A novel 3-D culture system to study bovine oviduct physiology, gamete interaction and early embryo development

Abstract

Successful fertilization depends on processes that take place in the oviduct. Due to its intra-abdominal location, it is difficult to study intra-oviductal processes in vivo in mammals. Instead, in vitro models that retain essential cell morphological and functional characteristics are being developed. In culture, bovine oviduct epithelial cells (BOECs) rapidly lose differentiated cell properties (e.g. secretory activity and cilia), while suspended cells have a limited lifespan. Progress with insert culture models and 3-D printing technologies prompted us to develop two independent BOEC culture systems, in which in vivo-like differentiation and function is re-established, to study oviduct physiology: (i) 3-D printed U-shaped inserts mounted with PET membranes with 0.4 µm pores (3D Ushaped culture) and (ii) hanging inserts (polycarbonate with 0.4 µm pores) containing 150 µL of Matrigel (3D culture). BOECs were harvested by scraping, and cultured for 24h to agglomerate into floating vesicles with outwardly oriented cilia. The vesicles were plated and, 7 days later, the resulting monolayers were scraped, washed and seeded onto the 2 systems described above and cultured at an air-liquid interface. For comparison, BOECs were also seeded onto coverslips as monolayers (2D culture). After 28 days, the apical side of all BOEC monolayers was washed to harvest secreted proteins, and the inserts were fixed for immunocytochemistry. Proteins (20 µg) were separated by SDS-PAGE and visualized by silver staining, or blotted onto nitrocellulose and immunostained for oviduct specific glycoprotein (OVGP1). Epithelial cell differentiation was indicated by immunodetection of laminin and the presence of primary cilia. Ciliated cell presence (acetylated α-tubulin) and secretory activity (OVGP1) characteristics of BOECs in 3D cultures were comparable to freshly harvested BOECs. The 3D culture yielded 46 silver-stainable protein bands versus 30 in 2D cultures (n=3 per system). In 3D U-shaped cultures, the polarized state (laminin and primary cilia) and their amenability to direct fluorescence microscopy (allowing live cell imaging) are currently determined. In conclusion, 3D culture methods promote polarization and differentiation of BOECs. The extent to which physiological function is maintained is under investigation. Studies in progress to assess the BOEC differentiation using the 3D U-shaped cultures include basolateral co-culture of stromal cells. Ultimately, we aim to develop an oviduct-like environment to study gamete activation, fertilization and early embryo development in situ.