Relative expression of growth differentiation factor-9 (GDF9) during canine oocyte in vitro 3D culture

High rates of full in vitro maturation of canine oocytes are still far to be obtained. The intrinsic quality of female gametes deeply influences their competence to develop into a fertilizable oocyte and into an embryo; in this scenario the biological activity of several oocyte-secreted factors (OSFs) has a pivotal role. Amongst OSFs, the growth differentiation factor-9 (GDF9) was identified in dog ovaries [1], and the proprotein was immunolocalized in canine oocytes and cumulus cells during in vitro maturation in two-dimensional systems [2]. Data on RNA transcription of specific OSFs are not available and, in this study, the GDF9 relative expression was analyzed in canine oocytes matured in a three-dimensional (3D) culture condition (barium alginate microcapsules) at different time intervals by Real-Time PCR. Grade I COCs (n=332) were collected from ovaries (n=26; replicates=17) of domestic bitches after routine surgery. The oocytes were encapsulated in barium alginate microcapsules and in vitro matured in a controlled atmosphere. At different time intervals (0-24-48-72 hours), the oocytes were enzymatically denuded and the total RNA was isolated using the RNeasy Micro Kit. RNA was reverse transcribed to cDNA using QuantiTect® Reverse Transcription kit. A primer pair was designed for the amplification of canine GDF9 gene, and GAPDH was selected as normalizer gene using the primer pairs by [3]. Relative expression of GDF9 was assessed by Real-Time PCR amplification using SsoFastTM EvaGreen® Supermix on iQTM5 Multicolor Real-Time PCR Detection System. To check the specificity of the GDF9 analysis protocol, amplicons were sequenced and the obtained sequences were aligned to the expected target sequences using BLAST program. Data were analyzed by Mann–Whitney U test, and the level of significance was set at p<0.05. The results showed that after 72 h of IVM, the GDF9 relative expression was significantly lower (p=0.018) compared to that evaluated at 24 or 48 h. No differences were observed in the RNA quantification of oocytes matured for 24 or 48 h (p=0.1). This study demonstrated the gene expression of GDF9 in canine oocytes and the ability of 3D microcapsules to preserve the transcriptional activity of this relevant gene. A decrease of GDF9 relative expression, linked to oocyte full maturation in other species, occurred after 48 h in canine oocytes. This finding confirmed that an extended time for meiosis resumption is required by the oocytes of this species, as evidenced by previous studies on the evaluation of nuclear stage after culture. These results together with further evaluations of other OSFs could provide significant information on the intrinsic quality of female gametes. [1] Hashimoto O, Takagi R, Yanuma F et al. Identification and characterization of canine growth differentiation factor-9 and its splicing variant. Gene 2012; 499: 266–272. [2] De los Reyes M, Rojas C, Parraguez V et al. Expression of growth differentiation factor 9 (GDF-9) during in vitro maturation in canine oocytes. Theriogenology 2013; 80: 587–596. [3] Mortarino M, Gelain ME, Gioia G et al. ZAP-70 and Syk expression in canine lymphoid cells and preliminary results on leukaemia cases. Vet Immunol Immunopathol 2009; 128: 395–401.