Identification of extracellular vesicles involved in chicken eggshell

Identification of extracellular vesicles involved in chicken eggshell biomineralization Joël GAUTRON, Lilian STAPANE, Nathalie LE ROY, Jacky EZAGAL Valérie LABAS, Lucie COMBES-SOIA A. RODRIGUEZ-NAVARRO M. T. HINCKE

Avian eggshell biomineralization Calcitic eggshell Extracellular biomineralization (uterine fluid) ACC deposition Eggshell membranes ACC transformation into calcite aggregates Crystalline calcite units formation Preferential crystal orientation and growth Cuticle deposition Mammillary knobs 5 h 6 h Initiation of mineralization 7 h 10 h Rapid growth ACC (amorphous calcium carbonate) calcite Gautron et al. , 2016 ; Rodriguez-Navarro et al. , 2015 ; Marie et al. , 2015 23 h End phase

Eggshell structure and organic matrix Ca. CO₃ 95% (calcite) Organic matrix 3, 5% Eggshell ultrastructure and mechanical properties Key roles of the organic matrix: Stabilize ACC, determine Ca. CO 3 polymorph and crystal morphology Gautron et al. , 2016 ; Marie et al. , 2015 ; Rodriguez-Navarro et al. , 2015 ; Nys et al. , 2010

in silico analysis: EDIL 3 and MFGE 8 evolution edil 3 et mfge 8 : localization Role in mineralization EDIL 3 MFGE 8 HAPLN 3 HAPLN 1 Identical arrangement of the 2 chromosomes Stapane et al. , 2019, J. Biol. Chemistry EDIL 3/MFGE 8 and the nested genes are involved in bone formation HAPLN 1 HAPLN 3 ACAN VCAN EDIL 3 MFGE 8

Characterization of Gallus gallus EDIL 3 and MFGE 8 proteins Stapane et al. , 2019, J. Biol. Chemistry

EDIL 3 and MFGE 8 : biomineralization Presence in bone/cartilage vesicles (chicken, human, mouse) Balcerzak et al. , 2007; Rosenthal et al. , 2011; Shapiro et al. , 2015 annexins vesicles integrins chicken uterus (Waqas et al. , 2016; Dennis et al. , 1996; Fraser et al. , 1998) (Gautron, 2016; Marie et al. , 2015) Matrix vesicles identified in Ca. CO₃ biomineralization organisms: sea urchin, molluscs, … (Vidavsky et al. , 2014; Bahn et al. , 2017)

Characterization of Gallus gallus EDIL 3 and MFGE 8 proteins annexins Ca 2+ Marie et al. , 2015 EDIL 3/MFGE 8 bind to vesicles ? Stapane et al. , 2019, J. Biol. Chemistry βα integrins

Are vesicles involved in avian eggshell formation? 1 Vesicle presence? Uterine cells Uterine fluid β Phosphatidylserine α Integrins 2 EDIL 3 and MFGE 8 on vesicles ? Mineralization site 3 Ca. CO 3 inside vesicles? ACC ?

1 Presence of vesicles in chicken eggshell biomineralization White Red Uterus isthmus Magnum Bone Duodenum Kidney Liver Strong vesicular activity in oviduct parts where mineralization occured Stapane et al. , Cellular & Molecular Life Sciences, submitted

1 Presence of vesicles in chicken eggshell biomineralization Exocytose Uterine cell Vesicle Exocytose Uterine fluid accumulates vesicles budding from uterus Stapane et al. , Cellular & Molecular Life Sciences, submitted

2 EDIL 3 and MFGE 8 are present in uterine fluid vesicles ? Pure fluid Fluid Ø vesicles Vesicles X 1 2 3 Pure fluid Fluid Ø vesicles Vesicles 1 2 3 X Pure fluid 1 Fluid Ø vesicles Vesicles 2 3 X EDIL 3 and MFGE 8 are vesicular proteins Stapane et al. , Cellular & Molecular Life Sciences, submitted

3 Uterine fluid vesicles and calcium carbonate Transmission electron microscopy (TEM) on uterine fluid and energy dispersive Xray Spectroscopy analysis (EDS) Stapane et al. , 2019, J. Biol. Chemistry Uterine fluid vesicles enclose Ca. CO 3

3 Uterine fluid vesicles and calcium carbonate Transmission electron microscopy on uterine fluid And elemental mapping Calcium SAE-Diffraction Calcium Ca. CO 3 Carbon Oxygen Carbone Ca. CO 3 Oxygène MET-Electron dispersive X-ray spectroscopy Amorphous calcium carbonate Stapane et al. , Cellular & Molecular Life Sciences, submitted Uterine fluid vesicles enclose Ca. CO 3 on amorphous form

Other proteins involved in the vesicular transport PDCD 6 IP Uterus EDIL 3 ANXA 1 ANXA 2 ANXA 8 Ezrin proteomics Syntenin-1 CA 4 ANXA 1, 2, 8 CA 4 CO 2 HCO 3 Ca 2+ Ezrin Syntenin-1 Ca. CO 3 β α PDCD 6 IP Stapane et al. , Cellular & Molecular Life Sciences, submitted

Summary-Conclusion Blood vessels HCO 3 Ca 2+ Uterine cells Uterine fluid CO 2 Carbonic anhydrase 2 Claudin Carbonic anhydrase 4 ? Paracellular SLC 4 A 4 -A 5 -A 10 JAM Mineralisation site SLC 8 A 1 -A 3 Vesicular SLC 26 A 9 ATP 2 B 1 -B 2 Transcellular Pathway Paracellular Pathway ? ACC Calcite Extra/intra-cellular vesicle TRPV 2 -3 Calbindin-1 Occludin Diffusion Transcellular EDIL 3/MFGE 8 Annexins Vesicular Pathway

Acknowledgements Thank you for your attention Infun dibu • • lum DOVE BOA PAIB 2 PEAT Ge. T-Genotoul Universidad de Granada University of Ottawa s Isthmu