329 A NOVEL SUSPENSION CULTURE SYSTEM FOR BOVINE OVIDUCT EPITHELIAL CELLS

Abstract

Bovine oviduct epithelial cells (BOEC) are difficult to culture without dedifferentiation. Frequently observed changes in morphology during culture suggest that gene expression patterns are affected as well. We explored a novel short-term culture system for BOEC – suitable for co-culture experiments with embryos – and evaluated the cells with respect to morphological criteria, candidate gene expression, and hormone responsiveness. Simmental heifers were slaughtered on Day 3.5 after standing heat and BOEC were obtained by squeezing along the ampulla with forceps. The cell sheets were separated mechanically by repeated passages through syringes and pipetting, and recovered by sedimentation. Cells from the ipsi- and contralateral oviduct were cultured separately at a density of 106 cells per well in 24-well plates with 800 μL TCM-199 supplemented with 2% OCS (estrous cow serum, as used in embryo culture) or CS 3.5 (cow serum, Day 3.5 after standing heat, adequate to the cycle stage in which cells were obtained) and 0.25 mg/mL gentamicin. For cell counting, an aliquot was further disaggregated by passing 15 times through a 30-gauge needle to achieve a single-cell suspension. Culture took place at 38°C in a humidified atmosphere of 5% CO2 in air. Cells were examined by light microscopy at seeding and after 6, 12, 24, and 48 h. Cell aggregates showed a worm-like structure, displaying numerous vigorously beating cilia on their surfaces throughout the culture period. Trypan blue staining indicated that cells contained in aggregates were viable while single cells stained predominantly positive (non-viable). The purity of the epithelial cell culture was >95%, as determined by immunohistochemistry using antibodies against vimentin and cytokeratin. For electron microscopic investigations, BOEC were sampled at seeding and after 24 h in culture. Cultured BOEC showed a morphology highly similar to that of BOEC in vivo. Both secretory cells with numerous secretory granules and mitochondria, and ciliated cells with long, well developed and actively moving kinocilia were visible. RT-PCR data for candidate genes (ERα, ERβ, HMGCR, PHGPx, PR) obtained from BOEC samples at seeding and after 6, 12, 24, and 48 h in culture showed that gene expression was stable for the majority of transcripts after 6 h in culture. There was no significant difference between cells cultured with OCS or CS 3.5 and no difference between cells obtained from the ipsi- or contralateral oviduct. Estradiol-17β (E2, 10 pg/mL) or progesterone (P4, 10 ng/mL) stimulation showed that the cultured BOEC are able to respond to hormonal signals in a manner similar to their reaction in vivo (Ulbrich et al. 2003 J. Steroid. Biochem. Mol. Biol. 84, 279–89). Progesterone receptor mRNA was up-regulated by E2 and estrogen receptor β mRNA was up-regulated by P4. The culture system for bovine oviduct epithelial cells thus provides an adequate tool to investigate mechanisms of the embryo-maternal communication in cattle. This work was supported by the Deutsche Forschungsgemeinschaft (Research Unit “Mechanisms of Embryo-Maternal Communication”; FOR 478/1).