90 CHARACTERIZATION OF A PRIMARY CULTURE OF OVIDUCTAL CELLS IN THE BITCH

Abstract

The oviduct is of particular importance in canine reproduction as it supports oocyte meiosis resumption, sperm capacitation and storage, fertilization and embryo development to the morula/blastocyst stage for 8 to 10 days post-ovulation. A long-time co-culture with oviducal cells could be employed to improve the yield of reproductive biotechnologies in this species, but no characterisation of canine oviduct cells in vitro has been reported to date. The objectives of this study were to (1) evaluate the viability and proportion of epithelial/fibroblast cells in a primary culture of canine oviducal cells collected around ovulation; (2) study the responsiveness of the cultured cells to steroids. Beagle bitches (n = 9) were ovariectomized between Day –1 and Day +1 around ovulation, and their oviducts were sectioned at the ampulla-isthmus junction. Mucosal cells (including stromal and epithelial cells) were collected by squeezing from the ampulla and isthmus sections and cultured separately at a concentration of 5 × 10⁵ cells/well in 500 μL of M199 + 10% FCS at 39°C for 9 days. At Days 3 and 6, 1 × 10⁶ cells were stimulated with 17β-oestradiol (E2, 20 pg mL^–1) or progesterone (P4, 20 ng mL^–1) for 6 h. At Days 3, 6, and 9 of culture, the viability of the cells was evaluated using the Live/Dead kit (Invitrogen, Carlsbad, CA, USA), and proportions of fibroblast and epithelial ciliated cells were evaluated by immuno-cytochemistry using anti-vimentin and anti-tubulin antibodies, respectively. At Days 0, 3 and 6, the total RNA was extracted from cells and mRNA levels of the oviduct-specific glycoprotein (OVGP, synthesised by nonciliated epithelial cells), E2 (ERα, ERβ) and P4 (PR) receptors were evaluated by RT-qPCR. The effects of the day of culture and of steroid exposure on mRNA levels were analysed by ANOVA followed by a Tukey test. Cell confluence was observed around Day 6 of culture and more than 90% of cells survived during the 9-day culture period. From Day 3 to Day 9, the proportion of vimentin-positive (fibroblast) cells was greater than 68% in both ampulla and isthmus cells. In contrast, the proportion of epithelial ciliated cells was low at Day 3 (9% in ampulla, 12% in isthmus) and null at Days 6 and 9 in both regions. The mRNA levels of OVGP, ER, and PR decreased significantly after 3 days of culture, and then remained stable in both ampulla and isthmus cells (P < 0.001). The steroid exposure had no effect on gene expression, except for ERα mRNA levels at Day 3, which was increased by E2 and reduced by P4 (P < 0.05). In conclusion, the method of collection did not allow us to collect a high proportion of epithelial oviducal cells. However, the relatively stable gene expression of PR and ER during the culture period provides us with a useful tool to study the steroid regulation of canine oviduct mucosal cell functions.