Effect of hyaluronan on developmental competence and quality of oocytes and obtained blastocysts from in vitro maturation of bovine oocytes

Abstract

The objective of the present study was to evaluate the effect of hyaluronan (HA) during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC), and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P < 0.001) was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P < 0.01). Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

Figure 1

Bovine oocyte after TUNEL staining. (a) DAPI staining of chromatin in metaphase II stage and the 1st polar body; (b) absence of green fluorescence after FITC staining of the same oocyte; (c) photos (a) and (b) merged.

Figure 2

Bovine blastocyst after TUNEL staining. (a) DAPI staining of all blastomere nuclei; (b) green FITC staining of apoptotic nuclei; (c) photos (a) and (b) merged.

Figure 3

Relative mRNA expressions (mean ± SD) for Bax in granulosa cells which served as coculture during IVM of oocytes cultured with addition of 0.035% HA, 0.07% HA, and control. The significant differences were noted **(P < 0.01), *(P < 0.05) (ANOVA followed by Tuckey's post hoc test)

Figure 4

Relative mRNA expressions (mean ± SD) for Bcl-2 in granulosa cells which served as coculture during IVM of oocytes. The expression of levels of Bcl-2 in granulosa cells was measured after the addition of 0.035% HA, 0.07% HA, and at control levels. No significant differences were noted (ANOVA followed by Tuckey's post hoc test).