Profiling of follicular fluid microRNAs in high and low Antral Follicle Count ovaries in cattle Rolando Pasquariello1,2, Nadia Fiandanese2, Andrea Viglino2, Paola Pocar 3, John L. Williams 4, Fulvio Gandolfi 1 1 Department of Veterinary Sciences for Healt,h, Animal Production and Food Security – University of Milan 2 Parco Tecnologico Padano, Lodi – Italy 3 Department of Veterinary Sciences and Public Health – University of Milan 4University of Adelaide – Australia e-mail:rolando.pasquariello@unimi.it Veterinary and Animal Science PhD Course Annual Meeting, 15-17 July 2015, Milan, Italy

Cattle with a low AFC have poor fertility INTRODUCTION Ovaries are classified into low and high antral follicle count (AFC) depending on less or more than 10 mid-andral follicles (2 to 6 mm) and absence/presence of dominant follicles (>8mm) Cattle with a low AFC have poor fertility Altered level of hormones in follicular fluid Low pregnancy rate Low ovarian size Altered follicle vascularization Low circulating concentrations of P4 Low response to superovulation Low number of healthy follicles

INTRODUCTION Altered ovarian function affects oocyte quality, which have much lower developmental competence when they are fertilized in vitro Low AFC ovary oocytes High AFC ovary oocytes DAY 0 DAY 6-7 DAY 3-5 DEVELOPMENT DAY 1-2 Mature oocyte Fertilized oocyte 2-cell embryo 4-cell embryo Blastocyst Morula 8-cell embryo In human low AFC is associated with women affected by premature ovarian failure (POF).

INTRODUCTION MicroRNAs (miRNA) are short non-coding RNAs involved in post-transcriptional regulation of gene expression. These molecules are also involved in controlling reproductive functions, including diverse ovarian biological processes

AIM AIM OF THE RESEARCH To identify key miRNAs involved in molecular mechanisms of ovarian follicle development related to high and low follicle count ovaries

Sampling and small RNA extraction MATERIALS AND METHODS Sampling and small RNA extraction 210 Ovaries collected at diverse slaughtering days High AFC Low AFC Ovaries were classified in low and high AFC Follicular fluids, collected by aspiration, were cleaned out from blood cells and cellular debris by centrifugation (10 minutes at 1500Xg) n = 144 n = 66 Small RNA extraction was carried out by MiRNA plasma (Mercherey Nagel ) using 300 μl of Follicular Fluid

MATERIALS AND METHODS Sequencing Truseq Small RNA kit (Illumina Inc., USA) for Library preparation Libraries were size-selected in order to sequence only microRNAs of 20-30 bp 3 samples of each experimental group during different slaughtering days (6 libraries sequenced) Deep sequencing on Illumina Hiseq2000 with 50 Single-Read module

Data analysis MATERIALS AND METHODS BIOINFORMATIC ANALYSIS ‘MiRDeep2’ for Novel small-RNA discovery Edger R Package - relevant microRNAs for differential expression among the two conditions Homologous human microRNAs and gene union options KYOTO ENCYCLOPEDIA OF GENE AND GENOME (KEGG) PATHWAYS ANALYSIS GENE TARGET PREDICTION GENE ONTOLOGY (GO) ANALYSIS CYTOSCAPE v3.2.1. – Bovine GO consortium database BIOLOGICAL PROCESSES

RESULTS AND DISCUSSION RESULTS Follicular fluids were enriched by microRNAs (20-30 nucleotide sequences) Electrophoresis of small RNAs in follicular fluids Sequencing allowed to analyze a total of 1279 microRNAs 289/481 (60%) of novel miRNA were not annotated in miRbase

* Differentially expressed microRNAs in Low versus high AFC ovaries RESULTS AND DISCUSSION RESULTS Differentially expressed microRNAs in Low versus high AFC ovaries * Down-regulated Up-regulated ✔ 27 miRNAs differentially expressed (FDR≤0.001) between high and low quality ovaries ✔ 18 known and 9 novel bovine miRNAs, 2 of which were not annotated in miRBase ✔miR-320* has been associated to premature ovarian senescence in human and embryonic developmental failure in mouse

RESULTS DISCUSSION RESULTS MOST SIGNIFICANT (P<0.0001) KEGG PATHWAYS WERE TOOK INTO ACCOUNT Genes known to be involved in follicle physiology 27 microRNAs TARGET PREDICTION = 108 GENES 16 GENES Target prediction identified 108 genes as regulated by differentially expressed microRNAs

- PI3K (phosphatidylinositol 3-kinases) - ERBB signaling pathway RESULTS RESULTS GO OF CANONICAL PATHWAYs ENRICHED BY TARGETED GENES USING IN CYTOSCAPE AND BOVINE DATABASE OF GO CONSORTIUM These genes enriched important GO canonical signaling pathways in follicle physiology - PI3K (phosphatidylinositol 3-kinases) - ERBB signaling pathway - JAK-STAT cascade - Wnt signaling pathway - restricted SMAD protein phosphorylation - ERK1 and ERK2 cascade

CONCLUSIONS RESULTS 1) Follicular fluids of low AFC ovaries have a set of 27 microRNAs that regulate 108 important genes involved in cell proliferation and differentiation, apoptosis, cellular communication and endocrine signaling according to target prediction using DIANA. 2) Follicular fluids of low AFC ovaries are characterized by altered molecular mechanisms in several GO canonical signaling pathways: phosphatidylinositol 3-kinase important in regulation of primordial follicle survival and activation; ERBB controlling differentiation of granulosa cells; Wnt, identified as target of miR-320 and down-regulated in low AFC ovaries. This miRNA has been associated to premature ovarian senescence in human and embryonic developmental failure in mouse. 3) MicroRNAs are non-invasive biomarkers and could be potentially used to evaluate healthy follicles and oocyte quality during assisted reproductive techniques

…AND FOR YOUR ATTENTION!!! THANK YOU… Fulvio Gandolfi Paola Pocar John L. Williams Nadia Fiandanese Andrea Viglino …AND FOR YOUR ATTENTION!!! THIS RESEARCH WAS SUPPORTED BY FP7-KBBE-2012-FECUND-312097