209 and 2186 genes respectively when compared to 20% O . were able to identify 21 times more peptide-and proteoforms in the mass range 2 of spermatozoa ICM-MS spectra by using our optimized top-down MS protocol. Interestingly functional analysis using DAVID software pointed to different Different masses of interest were detected as potential candidates for fertility biological pathways according to the cell types used for co-culture. Further biomarkers, and were confidently identified by top-down MS analysis. In all, here analyses at the blastocyst stage will be necessary to clarify the differential we demonstrate that ICM-MS coupled with adapted mathematic models and top- impact of cell types used in co-culture systems on embryo quality. down protein identification is an accurate tool to discriminate fertile and sub- Supported by the EU FP7 KBBE FECUND project fertile chicken males and to investigate the molecular basis of fertility. 85 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Murine model invalidated for LXR: dyslipidemia and The ReproGenomics Viewer: an integrative cross-species capacitation impairment toolbox for the reproductive science community 2015

l M Whitfield*, C Soubeyrand, H Pons-Rejraji, L Janny, R Cadet, R Guiton, A Thomas A. Darde1,2, Olivier Sallou2, Emmanuelle Becker1, Bertrand Kocer, JR Drevet, F Saez. Evrard1, Cyril Monjeaud2, Yvan Le Bras3, Bernard Jégou1,3, Olivier Collin2, Team « Mécanismes Post-testiculaires de l’Infertilité » Laboratoire GReD Antoine D. Rolland1 and Frédéric Chalmel1,* (Génétique Reproduction et Développement) UMR CNRS 6293 – INSERM U1103, Clermont Université 24 avenue des Landais, BP80026, 63171 1 Inserm U1085-Irset ; Université de Rennes 1; F-35042 Rennes, France. Aubière Cedex, France. 2 Institut de Recherche en Informatique et Systèmes Aléatoires [email protected] (IRISA/INRIA) - GenOuest platform, Université de Rennes 1; F-35042 Rennes, France. Aim: Disorders of lipid metabolism (dyslipidemia) constitute one of the male 3 Ecole des Hautes Études en Santé Publique, Avenue du Professeur Léon- infertility origins, but studies on this topic have generally focused on avri 15 au 13 du Rennes Bernard, F-35043 Rennes, France. endocrine pathologies effects. The murine model invalidated for LXR (Liver * To whom correspondence should be addressed. Tel: +33 (0)2 X Receptor, cholesterol homeostasis regulator) allows to study the post- 23 23 58 02; Email: [email protected] testicular infertility in dyslipidemic context (high plasma LDL concentration, Low Density Lipoprotein). LXR-deficient males are sterile starting from 10 months and characterized by testicular and epididymal defects associated We report the development of the ReproGenomics Viewer with dyslipidemia. Interestingly, epididymal phenotype is preferentially (RGV), a multi- and cross-species working environment for the induced by feeding young fertile males (three months old) with a high cholesterol diet (HCD) for four weeks, leading to early sterility. The impact visualization, mining, and comparison of published omics of this diet on gametes lipid composition and function has been studied. datasets for the reproductive science community. The system Methods: Their lipid composition was thus determined by liquid currently embeds 15 published datasets related to chromatography. The ability of sperm to realize capacitation was assessed after incubation in medium supporting capacitation, followed by anti- gametogenesis from nine model organisms. Datasets have been phosphotyrosine western blot (capacitation process terminals markers). curated and conveniently organized into broad categories The intracellular calcium concentration and membrane fluidity were studied including biological topics, technologies, species, and by flow cytometry using the Fluo-4 AM and merocyanine 540 probes respectively. publications. RGV’s modular design for both organisms and Results: Our results show that HCD diet increases the cholesterol / genomic tools enables users to upload and compare their data phospholipids ratio in the membrane of male gametes lxrα;β-/-. This with that from the datasets embedded in the system in a cross- change is associated with a decrease in gametes membrane fluidity, affecting the in vitro capacitation progress, characterized by tyrosine species manner. The ReproGenomics Viewer is freely available phosphorylation decrease and calcium influx disturbance. at http://rgv.genouest.org. Conclusion: Thereby, dyslipidemia seems to affect the gametes epididymal maturation, resulting in lipid composition and membrane dynamic alterations. This ultimately leads to disruption of sperm function, characterized by an alteration of the essential molecular capacitation process. These results open then new perspectives for the study and treatment of infertility in dyslipidemic men.

86 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Mass Spectrometry Imaging of lipids reveals the differences in fatty acid metabolism between ovarian and follicular compartments in porcine, bovine and ovine ovaries.

Svetlana Uzbekova1,2, Ana-Paula Teixeira-Gomes2,3, Sebastien Elis1, Alice Desmarchais1, Virginie Maillard1 and Valerie Labas 1,2. 1 Team BINGO (Biologie Integrative de l’Ovaire), INRA PRC (Physiologie de la Reproduction et des Comportements), UMR7247, Nouzilly, France 2 Laboratoire de la Spectrométrie de Masse, plate-forme PAIB2, INRA PRC, Nouzilly, France 3 INRA, Infectiologie et Santé Publique, UMR1282, Nouzilly, France [email protected] In mammals, an oocyte develops inside of ovarian follicle and this process is strongly supported by surrounding follicular environment consisting of somatic cells and follicular fluid. In antral follicle, the final stages of oogenesis require a lot of energy which is produced by follicular cells from different substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring of oocyte developmental competence in human and in domestic animals, we aimed to elucidate the specificity of FA metabolism at ovarian level by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. By using MALDI Mass Spectrometry Imaging (MSI) of lipids, the images of porcine, bovine and ovine ovarian sections were reconstructed from lipid ion signals acquired using UltrafleXtrem MALDI-TOF/TOF spectrometer (Bruker) at 22-50µM resolution. Hierarchical cluster analysis of ion spectra revealed different spatial distribution and abundance of numerous lipid species between the ovarian compartments, notably between the follicles and interstitial tissue, and also between the follicles. Inside of the follicle, follicular fluid, granulosa and theca cells and oocyte-cumulus complex were clearly discriminated by clustering and principle component analysis of MALDI MSI spectra. A number of lipid species were differently abundant between follicular cells and follicular fluid as shown in porcine (p<0.01). By real time PCR, we showed that gene expression of five key enzymes of FA metabolism (ACC, CPT1, FABP5, PLIN2 and CD36) significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte (p<0.05). In conclusion, FA metabolism differs between ovarian compartments and between different follicular cells. Supported by INRA and Val-de-Loire Region.

87 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

15avril

2015

Rennes du 13 au 13 du Rennes

Liste des Auteurs

Classement par ordre alphabétique

88 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Barrier-Battut I. Page 67 Abby E. Page 39 Bartzen-Sprauer J. Pages 48-49 Adeline B. Pages 74-75 Bashamboo A. Page 77 Adenot P. Page 58 Batailler M. Page 52 Alfaia C. Page 47 Baumard Y. Page 60 Allais-Bonnet A. Pages 62-69 Baur A. Page 32 Allet C. Page 83 Becker E. Pages 55-69-85 Alvarenga R. Page 77 Bellaiche J. Page 48 Alves S. Page 63 Beltramo M. Pages 48-49-58-72 Amaral A. Page 53 Ben Maamar M. Pages 49-59 Anastasiadou M. Page 63 Benachi A. Page 62 Andréa M. Page 55 Bensalah K. Pages 59-77 Anger K. Pages 48-49-58 Bernex F. Page 81 Archilla C. Page 84 Berthaut I. Page 31 Aucagne V. Page 58 Berthelin C.H. Pages 74-75 Auger J. Page 31 Bessonnard S. Page 50 Auguste A. Page 57 Bienvenu T. Page 63 Avet C. Page 47 Bjôrkgren I. Page 53 Bachelot A. Page 44 Blesbois E. Page 84 Baert Y. Page 53 Bobe J. Page 31 Barbat A. Page 32 Boichard D. Page 32 Barnea E. Pages 34-78 Boichon, D. Page 32 Baroncini M. Page 55 Boitrelle F. Page 34 Barral S. Page 38 89 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Boizet-Bonhoure B. Page 81 Buffat C. Page 61 Bonhomme J. Page 43 Bujan L. Page 31 Bonnamy P-J. Page 73 Butruille L. Page 52

Bonnard I. Page 64 Cacialli P Page 52 Bonnard M. Page 64 Cadet R. Page 85 Bonnet A. Page 53 Cadoret V. Pages 40-53-57-72 Borgmann J. Page 53 Caillet A. Page 83 Bosseboeuf A. Page 68 Calicchio R. Page 61 Boudry P. Page 74 Calvel P. Pages 53-77 Boulanger G. Page 50 Canepa S. Page 82 Boulay I. Page 82 Cano-Nicolau J. Page 54 Bouraima-Lelong H. Pages 51-67 Capitan A. Page 32 Boussouar F. Page 38 Caraty A. Page 58 Bovet-Courtois E. Page 51 Casoni F. Page 55 Bozon V. Pages 72-75 Castaldo L. Page 52 Briant E. Page 60 Castille J. Page 61 Brillard J-P. Page 63 Castillo J. Page 53 Brion F. Pages 37-54-65 Catteau A. Page 42 Brionne A. Page 80 Catteau-Jornard S. Page 55 Brisset S. Page 79 Cayla X. Pages 66-82 Brouard V. Page 51 Chalmel F. Pages 32-69-81-85 Brugnon F. Page 31 Chamero P. Page 38 Buchou T. Page 38 Champroux A. Page 54

90 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Charpigny G. Page 76 Da Silva N. Page 63 Chazaud C. Page 50 Dacheux JL. Page 83 Chesneau D. Pages 38-75 Dalbies-Tran R. Page 57 Chianese C. Page 53 Daniel-Carlier N. Page 57 Chocu S. Page 32 Darde T. A. Page 85 Chong S. Page 75 Dardente H. Page 48 Ciancia M. Page 39 Daudin M. Page 31 Cibois M. Page 50 David L.. Page 73

Cimino I. Page 55 De Girolamo P. Page 52 Clement F. Page41 De Graaf S.P. Page 83 Cocquet J. Pages 78-79-82 Decourt C. Pages 48-49-58 Cohen-Tannoudji J. Pages 47-64-73-80 Degrelle S-A. Page 58 Coiffec I. Pages 49-67 Dejucq-Rainsford N. Pages 32-49-59-77 Collier F. Page 55 Delagrange P. Page 72 Collin O. Page 85 Delahaut L. Page 64 Com E. Page 32 Delalande C. Pages 51-67-68-70 Combarnous Y. Page 56 Deleage C. Page 77 Congar P. Page 62 Delmas A. Page 58 Corbin E. Page 84 Dennefeld C. Page 35 Corfas G. Page 83 Denoyelle C. Page 47 Cotinot C. Page 78 De Roux N. Page 33 Curran E. Page 56 Deschamps S. Page 50 D’angelo L. Page 52 Desdoits-Lethimonier C. Pages 49-59

91 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Desmarchais A. Page 86 Egeberg D-L. Page 53 Deville M. Page 59 El Hachem H. Page 80 Dewailly D. Page 55 El Khouri E. Page 63 Dheilly N Page 74 Eladak S. Page 62 Di Clemente N. Pages 33-80 Elis S. Page 40-86 Dieudonné M-N. Pages 34-61-78 Elzaiat M. Pages 62-69 Diot M. Page 60 Eozenou C. Page 76 Dirami T. Page 63 Evain-Brion D. Page 58 Doridot L. Page 61 Evrard B. Pages 32-81-85 Dos Santos E. Pages 34-61-78 Faure M. Pages 47-63 Drevet J-R. Page 54 Favrel P. Page 74 Drevet J. Page 85 Féret B. Page 35 Drévillon L. Page 79 Fontaine I. Page 65 Druart X. Pages 80-83 Fontaine J. Page 82 Ducat A. Page 61 Fostier A. Page 50 Duchesne V. Page 31 Foulon-Gauze F. Page 66 Dufourny L. Page 72 Fournier T. Pages 35-58 Dulioust E. Page 63 Franceschini I. Page 47 Dupont J. Page 60 Fanchin R. Page 80 Dupont M. Page 60 Franco A. Page 64 Duquenne C. Page 39 Francois C. Pages 64- Duranthon V. Pages 34-84 Frapsauce C. Page 53 Duval F. Page 61 Fréour T. Page 42

92 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Fréret S. Page 40 Gennetay D. Page 75 Fritz S. Page 32 Gérard M. Page 38 Froment P. Page 63 Gerard N. Page 80 Fumel B. Page 65 Ghyselinck NB. Page 35 Gacon G. Page 63 Giacobini P. Pages 36-55 Gaggiotti O. Page 43 Giton F. Page 64 Galibert M. Page 58 Gobé C. Page 69 Gargaros A. Pages 72-80 Gonçalves R. Page 70 Garoche C. Page 65 Goudarzi A Page 38 Garrel G. Pages 47-73 Goupil A.S. Page 48 Gassem R. Page 66 Goux D. Page 75 Gatti J.L. Page 83 Grasseau I. Page 84 Gaucher J. Page 38 Grisin T. Page 62 Gauderat G. Page 66 Grison S. Page 74 Gaudriault P. Page 67 Griveau JF. Page 59 Gauer F. Page 70 Grynberg M. Page 80 Gautier A. Pages 66-68 Gschloessl B. Page 50 Gautier C. Page 68 Guénon I. Page 73 Gautier-Courteille C. Page 50 Guérif F. Page 57 Gautron J. Page 80 Guérin Y. Page 83 Gayrard V. Page 66 Guerquin MJ. Pages 39-52 Geffard A. Page 64 Guézanec L. Page 77 Gely-Pernot A. Pages 35-55-69 Guibert E. Page 63

93 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Guigon C-J. Page 64 Jacques S. Page 61 Guiguen Y. Page 37 Jaquiery J. Page 43 Guillaume F. Pages 32-72 Jamin S-P. Pages 55-71 Guillaumet G. Page 72 Janny L. Page 85 Guillou F. Page 66 Jarrier P. Page 53 Pages 32-49-55-59-67- Guiton R. Pages 54-85 Jégou B. 69-77-81-85 Habert R. Pages 39-62 Joachim S. Page 64 Hamamah S. Page 37 Jolivet G. Page 57 Hao C. Pages 55-69 Jørgensen A. Page 53 Harichaux G. Pages 80-82-83-84- Jouanny C. Page 56 Harscoët E. Page 57 Jouaux A. Page 74 Healey G. Page 76 Jouhanneau M. Page 38 Hennebicq S. Page 31 Jouneau L. Pages 62-84 Henningsen B-J. Page 70 Jouve G. Page 59 Henry-Berger J. Page 54 Kah O. Pages 52-54-65 Herbison A-E. Page 55 Kazdar N Pages 59-71 Hernio N. Page 32 Keller M. Page 8 Hinfray N. Pages 37-65 Kellner K. Pages 74-75 Hozé C. Page 32 Kervarrec C. Pages 55-69-71 Hue I. Page 58 Khochbin S. Page 38 Husson F. Page 76 Klopfenstein M. Page 35 Ialy-Radio C. Page 82 Klosen P. Page 39 Izard V. Page 79 Kocer A. Pages 54-85 94 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Kohnke PL. Page 83 Lépinay J. Page 75 Kristensen D-M. Pages 49-59 Leroy D. Page 83 L’hôte D. Page 73 Lesage-Padilla A. Page 76 Pages 40-72-80-82-83- Labas V. Lesné L. Pages 49-59 84--86 Lestaevel P. Page 74 Lacroix M. Page 66 Lesueur E. Page 76 Laffont L. Page 84 Leterme N. Page 43 Lagaraine C. Page 72 Letourneur F. Page 61 Laissue P. Page 61 Levallet J. Page 73 Lammers J Page 42 Leveque J. Page 59 Lannes J. Page 73 Liu X. Page 55 Laran-Chich M-P. Page 39 Livera G. Pages 39-62 Lardenois A. Page 81 Locatelli Y. Page 53 Lareyre J-J. Pages 48-56 Loeuillet L. Page 78 Larose C. Page 43 Logeart-Avramoglou D. Page 82 Laverrière J-N. Page 73 Lomet D. Pages 49-58 Lavoué V. Pages 49-67 Lopes M. Page 59 Le Bouffant R. Page 39 Lores P. Page 63 Le Bras Y Page 85 Loyens A. Page 83 Le Gac F. Pages 48-56 Luangpraseth-Prosper A. Page 76 Le Masson J. Page 73 Lucini C. Page 52 Legeai F. Page 43 Madinier J-B. Page 58 Legendre A. Page 74 Magniez G. Page 64 Lelong C. Pages 74-75 95 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Magre S. Page 64 Michel P. Page 41 Mahé D. Page 77 Michot P. Page 32 Maheo F. Page 43 Mieuzet L. Page 43 Maillard V. Pages 40-53-86 Migaud M. Page 52 Mandon-Pépin B. Page 76 Mikkelsen J-D Page 70 Marceau P. Page 58 MIlési S. Page 39 Mariot J. Page 56 Miralles F. Page 61 Mark M. Page 35 Moinard N. Page 31 Martinez G Page 31 Moindjie H. Pages 34-78 Martinez A.S. Page 74 Moison D. Pages 39-62 Massart C. Page 50 Monget P. Pages 40-53 Matusali G. Page 77 Monjeaud C. Page 85 Mazancourt P. Pages 61-78 Monniaux D Pages 41-53 Mazaud-Guittot S. Pages 49-59-67 Montellier E Page 38 McElreavey K. Page 77 Morcel K Page 59 Méhats C. Page 61 Moretti C. Pages 78-79-82 Melaine N. Page 34 Mouka A. Page 79 Mellouk N. Page 77 Moussu C. Page 38 Mermillod P. Page 84 Myamoto A. Page 78 Messiaen S. Page 39 Noly A. Page 66 Messina A. Page 55 Nouhaud P Page 43 Meunier N. Page 625 Ojeda S. Page 83 Michailidis G. Page 63 Oudin J-F. Page 76

96 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Pailhoux E. Pages 57-62-69 Primig M. Pages 32-69-71 Paillard L. Page 50 Puel S. Page 66 Pannetier M. Pages 62-69-76 Quérat B. Page 73 Parkash J. Page 55 Racine C. Page 80 Passet B. Page 62 Rafert S. Page 56 Pastezeur S. Page 50 Ramé C. Page 60 Pellegrini E. Page 52 Randriamanantena A. Page 81 Pellegrino G. Page 83 Rasri K. Page 39 Petit F-G. Page 71 Ravel C. Pages 59-71 Petit F. Page 64 Raverdeau M Page 35 Picard-Hagen N. Page 66 Regina Mena Barreto Silva F. Page 70 Picot M. Page 65 Reignier A. Page 42 Pierre A. Page 80 Reinaud P. Page 76 Pierre Henri Gouyon Page 36 Reverchon M. Page 60 Pineau C. Page 32 Richard C. Page 58 Platel C. Page 59 Rioux-Leclercq N. Page 32 Poirel V-J. Page 70 Rispe C. Page 43 Pons-Rejraji H. Page 85 Rives N. Page 31 Porcher J-M. Page 64-65 Rivière G. Page 74 Pouchin P. Page 50 Robert V. Pages 47-48-49-58 Poulat F. Page 81 Rode B. Page 63 Pozzi-Gaudin S. Page 62 Rolland A. Pages 32-49-81-85 Prevot V. Pages 55-83 Rossitto M. Page 81 97 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rouiller-Fabre V. Pages 39-62 Siepmann J. Page 83 Roulet A. Page 53 Simo J-C. Page 43 Rousseaux S. Page 38 Simon V. Page 47 Roy S. Page 65 Simonneaux V Pages 39-70 Royere D. Page 53 Simony-Lafontaine J. Page 81 Ruffini S. Page 84 Smagulova F. Pages 55-69 Saez F. Page 54-85 Soleilhavoup C Page 83 Saintilan R. Page 32 Soler Vasco L. Page 84 Saias J. Page 31 Soubeyrand C. Page 85 Sallon C. Page 82 Souidi M. Page 74 Sallou O. Page 85 Souquet B Page 39 Sambroni E. Page 48 Sourdaine P. Page 68 Sandra O. Pages 42-76 Sow A. Page 65 Satie A-P. Page 77 Splingart C Page 42 Schibler L. Page 32 Stiehl T. Page 41 Schulz R.W. Page 37 Stoeckel S. Page 43 Sébert M-E. Page39 Stuparevic I. Page 69 Serazin V. Page 34 Suzenet F. Page 72 Serrentino M-E. Pages 78-79-82 Szerman-Poisson E. Pages 31-51 Sharif A. Page 83 Szymanski L. Page 38 Sheldon M. Page 76 Tachdjian G. Page 79 Shimizu T. Page 76 Tack K. Page 74 Shiota H. Page 38 Tagu D. Page 43

98 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Taieb J. Page 80 Vernet N. Page 35 Taragnat C. Pages 75-82 Vialard F. Pages 34-61-78 Teixeira-Gomes A-P. Pages 40-72-84 Viard P Pages 59-71 Teletin M. Page 35 Viet J. Page 50 Thélie A. Page 84 Viguié C. Page 66 Thépot D. Page 57 Vilotte JL. Page 61 Teixeira Gomes AP. Page 86 Vincent R. Pages 47-48-49-58 Tores F. Page 79 Vitorino Carvalho A. Page 84 Tosca L. Page 79 Walschaerts M. Page 31 Tostivint H. Page 43 Welsh M. Page 53 Touraine P. Page 44 Whitfield M. Page 85 Toure A. Page 63 Wolf J-P. Page 63 Tourpin S. Page 39 Zanatta A-P. Page 70 Toutain P-L. Page 66 Zanatta L. Page 70 Touzé J-L. Page 60 Zhao Y. Page 38

Travers R. Page 75 Tsikis G. Page 83 Tymen M. Page 74 Uzbekova S. Pages 40-57-72-86 Vaiman D. Pages 61-76 Valour D. Page 32 Vanneste M. Page 73 Veau S. Page 59

99 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

100 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

Rennes du 13 au 15 avril

2015

2015

Rennes du 13 au 15 avril 15 au 13 du Rennes

Notes

101 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

102 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

103 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

104 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

105 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015 - 1ères Journées ReproSciences 2015 – 1ères Journées ReproSciences 2015

106