Abstract
BackgroundOocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach.MethodsFor this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture.ResultsThe luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial activity, while inhibition of miR-130b during preimplantation embryo development significantly reduced morula and blastocyst formation.ConclusionThis study demonstrated that in vitro functional modulation of miR-130b affected granulosa and cumulus cell proliferation and survival, oocyte maturation, morula and blastocyst formation suggesting that miR-130b is involved in bovine oocyte maturation and preimplantation embryo development.
Highlights
Oocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs
The expression of miR-130b was relatively higher in granulosa cells (GC) compared to the cumulus cells surrounded the immature (IMCC) or cumulus cells surrounded the mature oocytes (MCC) (Fig. 1b)
The signal transduction pathways regulated by miR-130b during the oocyte maturation is not clear: when we look into the expression of pattern of the target genes, the expression level of both SMAD5 and MSK1 were significantly increased in oocytes with lower maturation rate suggesting that miR-130b could be involved in oocyte maturation by fine-tuning the SMAD5 and MSK1 genes
Summary
Oocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. The growth and development of the oocyte is influenced by the functional activities of the surrounding somatic cells (cumulus or granulosa and theca cells) These cells regulate various kinds of hormones, proteins, metabolites and regulatory molecules via gap junctions, leading to the development and maturation of the oocyte [3]. Identification and functional characterization of the miRNAs in oocyte maturation and preimplantation embryo development have been the focus of research interest On this regard, Byrne and his colleague [20] showed the involvement of miRNA in the regulation of genes associated with early embryonic development in mice and others reported embryonic lethality in mice due to functional loss of miR-290–295 cluster [21]. The appearance and disappearance of specific sets of miRNAs during embryonic development in various animal species have been described in many instances [18, 19, 22,23,24,25]
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