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 meeting of the Society for Cryobiology, CRYO-2012; 2012

Society for Cryobiology

Efficient utilization of bovine oocytes for in vitro embryo production requires successful cryopreservation. However, oocyte development to the blastocyst stage is low after oocyte cryopreservation (Martino et al., 1996). Linoleic acid (LA) is an essential long-chain unsaturated fatty acid (FA), with positive effects reported on cryopreservation. The use of this FA in the culture medium increased the survival rate 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 fluidity as a result of the incorporation of an unsaturated FA, preventing their rupture during freezing. According to Pereira and Marques (2008), cold cell damage is mainly due to physical changes experienced by lipids at low temperatures, in particular, intracellular lipids play a major role in the cryosensitivity of oocytes (Nagashima et al., 1995). The aim of this study was to evaluate the effect of LA on intracellular lipid droplets content and its impact on viability of vitrified oocytes. Cumulus oocyte complexes (COC) aspirated from ovaries recovered after slaughter were in vitro matured in a chemically defined maturation medium supplemented with LA at 9, 43 and 100 uM and were denuded and fixed. Oocytes were stained with 1 lg/ml Nile Red in PBS for 10 min. Digital photographs were taken using an epifluorescence microscope and fluorescence intensity was measured with the software Nis Elements Br 3.1. As validated by other authors (Barceló Fimbres and Seidel 2011), the intensity of fluorescence correlates mainly with the content of triacylglycerols in lipid droplets. Results showed an increase in the mean fluorescence at all concentrations of LA (9, 43 and 100 uM) corresponding to an increase of 17.3, 47.4 and 57.3%, over the control (p < 0.05), respectively. No differences in fluorescence intensity were observed between the last two concentrations assayed, suggesting that there is an optimum concentration beyond which there is no increase in neutral lipid content in droplets. In addition, incubation of oocytes with 100 uM of LA inhibited meiosis progression. This negative effect could be due to an excess of free LA in the cytoplasm. Previously, we have shown esterification of radiolabelled LA in triacylglycerols after maturation. Accordingly, LA stimulates neutral lipid accumulation in lipid droplets of bovine oocytes, increasing the unsaturation level of intracellular lipid droplets. Our work revealed a protective effect of LA, leading to its esterification in triacylglycerols. Preliminary results show that LA added to maturation media could be involved in the increased viability registered in postvitrified oocytes (OPS methodology).