In addition to their association with microvesicles/exosomes and non-vesiclar molecules componants, very recent report showed that miRNAs may present in blood plasma in association of High Density Lipoprotein and delivered them to recipient cells with functional capabilities. In the present study, in addition to the exosomal fraction, we demonstrated that miRNAs were also coupled with non-exosomal fraction of the follicular fluid. Subsequently, we have confirmed the presence of miRNAs in both exosomal and nonexosomal fraction of the follicular fluid using the Human miRNome PCR array platform. Despite using a heterologous approach, the quantitative real time PCR analysis shows detection of the majority of miRNAs in both fractions of follicular fluid indicating the cross species conservation feature of miRNAs between human and bovine as it has been observed in wide range of species. Of the total of 750 miRNAs in the PCR array panel, a total of 509 and 356 miRNAs were detected in exosomal and non-exosomal fraction of follicular fluid respectively. Among the detected miRNAs, 331 were commonly found in both fractions, while 178 and 25 miRNAs were detected only in the exosomal and non-exosomal fraction of follicular fluid, respectively. This shows exosome mediated transport of miRNAs is the dominant pathway in bovine follicular fluid compared to the non-exosomal way as observed previously in blood plasma and saliva. To explore the possible association of exosomal and non-exosomal miRNA expression with oocyte growth in the follicle, we examined the relative miRNA expression level in exosomal and non-exosomal fractions of bovine follicular fluid collected from follicles containing growing and fully grown oocyte. Subsequently we found 25 and 32 miRNAs to be differentially regulated in exosomal and non-exosomal fraction, respectively between two oocyte groups. The higher number of up-regulated miRNAs in both exosomal and non-exosomal fraction of the follicular fluid of growing oocyte group may indicate the presence of a higher degree of Ibrutinib transcriptional activity during the growth phase of the oocyte. In order to evaluate the potential role of these differentially expressed miRNAs, the target genes were predicted bioinformatically and their biological function and gene ontology was determined. The most dominant categories enriched by predicted genes are related to transcription and transport which may 
indicate that, growing oocytes have higher degree of transcription and translation resulting in efficient RNA management and storage in oocytes as maternal resource. Furthermore, the most significant pathways enriched by predicted targets for up-regulated exosomal miRNAs include ubiquitinmediated pathway, neurotrophin signaling, MAPK signaling and insulin signaling pathways. All these pathways are known to be involved in ovarian follicular growth and many CX-4945 developmental processes. The ubiquitin mediated pathway is known to modulate oocyte meiotic maturation, early mitotic division in developing embryos and plays important role in many cellular processes. While neurotrophin signaling pathway is reported to be important in regulation of oogenesis and follicle formation, the MAPK signaling mediates LH-induced oocyte maturation and its activation in cumulus cells is appears to require the permissive effect of the oocyte itself. Similarly, the overrepresented pathways in non-exosomal fraction were WNT signaling pathway and pathways in cancer and focal adhesion. WNT molecules are glycoproteins involved in fetal ovarian development and adult ovarian function including follicular growth, oocyte growth or maturation, steroidogenesis, ovulation and luteinization. Moreover, the higher number of common pathways in both exosomal and non-exosomal fraction may indicate that, miRNAs associated with either exosomes or Ago2 has a complementary function in follicular microenvironment.