We have investigated the expression of 8 SCH772984 selected candidate miRNAs in granulosa cells, theca cells and cumulus oocyte complex. The results revealed that all these miRNAs are detected in all follicular cell types with varying expression level. MicroRNAs like miR-640, miR-526b* and miR-381 were abundant at higher level in theca cells, while miR-373, miR-30e* and miR-19b-1* expressed more in COCs. However, no significant differences were observed across different cell types in the expression of miR-654-5p and miR-29c. Although the origin of extra-cellular miRNAs in body fluids remains elusive, several reports demonstrated that the origin of extra-cellular miRNAs is closely related to the surrounding cells. While a common set of miRNAs were found in equine follicular fluid and surrounding follicular cells, blood cells are reported to be the major contributor of circulating miRNAs in serum. Therefore, the SAR131675 1433953-83-3 presence of significant level of candidate extra-cellular miRNAs in the surrounding follicular cells could enable us to postulate that the majority of extra-cellular miRNAs in follicular fluid originated from various cell types in the course of their communication during oocyte growth. In order to elucidate the possible exchange of molecular signals including miRNAs in form of exosome between follicular cells we have investigated the ability of granulosa cells to take up exosomes isolated from follicular fluid. Prior to uptake experiment, the stability of exosome coupled miRNA under in vitro culture environment was determined and result showed that exosome coupled miRNAs were stable not only up to 24 hours but even until 48. Similar studies have also shown the stability of circulating serum miRNAs, breast milk exosomal miRNA at room temperature and multiple freeze-thaw cycle. This may provide a great potential for functional analysis of exosome mediated transport of molecules for various cellular processes under in vitro conditions. Following the confirmation of exosomal miRNA stability under culture conditions, we performed exosomes uptake experiment by primary granulosa cells. Fresh exosomes isolated from follicular fluid and labeled by PKH67 fluorescent dye were co-cultured with bovine primary granulosa cells in vitro for 24 hrs. The fluorescent microscopy observation of labeled exosomes co-cultured granulosa cells revealed the presence of green fluorescent exosomes in cultured primary granulosa cells. Similar observation was reported in equine granulosa cells, microglial cell line, immune cells and other cell lines. Fluorescent microscopy result also shows that the majority of the exosomes taken up by granulosa cells were gathered around perinuclear region. Importantly, there were no fluorescent signals in plasma membrane indicating that exosomes were internalized in to granulosa cells via endocytosis. If fusion was the dominant pathway for exosomes uptake, plasma membrane would contain fluorescent signal after co-culture of cells and labeled exosomes. Similar results were 
observed in exosomes internalization by resting PC12 cells. The consequence of transfection of exosome-coupled miRNAs in endogenous miRNA abundance in granulosa cells was investigated by expression analysis of candidate miRNAs in exosomes derived from follicular fluid of either BCB+ or BCB- oocyte origin. Results have evidenced increased level of endogenous miRNA in exosome transfected granulosa cells compared to the control ones. This could enable us to conclude that exosome mediated exchange of miRNAs in follicular microenvironment is an important way of communication between follicular cells. However, the transfer of other miRNAs through these exosomes and their enrichment in granulosa cells and their subsequent contribution to the regulation of the same target genes or other genes cannot be ruled out.