Factors affecting proliferation and dedifferentiation of primary bovine oviduct epithelial cells in vitro

Abstract

The oviduct is the physiological site for key events in reproduction, such as capacitation of spermatozoa, fertilization and early embryonic development. Interactions between oviduct epithelial cells and gametes or embryos cannot sufficiently be studied in vivo. Therefore, model systems are needed which mimic in vivo conditions most closely. In this study we optimised the method for isolating bovine oviduct cells and compared different cell support materials as well as two culture systems (perfusion vs static culture) for their ability to maintain characteristic morphological and functional features of oviduct cells. Out of nine different cell support materials tested, cellulose nitrate (0.45 micron pore size) was the most suitable to maintain cells in a manner similar to freshly isolated oviduct epithelial cells. Comparing static vs perfusion culture by electron microscopy, morphological differences of the cells were insignificant in the first days of culture, while they became more evident after 8 days. The cells in the static system lost typical characteristics such as columnar shape, cilia and secretory protrusions, while these features were still present in perfusion culture. In addition, intense ciliogenesis and cytoplasmic organelles for protein synthesis were found under perfusion conditions. These findings were underlined by differences in expression of the oviduct-specific oestrus-associated glycoprotein 85-97 kDa (GP 85-97) gene as revealed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The RNA levels of this specific gene were significantly higher in perfusion compared to the static culture system. Our data show clear advantages of perfusion vs static culture for primary bovine oviduct epithelial cells.

Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle

“…The complete organs were rinsed with PBS. After opening the oviduct longitudinally, epithelial cells were isolated by scraping the mucosal epithelial layer with a sterile glass slide (Reischl et al 1999). Within 10 min after death cells were transferred into cryotubes and immediately dropped into liquid nitrogen for transport, and were stored at 80 C until further processing.…”

A bovine oviduct epithelial cell suspension culture system suitable for studying embryo–maternal interactions: morphological and functional characterization

“…BOECs have been isolated by enzymatic, mechanical, or mixed enzymatic/mechanical procedures (Abe & Hoshi 1997, Reischl et al 1999, but cell yield per oviduct has only been assessed for procedures involving trypsinization, and were found to be rather limited (Thibodeaux et al 1991). BOECs have been cultured as monolayers or in suspension.…”

“…These include reduction of cell height, loss of cilia, and loss of secretory granules and bulbous protrusions (Thibodeaux et al 1992, Walter 1995. Perfusion culture of BOECs grown on cellulose-nitrate sheets sustained morphology, including ciliated cells in circumscribed regions, more faithfully, but nevertheless, the cells showed signs of dedifferentiation (Reischl et al 1999). In a direct comparison between BOECs grown in monolayer or in suspension culture, only suspended cells maintained cilia and secretory activity (Walter 1995).…”

“…In a direct comparison between BOECs grown in monolayer or in suspension culture, only suspended cells maintained cilia and secretory activity (Walter 1995). Semi-quantitative expression analysis of OVGP1, encoding the oviduct-specific glycoprotein 1, was used for quality assessment of cultured BOECs (Reischl et al 1999). We have developed a short-term 24 h culture system for BOECs isolated on day 3.5 of the estrous cycle as a tool for studying embryo-maternal interactions in the oviduct.…”

Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system

“…Factors influencing reproductive aging and the development of fertilized eggs were screened with anterior pituitary gland cells (Zheng et al 2007), oviduct epithelium (Reischl et al 1999) and endometrial cells (Tiemann et al 2005). New protocols for an optimal matrix coating and adaptation to continuous medium flow were elaborated for hepatocytes (Fiegel et al 2004;Schumacher et al 2007;Du et al 2008;Xia et al 2009).…”