Inhibition of germinal vesicle breakdown using IBMX increases microRNA-21 in the porcine oocyte

Abstract

Background

Germinal vesicle breakdown (GVBD) occurs during oocyte meiotic maturation, a period when transcriptional processes are virtually inactive. Thus, the maturing oocyte is reliant on processes such as post-transcriptional gene regulation (PTGR) to regulate the mRNA and protein repertoire. MicroRNA (miRNA) are a class of functional small RNA that target mRNA to affect their abundance and translational efficiency. Of particular importance is miRNA-21 (MIR21) due to its role in regulating programmed cell death 4 (PDCD4). The objective of this study was to characterize the abundance and regulation of MIR21 in relation to GVBD.

Methods

Oocytes were collected from aspirated porcine tertiary follicles. Relative abundance of mature MIR21 was quantified at 0, 8, 16, 24, 32, and 42 h of in vitro (IVM) with or without treatment with 3-isobutyl-1-methylxanthine (IBMX).

Results

IBMX increased abundance of MIR21 at 24 h approximately 30-fold compared to control oocytes (P < 0.05), and the induced increase in MIR21 abundance at 24 h was concomitant with premature depletion of PDCD4 protein abundance. To characterize the effect of artificially increasing MIR21 on oocyte competence without inhibiting GVBD, a MIR21 mimic, scrambled microRNA negative control, or nuclease free water was micro-injected into denuded oocytes at 21 h of IVM. The maturation rate of oocytes injected with synthetic MIR21 (63.0 ± 7.5%) was higher than oocytes injected with negative controls (P < 0.05).

Conclusions

Inhibition of nuclear meiotic maturation via IBMX significantly increased MIR21 and decreased its target, PDCD4. Injection of a MIR21 mimic increased oocyte maturation rate. Our results indicate MIR21 is active and important during meiotic maturation of the oocyte.