A short-term suspension culture system for bovine oviduct epithelial cells suitable for the study of embryo-maternal communication processes

摘要

A short-term (24 h) culture system for bovine oviduct epithelial cells (BOEC) suitable for co-culture experiments with embryos was established and evaluated. BOEC were obtained on Day 3.5 of the estrous cycle and processed by mechanical means only to obtain cell aggregates. Cell yields were 10-fold higher as described in former studies employing enzymatic treatment to achieve a single cell suspension for seeding to obtain a BOEC monolayer.Light microscopic examinations showed vigorously beating cilia on the apical side of the BOEC in aggregates and a rapid and constant motion of cell aggregates due to active ciliary beat. Scanning electron microscopy and transmission electron microscopy confirmed ultrastructural characteristics of BOEC at seeding and after 24 h in culture very similar to the situation in vivo. Both secretory cells with numerous secretory granules and ciliated cells with long, well-developed kinocilia were visible. The purity of the epithelial cell culture was > 95 %, as assessed by immunocytochemical methods.For further characterization of cultured BOEC, gene expression patterns were examined after different time spans in culture. Cultured BOEC isolated from ampullae ipsilateral to the ovulation site yielded significantly higher amounts of RNA than their contralateral counterparts (2.73 ± 0.98 versus 2.31 ± 0.14 µg per 10^6 cells). However, quantitative PCR did not detect significant differences in transcript levels between ipsi- and contralateral BOEC for the majority of marker genes (ESR1, ESR2, HMGCR, OVGP1, PGR, TRA1) throughout the 24 h culture period. The analysis of combined data obtained from different sampling time points during the culture period revealed an effect only for GPX4 (B. taurus non-selenium glutathione phospholipid hydroperoxide peroxidase), a gene known to be differentially expressed in vivo. Marker gene expression of five genes remained stable after 6h of cell culture, indicating only a short adaptation period of cultured cells. The use of two different sera (estrous cow serum versus cow serum obtained on Day 3.5 of the estrous cycle) in a concentration of 2 % did not affect gene expression patterns.Western blot analysis confirmed ESR1 (estrogen receptor α) and PGR (progesterone receptor) protein expression throughout the culture period. In agreement with cyclic differences in vivo, stimulation with 10 pg/ml estradiol-17β increased PGR transcript abundance in BOEC significantly. A response to the stimulation with 10 ng/ml progesterone was shown as INOS (inducible nitric oxide synthase) gene expression increased significantly after steroid treatment. Thus, the developed culture system provides functional BOEC with an unchanged morphology and maintained functionality as compared with cells in vivo. The system is rapidly available for use in co-culture experiments with bovine embryos and provides cultured cells in sufficient quantities for holistic transcriptome and proteome studies, thereby helping to decipher early embryo-maternal communication.