| dc.description.abstract | To date, most assisted reproductive technologies in canid species remain poorly successful. This may be attributed to physiological differences in the bitch; mainly, the meiotic resumption of the oocyte in the oviduct, after being released in germinal vesicle stage from the ovary. This represents a major challenge for harvesting mature oocytes; thus, in vitro maturation (IVM) of canine oocytes is required. Recent studies have shown that co- culturing immature oocytes with oviduct epithelial cells (cOEC) increases the chances of oocyte meiotic resumption in vitro. However, the current protocols for isolating and culturing cOECs have failed to provide a favorable environment for IVM. In the present study we aimed to optimize the isolation of cOEC and culturing a monolayer system with Transwell® inserts. We hypothesized that the introduction of an air-liquid interface, in combination with the inhibition of the Notch pathway, would stir cOEC differentiation towards a morphology that more closely resembles the in vivo oviduct epithelium. We successfully managed to isolate cOECs from 15 bitches by enzymatic digestion and mechanic squeezing of the ex vivo oviducts obtained by elective ovariectomy in anestrus. Cells were directly seeded in Transwell inserts and cultured, first, for 7 days in a liquid-liquid interface, which allowed them to de-differentiate and form a confluent monolayer; then, 16 days in an air-liquid interface where they polarized into a columnar shape and re-differentiated into ciliated and non-ciliated cells. After, 10 more days of culturing in an air-liquid interface with the addition of DBZ, to further stimulate ciliation. Confluence of the monolayers was assessed by measuring the transepithelial electrical resistance.
By immunofluorescent staining, we found two main morphological categories of monolayers, low and high cell- density monolayers. The former, characterized by the presence of flat, irregular-shaped, unpolarized cells. The latter, resembling the desired morphology the most, with polarized columnar cells; nevertheless, the presence of ciliated cells after introducing the air-liquid interface, remained below 20%.
Treatment with DBZ only enhanced ciliation significantly in monolayers containing >15% ciliated cells, preceding the treatment. To determine the effects of DBZ on ciliogenesis related transcription factors FOXJ1 and RFX1, and Notch primary target genes, we performed qRT-PCR assays on two high cell-density monolayer samples. Expression fold change profiles were mostly consistent with our theoretical hypothesis: upregulation in transcription factors FOXJ1 and RFX1 and a downregulation in target genes HES1 and HEY1.
Overall, high cell-density monolayers resembled the in vivo morphology the most. However, functionality and long-term sustainability of the model remain unknown. This study provides a good foundation for further optimization of the cOEC monolayer Transwell® model. | |
