_____________________________________________________________________________

BREAKTHROUGHS IN

REPRODUCTION AND DEVELOPMENT

·

NOUVELLES AVANCÉES

EN REPRODUCTION ET DÉVELOPPEMENT

Research Day

Centre for the Study of Reproduction (CSR) at McGill

Tuesday, May 19, 2015

McGill New Residence Hall

3625 Avenue du Parc

Montréal, Québec

______________________________________________________________________________

BREAKTHROUGHS IN REPRODUCTION AND DEVELOPMENT

Research Day 2015

Centre for the Study of Reproduction (CSR) at McGill

Tuesday, May 19, 2015

New Residence Hall, 3625, avenue du Parc, Montréal, Québec

8:00-9:00 Registration and coffee / Poster set-up

9:00-9:10 Opening remarks: Dr. Daniel Bernard

9:10-10:10 Trainee oral presentations (Chairs: Steven Jones and Océane Albert)

O-01. Lundi Ly, “Lifetime Folate Deficiency and Supplementation Induces Aberrant

Sperm DNA Methylation and Reproductive Health”

O-02. Rohini Bose, “Ubiquitin Ligase Huwe1 Modulates Male Germ Cell

Development by Regulating Meiotic Progression”

O-03. Stephany El Hayek, “Oocyte-Driven Remodeling of the Follicular

Microenvironment Builds the Platform for Essential Germ Line-Somatic

Communication”

10:10-10:40 Dr. Thomas Duchaine, Department of Biochemistry, McGill University, “What

MicroRNA-Mediated Silencing in the C. elegans Embryo Can Tell Us About Cancer” –

introduced by Dr. Loydie Jerome-Majewska

10:40-11:00

Health Break / Briefing of Judges

11:00-11:45

Dr. Mellissa Mann, Departments of Obstetrics & Gynecology, and Biochemistry,

Schulich School of Medicine and Dentistry, University of Western Ontario,

“Developmental Regulation of the Kcnq1ot1 Imprinted Domain” – introduced by Dr.

Hugh Clarke

11:45-11:55

Introductory Remarks about Clermont Endowment Fund and Lectureship: Dr. Carlos

Morales

11:55-12:45

12:45-14:10

Lunch

Poster Session

P-01. Gauthier Schang, “Effects of Organophosphate Flame Retardants on Leydig

Cell Function”

P-02. Steven Jones, “Differential Effects of Combined Genistein and DEHP on

Testicular Cell Lipid Homeostasis and Steroid Production”

P-03. Anne Marie Downey, “Cyclophosphamide (CPA) Treatment Alters the

Expression of Members of the Zip Family Zinc Transporters in Pachytene

Spermatocytes”

P-04. Karl-Frédéric Vieux, “The Cytoplasmic Deadenylase CNOT6 Regulates

Deadenylation of a Subset of Transcripts During Oocyte Meiotic Maturation”

P-05. Gurpreet Manku, “Acetaminophen Versus Ibuprofen: Effects on Neonatal

Testicular Gonocyte Development”

P-06. Paulo Roberto Antunes da Rosa, “EGFR Inhibition Prevents Meiotic

Resumption in Bovine Oocytes Cocultured With Follicular Hemisections”

P-07. Laleh Abbassi, “The Role of Yes-Associated Protein (YAP) in Regulation

of Ocyte Growth”

P-08. Johanna Selvaratnam, “The Effects of Catalase Overexpression on

Developing Male Germ Cells in the Aged Mouse”

P-09. Lorena Carvelli, “Heparin-Alpha-Glucosaminide N-Acetyltransferase

(Hgsnat) Gene Inactivation Affects the Reproductive Tract of Adult Mice”

P-10. Océane Albert, “Sperm Chromatin Quality Assessment: Optimization of

the High Throughput Comet Assay”

P-11. Enrique Gamero-Estevez, “Modifying the Claudin Binding Specificity of

the C-Terminus of Clostridium Perfringens Enterotoxin (C-CPE)”

P-12. Rodrigo Camponogara Bohrer, “Effects of Inhibiting DNA Repair

Pathways During Early Embryo Development”

P-13. Eskandari Shahraki Marzieh, “Knockout Mice Model to Study Functions

of BSPH1 and BSPH2 in Fertility”

P-14. Naomi Dicks, “Reduction of Endoplasmic Reticultum (ER) Stress by

Treatment With Tauroursodeoxycholic Acid (TUDCA) Rescues Developmentally

Incompetent Slow-Cleaving Porcine Embryos in Vitro”

P-15. Suhaib Khayat, “Accumulation of Mitochondrial DNA During Early

Murine Embryogenesis is Linked to the Resumption of Cell Growth”

P-16. Aaron Kwong, “FGF4 as a Possible Regulator for Naïve to Primed

Pluripotency Differentiation and Rossette Formation”

P-17. Martika Rodgers, “The Role of Angiomotin in Hippo Signaling Regulation

in the Early Mouse Embryo”

P-18. Wenyang Hou, “Explants of Pre-Attachment Chorion and Allantois Reveal

Extensive Mixing and a Requirement for the Allantois for Maintenance of Gcm1

and Tpbpα Expression”

P-19. Yassemine Khawajkie, “Dissecting the Genetic Susceptibility to Sporadic

Molar Pregnancies and Mechanisms of Their Formation”

P-20. Elie Akoury, “NLRP7 and KHDC3L, the Two Maternal-Effect Proteins

Responsible for Recurrent Hydatidiform Moles, Co-Localize to the Oocyte

Cytoskeleton”

P-21. Vafa Keser, “Genetic Models for Identifying the Molecular Basis of

Phenotypic Variability in 22q11.2 Deletion Syndrome”

P-22. Laura Whidden, “Combined Effects of DNA Methyltransferase 1o-

Deficiency and Ovarian Stimulation on DNA Methylation Patterning and

Embryonic Outcome at Mid-Gestation”

P-23. Jonas Brandenburg, “Epigenetic Effects of Prenatal Exposure to

Polycyclic Aromatic Hydrocarbons on CYP1A Expression and Inducibility”

P-24. Keith Siklenka, “Over-Expression of Kdm1a in Spermatogenesis Alters the

Sperm Epigenome and has Dire Consequences for Development of the Embryo”

P-25. Kai Sheng, “Huwe1 Function on Male Fertility and Epigenetic Regulation

During Spermiogenesis”

P-26. Cécile Adam, “Understanding the Regulation of Connexin 26 in the

Epididymis”

P-27. Luisina Ongaro, “Gonadotrope-Specific Deletion of Bmpr2 Does Not

Affect FSH Synthesis or Estrous Cyclicty in Adult Female Mice”

P-28. Yasaman Aghazadeh, “Translocator Protein (TSPO) Drug Ligands Induce

Testosterone Formation in GnRH Antagonist Castrated Rats”

P-29. Nancy Li, “Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein,

in Intracellular Cholesterol Transport for Steroid Biosynthesis”

P-30. Sathvika Venugopal, “Cholesterol Trafficking for Steroid Biosynthesis in

MA-10 Mouse Tumor Leydig Cells”

P-31. Enrico Campioli, “Global Knockout and Steroidogenic Cell-Targeted

Deletion of the Translocator Protein (18-kDa) Unveil its Crucial Role in Viability

and Hormone-Dependent Steroid Formation”

P-32. Yasmin Schuermann, “From the Dry to the Lactating Period: The

Struggles of the Dairy Cow”

P-33. Samin Sabouhi Zarafshan, “Mutations of Human Binder of Sperm

Homolog1 (BSPH1): A New Cause of Male Infertility?”

P-34. Swati Gupta, “Characterization of SEC23A and MAN1B1 Expression and

Function in a Family With Craniofacial Abnormalities and Mental Retardation”

P-35. Renata Bahous, “High Dietary Folate During Pregnancy and Lactation

Leads to Disturbances in Folate Metabolism, Pseudo-MTHFR Deficiency and

Short-Term Memory Impairment in Murine Offspring”

P-36. Lisa-Marie Legault, “Implication of a Transient Kdm1a-Loss on the

Embryonic Epigenetic Landscape”

14:10-15:10 Trainee oral presentations (Chairs: Laleh Abbassi and Enrico Campioli)

O-04. Chirine Toufaily, “Addition of a C-Terminal Tail Alters Mammalian GnRH

Receptor Signaling”

O-05. Thomas Nardelli, “In Vitro Assessment of Novel Green Plasticizers Using a

Toxicogenomic Approach in Immortalized Sertoli Cell Lines”

O-06. Fatima Tokhmafshan, “Perturbations in the Extracellular Matrix of

Developing Ureterovesical Junction May Lead to Vesicoureteral Reflux”

15:10-15:55 Dr. Jon Oatley, Director, Center for Reproductive Biology, School of Molecular

Biosciences, College of Veterinary Medicine, Washington State University

“Molecular Control of the Stem Cell State in Mammalian Spermatogonia” –

introduced by Dr. Jacquetta Trasler

15:55-16:00 Concluding Remarks: Dr. Daniel Bernard

16:00-17:00 Cocktail / Awards Presentation / Take down posters

NOUVELLES AVANCÉES EN REPRODUCTION ET DÉVELOPPEMENT

Journée de recherche 2015

Centre d’études sur la reproduction (CER) à McGill

le mardi 19 mai 2014

New Residence Hall, 3625, avenue du Parc, Montréal, Québec

8 h Inscription et café / Installation des affiches

9 h Mot de bienvenue : Dr. Daniel Bernard

9 h 10 Présentations orales (Modérateurs: Steven Jones et Océane Albert)

O-01. Lundi Ly, “Lifetime Folate Deficiency and Supplementation Induces Aberrant

Sperm DNA Methylation and Reproductive Health”

O-02. Rohini Bose, “Ubiquitin Ligase Huwe1 Modulates Male Germ Cell

Development by Regulating Meiotic Progression”

O-03. Stephany El Hayek, “Oocyte-Driven Remodeling of the Follicular

Microenvironment Builds the Platform for Essential Germ Line-Somatic

Communication”

10 h 10 Dr. Thomas Duchaine, Department of Biochemistry, McGill University, “What

MicroRNA-Mediated Silencing in the C. elegans Embryo Can Tell Us About Cancer”–

présenté par Dr. Loydie Jerome-Majewska

10 h 40 Pause santé / Briefing des juges

11 h 00 Dr. Mellissa Mann, Departments of Obstetrics & Gynecology, and Biochemistry,

Schulich School of Medicine and Dentistry, University of Western Ontario,

“Developmental Regulation of the Kcnq1ot1 Imprinted Domain” – présentée par Dr.

Hugh Clarke

11 h 45 Remarques introductives concernant le fond de dotation et conférencier Clermont: Dr.

Carlos Morales

11 h 55

Dîner

12 h 45 Session d’affiche

P-01. Gauthier Schang, “Effects of Organophosphate Flame Retardants on Leydig

Cell Function”

P-02. Steven Jones, “Differential Effects of Combined Genistein and DEHP on

Testicular Cell Lipid Homeostasis and Steroid Production”

P-03. Anne Marie Downey, “Cyclophosphamide (CPA) Treatment Alters the

Expression of Members of the Zip Family Zinc Transporters in Pachytene

Spermatocytes”

P-04. Karl-Frédéric Vieux, “The Cytoplasmic Deadenylase CNOT6 Regulates

Deadenylation of a Subset of Transcripts During Oocyte Meiotic Maturation”

P-05. Gurpreet Manku, “Acetaminophen Versus Ibuprofen: Effects on Neonatal

Testicular Gonocyte Development”

P-06. Paulo Roberto Antunes da Rosa, “EGFR Inhibition Prevents Meiotic

Resumption in Bovine Oocytes Cocultured With Follicular Hemisections”

P-07. Laleh Abbassi, “The Role of Yes-Associated Protein (YAP) in Regulation

of Ocyte Growth”

P-08. Johanna Selvaratnam, “The Effects of Catalase Overexpression on

Developing Male Germ Cells in the Aged Mouse”

P-09. Lorena Carvelli, “Heparin-Alpha-Glucosaminide N-Acetyltransferase

(Hgsnat) Gene Inactivation Affects the Reproductive Tract of Adult Mice”

P-10. Océane Albert, “Sperm Chromatin Quality Assessment: Optimization of

the High Throughput Comet Assay”

P-11. Enrique Gamero-Estevez, “Modifying the Claudin Binding Specificity of

the C-Terminus of Clostridium Perfringens Enterotoxin (C-CPE)”

P-12. Rodrigo Camponogara Bohrer, “Effects of Inhibiting DNA Repair

Pathways During Early Embryo Development”

P-13. Eskandari Shahraki Marzieh, “Knockout Mice Model to Study Functions

of BSPH1 and BSPH2 in Fertility”

P-14. Naomi Dicks, “Reduction of Endoplasmic Reticultum (ER) Stress by

Treatment With Tauroursodeoxycholic Acid (TUDCA) Rescues Developmentally

Incompetent Slow-Cleaving Porcine Embryos in Vitro”

P-15. Suhaib Khayat, “Accumulation of Mitochondrial DNA During Early

Murine Embryogenesis is Linked to the Resumption of Cell Growth”

P-16. Aaron Kwong, “FGF4 as a Possible Regulator for Naïve to Primed

Pluripotency Differentiation and Rossette Formation”

P-17. Martika Rodgers, “The Role of Angiomotin in Hippo Signaling Regulation

in the Early Mouse Embryo”

P-18. Wenyang Hou, “Explants of Pre-Attachment Chorion and Allantois Reveal

Extensive Mixing and a Requirement for the Allantois for Maintenance of Gcm1

and Tpbpα Expression”

P-19. Yassemine Khawajkie, “Dissecting the Genetic Susceptibility to Sporadic

Molar Pregnancies and Mechanisms of Their Formation”

P-20. Elie Akoury, “NLRP7 and KHDC3L, the Two Maternal-Effect Proteins

Responsible for Recurrent Hydatidiform Moles, Co-Localize to the Oocyte

Cytoskeleton”

P-21. Vafa Keser, “Genetic Models for Identifying the Molecular Basis of

Phenotypic Variability in 22q11.2 Deletion Syndrome”

P-22. Laura Whidden, “Combined Effects of DNA Methyltransferase 1o-

Deficiency and Ovarian Stimulation on DNA Methylation Patterning and

Embryonic Outcome at Mid-Gestation”

P-23. Jonas Brandenburg, “Epigenetic Effects of Prenatal Exposure to

Polycyclic Aromatic Hydrocarbons on CYP1A Expression and Inducibility”

P-24. Keith Siklenka, “Over-Expression of Kdm1a in Spermatogenesis Alters the

Sperm Epigenome and has Dire Consequences for Development of the Embryo”

P-25. Kai Sheng, “Huwe1 Function on Male Fertility and Epigenetic Regulation

During Spermiogenesis”

P-26. Cécile Adam, “Understanding the Regulation of Connexin 26 in the

Epididymis”

P-27. Luisina Ongaro, “Gonadotrope-Specific Deletion of Bmpr2 Does Not

Affect FSH Synthesis or Estrous Cyclicty in Adult Female Mice”

P-28. Yasaman Aghazadeh, “Translocator Protein (TSPO) Drug Ligands Induce

Testosterone Formation in GnRH Antagonist Castrated Rats”

P-29. Nancy Li, “Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein,

in Intracellular Cholesterol Transport for Steroid Biosynthesis”

P-30. Sathvika Venugopal, “Cholesterol Trafficking for Steroid Biosynthesis in

MA-10 Mouse Tumor Leydig Cells”

P-31. Enrico Campioli, “Global Knockout and Steroidogenic Cell-Targeted

Deletion of the Translocator Protein (18-kDa) Unveil its Crucial Role in Viability

and Hormone-Dependent Steroid Formation”

P-32. Yasmin Schuermann, “From the Dry to the Lactating Period: The

Struggles of the Dairy Cow”

P-33. Samin Sabouhi Zarafshan, “Mutations of Human Binder of Sperm

Homolog1 (BSPH1): A New Cause of Male Infertility?”

P-34. Swati Gupta, “Characterization of SEC23A and MAN1B1 Expression and

Function in a Family With Craniofacial Abnormalities and Mental Retardation”

P-35. Renata Bahous, “High Dietary Folate During Pregnancy and Lactation

Leads to Disturbances in Folate Metabolism, Pseudo-MTHFR Deficiency and

Short-Term Memory Impairment in Murine Offspring”

P-36. Lisa-Marie Legault, “Implication of a Transient Kdm1a-Loss on the

Embryonic Epigenetic Landscape”

14 h 10

Présentations orales (Modérateurs: Laleh Abbassi et Enrico Campioli)

O-04. Chirine Toufaily, “Addition of a C-Terminal Tail Alters Mammalian GnRH

Receptor Signaling”

O-05. Thomas Nardelli, “In Vitro Assessment of Novel Green Plasticizers Using a

Toxicogenomic Approach in Immortalized Sertoli Cell Lines”

O-06. Fatima Tokhmafshan, “Perturbations in the Extracellular Matrix of

Developing Ureterovesical Junction May Lead to Vesicoureteral Reflux”

15 h 10 Dr. Jon Oatley, Director, Center for Reproductive Biology, School of Molecular

Biosciences, College of Veterinary Medicine, Washington State University

“Molecular Control of the Stem Cell State in Mammalian Spermatogonia” – présenté

par Dr. Jacquetta Trasler

15 h 55 Mot de conclusion : Dr. Daniel Bernard

16 h 00 Cocktail, présentation des prix et démontage des affiches

17 h 00 Au revoirs!

THOMAS F. DUCHAINE

DEPARTMENT OF BIOCHEMISTRY, ROSALIND AND MORRIS GOODMAN CANCER

RESEARCH CENTRE, MCGILL UNIVERSITY

WHAT MICRORNA-MEDIATED SILENCING IN THE C. ELEGANS EMBRYO

CAN TELL US ABOUT CANCER

The discoveries of the RNA interference (RNAi) phenomena, such as microRNA-mediated silencing,

forced a profound re-interpretation of how gene expression is controlled in metazoan. Our research

program employs and integrates classical genetics, biochemistry, as well as functional genomics and

proteomics in the model organism C. elegans to understand the mechanistic fundaments that underly

these phenomena, with a particular focus on the developing embryo. My presentation will revolve

mainly around two basic aspects of microRNA-mediated silencing that we’ve discovered here at

McGill. Firstly, we found that gene silencing instigated by microRNAs is profoundly cooperative, and

began deciphering how microRNA-binding sites functionally interact with one another when bound to

the 3’-untranslated regions (3’UTRs) of mRNAs. Secondly, we uncovered that through the use mRNA

alternative polyadenylation sites (APAs), 3’UTRs are remodelled so as to precisely modulate the

function and outputs of microRNAs in the embryo. I will substantiate on how these two properties, and

the underlying mechanisms, impact the functions of microRNAs in governing the expression of

tumour-suppressor and oncogenes in mammalian cells.

MELLISSA RW MANN

DEPARTMENTS OF OBSTETRICS & GYNECOLOGY, AND BIOCHEMISTRY, SCHULICH

SCHOOL OF MEDICINE AND DENTISTRY, UNIVERSITY OF WESTERN ONTARIO

DEVELOPMENTAL REGULATION OF THE KCNQ1OT1 IMPRINTED

DOMAIN

Genomic imprinting is an epigenetic process that restricts expression of specific genes to the

maternally or paternally-inherited allele; whereas the opposite parental copy is silent. Perturbations in

genomic imprinting can lead to imprinting defects that have severe consequences for growth and

development, including imprinting disorders such Beckwith-Wiedemann Syndrome, which result from

genetic and epigenetic defects at the KCNQ1OT1 imprinting domain. Current evidence indicates

complex regulation of paternal allelic silencing of imprinted genes within the Kcnq1ot1 domain,

through mechanisms that are not fully understood. In this study, we investigated the role of a novel

candidate, nucleoporin 107 (Nup107), that emerged from an RNA interference screen for epigenetic

factors involved in paternal allelic silencing at the Kcnq1ot1 imprinted domain in extraembryonic

endoderm cells. As a comparison, we also investigate three additional nucleoporins, Nup62, and Nup98

with documented chromatin association. We observed that depletion of Nup107 as well as Nup62

reactivated the normal silent paternal alleles of multiple imprinted genes in the Kcnq1ot1 domain. This

occurred by reducing Kcnq1ot1 ncRNA expression, diminishing nuclear ncRNA volume, decreasing

active and increasing repressive histone modifications at the Kcnq1ot1 ICR region but surprisingly

without gaining DNA methylation. Additionally, Nup107 and Nup62 depletion decreased

compartmentalizing of the paternal Kcnq1ot1 domain at the nuclear periphery, through decreased

chromatin interactions at Kcnq1ot1 ICR, putative enhancer in Kcnq1 intron 10 and Osbpl5 promoter.

By comparison, Nup98 did not play a role in paternal allelic silencing at the Kcnq1ot1 domain,

indicating that nuclear-cytoplasmic transport was not affected by nucleoporin depletion. Thus, we have

identified a novel mechanism of imprinted gene regulation, namely nucleoporin-mediated imprinted

gene regulation at the Kcnq1ot1 domain.

JON M. OATLEY

DIRECTOR, CENTER FOR REPRODUCTIVE BIOLOGY, SCHOOL OF MOLECULAR

BIOSCIENCES, COLLEGE OF VETERINARY MEDICINE, WASHINGTON STATE UNIVERSITY,

PULLMAN WA

MOLECULAR CONTROL OF THE STEM CELL STATE IN MAMMALIAN

SPERMATOGONIA

The continual spermatogenesis that generates millions of genetically unique gametes daily relies on the

activities of an undifferentiated spermatogonial population that consists of rare spermatogonial stem

cells (SSCs) and numerous progenitor spermatogonia. During steady-state conditions, self-renewal by

SSCs maintains a pool from which progenitors arise periodically and then transiently amplify in

number before committing to a pathway of terminal differentiation. Also, SSCs are capable of

regenerating spermatogenesis following transplantation into a recipient testis or cytotoxic damage that

eliminates the majority of the germline. The molecular mechanisms controlling the stem cell state in

mammalian spermatogonia are undefined. Our previous studies demonstrated that regenerative

capacity in spermatogonia is linked to expression of inhibitor of DNA binding 4 (ID4) and we

generated an Id4-Gfp reporter mouse line to study the population in more detail. We have found that

the ID4+ population is rare in number and a slow cycling subset of single spermatogonia during

steady-state conditions. Our studies of an Id4 null mouse line revealed defects in the maintenance of

the undifferentiated spermatogonial population leading to complete loss of the germline during aging, a

hallmark of impaired SSC functions. In recent studies, we generated a transgenic mouse line for

conditional ID4 overexpression and discovered that aberrant expression in spermatogonia leads to a

block in the formation of progenitor and differentiating spermatogonia. In addition, overexpression of

ID4 leads to an alteration of cell cycle progression. Furthermore, we discovered that the transcriptome

is greatly altered in spermatogonia with overexpression of ID4 overexpression including suppression

of genes known to be involved in progenitor spermatogonial progression and activation of genes with a

defined role in promoting stem cell maintenance. Collectively, our findings suggest that SSCs are a

quiescent subset of the undifferentiated spermatogonial population that is marked by high expression

of ID4 which plays a functional role in SSC maintenance by restricting cell cycle progression and

regulating expression of key genes that influence the transition between stem cell and progenitor states.

This research was supported by grant HD061665 awarded to J.M.O. from the National Institutes of

Health.

ORAL PRESENTATIONS

ORAL 1

LIFETIME FOLATE DEFICIENCY AND SUPPLEMENTATION INDUCES ABERRANT

SPERM DNA METHYLATION AND REPRODUCTIVE HEALTH

Lundi Ly1, Donovan Chan

2 , Mylene Landry

2 , Nathalie Behan

3 , Amanda MacFarlane

3 and Jacquetta

Trasler4

1Department of Human Genetics, McGill University, Montreal QC, Canada

2Research Institute of the McGill University Health Centre at the Montreal Children’s Hospital, McGill

University, Montreal QC, Canada 3Health Canada, Ottawa ON, Canada

4Departments of Human Genetics and Pediatrics & Pharmacology and Therapeutics, McGill

University, Montreal QC, Canada

Epigenetic modifications such as DNA methylation have an essential role in developmental programs.

Disruptions in gamete epigenetic reprogramming are associated with adult disease and

transgenerational effects. The fetal period is the key to DNA methylation pattern acquisition in

developing male germ cells and adequate supply of methyl donors is required. Previous studies showed

that postnatal folate deficiency (FD) or supplementation (FS) could alter the sperm epigenome. The

objective of this study was to determine if lifetime FS or FD induce an aberrant epigenetic landscape in

germ cells detrimental to offspring health. Female mice (n=15) were placed on one of four amino acid

controlled diets: control diet (FCD; 2mg folate/kg diet), 20−fold folate supplemented diet (20FS), 10−

fold folate supplemented diet (10FS) or 7−fold deficient diet (7FD). Females were mated to produce

F1 litters whose germ cells were exposed to the folate diets at through development. F1 males were

weaned onto their respective prenatal diets. F2 and F3 litters, unexposed to the folate treatments, were

subsequently generated. Tissues of interest were collected, and genome−wide DNA methylation

analysis by reduced representation bisulfite sequencing (RRBS) was performed. F2 litters derived from

7FD and 20FS exposed sperm were significantly smaller than FCD F2 litters at weaning. Preliminary

analysis of RRBS results from F1 sperm (n = 5) demonstrated that perinatal exposure to 7FD, 10FS,

and 20FS diets resulted in 153, 132 and 114 differentially methylated (DM) loci, respectively. Affected

regions included intergenic, intron, exon, promoter, 5’ and 3’ UTR sequences. These results suggest

that lifetime FD/FS can impact sperm development and offspring health.

(Supported by CIHR and CEEHRC).

ORAL 2

UBIQUITIN LIGASE HUWE1 MODULATES MALE GERM CELL DEVELOPMENT BY

REGULATING MEIOTIC PROGRESSION

Rohini Bose, Simon S. Wing

Department of Medicine, Research Institute of the McGill University Health Centre

Spermatogenesis involves crucial, highly regulated transitions between developmental programs such

as mitotic proliferation, meiosis and differentiation. How these switches are regulated remains to be

delineated. The ubiquitin proteasome system plays a significant role in protein turnover and cellular

remodeling and may be involved in these transitions during spermatogenesis. We previously identified

ubiquitin ligase Huwe1 in the testis and showed that inactivating it in gonocytes results in a delay in

their mitotic re-entry and leads to spermatogonial depletion. Here we examined the role of Huwe1 in

spermatogonial differentiation, meiotic entry and progression. We inactivated it in differentiating

spermatogonia by expressing Cre recombinase using the Stra8 promoter. Huwe1-/Y

males (KO) were

subfertile siring 33% smaller litters compared to the Huwe1flox/Y

(WT). The average testes weight of

adult KO was only 30% of the WT. Morphological analysis of adult testis revealed a heterogeneous

phenotype with tubules displaying fewer spermatocytes and spermatids. Since we observed fewer

spermatocytes, we looked at meiotic progression more closely. While Q-PCR analysis of markers of

early meiosis did not show any significant alterations, markers of sex chromosome inactivation

(Ube1x, Atp7A, Gla) failed to be inactivated in the KO. Chromosome spread analysis using SCP3

(marker of meiotic progression) revealed severe degeneration of spermatocytes (2.4% WT vs. 53.3%

KO) with percentage of zygotene and pachytene spermatocytes falling by 90% and 95% respectively.

Defects in meiosis were further confirmed using WIN 18,446/retinoic acid to synchronize the tubules.

Collectively, these results indicate a crucial role of Huwe1 in regulating meiotic progression.

Funding source: FRSQ, Genome Quebec, CIHR-China

ORAL 3

OOCYTE-DRIVEN REMODELING OF THE FOLLICULAR MICROENVIRONMENT

BUILDS THE PLATFORM FOR ESSENTIAL GERM LINE-SOMATIC COMMUNICATION

Stephany El-Hayek1,2,4

, Qin Yang 4, and Hugh Clarke

1,2,3,4

Departments of Obstetrics and Gynecology1, Biology2, and Experimental Medicine

3

McGill University. Research Institute of the McGill University Health Centre4

Germ cells develop in a microenvironment composed of the surrounding somatic cells. In female

mammals, granulosa cells enclose the developing oocyte. As the oocyte grows, it secretes an

extracellular matrix termed the zona pellucida that separates it from the granulosa cells. The granulosa

bypass the physical barrier imposed by the zona by means of narrow cytoplasmic extensions, termed

transzonal projections (TZPs), that traverse the zona and contact the oocyte. TZPs have been identified

in all mammalian species studied to date. As the sole means by which the granulosa cells establish

contact-dependent communication with the oocyte, the TZPs play a fundamental and indispensable

role in oocyte development. TZPs have been assumed to arise passively as the zona deposits around

the oocyte. In contrast, we found that TZPs increase both in number and density as the oocyte grows.

This thus implies that new TZPs must be actively generated as oocytes grow. Moreover, we found that

both mRNA and protein of known filopodial markers (MyoX, Fascin, and Daam1) can be detected in

granulosa cells surrounding oocytes, and within the zona. Intriguingly, both TZP numbers as well as

expression levels of filopodial markers are enhanced by the oocyte-secreted factor GDF-9. Our data

thus shows that TZPs are actively generated during oogenesis, under the stimulation of the oocyte

itself. The novel concept that these intercellular bridges, which are essential to produce a healthy

oocyte, are dynamically regulated during growth, sheds the light on potential directions for diagnosing

and treating infertility.

Funding: This work was supported by a grant from the Canadian Institutes of Health Research

CIHR).S.E.-H. received support from Réseau Québécois en Reproduction and the CIHR Training

Program in Reproduction, Early Development, and the Impact on Health.

ORAL 4

ADDITION OF A C-TERMINAL TAIL ALTERS MAMMALIAN GNRH RECEPTOR

SIGNALING

Chirine Toufaily1*

, Jérôme Fortin1*

, Évelyne Lapointe2, Derek Boerboom

2, Daniel J. Bernard

1

1Deptartment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

2Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, Canada

*These authors contributed equally to this work

The mammalian GnRH receptor (GnRHR) is unusual among G protein coupled receptors (GPCRs) in

lacking an intracellular C-terminus (C-tail). The C-tail in many GPCRs mediates agonist-induced

desensitization and receptor internalization. Indeed, the lack of a C-tail prevents homologous

desensitization and slows internalization of mammalian GnRHRs. To better understand the functional

significance of the loss of the C-tail in mammals, we generated knock-in mice expressing a chimeric

GnRHR. In this model, the chicken GnRHR C-tail was fused with the C-terminus of the endogenous

murine GnRHR (hereafter GnrhrCtail/Ctail

). Neither serum LH nor pituitary LHβ (Lhb) subunit mRNA

levels differed between GnrhrCtail/Ctail

and wild-type males. In contrast, both serum FSH and pituitary

FSHβ subunit (Fshb) expression were decreased in GnrhrCtail/Ctail

males. Pituitary expression of the

gonadotropin α subunit (Cga) and Gnrhr was also decreased in these mice. In contrast, gonadotropin

subunit and Gnrhr mRNA levels did not differ between intact GnrhrCtail/Ctail

and wild-type females on

metestrus. Following bilateral ovariectomy, pituitary Fshb, Lhb, and Cga mRNA levels as well as

serum LH increased significantly in females of both genotypes; however, the response was blunted in

GnrhrCtail/Ctail

mice. GnrhrCtail/Ctail

females exhibited abnormal estrous cyclicity and were subfertile.

Reduced litter sizes could derive from impaired FSH induced follicle maturation and/or from altered

LH surge dynamics. Based on the data collected thus far, we propose that the loss of the C-tail in

GnRHR evolution conferred a selective advantage by enhancing GnRH’s regulation of FSH rather than

by enabling the LH surge.

Supported by FQRNT Team Grant PR-174948 and CIHR MOP-123447 to DJB and DB

ORAL 5

IN VITRO ASSESSMENT OF NOVEL GREEN PLASTICIZERS USING A

TOXICOGENOMIC APPROACH IN IMMORTALIZED SERTOLI CELL LINES

Thomas C. Nardelli (a), Hanno C. Erythropel (b), Bernard Robaire (a,c)

(a) Department of Pharmacology and Therapeutics, McGill University

(b) Department of Chemical Engineering, McGill University

(c) Department of Obstetrics and Gynecology, McGill University

Phthalate esters are a family of compounds used in the manufacturing of PVC. Exposure to these

compounds has been correlated with a variety of phenotypes resulting from their endocrine disruption

properties. Some of the most prominent of these effects can be observed in the male reproductive

system where they cause, among other things, dysregulation of Sertoli cell physiology.

Our study aims to assess the safety of a replacement commercial plasticizer (DINCH), two alternative

plasticizers (1,4 butanediol dibenzoate (BDD), dioctyl succinate (DOS)), a predicted toxic plasticizer

(dioctyl maleate (DOM)) and the bioactive metabolite of DEHP (MEHP). Using a toxicogenomic

approach with the immortalized Sertoli cell line TM4, we treated cells for 48 hours with a series of

plasticizers and extracted their RNA for microarray analysis. We noted that our positive control

MEHP, DOM, and the commercial plasticizer DINCH, caused a large change both in number and in

fold-change of several genes. To better understand the physiological relevance of these changes, genes

that were significantly changed greater than 1.5 fold were analyzed using Ingenuity Pathway Analysis.

As previously reported, MEHP targeted several genes involved in cholesterol biosynthesis. DOM

upregulated genes involved in cell-cycle arrest and glutathione depletion. DINCH affected several

important signalling pathways such as Map, Erk and Rho signalling pathways. BDD and DOS did not

cause significant changes in gene expression following treatment. From these studies, it appears the

safety of the commercial plasticizer DINCH needs to be re-evaluated while BDD and DOS are suitable

candidates for extensive animal studies.

These studies are funded by a CIHR team grant awarded by the Institute of Human Development,

Child and Youth Health

ORAL 6

PERTURBATIONS IN THE EXTRACELLULAR MATRIX OF DEVELOPING

URETEROVESICAL JUNCTION MAY LEAD TO VESICOURETERAL REFLUX

Fatima Tokhmafshan1, Marie-Lyne Fillion

1, Jasmine El Andalousi

2,Rasheed A. Gbadegesin

3, Patrick

D. Brophy4, Indra R. Gupta

1,2.

1) Department of Human Genetics, McGill University, Montreal, Quebec, Canada

2) Department of Pediatrics, McGill University Health Center, Montreal, Quebec, Canada;

3) Department of Pediatrics, Division of Nephrology, Duke University Medical Center, Durham, NC,

USA

4) Department of Pediatrics, University of Iowa, Carver College of Medicine, Iowa City, IA, USA

Anomalous development of the ureterovesical junction (UVJ), which joins the ureter to the bladder, is

associated with vesicoureteral reflux (VUR), the retrograde flow of urine from the bladder to the

kidneys. The muscular layer of the ureter and bladder produce a valve-like effect that occludes the UVJ

during voiding thereby preventing VUR. Human studies have demonstrated that refluxing UVJs have

elevated levels of fibrillary collagens, and degeneration of the ureteral smooth muscle, suggesting that

the integrity of the extracellular matrix (ECM) is crucial for the competence of the UVJ. Longitudinal

studies of children with VUR have demonstrated that up to 65% will undergo spontaneous resolution

of VUR. This could be due to changes in the ECM of the UVJ that result in resolution of VUR. A large

kindred was recently discovered in which patients with VUR and joint hypermobility (JH) have

heterozygous missense mutations in Tenascin-XB (TNXB), an ECM glycoprotein that regulates proper

assembly and deposition of fibrillary collagens, and elastin deposition through its FNIII domain. In

addition to joint laxity, these individuals also have hyperextensible and fragile skin, suggesting a

global derangement in ECM composition. My preliminary data indicates that TNXB expression is

upregulated in the urinary tract of newborn mice. This data also shows that the ECM matures with age

in mice as indicated by the increase in the amount of collagen deposited in their UVJ and bladder. The

TNXB gene was sequenced in 96 children with VUR, and novel missense and deleterious mutations in

the FNIII domain of TNXB have been identified. These patients are currently being tested for JH using

the Beighton scoring system.

Funding Sources: Supported by CIHR

POSTER PRESENTATIONS

ANNOUNCING AUDIO PRESENTATION OF POSTERS

Do you like to visit posters during off hours to avoid crowds and check out promising

presentations; but the presenter isn’t there? Do you hate to fight the crowd that mobs the posters

that you were the most keen to visit? Do you want to get a quick review of a hot poster but the

presenter is fully occupied by someone who wants seemingly endless personal attention? The

CSR meeting this year will feature an exciting new way to preview poster presentations: a 5-

minute QR-coded audio teaser that will not only encapsulate the poster’s facts and figures but also

give insights into how the poster presenters view their conclusions, the surprises and potential

applications. We urge all poster presenters to participate and let your personality, as well as your

experimental results, draw attention to you and your work.

Participant presenters will display a QR code in the upper right corner of their poster. You will be

able scan the QR code with your smart phone’s QR reader and listen to the presentation with ear

buds as you stand in front of the poster and follow along. You can then plan to visit the poster or

presenter at a more opportune time to get details, clarifications, or just get to know the presenter

better.

We suggest that you download the Kaywa Reader and/or QRReader to your smart phone and

bring ear buds to the poster sessions. If you forget your ear buds, free buds will be available at the

meeting. These QR readers are available for virtually all smart phones and they work with the QR

codes used for access the audio presentations. Most other readers also work. There will be

volunteers available to assist you if you encounter any difficulties. Here is a QRcode for you to

scan and test your QR reader and smart phone audio before you head out for the meeting. We

recommend listening to the entire recording.

Poster Session Schedule CSR Research Day 2015

BREAKTHROUGHS IN REPRODUCTION AND DEVELOPMENT

May 19th

2015

Presentation

Time Group 2 Presenter Group 3 Presenter Group 4 Presenter Group 5 Presenter Group 6 Presenter Group 7 Presenter

12:50-12:57 P-11 Enrique Gamero-

Estevez

P-36 Lisa-Marie

Legault P-01 Gauthier Schang

P-12 Rodrigo

Camponogara Bohrer P-24 Keith Siklenka P-05 Gurpreet Manku

13:00-13:07 P-15 Suhaib Khayat P-22 Laura Whidden P-02 Steven Jones P-13 Eskandari

Shahraki Marzieh P-26 Cécile Adam P-09 Lorena Carvelli

13:10-13:17 P-16 Aaron Kwong P-23 Jonas

Brandenburg

P-03 Anne Marie

Downey P-14 Naomi Dicks

P-30 Sathvika

Venugopal P-10 Océane Albert

13:20-13:27 P-17 Martika Rodgers P-25 Kai Sheng P-04 Karl-Frédéric

Vieux P-18 Wenyang Hou

P-32 Yasmin

Schuermann P-27 Luisina Ongaro

13:30-13:37 P-19 Yassemine

Khawajkie P-29 Nancy Li

P-06 Paulo Roberto

Antunes da Rosa P-20 Elie Akoury

P-33 Samin Sabouhi

Zarafshan

P-28 Yasaman

Aghazadeh

13:40-13:47

P-07 Laleh Abbassi P-21 Vafa Keser P-34 Swati Gupta P-31 Enrico Campioli

13:50-13:57 P-08 Johanna

Selvaratnam P-35 Renata Bahous

POSTER 1

EFFECTS OF ORGANOPHOSPHATE FLAME RETARDANTS ON LEYDIG CELL FUNCTION Gauthier Schang

1, Babara F. Hales

1, Bernard Robaire

1,2

1 Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

2 Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada

Organophosphate flame retardants (OPFRs) serve as alternatives to replace banned brominated flame

retardants, but little is known about their toxicity. We determined the effects of seven common OPFRs

on Leydig cells, the main source of androgen in males. BDE−47, a brominated diphenyl ether flame

retardant, served as a reference compound. MA−10 Leydig cell mitochondrial activity was reduced by

treatment with 100 μM BDE-47 as well as with all OPFRs. Lower BDE-47 concentrations (1-20 μM)

did not affect mitochondrial activity; in contrast, <20 μM of any OPFR, with the exception of triphenyl

phosphate, significantly reduced mitochondrial activity. Leydig cell numbers were decreased to ~60%

of control by treatment with 50 μM BDE-47; IC50 values for the OPFRs ranged from 10.3 to 27.5 μM.

Superoxide production was significantly increased (1.7 to 4.4-fold) by all seven OPFRs at 10 μM,

suggesting oxidative stress; 10 μM BDE-47 had no effect. Basal progesterone production was

significantly increased (1.5 to 3-fold) by 10 μM of 2-ethylhexyl diphenyl phosphate, isodecyl diphenyl

phosphate, isopropylated phenyl phosphate, tert-butylphenyl diphenyl phosphate or tricresyl

phosphate; BDE-47, triphenyl phosphate and tri-o-cresyl phosphate had no effect. Interestingly,

dbcAMP-stimulated progesterone production was further enhanced by treatment with isopropylated

phenyl phosphate (2-fold), while LH-stimulation decreased by tri-o-cresyl phosphate (~ 2/3). Thus, all

OPFRs affected mitochondrial activity, cell survival and superoxide production. Only BDE-47 and

triphenyl phosphate did not affect basal or stimulated steroid production. Significant increases in basal

steroid production or decreases in responsiveness to stimuli may have long term consequences on male

hormone functions.

Funding sources: supported by REDIH and CIHR.

POSTER 2

DIFFERENTIAL EFFECTS OF COMBINED GENISTEIN AND DEHP ON TESTICULAR

CELL LIPID HOMEOSTASIS AND STEROID PRODUCTION

Steven Jones MSc1, Annie Boisvert MSc

1, Gurpreet Manku PhD

2, Francoise Hullin−Matsuda PhD

3,

Peter Greimel PhD3, Toshihide Kobayashi PhD

3 and Martine Culty PhD

1,2

1 Department of Experimental Medicine, McGill University, Montreal Quebec, Canada

2 Dpartment of Experimental Medicine, McGill University, Montreal Quebec, Canada

3 Lipid Biology Laboratory, RIKEN, Wakoshi Saitama, Japan

Previous work in our laboratory demonstrated the ability of in utero exposure to a mixture of the

phytoestrogen, Genistein (G), and plasticizer, DEHP (D), to induce long term alterations in gene and

protein expression that are substantially different from individual exposures. Recent data identified

fetal –type and adult Leydig cells and germ cells, as well as their progenitors as sensitive targets for

low dose ED mixtures.

To further investigate the direct effects of G and D and elucidate specific mechanisms of toxicity,

MA−10 Leydig cells and isolated primary rat gonocytes were exposed in−vitro to varying

concentrations of G and MEHP (M), the principle bioactive metabolite of DEHP. Thin layer

chromatography demonstrated the ability of combined 10μM G + M to increase levels of neutral-

(CHOL,FFA,TG) and phospholipid (SM,PC,PE,PS,PI) classes in MA−10 cells, indicating a

generalized deregulation of lipid homeostasis. In contrast, combined 10μM G + M reduced neutral and

phospholipid classes in primary gonocytes, suggesting a particular sensitivity of early germ cell

progenitors. Further investigation by qPCR analysis revealed concomitant alterations in MA−10

cholesterol (Hmgcoa) and phospholipid (Srebp1c,Fasn) mediator mRNAs, suggesting the possible

involvement of upstream LXRα agonism. Interestingly, 10μM combined G + M also had a stimulatory

effect (2 fold) on basal MA−10 progesterone production and a consistent increase in the mRNA of

steroidogenic mediators (Star,Tspo,Cyp11a,Srb1,Hsl) and upstream nuclear receptors (Coup−tfII,Sf1).

These results suggest a deregulation of testicular cell function in response to a combination of GEN +

MEHP. Further research is underway to elucidate the origins of differential lipid alterations and

stimulation of steroidogenesis. Taken more broadly, this research highlights the importance of

assessing the impact of ED mixtures in multiple toxicological models across a range of

environmentally relevant doses.

POSTER 3

CYCLOPHOSPHAMIDE (CPA) TREATMENT ALTERS THE EXPRESSION OF MEMBERS OF THE ZIP FAMILY ZINC TRANSPORTERS IN PACHYTENE SPERMATOCYTES. Anne Marie Downey

1, Barbara Hales

1, Bernard Robaire

1,2

1Department of Pharmacology and Therapeutics and of

2Department of Obstetrics and Gynecology,

McGill University, Montreal, Canada Previous studies have shown that paternal exposure to CPA, a chemotherapeutic agent and immunosuppressant, causes DNA damage and oxidative stress in the testis and has detrimental effects on sperm quality and progeny outcome. How CPA affects the developing germ cells and how they respond to this insult remain unresolved. The objective of this study was to test the hypothesis that CPA affects gene expression in pachytene spermatocytes and round spermatids, the meiotic and post-meiotic germ cells. Adult male Sprague-Dawley rats were gavaged daily with saline or CPA (6 mg/kg) for 4 weeks; pachytene spermatocytes and round spermatids were collected by unit gravity sedimentation of testicular cells. Using whole genome gene expression microarrays we have identified differentially expressed genes in both cell types. Interestingly, transcripts for two members of the ZIP family of metal ion transporters (ZIP5 and ZIP14), which play an important role in supplying zinc to cells, were up-regulated over 1.5 fold in pachytene spermatocytes after CPA treatment. Further analysis revealed that transcripts for two more ZIP transporters (ZIP6 and ZIP13) were also significantly up-regulated. The expression of ZIP transporters remained unchanged in round spermatids. PCR validation confirmed the microarray results. Preliminary protein analysis also indicated an increased expression of the corresponding proteins. These results suggest that zinc uptake is increased in pachytene spermatocytes in response to CPA treatment. As zinc plays an important role in antioxidant activity, enhanced uptake may reflect an increased demand for zinc in response to elevated oxidative stress following CPA treatment. Zinc is also an important trace element in spermatogenesis, particularly for proper compaction of chromatin. The poor sperm chromatin quality previously observed after CPA treatment may, in part, be due to altered zinc homeostasis in the germ cells. Funding Sources: Supported by CIHR and CSR

POSTER 4

THE CYTOPLASMIC DEADENYLASE CNOT6 REGULATES DEADENYLATION OF A

SUBSET OF TRANSCRIPTS DURING OOCYTE MEIOTIC MATURATION

Karl-Frederic VIEUX 1,2,3, Joao SUZUKI 3, Qin Yang1,3 and Hugh CLARKE 1,2,3

1Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada

2 Department of Biology, McGill University, Montreal, Quebec, Canada

3 Research Institute, McGill University Health Center, Montreal, Quebec, Canada

Precise regulation of gene expression is indispensable for proper cell function. This challenge is

particularly complex in oocytes where transcription is active during growth but is undetectable in fully

grown oocytes and during meiotic maturation. Transcripts required to drive oocyte maturation and

early post-fertilization embryogenesis are therefore synthesized in growing oocytes and kept

translationally silent until the appropriate time. In addition, the accumulated oocyte transcripts must

eventually be degraded to enable the embryonic genome to assume control of development after

fertilization. mRNA translation and stability are largely controlled by the 3’-poly(A) tail, whose length

in many cell types is regulated by the opposing actions of polyadenylases and deadenylases. Little is

known, however, of the identity or role of deadenylases regulating transcript function in mouse

oocytes. Here we report the expression and function of CNOT6, a key component of the CCR4-NOT

deadenylase complex, in oocytes. RT-PCR and immunoblotting detected the Cnot6 transcript and

protein in oocytes at all stages of growth and during meioticmaturation. Immunofluorescence revealed

that CNOT6 was localized in cortical foci that also contained GW182, a common marker of

cytoplasmic ribonucleprotein particles. When Cnot6 was depleted using siRNA, CNOT6 in RNP foci

was reduced and the deadenylation of a subset of transcripts, including Slbp and Orc6l, which

normally occurs during meiotic maturation, was inhibited. These results reveal a novel role or CNOT6

in regulating mRNA deadenylation during oocyte development.

Funding Sources: Supported by CIHR and RQR

POSTER 5

ACETAMINOPHEN VERSUS IBUPROFEN: EFFECTS ON NEONATAL TESTICULAR

GONOCYTE DEVELOPMENT

Gurpreet Manku1,2,3

, Philippos Papadopoulos1, and Martine Culty

1,2,3

The Research Institute of the McGill University Health Centre1, and the Departments of Pharmacology

& Therapeutics2, and Medicine

3, McGill University, Montreal, Quebec, Canada.

Newborn baby fever is often treated with acetaminophen (AC) (Tylenol®) or ibuprofen (IB)

(Motrin®). Both drugs inhibit cyclooxygenases (COXs), enzymes involved in platelet aggregation,

fever, and inflammation. There are two types of COX enzymes, COX1 and COX2. Although COX2 is

not commonly known for a role in male reproductive biology, it has been reported to play a role in the

steroidogenic function of Leydig cells. However, the possible role of COX2 in germ cells is not yet

known.

Here, we report that COX2 is abundantly expressed in postnatal day (PND) 3 rat gonocytes, the

precursor cells to spermatogonial stem cells which provide a life-long source for sperm production.

Interestingly, COX2 expression was downregulated in PND8 spermatogonia, indicating a possible role

in gonocyte development.

Here, our objective was to determine whether gonocyte proliferation or differentiation could be altered

upon exposure to either AC or IB. This is important as improper gonocyte development has been

suggested to lead to testicular tumor formation.

Isolated PND3 gonocytes were treated with either AC or IB alongside PDGF-BB and 17β estradiol

(PE; proliferation) or Retinoic Acid (RA; differentiation). Although neither drug had any effect on cell

survival, we found that both AC and IB stimulated gonocyte proliferation to levels similar to those

seen with PE treatment. Furthermore, IB reduced the effect of RA on mRNA expression of the

differentiation marker Stra8 (Stimulated by RA 8), indicating a negative effect of this COX inhibitor

on differentiation.

These data suggest that COX2 activity plays a dual role in gonocytes, being positively involved in

gonocyte differentiation, while preventing proliferation. Taken together, our data suggests that anti-

pyretic medications could disrupt neonatal gonocyte development, which could potentially lead to the

formation of testicular germ cell tumors.

POSTER 6

EGFR INHIBITION PREVENTS MEIOTIC RESUMPTION IN BOVINE OOCYTES

COCULTURED WITH FOLLICULAR HEMISECTIONS

Paulo Roberto Antunes da Rosa1, Andressa Minussi Pereira Dau

1, Matheus Pedrotti De Cesaro

1, Raj

Duggavathi2, Vilceu Bordignon

2,Paulo Bayard D. Gonçalves

1

1 Federal University of Santa Maria, Santa Maria, Brazil.

2 Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Canada

In this study, we evaluated the effect of EGFR inhibition on meiotic resumption and gene expression in

granulosa cells, cumulus cells and oocytes in cattle. In the first experiment, COCs (n=52/group) were

cultured for 15h in TCM199 medium with follicular hemisections in the presence of 0.05, 0.5, 5 or

50µM of the EGFR inhibitor, AG1478. Most of the oocytes exposed to 5µM (89.28%) and 50µM

(90.56%) remained at the germinal vesicle (GV) stage at the end of treatment, which was significantly

higher than those treated with the lower concentrations. In the second experiment, embryo culture

revealed that blastocyst formation rates were similar between groups. We assessed the transcript levels

by qPCR in granulosa cells (Pgr, Pgrmc1, Adamts1, Ptger2, Ptger4 and Ptgs2), cumulus cells (Ptgs2,

Tnfaip6, Has2, Cx43 and Pgr) and oocytes (Pde3a, Ccnb1, Stc1, Anxa1 and Serpine1) obtained from

5µM AG1478 treated groups. In cumulus cells, Tnfaip6 and Has2 mRNA was lower in AG1478

treated compared to negative control group. The Cx43 mRNA in negative control group was lower

when compared to positive control group and not different from AG1478 treated group. In granulosa

cells, mRNA levels of Pgr and Adamts1 were lower in AG1478 treated than control samples. These

findings indicate that: i) addition of EGFR inhibitor in the coculture system is an effective and

reversible method to maintain bovine oocyte at GV stage; ii) EGFR inhibition alters gene pathways in

both mural granulosa cells and cumulus cells, which might be involved in the arrest of meiotic

progress.

Funding Sources: Research supported by CAPES and CNPq – Brazil (PBDG) and NSERC – Canada

(VB).

POSTER 7

THE ROLE OF YES-ASSOCIATED PROTEIN (YAP) IN REGULATION OF OCYTE

GROWTH

Laleh Abbassi 1,2,3

, Hugh Clarke 1,2,3

1Department of Obstetrics and Gynaecology, McGill University, Montreal, Quebec, Canada

2Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada

3Research Institute, McGill University Health Center, Montreal, Quebec, Canada

Most oocytes within the ovary lie dormant within primordial follicles, and only a small number

undergo growth to yield fertilizable eggs. Recent work has shown that activation of YAP (Yes-

associated protein), a transcriptional co-activator that drives growth and proliferation in many cell

types, dramatically increases the number of growing oocytes, suggesting a new therapeutic strategy to

overcome human infertility. However, in the absence of knowledge of the normal function of YAP in

the follicle, our understanding of the mechanistic basis of these strategies remains very limited. We

found that Yap and YAP are expressed in growing and full-grown oocytes. Throughout growth, YAP is

phosphorylated on S127 and restricted to the cytoplasm. To identify the mechanism of

phosphorylation, we manipulated the activity of cAMP/PKA pathway, an upstream regulator of YAP

in somatic cells. In fully grown oocytes, YAP remained phosphorylated in the presence of the

analogue, dbcAMP, but became dephosphorylated in its absence. In growing oocytes, YAP remained

phosphorylated in the absence of dbcAMP, but became dephosphorylated in the presence of an

inhibitor of PKA. By pharmacologically trapping proteins in the nucleus, we found that YAP shuttles

between the nucleus and cytoplasm in growing but not fully grown oocytes and that PKA inhibition

increases shuttling activity. We also found that YAP is excluded from the nucleus of the granulosa

cells surrounding the oocyte. Our results suggest that the mechanism by which activators of YAP

induce oocyte growth does not involve activation of YAP-dependent transcription in the oocyte or

neighbouring granulosa cells.

Funding Sources: Supported by CIHR, McGill Faculty of Medicine and RQR

POSTER 8

THE EFFECTS OF CATALASE OVEREXPRESSION ON DEVELOPING MALE GERM

CELLS IN THE AGED MOUSE

J. Selvaratnam & B. Robaire

Department of Pharmacology and Therapeutics and of Obstetrics and Gynecology, McGill University,

Montreal, QC, Canada

Males produce germ cells continually throughout life; however, the quality of these germ cells

decreases with advancing age. Germ cells from aged rats have increased reactive oxygen species

(ROS) and reduced antioxidant activity, thus indicating a reduced capacity to deal with oxidative

stress. The peroxisomal antioxidant enzyme, catalase (Cat), plays a major role in regulating cellular

ROS by decomposing hydrogen peroxide, known to damage cellular components. Mice over-

expressing Cat (CatOE) show reduced ROS toxicity and increase longevity; however, the effects of

increased Cat in aging male germ cells remains unknown. We hypothesize that CatOE mice will

display decreased ROS and damage in developing germ cells. Mice were bred, genotyped, and aged to

3-mo and 18-mo. Groups of wild-type (WT)-3mo, WT-18mo, CatOE-3mo, and CatOE-18mo mice

were injected with either saline or 30mg/kg tert-butyl hydroperoxide (tBH). 1-hr later mice were

euthanized, with body, testis, epididymal, and seminal vesicle weights collected. Testicular germ cells

were extracted and incubated with CellROX® Deep Red (InvitrogenTM

Life) to detect cytoplasmic

ROS. Fluorescent signals were captured using Operetta® High Content Imaging System

(PerkinElmer), spermatocytes were localized and signals quantified using Columbus© (PerkinElmer).

We found that body and seminal vesicle weights increased in WT-18mo and CatOE-18mo. Atrophied

tubules increased in aged mice, and CatOE-18mo had higher atrophied tubules than WT-18.

Spermatocyte ROS was unaltered in all saline treated groups, tBH treatment increased ROS in WT-

18mo vs. WT-3mo. CatOE-18mo displayed decreased ROS vs. CatOE-3mo. In conclusion, CatOE

spermatocytes show alleviated ROS in aged mice, but greater germ cell atrophy.

Funding Source: Supported by CIHR

POSTER 9

HEPARIN-ALPHA-GLUCOSAMINIDE N-ACETYLTRANSFERASE (Hgsnat) GENE

INACTIVATION AFFECTS THE REPRODUCTIVE TRACT OF ADULT MICE

Lorena Carvelli1, Alexey V. Pshezhetsky

2, Louis Hermo

1, Yan Zhang

1. and Carlos R. Morales

1

1Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

2Department of Pediatrics and Biochemistry, McGill University, Montreal, Quebec, Canada

Introduction: Spermatozoa mature in the epididymis under the control of the lining epithelial cells.

Heparan sulfate (HS) is an abundant proteoglycan in the testis and epididymis. HS is endocyted and

degraded in a stepwise fashion in lysosomes by the action heparin-alpha-glucosaminide N-

acetyltransferase (HGSNAT) and other enzymes. A deficiency in HGSNAT results in a variant type of

Sanfilippo syndrome (MPSIIIC). In mice, inactivation of the Hgsnat gene leads to a mild form of

MPSIIIC, with animals at late ages showing reduced liter sizes. Objectives: Determine the

morphological effects of Hgsnat inactivation on epithelial cells of the testis and epididymis.

Methodology: The testes and epididymides of both wild type and Hgsnat -/-

adult mice at different ages

were removed and fixed for LM routine and immunocytochemical studies. Results and discussion: In

Hgsnat deficient mice, some seminiferous tubules were smaller in diameter, had vacuolated areas,

and/or presented partial germ cell depletion. In the epididymis of Hgsnat deficient mice, basal cells

were greatly enlarged in size and contained numerous empty looking vacuoles, reactive for cathepsin

D and prosaposin, suggesting that they are lysosomal in nature. HS also accumulates in principal cells

of the Hgsnat deficient mice, especially in the corpus region, but not surprisingly in basal cells.

Although, spermatozoa were present in the lumen of the epididymis of knockout mice, small spherical

cells and debris were also noted. All of the above abnormalities increased with the age. Our results

provide the first evidence that glycan metabolism is important for normal male reproductive functions.

Funding Sources: Supported by NSERC and CIHR.

POSTER 10

SPERM CHROMATIN QUALITY ASSESSMENT: OPTIMIZATION OF THE HIGH

THROUGHPUT COMET ASSAY

Océane Albert1, Robert G. Berger

1, Wolfgang Reintsch

2, Barbara F. Hales

1, Bernard Robaire

1,3

1Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

2Green

Chemistry CFI Platform, Department of Pharmacology and Therapeutics, McGill University,

Montreal, Quebec, Canada 3Department of Obstetrics & Gynaecology, McGill University, Montreal,

Quebec, Canada

To date, the standard semen parameters used to assess fertility, as described by the World Health

Organization, do not include any information about the quality of sperm nuclear material. However, a

number of studies show that there is sperm DNA damage in men with apparently normal standard

semen parameters, and that this damage can imperil the outcome of pregnancy. The COMET assay

(single cell gel electrophoresis) involves the collection of data on sperm DNA damage at the level of

the single cell, allowing the use of samples from severe oligospermic patients. However, this

particularity makes comet scoring a low throughput procedure that renders large cohort analyses

tedious. Our objective is to develop a standardized high throughput COMET assay for human sperm

that will increase both its accuracy and efficiency. The assay we have developed includes (i) automated

mixing and distribution of sperm and low melting point agarose on a 96-well plate by the Janus®

workstation, to ensure evenness across the plate and avoid artifactual DNA damage trends; (ii)

optimized cell lysis and DNA decondensation treatment parameters for human sperm; (iii) optimal

horizontal electrophoresis conditions; (iv) automated detection of comets by the Operetta® high content

imaging system; and (v) automated scoring of comets by the Columbus™ image data analysis system

that compares to the broadly used Komet™ software. The use of the standardized high throughput

COMET assay on 15 patients from different fertility classes confirmed that sperm concentration is not

correlated with DNA damage rates.

Funding Sources: Supported by RHF100625 from the CIHR Institute of Human Development, Child

and Youth Health, the CIHR Training Program in Reproduction, Early Development, and the Impact

on Health, and the Fonds de la Recherche du Québec en Santé.

POSTER 11

MODIFYING THE CLAUDIN BINDING SPECIFICITY OF THE C-TERMINUS OF

CLOSTRIDIUM PERFRINGENS ENTEROTOXIN (C-CPE).

Enrique Gamero Estevez1, Amanda Baumholtz

1, Makoto Nagano

2, Aimee K. Ryan

1,2.

1Department of Human Genetics, McGill University, Montreal, Quebec, Canada.

2RI-MUHC, Montreal, Quebec, Canada.

Claudins are essential for formation and function of tight junctions and the combination of claudins

present within an epithelial cell layer determines its paracellular barrier properties. Claudins have two

extracellular loops and intracellular N- and C-termini. The first extracellular loop determines the size

and charge selectivity of the paracellular barrier and the second interacts with claudin molecules on

adjacent cells. The non-toxic C-terminal domain of C. perfringens enterotoxin (C-CPE) can bind to

the second extracellular loop of Claudin-3, -4, -6, -7, -8 and -14 and remove them from tight junctions

via internalization of the claudin-C-CPE complex. We showed that incubating gastrulation stage chick

embryos with C-CPE causes neural tube defects as a result of removing Claudin-3, -4 and -8 from tight

junctions. We also determined that incubating murine inner medullary collecting duct (mIMCD) cells

with EL2 peptides transiently opened the TJ barrier within 1 hour of treatment. Based on these

findings we have designed C-CPE variants where the claudin binding domain has been replaced with

the EL2 amino acid sequence of specific claudin family member. We predict that these C-CPE

variants will have increased affinity for individual claudin family members and can be used to

specifically target the removal of individual claudins from tight junctions.

Funding sources: Supported by CIHR

POSTER 12

EFFECTS OF INHIBITING DNA REPAIR PATHWAYS DURING EARLY EMBRYO

DEVELOPMENT

Rodrigo C. Bohrer, Naomi Dicks, Eliza R. Komninou, Karina Gutierrez, Werner G. Glanzner, Raj

Duggavathi and Vilceu Bordignon

Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, Quebec, Canada

DNA double-strand breaks (DSBs) are known to affect early embryo development in multiple species.

In our previous studies we have shown that DSBs affect embryo cleavage kinetics, somatic cell

reprogramming, gene expression and blastocyst formation in porcine embryos. Two main DNA

damage repair pathways, the homologous recombination (HR) and the non-homologous end-joining

(NHEJ), are activated for DSBs repair. Although genes and proteins participating in the two repair

pathways have been shown to be regulated in response to DSBs in early developing embryos, it has not

been determined if both pathways are required for DSBs repair during early embryo development. In

this study we used specific inhibitors of the HR and the NHEJ repair pathways to investigate the

importance of each pathway on embryo development, presence of DSBs and cell death. In this study,

porcine embryos were in vitro cultured in the presence of inhibitors of DNA repair by HR, NHEJ and

their combination. Our findings indicate that: a) both HR and NHEJ DNA repair pathways are

important for early embryo development; b) the inhibition of DNA repair pathways results in DSBs

accumulation during embryo development; c) the inhibition of DNA repair pathways increases cell

apoptosis in developing embryos; and d) the HR pathway seems to be more important than the NHEJ

pathway for DNA repair in developing embryos, especially in embryos with higher number of DSBs.

Funding Sources: Supported by a NSERC Discovery Grant to V.B., and a scholarship from CNPq,

Brazil to R.C.B.

POSTER 13

KNOCKOUT MICE MODEL TO STUDY FUNCTIONS OF BSPH1 AND BSPH2 IN

FERTILITY.

Marzieh Eskandari-Shahraki1,2

, Bruno Prud’homme1, Geneviève Plante

1,2, Qinzhang Zhu

3 and

Puttaswamy Manjunath1,2

1Maisonneuve-Rosemont Hospital Research Centre, Montréal, Québec, Canada

2Departments of Medicine and of Physiology, Faculty of Medicine, University of Montréal, Montréal,

Québec, Canada. 3Institut de Recherche Clinique de Montréal (IRCM), Montréal, Québec, Canada

Mammalian testicular sperm are immotile and are unable to fertilize eggs. The fertilizing ability is

gained through the acquisition of secreted molecules in the male and female reproductive tracts, which

induce important changes in the lipid composition of the sperm membrane. Two of the main post-

testicular processes leading to sperm fertilizing ability are called epididymal sperm maturation and

capacitation. Our laboratory has identified a family of proteins named, the Binder of Sperm (BSP)

proteins, known to be involved in sperm capacitation. Two members of this family (BSPH1 and

BSPH2) are expressed in murine epididymis. To elucidate the functions of these BSP proteins in vivo,

we generated mice carrying single and double mutations in Bsph1 and Bsph2 genes using a

revolutionary new technology, the RNA-guided genome editing tool, the Clustered Regularly

Interspaced Short Palindromic Repeats/CRISPR associated nuclease 9 (CRISPR/Cas9) systems. The

progeny carrying the mutated Bsph1 and Bsph2 alleles was screened by PCR and the targeted deletion

and/or recombination by guided RNA was determined. To study the effect of the deletion of Bsph1 and

Bsph2 genes, we have begun mating of the single and double knock-out mice with wild-type mice.

Further studies will be performed to evaluate fertility and sperm functions (motility, viability,

capacitation, acrosome reaction) of those single and double knockout mice, and to assess sperm and

reproductive organs morphology. This model should help elucidate the in vivo functions of mouse BSP

proteins, which are the counterparts of the human (BSPH1). (Supported by CIHR)

POSTER 14

REDUCTION OF ENDOPLASMIC RETICULTUM (ER) STRESS BY TREATMENT WITH

TAUROURSODEOXYCHOLIC ACID (TUDCA) RESCUES DEVELOPMENTALLY

INCOMPETENT SLOW-CLEAVING PORCINE EMBRYOS IN VITRO

Naomi Dicks

1, Rodrigo C. Bohrer

1, Eliza Komninou

4, Karina Gutierrez

1, Monique Rovani

5, Gustavo

Ilha5, Joao Ricardo Souza

6, Joanna Jung

3, Marek Michalak

3, Luis B. Agellon

2, Vilceu Bordignon

1

1Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada

2School of Dietetics and Human Nutrition, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada

3Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

4Centro de Desenvolvimento Tecnologica, Universidade Federal de Pelotas, Pelotas, Brazil

5Departamento de Clinica de Grandes Animais, Universidade Federal de Santa Maria, Santa Maria,

Brazil 6Faculdade de Veterinaria, Universidade Federal de Pelotas, Pelotas, Brazil

Studies from different species have revealed that fast-cleaving embryos are more likely to develop to

the blastocyst stage whereas slow-cleaving embryos fail to develop. Also, induction of ER stress in

cultured embryos has caused reduced blastocyst rate and quality. Systematic characterization of ER

stress effects on embryo cleavage kinetics, development and quality has yet to be completed. This

study aimed to determine if treatment of slow-cleaving embryos with TUDCA can improve

developmental competence and embryo quality. Parthenogenetically-activated porcine embryos were

separated into fast-cleaving (<1 day of activation) and slow-cleaving (1-2 days after activation) groups

and supplemented with either 50µM TUDCA or vehicle at day 2 of culture. Embryos were collected at

days 3, 5 and 7 to evaluate development, total cell number and ER stress status. TUDCA treatment

increased the blastocyst rate and total cell number in both embryo groups. Slow-cleaving embryos

treated with TUDCA had a similar blastocyst rate, but higher cell number, compared with untreated

fast-cleaving embryos. Abundance of XBP1s mRNA, an ER stress marker, was higher in all untreated

embryos at day 3 and 5, but not 7. GRP78 ER stress chaperone abundance, determined by

immunofluorescence, was increased at day 3, but not 5 and 7. ER stress was highest prior to porcine

zygotic genome activation, but only persisted in untreated slow-cleaving embryos. The results of this

study demonstrate that slow-cleaving embryos display increased ER stress which can be decreased by

TUDCA treatment. The treatment also improves embryo quality to a level superior to untreated fast-

cleaving embryos.

Funding sources: This project was supported by an NSERC Alexander Graham Bell Doctoral

Scholarship.

POSTER 15

ACCUMULATION OF MITOCHONDRIAL DNA DURING EARLY MURINE

EMBRYOGENESIS IS LINKED TO THE RESUMPTION OF CELL GROWTH

Suhaib KHAYAT 1,2,3,4 and Hugh CLARKE 1,2,3

1Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada

2 Department of Experimental Medicine, McGill University, Montreal, Quebec, Canada

3 Research Institute, McGill University Health Center, Montreal, Quebec, Canada

4 Saudi Arabian Cultural Bureau

Mitochondria play a major role in providing the energy required for various cellular functions.

Mitochondrial DNA is commonly used to assess cellular mitochondrial content. In mammalian

oocytes, mtDNA accumulate during oocyte growth and this accumulation stops when oocyte growth

ceases. Following fertilization, mtDNA levels remains constant during early embryogenesis, then

increase after implantation. Yet the mechanism behind the resumption of accumulation remains

unknown. In the current study, we aim to identify the pattern, mechanism and timing of mtDNA

accumulation during embryogenesis.

qPCR analysis of mtDNA levels showed that in preimplantation embryos ( 1CE, 2CE, early- and late-

blastocysts), mitochondrial content is comparable to that of full-grown oocytes. Next, to assess levels

at implantation, we cultured blastocyst outgrowths in vitro, thus mimicking the process of

implantation. We found a significant increase in levels of mtDNA in blastocysts outgrowth compared

to late blastocyst controls. Moreover, we also detected a significant increase in mRNA levels of

replication factors (Tfam, Nrf1, Polga, Polgb, Twinkle and mtSsb) as the embryo develops from the 2-

cell to the blastocyst stage. Our results demonstrate that the of levels mtDNA, which reflects cellular

mitochondrial content, remain constant at the early stages of embryogenesis and significantly increase

after implantation. This data also suggests that this increase is associated with an enhanced expression

of replication factors required for mtDNA replication. Future work will focus on determining the

trigger that allows resumption of mitochondria accumulation during post-implantation development.

Funding Sources: Scholarship from Saudi Arabian Cultural Bureau. NSERC

POSTER 16

FGF4 AS A POSSIBLE REGULATOR FOR NAÏVE TO PRIMED PLURIPOTENCY

DIFFERENTIATION AND ROSSETTE FORMATION

Kwong, A.M.1,2

, Honma-Yamanaka, N.1, Yamanaka, Y.

1,2

1Goodman Cancer Research Centre, McGill University, 1160 Pine Avenue West, Room 419, Montréal,

QC H3A 1A3, Canada 2Department of Human Genetics, McGill University, 1160 Pine Avenue West, Room 419, Montréal,

QC H3A 1A3, Canada.

During blastocyst implantation, naïve epiblast cells epithelializes into a primed-pluripotent rosette

which then hollows to form the egg cylinder cone. While previous gene knockout models targeting

MEK/ERK pathway have demonstrated to inhibit naïve-primed pluripotency transition, the dynamics

of this pathway in regards to cluster epithelialization and cooperative integration into a rosette remain

unclear. Here we describe the an in vitro 3D culturing method for primed pluripotent differentiation of

mouse embryonic stem cells (mESC) and autonomous rosette reorganization to investigate the role of

the MEK/ERK pathway during this transition.

mESC were cultured in a single cell suspension on top of a membrane of growth-factor reduced

matrigel with additional supplemented matrigel in the differentiation media for 48 hours. Cell cluster

morphology was classified into five classes: EpiLC (Epiblast like-cell) monolayer, disordered cluster

(DC) with and without actin foci, ordered cluster (OC) with and without actin foci, and superclusters.

OCs were characterized as a rosette or monolayer cyst which may contain a central actin foci, in

contrast to DCs with a disordered cellular configuration with or without either a peripheral or non-

central actin foci. Superclusters were classified as post/peri merged rosettes. Interestingly, cluster-

migration was observed while retaining conformation, however after 3 days of culturing these clusters

reorganized into a monolayer which migrated towards neighboring clusters and appear to simulate

their reorganization into a monolayer concordantly. Our objective with this culturing model is to

investigate the dependence of MEK/ERK pathway on rosette formation in Grb2-/-

mESC.

Funding Sources: Supported by NSERC and CIHR.

POSTER 17

THE ROLE OF ANGIOMOTIN IN HIPPO SIGNALING REGULATION IN THE EARLY

MOUSE EMBRYO

Martika Rodgers1, Yojiro Yamanaka

2,3

1McGill University, Montreal, Quebec, Canada

2Department of Human Genetics, McGill University, Montreal, Quebec, Canada

3Goodman Cancer Research Centre, McGill University, Montreal, Quebec, Canada

In the early mouse embryo, the 8-to-16 cell division is crucial for forming the first two distinct lineages

in the embryo, polar and apolar cells. These lineages become the placental (trophectoderm) and

embryonic (inner cell mass) lineages, respectively. Although the polarity-dependent Hippo signaling

pathway has been shown to be involved in this process, upstream regulation of this pathway has yet to

be elucidated. It is known that cell fate is orchestrated through a transcriptional activator, Yap, which

promotes specification of the trophectoderm in polar cells where Hippo signaling is inactive. Active

Hippo signaling leads to the phosphorylation of Yap in apolar cells, which allows the cell to acquire

the inner cell mass fate. Amot (angiomotin), a junction-associated protein, has been shown to localize

at cell-cell contacts and be necessary for Yap phosphorylation. The current model for Amot-dependent

Hippo signaling regulation suggests that the apical domain is crucial in sequestering Amot to suppress

Hippo signaling. We used blastomere isolation and immunostaining examine the importance of Amot’s

subcellular localization in Yap phosphorylation. Interestingly, it was found that Yap phosphorylation

could occur in apolar cells before subcellular Amot patterns were established. In addition, image

analysis suggested that Amot was able to localize to non-apical, non-contact surfaces of apolar cells

with similar levels to the apical domains of polar cells. These findings question the current

sequestration model of Amot and urge us to explore other alternatives.

POSTER 18

EXPLANTS OF PRE-ATTACHMENT CHORION AND ALLANTOIS REVEAL EXTENSIVE

MIXING AND A REQUIREMENT FOR THE ALLANTOIS FOR MAINTENANCE OF GCM1

AND TPBPΑ EXPRESSION

Wenyang Hou1, Didem P. Sarikaya

1,3, Loydie A. Jerome-Majewska

1,2

1) Department of Human Genetics, McGill University, Montreal, QC, Canada; 2) McGill University

Health Centre at Glen, Montreal, QC, Canada; 3) Organismic and Evolutionary Biology Department,

Harvard University, Cambridge, Massachusetts, USA

Development of the mouse placenta proceeds rapidly after the allantois and chorion attach through a

process called chorioallantoic attachment. As a consequence of chorioallantoic attachment, a subset of

chorionic cells in proximity of the allantois differentiate into syncytiotrophoblast cells and form

invaginations into which allantoic-derived embryonic blood vessels migrate to form the labyrinth

layer. Chorioallantoic attachment and subsequent differentiation events required for placental

development have remained poorly investigated partly due to being inaccessible to ex vivo analysis.

Here, we report conditions for ex vivo culture of pre-attachment chorion and allantois. Under these

culture conditions, explants of pre-attachment allantois and chorion attached and showed extensive

mixing of chorionic and allantoic cells. We confirmed that the allantois was required for expression of

the syncytiotrophoblast cell marker Gcm1. In addition, we found that maintained expression of the

spongiotrophoblast cell marker Tpbpα also depended on chorioallantoic attachment. We tested the

efficacy of this ex vivo model by examining the tissue-specific requirement for Tmed2, a member of

the transmembrane emp24 domain (TMED) protein family, required for normal placental

development. Recombinant cultures of Tmed2 null and wild type chorion/allantois revealed a role for

Tmed2 in cell survival and for mixing of chorionic and allantoic cells. Thus we report the first

successful ex vivo model of pre-placantal tissues before chorioallantoic attachment, and show that this

explant system can be used to reveal tissue-specific requirements of genes required for placental

development.

POSTER 19

DISSECTING THE GENETIC SUSCEPTIBILITY TO SPORADIC MOLAR PREGNANCIES

AND MECHANISMS OF THEIR FORMATION

Y. Khawajkie1, NMP. Nguyen

2, P. Sauthier

3, W. Buckett

3, M. Lemoine

2, Zhu Xiao Xu, M. Breguet

3,

R. Slim1,2,3

1Departments of Experimental Medicine,

2Human Genetics, and

3Obstetrics and Gynecology, McGill

University Health Centre, Montreal H3G 1A4, Canada

A hydatidiform mole is an abnormal human pregnancy characterized by absence of, or abnormal,

embryonic development, excessive trophoblastic proliferation, and hydropic degeneration of placental

villi. The common types of moles are sporadic and affect 1 in 600 pregnancies in western countries.

Among patients with one mole, up to 20% have a second reproductive loss (in the form of a

spontaneous abortion), which is higher than the incidence of recurrent reproductive losses in the

general population (2-5%). This indicates that these patients are genetically susceptible to reproductive

loss. Based on the parental contribution to sporadic molar tissues, there are three main genotypic types

of moles: diploid androgenetic monospermic, diploid androgenetic dispermic, and triploid dispermic.

However, it is not known whether patients with these three genotypic entities have the same genetic

predisposition to reproductive loss. In addition, it is not known whether the other forms of reproductive

loss in women with one mole are caused by the same or by a different mechanism as their mole. To

address these questions, we compared the reproductive histories of patients with the three genotypic

types of moles amongst each other. We also studied conceptions of patients with at least three

spontaneous abortions to characterize their parental contribution, categorize them, and better

understand the mechanisms underlying them. We used flow cytometry, fluorescent microsatellite

genotyping, and fluorescent in situ hybridization to determine the parental contribution to all their

available products of conception, and consequently, to uncover the mechanism underlying their

occurrence.

Funding Sources: supported by CSR and RI MUHC

POSTER 20

NLRP7 AND KHDC3L, THE TWO MATERNAL-EFFECT PROTEINS RESPONSIBLE FOR

RECURRENT HYDATIDIFORM MOLES, CO-LOCALIZE TO THE OOCYTE

CYTOSKELETON

Elie Akoury1, 2

, Li Zhang2, Asangla Ao

2, Rima Slim

1, 2

1Department of Human Genetics, McGill University Health Center, Montreal, Quebec, Canada

2Department of Obstetrics and Gynecology, McGill University Health Center, Montreal, Quebec,

Canada

Hydatidiform mole (HM) is an aberrant human pregnancy with abnormal embryonic development and

excessive trophoblastic proliferation. Recessive mutations in the maternal-effect genes, NLRP7 or

KHDC3L, are responsible for recurrent HMs (RHMs). However, the exact roles of NLRP7 and

KHDC3L in this condition are not fully understood. To gain insights into their functions, we

characterized their subcellular localizations in human oocytes and early embryos using regular and

confocal immunofluorescence and electron microscopies. We found that in oocytes, from the germinal

vesicle until the formation of the zygote, NLRP7 co-localized with KHDC3L mainly to the cortical

region. Within this region, electron and high resolution confocal microscopies confirmed the co-

localization of NLRP7 and KHDC3L between cortical granules, mitochondria, and other organelles on

some cytoskeletal structures that did not overlap exactly with the α-tubulin microtubule network or

display similar pattern by immunofluorescence. As the embryo completed its first division, NLRP7

and KHDC3L became excluded from the cell-to-cell contact region, restricted to the outer cortical

regions, and were part of some structural complexes that are not E-cadherin dependent. During early

cleavage stages, the two proteins displayed different localization patterns. While NLRP7 maintained its

polarity until the blastocyst stage where it became homogeneously distributed in the cytoplasm of cells

from the inner cell mass and trophectoderm, KHDC3L translocated to the nuclei of cells from both the

inner cell mass and trophectoderm at the morula stage.

To better understand how the two oocyte cytoskeletal proteins, NLRP7 and KHDC3L, play a role in

cellular proliferation, one of the fundamental aspects of hydatidiform moles, we characterized their

subcellular localizations during the cell cycle of somatic cells. Our study is the first comprehensive

high resolution localization of the only two known maternal-effect proteins, NLRP7 and KHDC3L, in

human oocytes, preimplantation embryos, and somatic cells and will contribute to a better

understanding of their roles in all aspects of the pathology of hydatidiform moles.

Funding Sources: supported by the Canadian Institute of Health Research, Research Institute of the

McGill University Health Centre and a CREATE award from the Réseau Québécois en Reproduction.

POSTER 21

GENETIC MODELS FOR IDENTIFYING THE MOLECULAR BASIS OF PHENOTYPIC

VARIABILITY IN 22q11.2 DELETION SYNDROME

Vafa Keser1 and Loydie A. Jerome-Majewska

1, 2, 3

1Department of Human Genetics, McGill University, 1205 Avenue Docteur Penfield, N5/13, Montreal,

Quebec H3A 1B1, Canada 2Department of Pediatrics, McGill University Health Centre Glen Site 1001 Decarie Blvd, EM02210

Montreal, Quebec H4A 3J1, Canada 3Department of Anatomy and Cell Biology, McGill University, Strathcona Anatomy and Dentistry

Building, 3640 University Street, Montreal, Quebec H3A2B2, Canada

22q11.2 deletion syndrome (22q11.2DS) is a contiguous gene syndrome that characterized by 1.5–3

MB deletions of chromosome 22q11.2. The main aspect of this syndrome is the phenotypic

heterogeneity, which includes craniofacial, heart, palatal anomalies, immunodeficiency, and

hypoparathyroidism although other various phenotypes observed also (renal, skeletal etc.). About 90%

of patients have common 3 Mb deletion which carry more than 35 genes including TBX1, SNAP29,

and SCARF2, genes that have been sown to contribute to developmental syndromes. Although TBX1 is

considered as the main causal gene for the syndrome and mouse studies phenocopy the effect of the

deletions in human, a spectrum of anomalies are not observed in these mice lead to the idea that other

genes contribute to the disease manifestation. Our laboratory and other laboratories showed that

22q11.2 deletion unmasks rare variants in the 22q11.2 region of the intact chromosome, which resulted

in manifestations of recessive disorders caused by two other genes; SCARF2 and SNAP29. Thus,

mutations in these 3 genes are associated with abnormal human development and commonly deleted in

22q11.2DS.

We postulate that haploinsufficiency for TBX1 and SNAP29 or TBX1 and SCARF2 might explain

phenotypic variability found in 22q11.2DS patients. To test this hypothesis we cloned and

characterized expression of Scarf2 and Snap29 during mouse development, and showed that these two

genes have tissue-specific expression during development. In addition, we have found that these genes

are co-expressed with Tbx1 in the craniofacial region suggesting that these genes may genetically

interact in development of this region.

Funding sources: None

POSTER 22

COMBINED EFFECTS OF DNA METHYLTRANSFERASE 1o-DEFICIENCY AND

OVARIAN STIMULATION ON DNA METHYLATION PATTERNING AND EMBRYONIC

OUTCOME AT MID-GESTATION

Laura Whidden1, Josée Martel2, Donovan Chan2 and Jacquetta Trasler3

1Department of Pharmacology & Therapeutics, McGill University; 2 McGill University Health Centre

Research Institute; 3Departments of Pediatrics, Human Genetics and Pharmacology & Therapeutics,

McGill University; Montreal, QC, Canada.

The use of assisted reproductive technologies (ARTs) has been linked with an increased incidence of

growth and genomic imprinting disorders in children, shown in some cases to be the result of aberrant

DNA methylation. DNA methylation is a well-characterized epigenetic mechanism catalyzed by DNA

methyltransferases (DNMTs). Mouse studies have shown reduced expression of DNMTs in the

oocytes of aging females. We propose that factors related to underlying infertility (i.e. reduced

expression of DNMTs) will increase offspring susceptibility to aberrant DNA methylation patterning,

exacerbated by ARTs. Blastocysts were collected from superovulated control and Dnmt1o-

heterozygote females (5.0IU PMSG/hCG) and transferred non-surgically to recipients. Mid-gestation

embryos and placentas were collected and assessed for developmental delays and morphological

abnormalities. DNA methylation was examined both at imprinted genes (pyrosequencing) and

genome-wide (Reduced Representation Bisulfite Sequencing).

Similar rates of pre- and post-implantation loss were observed between groups (n=39-40), with a trend

towards an increased proportion of delayed and abnormal embryos in the Dnmt1o-heterozygote group.

DNA methylation at examined imprinted genes was not affected by DNMT1o-deficiency, however

RRBS analysis of female embryo and placenta (n=4/group) revealed 537 and 1685 differentially

methylated tiles (DMTs), respectively. These DMTs resulted from both loss and gain of methylation,

mapped predominantly to intergenic regions, introns and exons, and were distributed across all

chromosomes. Greater methylation variability was observed in the placentas of Dnmt1o-heterozygotes

compared to controls. These preliminary results indicate that DNMT1o-deficiency exacerbates

genome-wide DNA methylation abnormalities induced by ovarian stimulation and may be involved in

mediating poor embryonic outcome. (Supported by CIHR, RQR and RI-MUHC funding)

POSTER 23

EPIGENETIC EFFECTS OF PRENATAL EXPOSURE TO POLYCYCLIC AROMATIC

HYDROCARBONS ON CYP1A EXPRESSION AND INDUCIBILITY.

Jonas Brandenburg, Jessica Head

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been

reported to adversely affect embryonary development. Toxicity of PAHs has, at least partly, been

linked to the induction of two Cytochrome P450 (CYP) isoforms: 1A1 and 1A2 (1A4 and 1A5 in

birds). Recent reports linked prenatal PAH exposure to changes in DNA methylation and gene

expression in humans, rats and fish.

We hypothesize that in ovo exposure of chicken embryos to PAHs causes persistent changes in

CYP1A gene expression and inducibility by changing the methylation pattern of CYP1A4 and

CYP1A5. As epigenetic modifications established early in development can last throughout an

individuals’ life, a change in the capacity to induce CYP isoforms could have a mayor impact on the

tolerance of an organism to exposure to PAHs and similar compounds later in life. CYP1A isoforms

are major players in the xenobiotic biotransformation response, and epigenetic regulation of these

genes may have important implications for human and environmental health.

To test our hypothesis, we injected PAHs into fertilized chicken eggs prior to incubation. Liver

samples were taken at embryonic day 10, and expression of the CYP1A4 and CYP1A5 were assessed

by qPCR. At day 10, both genes showed not only an important induction (27- fold and 16-fold) at high

doses (> LD50), but also at low doses (4 –fold) that do not cause embryomortality. Further

investigations will address the question whether PAH exposure, especially at low doses, results in

changes in epigenetic regulation of CYP1A gene expression.

Funding sources: Supported by FRQS.

POSTER 24

OVER-EXPRESSION OF KDM1A IN SPERMATOGENESIS ALTERS THE SPERM

EPIGENOME AND HAS DIRE CONSEQUENCES FOR DEVELOPMENT OF THE

EMBRYO

Keith Siklenka1*

, Serap Erkek2*

, Maren Godmann1, Romain Lambrot

1, Serge Mcgraw

1, Christine

Lafleur1, Tamara Cohen

1, Jianguo Xia

1, Matthew Suderman

3, Mike Hallett

1, Jacquetta Trasler

1,

Antoine Peters2 and Sarah Kimmins

1

1 McGill University, Montreal, Canada

2 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland

3 University of Bristol, Bristol, United Kingdom

Paternal effects can influence health and development of generations to come through a process called

epigenetic inheritance. The mechanisms underlying epigenetic inheritance are largely unknown but

may involve transmission via the sperm epigenome. Recent studies have shown that sperm histones

are retained at genomic regions high in CpG, and activating histone marks such as histone H3 lysine 4

dimethylation (H3K4me2) are retained at gene promoters implicated in embryonic development. Thus,

we hypothesized that the epigenetic marks on sperm histones play a major role in the development of

offspring, and serve to influence the inheritance of non-genetic information in future generations. To

test this hypothesis we designed a transgenic inbred mouse model with a disturbed sperm-epigenome

induced by over-expression of the human histone demethylase KDM1A in the testes. Offspring sired

by transgenic males have a range of gross abnormalities and increased frequency of death.

Importantly, the offspring sired by the wild type littermates (nonTG) also exhibited the same

phenotype, which persisted for three generations. To elucidate a potential mechanism for epigenetic

inheritance we employed epigenomic techniques and affymetrix arrays to profile histone marks, DNA

methylation and the RNA population in sperm from TG and nonTG males. We also analyzed gene

expression two-cell embryos to associate changes in gene expression with an altered sperm epigenome.

Our data reveals a possible complex collaboration between sperm chromatin and RNA to regulate not

only offspring health, but a persistent epigenetic memory. This research was funded by CIHR,

Genome Quebec, RQR, FQRNT, Swiss National Science Foundation, and the Novartis Research

Foundation

POSTER 25

HUWE1 FUNCTION ON MALE FERTILITY AND EPIGENETIC REGULATION DURING

SPERMIOGENESIS

Kai Sheng1, Rohini Bose

1, Simon Wing

1

1.Dept. of Medicine, McGill University & McGill University Health Centre Research Institute,

Montreal, Quebec, Canada H4A 3J1

An increasing number of studies have demonstrated the importance of ubiquitination in

spermatogenesis and fertilization. For example, during the remodeling phase, the replacement of

histones initially by transition proteins and subsequently by protamines is vital for normal sperm

formation, and involves the ubiquitin-proteasome system. This chromatin reorganization is thought to

allow compaction of the paternal genome into the sperm head and to protect the DNA from damaging

agents. The ubiquitin ligase Huwe1 is a large protein (480 kDa) whose functions remain to be

completely defined. Our laboratory was the first to describe Huwe1 through purification from the testis

and to identify its ubiquitinating activity towards all core histones. We hypothesize that Huwe1 is

responsible for histone ubiquitination and degradation by the proteasome during sperimogenesis. To

testify our hypothesis, we crossed conditional Huwe1 knockout female mice (Huwe1flox/flox

) with males

hemizygous for Stra8-Cre, which expresses Cre recombinase when spermatogonia undergo

differentiation. Huwe1 KO male mice were sub-fertile. The average weight of the testes of adult

Huwe1 KO mice was 30 % of WT controls. The epididymal sperm concentration of KO mice was

decreased by 80%. Morphological analysis suggested abnormalities of the sperm head. The KO sperm

also exhibited low motility as revealed by Computer Assisted Sperm Analyzer. These data demonstrate

that Huwe1 is required for normal spermatogenesis and fertility. The ongoing experiments aim to

examine for residual histones in KO sperm and to correlate it with DNA integrity in order to

understand the effects of loss of Huwe1 on spermiogenesis.

POSTER 26

UNDERSTANDING THE REGULATION OF CONNEXIN 26 IN THE EPIDIDYMIS.

C. Adam1, D.G. Cyr

1

1 INRS-Institut Armand-Frappier, Laval, QC

Connexins (Cxs) are proteins that form gap junctions allowing neighboring cells to communicate by

the diffusion of ions and small molecules (<1 kDa). Our laboratory has previously reported that

Cx30.3, 31.1 and 32 are expressed in adult rat epididymis, whereas Cx26 is expressed in young animal

when the epithelium is undifferentiated; suggesting a role for Cx26 in the differentiation of the

epididymal epithelium. The regulation of the Cx26 gene in the epididymis is unknown. The present

objective was to elucidate the mechanisms regulating the Cx26 gene in the epididymis through the

characterization of its promoter. RLM-RACE revealed a single major transcription start site (tss) for

Cx26 at position -3829 relative to the ATG. A 1.7kb fragment of the Cx26 promoter was amplified and

cloned into a luciferase reporter vector. Several constructs were generated by deletions of the promoter

and transfected into RCE cells. The luciferase assays revealed the presence of two binding sites

necessary for the expression of Cx26: an AP2/SP1 site and an SP1 site. The implication of those two

sites was confirmed by directed mutagenesis. ChIP analysis confirmed that these factors bind to the

DNA in vivo and indicated that this binding decreases as a function of age when Cx26 mRNA levels

decrease. DNA methylation status was assessed in young and pubertal rats. Results indicated that there

were no changes in DNA methylation in the promoter of young versus pubertal animals. These results

indicate that the transactivation of the Cx26 gene is regulated by AP2 and SP1 sites. The decrease in

Cx26 mRNA levels is correlated with a decrease in AP2 and SP1 binding to the promoter and this is

not the result of changes in DNA methylation.

Supported by NSERC.

POSTER 27

GONADOTROPE-SPECIFIC DELETION OF BMPR2 DOES NOT AFFECT FSH

SYNTHESIS OR ESTROUS CYCLICTY IN ADULT FEMALE MICE

Luisina Ongaro, Xiang Zhou, Daniel J. Bernard

Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.

The TGFβ superfamily ligands, activins and inhibins, were characterized as selective regulators of

pituitary FSH secretion. More recently, other ligands in the family, the bone morphogenetic proteins

(BMPs), were also implicated in FSH regulation; acting independently and synergistically with

activins to simulate FSHβ subunit (Fshb) transcription in immortalized gonadotrope cells. According

to in vitro observations, both BMP2 and activin A signal via the type II receptors BMPR2 and ACVR2

to regulate Fshb. Consistent with these data, Acvr2 knockout mice are FSH-deficient. BMPR2’s role in

vivo, however, was previously unknown. We therefore generated mice carrying loss of function

mutations in Bmpr2 specifically in gonadotropes. We crossed Bmpr2fl/fl

and GnrhrGRIC

mice to

generate conditional knockouts (Bmpr2fl/fl

;GnrhrGRIC/+

) and littermate controls (Bmpr2fl/fl

). We

measured puberty onset and estrous cyclicity, either of which different between genotypes. At

approximately 9-10 weeks of age, pituitaries and reproductive organs were collected at 07h00 the

morning of estrus (i.e, at the time of the secondary FSH surge). Pituitary Fshb expression as

determined by RT-qPCR did not differ between genotype. There was a non-significant trend toward

increased ovarian weights in KO relative to control mice, whereas uterine weights were

indistinguishable between genotypes. The data collected thus far suggest that BMPR2 may be

dispensable for pituitary FSH synthesis in female mice. We have not yet determined whether ACVR2

compensates for the loss of BMPR2.

Funding Sources: CIHR MOP-133394 to DJB and a CSR PDF Scholarship to LO.

POSTER 28

TRANSLOCATOR PROTEIN (TSPO) DRUG LIGANDS INDUCE TESTOSTERONE

FORMATION IN GnRH ANTAGONIST CASTRATED RATS

Yasaman Aghazadeh1, Barry Zirkin

2 and Vassilios Papadopoulos

1

1Research Institute of the McGill University Health Centre and Department of Medicine, McGill

University, Montreal, Quebec, H3G 1A4, Canada 2Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of

Public Health, Baltimore, Maryland 21205, USA

Low testosterone (T) is a major cause of male hypogonadism and infertility and is linked to

osteoporosis, reduced bone-mass index and aging. T-replacement therapy, has been linked to

increased risk of prostate cancer, LH suppression, increased mortality rates and skin irritation. The

principal site of regulation of steroid hormone biosynthesis is at the mitochondria where the lipophilic

cholesterol moves across the aqueous intramembranous space of this organelle. This transport is

hormone-dependent and mediated through a multi-protein complex, the transduceosome. This complex

is assembled from proteins such as steroidogenic acute regulatory (STAR) protein, translocator protein

(TSPO) and voltage-dependent anion channel 1 (VDAC1). Studies showed that in the presence of 8-

Br-cAMP, TSPO and STAR interact directly after 15 min, due to assembly of transuceosome but this

interaction is further lost. However, VDAC1-TSPO interaction is time sensitive is regulated by 14-3-

3ε. Manipulating these interactions with a synthetic peptide increases steroidogenesis rates (Mol

Therapy 2014; 22:1779). We further tested drug candidates that target TSPO on T formation in

chemically castorated rats. The GnRH antagonist Cetrorelix was used for castoration which completely

suppress T production.Various classes of well-characterized high affinity TSPO drug ligands,

including PK 11195, FGIN-1-27 and XBD173 were administered by intratesticular injection to one

testis per animal. The results obtained showed a significant increase in intratesticular T levels in

Cetrorelix-treated rats by all TSPO drug ligands. These results suggest that the mitochondrial

transduceosme is a viable pharmacological target for treating primary hypogonadism and for

maintaining physiological T levels without exogenous administration of T.

POSTER 29

STEROL CARRIER PROTEIN-2, A NONSPECIFIC LIPID-TRANSFER PROTEIN, IN

INTRACELLULAR CHOLESTEROL TRANSPORT FOR STEROID BIOSYNTHESIS

Nancy Li1,2

, Jinjiang Fan2,3

and Vassilios Papadopoulos1,2,3,4

1Department of Pharmacology & Therapeutics, McGill University, Montreal, Quebec, H3G 1A4,

Canada; 2The Research Institute of the McGill University Health Centre, Center for Translational

Biology, 1001 Boul. Décarie, Montreal, QC H4A 3J1, Canada; 3Department of Medicine, McGill

University, Montreal, Quebec, H3G 1A4, Canada; 4Department of Biochemistry, McGill University,

Montreal, Quebec, H3G 1A4, Canada

Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major

role in intracellular lipid transport and metabolism, and has been associated with diseases involving

abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa

sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13k Da SCP2

domain in their C-termini. Using 22-NBD-cholesterol, a fluorescent analog of cholesterol, this study

seeks to further our understanding of the role of these proteins in the intracellular cholesterol transport

required for steroid biosynthesis. Immunofluorescence staining of MA-10 mouse tumor Leydig cells

and cryosections of mouse testis showed that SCPX and SCP2 are present in both MA-10 cells and in

mouse testicular interstitial tissue. Fluorescent fusion proteins of SCPX and SCP2 and confocal live

cell imaging were used to investigate the subcellular targeting of these proteins to mitochondria and

peroxisomes in the MA-10 cells. The results obtained showed that SCPX and SCP2 are localized to

peroxisomes, targeted by the C-terminal PTS1 domain, but the N-terminal mitochondrial-targeting

sequence is not potent enough to solely lead SCPX and SCP2 to mitochondria. Homology modeling

and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks the

specificity of binding and/or transport. These findings further our understanding of the role of SCPX

and SCP2 in intracellular cholesterol transport, and present a new point of view challenging previous

reports on the role of these proteins in cholesterol trafficking.

Funding Sources: Supported by a grant from the Canadian Institutes of Health Research (MOP125983)

and a Canada Research Chair in Biochemical Pharmacology.

POSTER 30

CHOLESTEROL TRAFFICKING FOR STEROID BIOSYNTHESIS IN MA-10 MOUSE

TUMOR LEYDIG CELLS

Sathvika Venugopal1, Seimia Chebbi

1, Francoise Hullin-Matsuda

2, Toshihide Kobayashi

2 and

Vassilios Papadopoulos1.

1Research Institute of the McGill University Health Centre and Department of Medicine, McGill

University, Montreal, Quebec, Canada and 2Lipid Biology Laboratory, RIKEN Advanced Science

Institute, Wako, Saitama, Japan.

The hormone-sensitive and rate-limiting step in steroid biosynthesis is the movement of cholesterol

from intracellular sources to the inner mitochondrial membrane (IMM). Despite the numerous studies

on cholesterol trafficking in steroidogenesis, the exact source and mechanism by which cholesterol is

transported to IMM remains to be elucidated. D4 is the fourth domain of perfringolysin O protein,

which has the ability to bind membranes containing greater than 30 mol% cholesterol of total lipid

concentration with high affinity. mCherry-tagged D4 was used to visualize cholesterol trafficking in

MA-10 cells. Confocal microscopy showed D4-mCherry localized at the plasma membrane, but upon

45 minutes treatment with dibutyryl-cAMP (dbcAMP) a significant reduction in plasma membrane

labeling was observed. Functional inhibitors of proteins involved in cholesterol import into

mitochondria and metabolism, blocked steroid formation and slowed down the movement of D4-

mCherry from the plasma membrane. Recombinant D4-GFP protein readily bound the outer leaflet of

the plasma membrane even after 120 minutes of dbcAMP stimulation, indicating that cholesterol was

trafficked from the inner leaflet of the plasma membrane. D4-mCherry also localized the late

endosomes upon dbcAMP stimulation suggesting a route for the cholesterol from plasma membrane to

mitochondria. These data suggest that the bulk of the steroidogenic pool of cholesterol, mobilized by

cAMP for acute steroidogenesis, likely originates from the inner leaflet of the plasma membrane.

Funding sources: CIHR, CRC, RIKEN, and CSR.

POSTER 31

GLOBAL KNOCKOUT AND STEROIDOGENIC CELL-TARGETED DELETION OF THE

TRANSLOCATOR PROTEIN (18-KDA) UNVEIL ITS CRUCIAL ROLE IN VIABILITY AND

HORMONE-DEPENDENT STEROID FORMATION

Enrico Campioli, Jinjiang Fan, Andrew Midzak, Martine Culty, and Vassilios Papadopoulos

The Research Institute of the McGill University Health Centre and Department of Medicine, McGill

University, Montreal, Quebec, Canada

Translocator protein (TSPO, 18 kDa) is an outer mitochondrial membrane high affinity cholesterol-

and drug-binding protein abundant in steroid synthesizing cells. Previous pharmacological,

biochemical and genetic studies, as well as in vivo experiments, provided several lines of evidence

demonstrating that TSPO is a key member of the mitochondrial cholesterol transport complex in

steroidogenic tissues. Moreover, in vitro and in vivo studies, performed in various species,

demonstrated the crucial role of TSPO in mitochondrial function and cell viability. However, the

recently reported genetic ablation of Tspo gene in mice, via either conditional (using the Amhr2-Cre)

targeting to Leydig and Sertoli cells of the testis or global (using Tspofl/fl

female mice crossed with

Ddx4-Cre to generate a germ cell-specific Tspo) approaches reported that TSPO has no role in steroid

formation and viability (Morohaku et al., & Tu et al., 2014). To clarify the role of TSPO in

steroidogenesis and viability, we generated two lines of Cre-mediated Tspo conditional knockout

(cKO) in mice and two lines of zinc finger nuclease (ZFN)-based Tspo gene mutation in rat. Amhr2-

Cre mice were crossed with Tspofl/fl

mice for two rounds of breeding to obtain F1 Tspo cKO mice

(Tspofl/fl

; Amhr2Cre /+

) to study the role of Tspo under disturbed genetic condition compared to wild-

type (wt) mice. Six cKO out of 135 mice were obtained indicating an unexpected Mendelian ratio of

4.4% instead of 25% for the cKO mice. To confirm the observed abnormal Mendelian ratio, we

genotyped embryos obtained at dpc 12.5, when Amhr2-Cre is expressed in gonads. The results

obtained showed that there was only one Tspo cKO in 22 embryos obtained, corresponding to the same

4.5% ratio seen in adults. The normal embryonic development in the uteri extirpated on dpc 12.5

suggests that there is a Tspo-dependent preimplantation selection. Interestingly, using ZFN technology

in rats to develop a global rat cKO for Tspo, we obtained an unexpected low birth rate of 1.2 and 3.5%

cKO rats using two different mRNAs leading to 0 and 2 mutant survivors, respectively. Taken

together, these findings indicate embryonic lethality when Tspo is removed or mutated although there

are rare cases of survival, likely through adaptation. To further establish Tspo steroidogenic cell-

specific cKO mice, we used the Nr5a1-Cre (Sf1-Cre) mice crossed with Tspofl/fl

mice for two rounds of

breeding to obtain the F1. The resulting 36 Tspo cKO (Tspofl/fl

; Nr5a1Cre /+

) from a total of 185 F1 mice

showed a normal Mendelian ratio and steroidogenic tissue-specific reduction of Tspo mRNA and

protein. Tspo cKO mice lost their ability to produce increased corticosterone levels in response to

ACTH treatment. Testosterone production in response to hCG treatment was highly variable. Taken

together these studies demonstrate that TSPO is required for embryo development and confirm its

critical role in mediating steroid hormone formation.

Funding Sources: Supported by CIHR, Canada Research Chair in Biochemical Pharmacology.

POSTER 32

FROM THE DRY TO THE LACTATING PERIOD: THE STRUGGLES OF THE DAIRY

COW

Yasmin Schuermann1, Gerald Welsford

1, Evan Nitschmann

2, Ermano Lucena De Oliveira

1, Lucio Da

Cunha Oliveira1, Bruno Milen Varjao

1, Linda J. Wykes

2, Luis B. Agellon

2 and Raj Duggavathi

1

1Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9

2School of Dietetics and Human Nutrition, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9

Dairy cows in the top 50% of Canadian farms are culled after an average of 1.6 lactations mainly due

to infertility. During the peri-calving period, dairy cows experience metabolic stress, which has been

linked to ovarian dysfunction and infertility. Systematic cataloging of metabolic indicators in

circulation is fundamental to better understand the molecular basis of infertility during early lactation.

We collected weekly blood samples from dairy cows (N=15) from three weeks before until 12 weeks

after calving. There was a gradual decline in the level of circulating triglycerides from 3 weeks before

to 1 week after calving, while the first significant increase occurred at 5 weeks post-calving (P<0.05).

Glucose levels reduced from calving to week 1 post-calving and reached a nadir at 3 weeks of lactation

and the first significant increase in glucose concentration occurred at 10 weeks into lactation (P<0.05).

Total cholesterol concentrations increased steadily from the 3rd

to 7th

week post-calving (P<0.05).

Total bile acid levels increased from 3 weeks pre-calving to 2 weeks before calving and stayed

elevated throughout the sampling period. β-hydroxybutyric acid (BHBA) levels increased from calving

until week 3 of lactation (P<0.05) and subsequently returned to baseline. As for oxidative stress

markers, glutathione (GSH) levels declined to reach a nadir by 7 weeks in lactation, while ferric

reducing ability of plasma (FRAP) increased gradually after calving. Taken together, the cumulative

effect of lower blood glucose and GSH, and higher total cholesterol, total bile acids, BHBA and FRAP

may have negative effects on ovarian functions.

Funding Source: NSERC Discovery Grant (RD), RQR-CREATE Scholarship and McGill Graduate

Excellence Fellowship (YS).

POSTER 33

MUTATIONS OF HUMAN BINDER OF SPERM HOMOLOG1 (BSPH1): A NEW CAUSE OF

MALE INFERTILITY?

Samin Sabouhi Zarafshan, Geneviève Plante, Puttaswamy Manjunath

Maisonneuve-Rosemont Hospital Research Center, Department of Biochemistry and Molecular

Medicine, University of Montreal, Quebec, Canada

The latest statistics in Canada shows that ~8% of men of reproductive age seek medical attention for

fertility-related problems. Capacitation is a key step of sperm maturation that is essential for

fertilization.Our laboratory has demonstrated that a group of proteins from bovine seminal plasma

called Binder of Sperm (BSP proteins) bind to sperm membrane and promote capacitation. Recently,

one BSP-homologous gene was identified in human (BSPH1) and shown to be expressed in the

epididymis. In vitro experiments using a recombinant BSPH1 protein revealed that it could promote

sperm capacitation similar to the bovine proteins. Genetic abnormalities are known to be responsible

for many infertility cases. To this day, it is not known whether mutations exist in the BSPH1 gene and

if such mutations could affect sperm capacitation and male fertility. The goal of the current project is

to identify new mutations or polymorphisms in the BSPH1 gene and investigate the impact of these

genetic differences on fertility. To do so, the exons coding for BSPH1 will be sequenced in the

genomic DNA of patients with idiopathic infertility and compared with sequences obtained from DNA

of healthy fertile men. To identify putative problems linked with the regulation, expression or secretion

of BSPH1, proteins will be extracted from spermatozoa and seminal plasma of the same patients.

Using monoclonal antibody, BSPH1 will be immunoprecipitated and quantified to detect any increase

or decrease in the semen of infertile patients. Methods to extract genomic DNA and to amplify and

sequence BSPH1 exons have already been established. Sensitivity and specificity of the monoclonal

antibodies have also been tested. Identification of infertility due to mutation(s) related to BSP genes

could provide new diagnostic tool and therapeutic applications.

Funding sources: Supported by CIHR and HMR Foundation.

POSTER 34

CHARACTERIZATION OF SEC23A AND MAN1B1 EXPRESSION AND FUNCTION IN A

FAMILY WITH CRANIOFACIAL ABNORMALITIES AND MENTAL RETARDATION

Swati Gupta1, Somayyeh Fahiminiya

1, Tracy Wang

1, Laura Dempsey Nunez

1, David S. Rosenblatt

1,2,

William T. Gibson3, Brian Gilfix

4, John J. M. Bergeron

4, Loydie A. Jerome-Majewska

1,2,5 *

1 Department of Human Genetics, McGill University, Montreal, Quebec, Canada

2 Department of Pediatrics, McGill University, McGill University Health Centre Glen Site, Montreal,

Quebec, Canada

3 Department of Medical Genetics, Child and Family Research Institute, Vancouver, British Columbia,

Canada

4 Department of Medicine, McGill University, Montreal, Quebec, Canada

5 Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

SEC23A and MAN1B1 are essential genes involved in protein secretory pathway. SEC23A is an

essential component of COPII-coated vesicles that transport secretory proteins from the endoplasmic

reticulum (ER) to the Golgi complex and Alpha 1,2-mannosidase (MAN1B1) is an essential enzyme

required for targeting proteins into the Endoplasmic-reticulum-associated protein degradation pathway.

Mutation in SEC23A is associated with craniolenticulosutural dysplasia (CLSD) whereas mutation in

MAN1B1 is associated with nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and

congenital disorders of glycosylation (CDG)-II. Mutations in SEC23A or MAN1B1 are extremely rare

and have only been reported in a handful of patients. Our group identified a novel missense mutation in

SEC23A c.1200G>C (p.M400I) and a previously identified mutation in MAN1B1 c.1000C>T

(p.R334C) using whole exome sequencing in two boys suffering from moderate global developmental

delay, tall stature, obesity, macrocephaly, maloccluded teeth and intellectual disability. The parents are

first cousins. In this poster we present that cells with mutation in SEC23A alone showed distended ER

membranes, fewer and more compacted Golgi, and increased pro-collagen 1 secretion (a SEC23A

cargo protein). Fibroblasts with heterozygous and homozygous (mutant fibroblasts) mutations in both

SEC23A and MAN1B1 resembled SEC23A mutant cells, but showed decreased MAN1B1 level.

Furthermore, both patients had N-glycan remodeling defects and fibroblasts from these patients had

increased levels of intracellular and secreted pro-collagen1. Thus, our data support a combination of

abnormal N-glycan remodeling and protein transport as the primary cause of abnormalities in these

patients. We postulate that novel phenotypes found in our affected patients that were not reported in

patients with mutations in either SEC23A or MAN1B1 are due to genetic interaction between these two

genes.

Funding Sources: Supported by CIHR and Fonds de recherché Santé.

POSTER 35

HIGH DIETARY FOLATE DURING PREGNANCY AND LACTATION LEADS TO

DISTURBANCES IN FOLATE METABOLISM, PSEUDO-MTHFR DEFICIENCY AND

SHORT-TERM MEMORY IMPAIRMENT IN MURINE OFFSPRING.

Renata H. Bahous 1, Nafisa M. Jadavji

2, Liyuan Deng

1, Olga Malysheva

3, Marie Caudill

3, Rima

Rozen 1,4

1 Department of Human Genetics, McGill University, Montreal, Quebec, Canada

2 Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada

3 Division of Nutritional Sciences and Genomics, Cornell University, Ithaca, NY, USA

4 Department of Pediatrics, McGill University Health Center, Montreal, Quebec, Canada

Funding source: Supported by CIHR

Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) in mice is associated with brain

dysfunction, short-term memory impairment and disturbed choline/acetylcholine metabolism. With the

increased folate intake, particularly in women of childbearing age, due to food fortification and use of

high dosage vitamin supplements, we have been studying the impact of high dietary folate (HFD) on

health. We reported that male mice fed HFD had a pseudo-MTHFR deficiency.

In this study, we wanted to determine whether HFD during pregnancy and lactation would affect brain

function in offspring. At weaning, female mice were placed on a control diet (CD; recommended level

of folate for rodents) or folic acid-supplemented diet (FASD; 10-fold higher folate than recommended

level) for 5 weeks prior to mating and maintained on the same diet during pregnancy and lactation.

Male offspring were evaluated for memory impairment at 3 weeks of age. Pups from FASD mothers

showed short-term memory impairment using the novel object recognition test (p<0.005, t-test).

MTHFR protein levels in FASD maternal liver were significantly reduced (p=0.005, t-test) and there

was an increase in the ratio of the phosphorylated (less active): non-phosphorylated MTHFR isoform

(p<0.005). We observed a trend towards decreased glycerophosphocholine in maternal plasma and in

offspring hippocampus (p=0.06). To assess these processes earlier in development, we followed the

same experimental design and collected embryos and maternal tissues at E17.5. We observed growth

delay and decreased MTHFR protein in livers of embryos from FASD dams (p<0.05, t-test). We

suggest that HFD during pregnancy leads to a pseudo-MTHFR deficiency in dams and embryos

disturbs choline metabolism and results in memory impairment in offspring.

POSTER 36

IMPLICATION OF A TRANSIENT KDM1A-LOSS ON THE EMBRYONIC EPIGENETIC

LANDSCAPE

Lisa-Marie Legault1,2

, Perrine Gaub1, Serge McGraw

1,3

1Centre de Recherche du CHU Sainte-Justine, Montréal, Québec, Canada

2Département de Biochimie, Faculté de Médecine et Sciences de la Santé, Université de Sherbrooke,

Sherbrooke, Québec, Canada 3Département Obstétrique-Gynécologie, Faculté de Médecine, Université de Montréal, Québec,

Canada

During prenatal life, adverse in utero conditions via environmental factors are thought to perturb

epigenetic marks in the embryonic program that could potentially enhanced the risk of

neurodevelopmental disorders in children. To date, we have little information if a temporary deficiency

in histone modifying enzymes in early development could initiate inherited epigenetic dysregulation on

histone residues and compromise future genes regulations. Here we propose to induce perturbations

onto the epigenetic program of embryonic stem cells (ES) by targeting Kdm1a (lysine specific

demethylase-1a) to identify which epigenetic modifications and interactions associated with brain

development processes are susceptible to inherited dysregulation, i.e. the cell-to-cell transmission of

epigenetic errors. We will use a tetracycline-controlled transcriptional (tet-off) system to induce a

transient Kdm1a, a regulator targeting H3K9me1/2 and H3K4me1/2, in embryonic stem cells. Kdm1a

is also fundamental for both embryo and brain development. We postulate that the transient loss of

Kdm1a will remodel epigenetic interactions in the embryonic epigenome and introduce inherited

dysregulation on regulatory networks related to neuronal cell fates. My goals are to i) Generate an ES

cell line with malleable Kdm1a expression and to ii) Determine the impact of transient Kdm1a

expression on the embryonic epigenetic landscape. These experiments will allow us to identify which

epigenetic marks and interactions associated with brain development processes are the most vulnerable

to dysregulation of Kdm1a activity during early life.

Funding Sources: Supported by the CHU Sainte-Justine

PARTICIPANTS

Abbassi Laleh laleh.abbassi@mail.mcgill.ca

Adam Cécile cecile.adam@iaf.inrs.ca

Aghazadeh Yasaman yassaman.aghazadeh@mail.mcgill.ca

Akoury Elie elie.akoury@mail.mcgill.ca

Albert Océane oceane.albert@mcgill.ca

Antunes da Rosa Paulo Roberto paulorobertoadr@gmail.com

Ao Asangla asangla.ao@muhc.mcgill.ca

Bagheri Negar negar.bagheri-sereshki@mail.mcgill.ca

Bahous Renata renata.bahous@mail.mcgill.ca

Bannoud Nadia nadiabannoud@gmail.com

Bernard Daniel daniel.bernard@mcgill.ca

Boisvert Annie annieboisvert@hotmail.com

Boivin-Ford Elise elise.boivin-ford@mcgill.ca

Bolze Pierre Adrien bolzepa@gmail.com

Bose Rohini rohini.bose@mail.mcgill.ca

Brandenburg Jonas jonas.brandenburg@mail.mcgill.ca

Campioli Enrico enrico.campioli@mail.mcgill.ca

Camponogara Bohrer Rodrigo rodrigocbohrer@hotmail.com

Carvalho Pereira de Sa Ana Karolina ana.carvalhopereiradesa@mail.mcgill.ca

Carvelli Flavia Lorena lorena.carvelli@mcgill.ca

Chan Donovan donovan.chan@mail.mcgill.ca

Chang Ching-Wen ching-wen.chang@mail.mcgill.ca

Chung Yuri yuri.chung@mail.mcgill.ca

Chian Ri-Cheng ri-cheng.chian@mcgill.ca

Clarke Hugh hugh.clarke@mcgill.ca

Cote Nancy nancy.cote.3@ulaval.ca

Culty Martine martine.culty@mcgill.ca

Currin Luke luke.currin@mail.mcgill.ca

de Moraes Ourique Giovana giovanaourique@gmail.com

de Oliveira Regiana Lucia biotom18@yahoo.com.br

Desmarais Joelle joelle.desmarais@mail.mcgill.ca

Dicks Naomi naomi.dicks@gmail.com

Downey Anne Marie anne.downey@mail.mcgill.ca

Duchaine Thomas thomas.duchaine@mcgill.ca

Duggavathi Raj raj.duggavathi@mcgill.ca

El Hayek Stephany stephany.el-hayek@mail.mcgill.ca

El Husseini Nazem nazem.elhusseini@mail.mcgill.ca

Elzhein Samar samar.elzein@mail.mcgill.ca

Eskandari Shahraki Marzieh marzieh.eskandari78@gmail.com

Essagian Charles charles.essagian@muhc.mcgill.ca

Fan Jinjiang jinjiang.fan@mcgill.ca

Fulton Debra debra.fulton@mcgill.ca

Gamero-Estevez Enrique egameroestevez@gmail.com

Gaub Perrine perrine.gaub@gmail.com

Glanzner Werner werner.glanzner@mail.mcgill.ca

Graveline Richard richard.graveline@mcgill.ca

Gregory Mary mary.gregory@iaf.inrs.ca

Gupta Swati 29swatigupta@gmail.com

Gupta Indra indra.gupta@muhc.mcgill.ca

Gutierrez Karina karina.gutierrez@mail.mcgill.ca

Hales Barbara barbara.hales@mcgill.ca

Head Jessica jessica.head@mcgill.ca

Hermo Louis louis.hermo@mcgill.ca

Hou Dominic wenyang.hou@gmail.com

Huang Tiffany tiffany.huang@mail.mcgill.ca

Jerome-Majewska Loydie loydie.majewska@mcgill.ca

Jones Steven steven.jones@mail.mcgill.ca

Keser Vafa vafa.keser@mail.mcgill.ca

Khawajkie Yassemine yassemine@gmail.com

Khayat Suhaib suhaib.khayat@mail.mcgill.ca

Kimmins Sarah sarah.kimmins@mcgill.ca

Kong Chi Chon (Joshua) chi.kong@mail.mcgill.ca

Kwong Aaron aaron.kwong@mail.mcgill.ca

Lambrot Romain romain.lambrot@mcgill.ca

Landry Mylène mylene.landry@mail.mcgill.ca

Laplante David david.laplante@douglas.mcgill.ca

Lau Matthew matthew.lau@mail.mcgill.ca

Lee Daniel sunghoon.lee2@mail.mcgill.ca

Legault Lisa-Marie Lisa-Marie.Legault@usherbrooke.ca

Li Xingfang xinfangli@yahoo.ca

Li Nancy nancy.li775@gmail.com

Liu Richard richard.liu@mail.mcgill.ca

Lusignan Marie-France lusignanmf@sympatico.ca

Ly Lundi lundi.ly@mail.mcgill.ca

Madogwe Ejimedo ejimedo.madogwe@mail.mcgill.ca

Manjunath Puttaswamy puttaswamy.manjunath@umontreal.ca

Manku Gurpreet gurpreet.manku@mail.mcgill.ca

Mann Mellissa mmann22@uwo.ca

Maria Santos Santana Karoline karoline.mariasantossantana@mail.mcgill.ca

Martel Josee josee.martel@mail.mcgill.ca

Martinez Daniel bmartine1978@gmail.com

Mazzarella Rosane rosane.mazzarella@mail.mcgill.ca

McCaffrey Charlotte charlotte.mccaffrey@mcgill.ca

McGraw Serge Serge.McGraw@recherche-ste-justine.qc.ca

Mechtouf Nawel Nawel.Mechtouf@MUHC.MCGILL.CA

Michalovic Laura laura.michalovic@mail.mcgill.ca

Morales Carlos carlos.morales@mcgill.ca

Nagano Makoto makoto.nagano@muhc.mcgill.ca

Nardelli Thomas thomas.nardelli@mail.mcgill.ca

Nguyen Ngoc Minh Phuong phuong2611@hotmail.com

Noblanc Anaïs anais.noblanc@mcgill.ca

Oatley Jon joatley@vetmed.wsu.edu

O'Flaherty Cristian cristian.oflaherty@mcgill.ca

Ongaro Gambino Luisina luisina.ongarogambino@mail.mcgill.ca

Papadopoulos Vassilios vassilios.papadopoulos@mcgill.ca

Pedrotti De Cesaro Matheus vetmatheus@hotmail.com

Prud'homme Bruno bruno.prudhomme12@gmail.com

Reddy Ramesh ramesh.reddy@mail.mcgill.ca

Robaire Bernard bernard.robaire@mcgill.ca

Rodgers Martika martika.rodgers@mail.mcgill.ca

Ryan Aimee aimee.ryan@mcgill.ca

Sabouhi Zarafshan Samin samin.sabouhi@yahoo.com

Schang Gauthier gauthier.schang@mail.mcgill.ca

Schuermann Yasmin yasmin.schuermann@mail.mcgill.ca

Selvaratnam Johanna johanna.selvaratnam@mail.mcgill.ca

Sheng Kai kai.sheng@mail.mcgill.ca

Siklenka Keith keith.siklenka@mail.mcgill.ca

Slim Rima rima.slim@mcgill.ca

Taibi Milena milena.taibi@mail.mcgill.ca

Taketo Teruko teruko.taketo@mcgill.ca

Tanphaichitr Nongnuj ntanphaichitr@ohri.ca

Tokhmafshan Fatima fatima.tokhmafshan@mail.mcgill.ca

Toufaily Chirine chirine.toufaily@mail.mcgill.ca

Trasler Jacquetta jacquetta.trasler@mcgill.ca

Turgeon Marc-Olivier marc-olivier.turgeon@mail.mcgill.ca

Vaz Brandon brandon.vaz@gmail.com

Venugopal Sathvika sathvika10@gmail.com

Vieux Karl-Frédéric omar.vieux@mail.mcgill.ca

Wang Ying ywdearfriends@yahoo.com

Whidden Laura Laura.Whidden@mail.mcgill.ca

Wing Simon simon.wing@mcgill.ca

Xu Yixin xuyixin39@gmail.com

Yamanaka Nobuko nobukoyamanaka@gmail.com

Yamanaka Yojiro yojiro.yamanaka@mcgill.ca

Yan Han han.yan@mail.mcgill.ca

Yang Qin qin.yang@mail.mcgill.ca

Zhang Yan yan.zhang10@mcgill.ca

Zhang Li li.zhang@muhc.mcgill.ca

Zhang Xiangfan xiangfan.zhang@muhc.mcgill.ca

Zhang Xiaoyun ziaoyun.zhang@muhc.mcgill.ca

Zhou Xiang xiang.zhou3@mcgill.ca

Breakthroughs in reproduction and development

Research Day of the Centre for the Study of Reproduction (CSR) at McGill

Tuesday, May 19, 2015

EVENT EVALUATION SURVEY

Thank you for attending our Research Day. We’d like to hear your impression on the various aspects

of the event. We will use your responses to help tailor our next Research Day to deliver an enjoyable

experience for all attendees.

Using a scale of 1 to 5 with 1 indicating well below your expectations and 5 well above your

expectations, please rate each of the following:

The registration process 1 2 3 4 5

The guest speakers 1 2 3 4 5

The oral presentations 1 2 3 4 5

The poster presentations 1 2 3 4 5

The content of the sessions 1 2 3 4 5

The length of the sessions 1 2 3 4 5

The facility 1 2 3 4 5

The refreshments 1 2 3 4 5

Overall 1 2 3 4 5

Comments/Suggestions:

Nouvelles avancées en reproduction et développement

Journée de recherche du Centre d’études sur la reproduction (CER) à McGill

Mardi le 19 mai 2015

FORMULAIRE D’ÉVALUATION

Merci d’assister à notre journée de recherche. Nous aimerions avoir vos commentaires sur différents

aspects de l’événement. Vos réponses et commentaires nous aideront à améliorer notre prochaine

journée de recherche.

Sur une échelle de 1 à 5, avec 1 étant « pas du tout satisfait » et 5 étant « très satisfait »,

veuillez indiquer votre degré de satisfaction concernant les aspects suivants :

Le processus d’inscription 1 2 3 4 5

Les présentateurs invités 1 2 3 4 5

Les présentations orales 1 2 3 4 5

La présentation des affiches 1 2 3 4 5

Le contenu des sessions 1 2 3 4 5

La durée des sessions 1 2 3 4 5

L’emplacement 1 2 3 4 5

La nourriture et les breuvages 1 2 3 4 5

En général 1 2 3 4 5

Commentaires/Suggestions: