Linoleic acid increases neutral lipid storage during bovine oocyte maturation

CARRO, M.M.; RIOS, G.; ORESTI, G.M.; BUSCHIAZZO, J.; ALBERIO, R.

Rosario

Congreso; 49th Annual Meeting of the Society for Cryobiology; 2012

Society for Cryobiology- Universidad Nacional de Rosario

Efficient utilization of bovine oocytes for in vitro embryo production requiressuccessful cryopreservation. However, oocyte development to the blastocyst stageis low after oocyte cryopreservation (Martino et al., 1996). Linoleic acid (LA) is anessential long-chain unsaturated fatty acid (FA), with positive effects reported oncryopreservation. The use of this FA in the culture medium increased the survivalrate of frozen-thawed enucleated oocytes and embryos (Hochi et al., 1999, 2000;Tominaga et al., 1999). This effect could be due to an increase in membrane fluidityas a result of the incorporation of an unsaturated FA, preventing their rupture duringfreezing. According to Pereira and Marques (2008), cold cell damage is mainlydue to physical changes experienced by lipids at low temperatures, in particular,intracellular lipids play a major role in the cryosensitivity of oocytes (Nagashimaet al., 1995). The aim of this study was to evaluate the effect of LA on intracellularlipid droplets content and its impact on viability of vitrified oocytes. Cumulusoocyte complexes (COC) aspirated from ovaries recovered after slaughter werein vitro matured in a chemically defined maturation medium supplemented withLA at 9, 43 and 100 uM and were denuded and fixed. Oocytes were stained with1 ug/ml Nile Red in PBS for 10 min. Digital photographs were taken using an epifluorescencemicroscope and fluorescence intensity was measured with the softwareNis Elements Br 3.1. As validated by other authors (Barceló Fimbres andSeidel 2011), the intensity of fluorescence correlates mainly with the content oftriacylglycerols in lipid droplets. Results showed an increase in the mean fluorescenceat all concentrations of LA (9, 43 and 100 uM) corresponding to an increaseof 17.3, 47.4 and 57.3%, over the control (p < 0.05), respectively. No differences influorescence intensity were observed between the last two concentrations assayed,suggesting that there is an optimum concentration beyond which there is noincrease in neutral lipid content in droplets. In addition, incubation of oocytes with100 uM of LA inhibited meiosis progression. This negative effect could be due to anexcess of free LA in the cytoplasm. Previously, we have shown esterification ofradiolabelled LA in triacylglycerols after maturation. Accordingly, LA stimulatesneutral lipid accumulation in lipid droplets of bovine oocytes, increasing the unsaturationlevel of intracellular lipid droplets. Our work revealed a protective effect ofLA, leading to its esterification in triacylglycerols. Preliminary results show that LAadded to maturation media could be involved in the increased viability registered inpostvitrified oocytes (OPS methodology).