Fachbereich Veterinärmedizin



    Characterization of microRNA (miRNA) profiles in selected female reproductive tissues of cattle: prediction of fertility associated pathways (2016)

    Palma Vera, Sergio Eliseo (WE 3)
    Berlin, 2016 — IV, 49 Seiten
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000104293
    Institut für Veterinär-Biochemie

    Oertzenweg 19 b
    14163 Berlin
    Tel.+49 30 838 62225 Fax.+49 30 838-62584

    Abstract / Zusammenfassung

    MicroRNAs (miRNAs) are small non coding RNAs, able to bind to mRNA and silence gene expression. They have been described to have roles in physiological, as well as in pathological reproductive processes. Reproductive performance in cattle has been steadily decreasing over the last decades. The reasons for this have not been completely defined, but poor embryo development and implantation are among the main causes. Both processes depend on embryo interactions with the maternal oviduct and endometrium. Therefore, the aim of this study was to characterize the role of miRNAs in these reproductive tissues and to predict their function in the context of fertility.

    First, a bovine oviductal epithelial cell culture (BOEC) system was developed. The purpose of this was to establish an in vitro model that can resemble the oviductal epithelium. To do this, an air liquid interphase system was implemented and cells were grown for up to six weeks. As a result, cells exhibited polarization and markers of differentiation. However, ciliated cells were not observed. Insulin, a common supplement used to culture BOECs, was detected to significantly impact cell morphology, improving the quality of cultures. Additionally, oviductal miRNAs were characterized in native tissue and then validated in the established air-liquid interphase BOEC system. Among the RNAseq-detected oviductal miRNAs, let-7a-5p and let-7c were found to be regulated by insulin in BOECs. Their targets were mapped to MAPK signaling pathway, a downstream mechanism in the insulin signaling, controlling cell proliferation and differentiation.

    Secondly, miRNAs were characterized in endometrial cells. Estrous cycle was divided into four stages: post-ovulatory, early luteal, late luteal and pre-ovulatory phases. RNA samples of four animals were pooled and the miRNA expression pattern analyzed via RNAseq. Results demonstrated a high similarity in miRNA expression profiles across the estrous cycles in bovine endometrium. These miRNAs were predicted to regulate pathways involved in cell proliferation, differentiation, transport and catabolism, among them, MAPK signaling pathway. To test for functional regulations in vitro, endometrial miRNAs were analyzed using a commercial bovine endometrial cell line (BEND), where activation of MAPK signaling pathway can be counter-regulated by interferon-tau (IFNT), mimicking the process of embryo implantation. As result, miR-106a responded to IFNT alone and in combination with progesterone, a strong indicator of the relevance of this miRNA for embryo implantation.

    In summary, the present study characterized miRNAs expressed in key reproductive tissues for embryo development. Upon validation using in vitro systems, regulated miRNAs were detected. In BOECs let-7a-5p and let-7c were regulated by insulin and in BEND cells, miR-106a was regulated by IFNT. Interestingly, in both situations, MAPK signaling pathway plays a central role. Future studies will define the role of miRNAs in the control of oviductal and endometrial function through MAPK signaling pathway.