Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro.

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes.

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.