Nuclear competence and genetic expression of growth differentiation factor-9 (GDF-9) of canine oocytes in 3D culture.

Abstract

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 72hr, 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 72hr of IVM, the GDF-9 transcription significantly dropped (p=0.018) compared to 24 and 48hr. 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.