Morselli Maria Giorgia, Loiacono Monica, Colombo Martina, Mortarino Michele, Luvoni Gaia Cecilia
Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Università degli Studi di Milano, Milan, Italy.
Dipartimento di Medicina Veterinaria, Università degli Studi di Milano, Milan, Italy.
Reprod Domest Anim. 2018 Nov;53 Suppl 3:117-124. doi: 10.1111/rda.13336.
To evaluate the ability of a 3D culture system in improving the nuclear and molecular competence of canine oocytes, barium alginate microcapsules were used for in vitro maturation (IVM) and the expression profile of one selected oocyte-secreted factor, the growth differentiation factor-9 (GDF-9) was analysed. In Experiment I, canine grade I cumulus-oocyte complexes (COCs) were in vitro matured in 3D microcapsules in a controlled atmosphere for 72 hr, and meiosis resumption rates were compared to those of oocytes cultured in traditional 2D microdrops of medium. In Experiment II, a primer pair specific for canine GDF-9 was designed, and preliminary tested in conventional PCR on genomic DNA. Total RNA content was isolated from oocytes at different time intervals (T0-T24-T48-T72) during in vitro 3D culture, and a reverse transcription to cDNA was performed. The expression of target gene was assessed by quantitative Reverse Transcription Real-Time PCR (qRT-PCR), and the obtained amplicons were sequenced to check the specificity of the analysis. Canine COCs resumed meiosis at higher rates in 3D microcapsules than in 2D microdrops (p < 0.05), even though no significant differences in the proportions of oocytes achieving full maturational stages were obtained. A significant dynamic decrease in GDF-9 expression was recorded during culture: after 72 hr of IVM, the GDF-9 transcription significantly dropped (p = 0.018) compared to 24 and 48 hr. In conclusion, in vitro 3D culture represents an efficient system for IVM of canine oocytes, and the expression profile of GDF-9 well reflects temporal dynamics for the acquisition of developmental competence in this species.
为评估三维培养系统提高犬卵母细胞核及分子水平成熟能力的效果，采用海藻酸钡微胶囊进行体外成熟培养（IVM），并分析了一种选定的卵母细胞分泌因子——生长分化因子9（GDF-9）的表达谱。在实验I中，将犬I级卵丘-卵母细胞复合体（COCs）在三维微胶囊中于可控环境下进行体外成熟培养72小时，并将减数分裂恢复率与在传统二维培养基微滴中培养的卵母细胞进行比较。在实验II中，设计了一对针对犬GDF-9的引物对，并在常规PCR中对基因组DNA进行了初步检测。在体外三维培养的不同时间间隔（T₀-T₂₄-T₄₈-T₇₂）从卵母细胞中分离总RNA含量，并进行逆转录生成cDNA。通过定量逆转录实时PCR（qRT-PCR）评估靶基因的表达，并对获得的扩增子进行测序以检查分析的特异性。犬COCs在三维微胶囊中的减数分裂恢复率高于二维微滴（p < 0.05），尽管在达到完全成熟阶段的卵母细胞比例上未获得显著差异。在培养过程中记录到GDF-9表达显著动态下降：体外成熟培养72小时后，与24小时和�小时相比，GDF-9转录显著下降（p = 0.018）。总之，体外三维培养是犬卵母细胞体外成熟的有效系统，GDF-9的表达谱很好地反映了该物种获得发育能力的时间动态。
