269 RELATIVE TRANSCRIPT ABUNDANCE OF EIGHT MARKER GENES IN OOCYTES OF PREPUBERTAL AND CYCLIC GILTS

Abstract

Oocytes from prepubertal (P) females usually show impaired quality when compared with those of adult animals. Recently, we have shown that although P oocytes mature in vitro at similar rates as oocytes from cyclic (C) gilts, they are more often chromosomally unbalanced. We have also reported some impaired processes in cytoplasmic maturation of the P oocytes. This was especially evident in cases of a disturbed mitochondria redistribution and more frequent apoptosis. It is therefore hypothesised that incomplete cytoplasmic maturation is responsible for the decreased developmental competence of porcine oocytes. A factor reflecting the status of cytoplasmic maturation is the relative transcript abundance (RA) of developmentally important genes. We investigated the RA of 8 genes known as markers of oocyte quality. Ovaries of unknown origin were collected from P and C gilts in a local slaughterhouse. Cumulus–oocyte complexes (COC) were aspirated from 2- to 6-mm follicles and evaluated morphologically. Pools of 25 denuded oocytes with a proper morphology were frozen in liquid nitrogen (pre- and post-IVM). Altogether, 4 groups of oocytes were analysed: P pre-in vitro maturation (IVM), P post-IVM, C pre-IVM, and C post-IVM. A two-step IVM protocol was performed in NCSU23 medium for 44 h. Transcript analysis included total RNA isolation followed by reverse transcription and real-time PCR using fluorescence resonance energy transfer probes for 8 genes (BMP15, GDF9, ZAR1, ATP5A1, EEF1A1, GSTA2, TOP2B, SMARCA4). Each of the four groups was represented by 6 independent oocyte pools (6 × 25 oocytes × 4 groups = 600 oocytes), which were analysed in duplicate. Among the pre-IVM oocytes, no significant differences in RA were observed. However, the post-IVM oocytes of C gilts were characterised by higher RA of 3 marker genes [BMP15 (P < 0.05), GDF9 (P < 0.01), ZAR1 (P < 0.05)]. The RA of the GSTA2 and ATP5A1 genes showed a tendency to be higher in C oocytes, whereas the RA of the EEF1A1, TOP2B, and SMARCA4 genes did not differ between P and C gilts. Although the reduced quality of P oocytes has been well documented, only 10% of genes were differentially expressed in oocytes of P and C gilts (Paczkowski et al. 2011). In this study, we demonstrated that the sexual maturity of the donor gilt affected transcript abundance of 3 out of 8 developmentally important genes. Our results corroborate the previous findings and extend them to new transcripts (GDF9, BMP15, ZAR1). We did not show differences in the RA of genes regulating energy production and transcription (ATP5A1, EEF1A1). Some genes, such as TOP2B (apoptosis control) and SMARCA4 (a chromatin remodelling factor), were expressed at similar levels. We suggest that the RA of the ZAR1, BMP15, and GDF9 genes may serve as markers of the reduced quality of oocytes from P gilts and, because of their function, might alter cytoplasmic maturation.

Funding was from the Polish Ministry of Science and Higher Education, COST GEMINI FA0702-DWM/N190/COST/2008.