| The development of follicle is a very complicated physiological process,which not only needs basic nutrients but also needs a variety of hormones,growth factors and fatty acids.Arachidonic acid(AA)is one of the unsaturated fatty acids with relatively high proportion,accounting for approximately 2.5% of the total fatty acid content of follicular fluid.However,the mechanism of the effect of AA on follicles is not clear.In this experiment,the effects of AA on bovine ovarian granulosa cells were studied by adding different concentrations of AA to cultured bovine ovarian granulosa cells in vitro.At the end of culture period,we analyzed the effects of different concentrations of AA(0.1% DMSO,1,10,50,100,200 ?M)on physiological function of ovarian granulosa cells and elucidated the molecular mechanism behind it.The ultimate goal is to provide data and theoretical guidance for improving the developmental potential of follicles and oocytes.Exp 1: Effects of AA on cell viability of bovine ovarian granulosa cellsIn this study,granulosa cells isolated from bovine ovaries were cultured and treated with different concentrations of AA.The cell viability of cultured bovine granulosa cells was investigated by CCK-8.The results showed that 50 ?M AA treated cells for 24 h significantly increased the viability of bovine ovarian granulosa cells.Moreover,this study also investigated the effects of AA on nuclear morphology,cell apoptosis rate and apoptotic-related gene expression of granulosa cells.The results showed that in comparison to 50 ?M AA treatment group,The nuclei of granulosa cells cultured in medium alone appeared to be with higher intensity of stain;The apoptosis rate of granulosa cells in the control group was 24.51±1.80%,while that in the 50 ?M AA treatment group was 18.26±1.57%,and the apoptosis rate of granulosa cells treated with 200 ?M AA was as high as 90.22±3.11%.The difference between the 50,100 ?M AA treatment group and the control group was statistically significant;50 ?M AA stimulated the expression of BCL-2 mRNA,while inhibited the expression of BAX mRNA in bovine ovarian granulosa cells(P<0.05).Exp 2: Effects of AA on lipid droplet formation of bovine ovarian granulosa cellsIn this study,granulosa cells isolated from bovine ovaries were cultured and treated with different concentrations of AA for 24 h.The lipid droplets were measured by oil red O stain,and the mRNA levels of fatty acid transporter genes was analyzed by quantitative real-time RT-PCR.The results showed that AA induced intracellular lipid droplet accumulation;In vehicle and the treatment group of 1 ?M and 10 ?M AA,intracellular red stain was visible,but the size of red stain was small,whereas numerous small lipid droplets were stained in cells that had been incubated with 50 and 100 ?M AA.However,both the quantity and size of lipid droplets were decreased in the granulosa cells incubated with 200 ?M AA.The abundance of FABP3 and CD36 increased with the AA concentration went up from vehicle to 100 ?M in a dose-dependent manner;50 ?M AA up-regulated the expression level of SLC27A1,and 200 ?M AA down-regulated the expression of the CD36 in bovine ovarian granulosa cells(P<0.05).Exp 3: Mechanism of AA on bovine ovarian granulosa cellsTo study the influence mechanism of AA on bovine ovarian granulosa cells,in this study,granulosa cells isolated from bovine ovaries were cultured and treated with AA for 24 h,the amounts of estradiol and progesterone in cultured media were measured by radioimmunoassay,the mRNA levels of steroidogenic-related genes were analyzed by quantitative real-time RTPCR and the phosphorylation of AKT and ERK 1/2 was measured by Western Blot.The results showed that compared with the vehicle group,higher concentration of AA,at 100 and 200 ?M,decreased the production of estradiol(P < 0.05),but lower concentration of AA did not change the secretion of estradiol in cultured granulosa cells;In contrast,AA stimulated the production of progesterone in cultured granulosa cells in a dose-dependent manner;Compared with the vehicle group,50,100 and 200 ?M of AA significantly decreased the abundance of CYP19A1,FSHR and HSD3B1 mRNA(P < 0.05),and AA decreased the level of STAR mRNA(P < 0.05);50 ?M AA significantly increased the phosphorylation levels of AKT and ERK 1/2.Conclusions:(1)Fifty ?M AA increased the proliferation of bovine ovarian granulosa cells through activating MAPK and PI3K/Akt signal pathways,while higher concentration of AA(200 ?M)induced granulosa cell apoptosis.(2)Fifty and one hundred ?M AA promoted the increase of lipid droplets in bovine ovary granulosa cells and thus resulting in the changes of cell morphology.(3)AA may regulate the production of steroid hormone in granulosa cells.Higher concentration of AA(100,200 ?M)significantly inhibited the production of estradiol in cultured granulosa cells.Whereas,AA stimulated the production of progesterone in a dose-dependent manner in cultured granulosa cells.. |
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