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 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.

One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation

“…In addition, our data showed unvaried expression of the OOEP transcript variants, OOEP1 and OOEP2. Our data on effects of 3D IVM on gene expression are in agreement with those by Morselli et al [34] who reported down-regulation of the growth differentiation factor-9 (GDF-9), another gene involved in oocyte developmental competence, in canine oocytes after 3D IVM.…”

“…Dorati et al [ 15 ] described alginate microbead preparation by the diffusion setting gelation technique in which, using a mouth-controlled hand-pulled glass Pasteur micro-pipette, single COCs were transferred into the alginate solution in culture medium and dropped into single wells of a 96-well plate containing the cross-linking agent. Morselli et al [ 32 ] developed a two-step technique in which barium alginate microcapsules were produced and subsequently injected feline and canine COCs into the inner core of the microcapsules [ 32 – 34 , 36 ]. Faustini et al [ 35 ] described a protocol for bioencapsulation of COCs and granulosa cells in which a suspension of granulosa cells in cross-linking solution was dropped in an alginate solution.…”

“…biomaterial has also been reported for oocyte IVM [15,[32][33][34][35][36]. The clinical interest in IVM is constantly urged by the World Health Organization since infertility affects about 15% of couples [15].…”

“…COC-microbeads and assessing its validity on oocyte nuclear and cytoplasmic maturation. Indeed, most of the studies published to date described two-step or manual procedures [32][33][34][35][36]. Dorati et al [15] described alginate microbead preparation by the diffusion setting gelation technique in which, using a mouth-controlled hand-pulled glass Pasteur micro-pipette, single COCs were transferred into the alginate solution in culture medium and dropped into single wells of a 96-well plate containing the cross-linking agent.…”

“…Among biocompatible materials, alginate hydrogels have been successfully used for culturing and studying different cell types [17] and have been widely employed in long-term in vitro culture of ovarian follicles [18][19][20][21][22][23][24][25][26][27][28][29][30][31]. In recent years, IVM culture of COCs in 3D alginate microenvironments has been reported in various animal models with encouraging results [18,[32][33][34][35][36]. Methods for fabricating microbeads containing a COC, namely "COC-microbeads", need to be chosen according to their ability to control microbead shape and dimensions and generate reproducible 3D IVMs.…”