256 HOXB9 KNOCK-DOWN DURING OOCYTE MATURATION IN CATTLE

Abstract

HOXB9 is a transcription factor playing a major role in patterning the main body-axis of vertebrate embryos from the gastrulation stage. HOXB9 transcripts have been detected in cattle oocytes and early embryos (Paul et al. 2011 Mol. Reprod. Dev. 78, 436–449), but their role at those stages has remained unknown. The aim of the study was to inject small interfering RNA (siRNA) in immature oocytes to investigate HOXB9 functions during oocyte maturation and early development. Immature cow oocytes were injected with 7 pL of a HOXB9 siRNA mixture (Integrated DNA Technologies, Coralville, IA, USA) or with a negative control siRNA (SCR) or were not injected (control). To allow microinjection, the size of the cumulus was reduced by pipetting; however, the corona radiata was left intact. The efficiency of the knock-down was measured 24 h post-injection using RT-qPCR, and the results were normalized with an internal normalization factor corresponding to the mean expression of three housekeeping genes (Goossens et al. 2005 BMC Dev. Biol. 5, 27). The injection of HOXB9 siRNA induced a 60.7% reduction of HOXB9 relative expression in mature oocytes by comparison with the injection of SCR siRNA (Kruskall-Wallis; P < 0.05). The nuclear maturation rates, assessed 24 h post-injection after Hoechst staining, were not statistically different between the HOXB9 group (48.8%) and the SCR group (53.6%; chi-square; P > 0.05). After in vitro fertilization and culture (Paul et al. 2011 Mol. Reprod. Dev. 78, 436–449), the developmental rates of the HOXB9 and the SCR groups were not statistically different in terms of achieving the first cleavage (64.4% v. 57.0%, respectively) and 5- to 8-cell stages (25.1% v. 25.3%, respectively) at 48 h post-insemination and the blastocyst stage on Day 7 (IVF = Day 0; 22.0% v. 20.6%, respectively) and Day 8 (26.9% v. 23.1%, respectively; two-way ANOVA; P > 0.05). Differential staining of Day 8 blastocysts showed no difference between the HOXB9 and the SCR groups regarding the mean number of cells in the inner cell mass (53.9 v. 49.4, respectively), the trophectoderm (125.5 v. 104.3, respectively), or the total number of cells (two-way ANOVA; P > 0.05). However, the microinjection procedure impaired the developmental competence after fertilization of the injected oocytes (HOXB9 + SCR) compared with the non-injected ones, as shown by their lower ability to cleave (60.7% v. 72.1%, respectively), to reach the 5- to 8-cell stage (25.2% v. 35.1%, respectively) or the blastocyst stage on Day 7 (21.3% v. 29.8%, respectively) and Day 8 (25.0% v. 35.8%, respectively; two-way ANOVA; P < 0.05). The injection did not affect blastocyst cell numbers on Day 8 (two-way ANOVA; P > 0.05). In conclusion, microinjection of siRNA in immature oocytes is a valuable method to efficiently knock down the maternal messenger RNA during oocyte maturation. Indeed, HOXB9 knock-down in immature oocytes led to a significant and important reduction in the amount of transcript in mature oocytes. The absence of effect of the knock-down on nuclear maturation and embryo development could be explained by the persistence of the maternal proteins.

This work was funded by the Fonds National de la Recherche Scientifique (Belgium).