Improvement of in vitro canine oocyte maturation by oviductal secretome

The in vitro maturation (IVM) of canine oocyte is problematic because it is difficult to reproduce the oviductal microenvironment, where the in vivo maturation occurs. Since cells are able to communicate with each other by paracrine action, oviductal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MVs), that represent a carrier for non-soluble molecules, including miRNAs. Aim of the present work was to investigate the effect of the addition of CM or MVs, secreted by oviductal cells, to the canine IVM medium. To generate CM, cells from oviducts of three animals in late estrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500g, and stored at –80°C. MVs were obtained by ultracentrifugation of CM at 100,000g for 1h at 4°C and measured for concentration and size by Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the estrous cycle. Cumulus–oocyte complexes (COCs) were released by slicing the ovarian cortex with a scalpel blade and only grade 1 COCs, (darkly granulated cytoplasm and surrounded by three or more compact cumulus cell layers), 110-120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24h in SOF with 5.0 μg/ml LH followed by 48h in SOF supplemented with 10% of estrous bitch serum and 10% CM or 50-75-100-150 x 106 MVs/ml labelled with PKH-26. Control (CTR) was the same medium without CM or MVs. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII) by Hoechst staining and the incorporation of MVs. Statistical analyses was performed by chi-squared test. Results show that canine oviductal cells secreted MVs of 234±23 nm in size underling that these MVs fall within the shedding vesicles category. The incorporation of labeled MVs occurred at first in cumulus cells, at 48h of maturation, and then, at 72h, in oocyte cytoplasm. These MVs had a positive effect on maturation rate (MII) at the concentration of 75 and 100 x 106 MVs/ml compared to CM and CTR (20.34% and 21.82% vs 9.09% and 3.95%, respectively). The concentration of 150 x 106 MVs/ml provided only 9.26% of MII. To understand the role of MVs, we assessed the expression of three miRNA (miRNA-30b, miR-375 e miR-503) that are involved in some key pathways (WNT, MAPK, ERbB and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375 that recent literature shows to suppress the TGFβ pathway leading to impaired oocyte maturation. In conclusion, the oviductal MVs, or specific miRNA, are involved in cellular trafficking during oocyte maturation and their possible use in vitro could facilitate the exploitment of canine reproductive biotechnologies