1. 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
Veterinary and Animal Science PhD Course Annual Meeting, July 2015, Milan, Italy

2. 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

3. 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).

4. 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

5. 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

6. 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

7. 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 bp
3 samples of each experimental group during different slaughtering days
(6 libraries sequenced)
Deep sequencing on Illumina Hiseq2000 with 50 Single-Read module

8. 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 v – Bovine GO consortium database
BIOLOGICAL PROCESSES

9. 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

10. * 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

11. 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

12. - 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

13. 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

14. …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