Fachbereich Veterinärmedizin



    In vitro mimicking of estrous cycle stages based on a comprehensive porcine oviduct epithelial cell culture model (2013)

    Chen, Shuai (WE 3)
    Berlin: Mensch und Buch Verlag, 2013 — 51 Seiten
    ISBN: 978-3-86387-385-1
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000095322
    Institut für Veterinär-Biochemie

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

    Abstract / Zusammenfassung

    The oviduct epithelium is an important reproductive venue where gamete transport, sperm capacitation, oocyte maturation, and early embryo development occur. To understand the fundamental molecular mechanisms of these events, as well as to provide a tool for reproductive toxicity testing, a standardized in vitro oviduct epithelium model is on request. Although oviduct epithelium is reported to be sensitive to changes in circulating steroid hormones during estrous cycle, cyclic cellular events, especially sperm-epithelium binding and cilia activity are not well understood yet. In vitro investigations are needed to reveal principle regulatory mechanisms of hormones on cellular functions.
    We firstly developed a validated, comprehensive culture model of POEC. After the systematical assessment of standard sera, 3T3 medium conditioning, culture duration, and effects of cryopreservation on cells from 25 donor animals, a dossier of validation on these parameters was provided. The results suggest that cells faithfully recapitulated the morphology, ultrastructure and functionality of oviduct epithelium in vivo. TEER measurement indicated that cells maintained tissue polarity and cell type specific tight junctions. Furthermore, the stable mucin secretion and consistent expression of functional markers revealed that cells maintained an in vivo-like, homeostatic status from 3w up to 6w.
    Based on the POEC model, we could simulate estrous stages by mimicking the endocrine profiles of E2 and P4 for physiological time period. Cells exhibited distinct changes in cellular height and cilia density, which directly proved that E2-domination (estrus) induces cell polarization and ciliogenesis, but P4-domination (diestrus) suppresses these effects. Changes in composition of cell population suggested that E2 and P4 affected the transformation of ciliated cells and secretory cells. The TEER measurement revealed the cyclic changes of oviductal electrophysiology. In addition, simulated diestrus decreased expression of hormonal receptors and other oviductal markers, while subsequent estrus up-regulated these genes. Moreover, increased sperm binding was observed in simulated estrus compared to simulated diestrus.
    Finally, we developed a novel approach, which could be routinely used to monitor the cilia activity. With the assistance of the commercial AndroVision™ software, we detected the typical movement patterns of apical fluid streams. Fixed flowing routes suggested the directivity of fluid stream along the epithelium lining. Driven by cilia, beads were travelling at high speed comparable to ex vivo studies. However, there was no difference in the transport speed during estrous simulation.
    To summarize, our model together with the present approach to monitor cilia activity is a promising tool for basic reproductive science, as well as for reproductive toxicity testing. We present the first in vitro simulation of estrus cycle stages on mammalian oviduct epithelial cells, and revealed roles of E2 and P4 on oviduct development and functionality.