Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

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Recent progress in high throughput sequencing and bioinformatic analysis and other biochemical methods have fuelled our appreciation for the important role of microRNAs (miRNAs) in disease, fertility and development. These tiny RNAs were found to be potentially involved in various aspects of cellular processes of reproductive tissues by posttranscriptional regulation of protein coding genes. Mammalian gonads which exhibit strictly regulated spatiotemporal gene expression patterns are also known to express unique sets of miRNAs and genes involved in the miRNA biogenetic pathway. Studies on miRNAs and their associated processing enzymes have evidenced the contribution of these small regulatory RNAs to germ cell differentiation, post-meiotic male germ cell function and growth, and development and maturation of oocytes through pertaining tightly regulated gene expression. The existence, preferential and temporal expression of miRNAs and their processing machinery genes in different stages of testicular and ovarian cellular development have evidenced the potential role of miRNAs in testicular and ovarian physiology. MiRNAs are also found to be associated with functional regulation of gonadal somatic cells, namely Leydig cells and Sertoli cells in testis and granulosa cells/cumulus cells in the ovary in steroid synthesis. Here, we review the recent works on the involvement and diverse roles of miRNAs in the development and physiology of gonadal cells in mammalian reproduction.

BackgroundDespite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract.MethodTwelve simmental heifers were estrous synchronized and six of them were hyperstimulated using FSH. Follicular fluid samples from experimental animals were collected using ovum pick up technique at day 0 of the estrous cycle and blood samples were collected at day 0, 3 and 7 of post ovulation. The expression profile of circulatory miRNAs in follicular fluid and blood plasma were performed using the human miRCURY LNA™ Universal RT miRNA PCR array system. A comparative threshold cycle method was used to determine the relative abundance of the miRNAs.ResultsA total of 504 and 402 miRNAs were detected in both bovine follicular fluid and blood plasma, respectively. Of these 57 and 21 miRNAs were found to be differentially expressed in follicular fluid and blood plasma, respectively derived from hyperstimulated versus unstimulated heifers. Bioinformatics analysis of those circulating miRNAs indicated that their potential target genes are involved in several pathways including TGF-beta signaling pathway, MAPK signaling pathway, pathways in cancer and Oocyte meiosis.Moreover, detail analysis of the mode of circulation of some candidates showed that most of the miRNA were found to be detected in both exosomal and Ago2 protein complex fraction of both follicular fluid and blood plasma.ConclusionOur data provide the consequence of hyperstimulation induced changes of extracellular miRNAs in bovine follicular fluid and blood plasma, which may have a potential role in regulating genes associated not only with bovine ovarian function but also involved in altering various physiological in bovine oocytes, embryos and modulating reproductive tract environment.

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Since the first evidence for the involvement of microRNAs (miRNAs) in various reproductive processes through conditional knockout of DICER, several studies have been conducted to investigate the expression pattern and role of miRNAs in ovarian follicular development, oocyte maturation, embryo development, embryo-maternal communication, pregnancy establishment and various reproductive diseases. Although advances in sequencing technology have fuelled miRNA studies in mammalian species, the presence of extracellular miRNAs in various biological fluids, including follicular fluid, blood plasma, urine and milk among others, has opened a new door in miRNA research for their use as diagnostic markers. This review presents data related to the identification and expression analysis of cellular miRNA in mammalian female fertility associated with ovarian folliculogenesis, oocyte maturation, preimplantation embryo development and embryo implantation. In addition, the relevance of miRNAs to female reproductive disorders, including polycystic ovary syndrome (PCOS), endometritis and abnormal pregnancies, is discussed for various mammalian species. Most importantly, the mechanism of release and the role of extracellular miRNAs in cell-cell communication and their potential role as non-invasive markers in female fertility are discussed in detail. Understanding this layer of regulation in female reproduction processes will pave the way to understanding the genetic regulation of female fertility in mammalian species.

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