Lange-Consiglio Anna, Perrini Claudia, Albini Giulia, Modina Silvia, Lodde Valentina, Orsini Eleonora, Esposti Paola, Cremonesi Fausto
Reproduction UnitCentro Clinico-Veterinario e Zootecnico-Sperimentale di Ateneo, Università degli Studi di Milano, Lodi, Italy
Department of Veterinary MedicineUniversità Degli Studi di Milano, Milano, Italy.
Reproduction. 2017 Aug;154(2):167-180. doi: 10.1530/REP-17-0117.
The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 and measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO and 5% of O in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 μg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25-50-75-100-150 × 10 MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect ( < 0.05) on maturation rate (MII) at the concentration of 75 and 100 × 10 MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 × 10 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible use could facilitate the exploitment of canine reproductive biotechnologies.
研究了输卵管细胞多细胞球体分泌的条件培养基（CM）或微泡（MVs）的作用以及一些微小RNA（miRNAs）在犬卵母细胞成熟过程中的参与情况。为了制备CM，将球体培养3天。通过在100,000下对CM进行超速离心获得MVs，并用NanoSight仪器测量其大小和浓度。卵丘-卵母细胞复合体（COCs）在双相系统中于38.5°C、含5% CO₂和5% O₂的合成输卵管液（SOF）中成熟：先在含5.0μg/mL促黄体生成素（LH）的条件下培养24小时，然后在含10%发情母犬血清的条件下再培养48小时。将SOF用作对照（CTR），或补充10% CM或用PKH-26标记的25 - 50 - 75 - 100 - 150×10⁶ MVs/mL。结果表明，多细胞聚集体分泌脱落囊泡。通过荧光显微镜观察，仅在72小时时可见标记的MVs进入卵母细胞细胞质。与CM和CTR相比，这些MVs在浓度为75和100×10⁶ MVs/mL时对成熟率（MII）有积极影响（P < 0.05）（分别为20.34%和21.82%，而CM和CTR分别为9.09%和8.66%）。150×10⁶ MVs/mL的浓度仅提供了9.26%的MII。研究了三种特定miRNA（miR-30b、miR-375和miR-503）的表达。MVs浓度较高时MII率较低可能是由于miR-375水平较高。总之，输卵管MVs可能参与卵母细胞成熟过程中的细胞运输，其可能的应用有助于犬类生殖生物技术的开发利用。
