| In mammalian ovaries,follicles usually undergo the stage of growth and development from small to large in size and are selected to go through two different fates--either to ovulating,or to going atresia under the interaction of many factors such as hormones.During follicular growth and development,more than ninety-nine percent of oocytes will disappear resulting from follicular atresia,only less than one percent of the follicles can ovulate,and the fate of the vast majority of follicles is degenerated through apoptosis.Previous studies on the regulatory mechanism of follicle atresia mostly focus on granulosa cell apoptosis,but few literature has been reported on the internal mechanism of follicular development and atresia from the aspect of microenvironmental changes in the follicle.Combined with the expression of genes involving glycolipid in granulosa cell and oxidative index in follicular fluid,the difference of endogenous metabolites in porcine follicular fluid during follicle development and atresia were studied based on gas chromatography time of flight mass spectrometry(GC/TOF-MS)metabolomics,so that we can screen the biomarkers related to follicular development and atresia,in order to provide new ideas and directions for improving animal reproductive rate and artificial assisted reproductive efficiency.Experiment 1:The difference of endogenous metabolites in porcine follicular fluid during follicle development and atresia.Based on GC/TOF-MS,follicular fluid collected from healthy and atretic follicles with different sizes of domestic pigs were measured,and the metabolite atlas was obtained.Then the endogenous metabolites significantly differently were selected by multidimensional and single dimensional statistical analysis.The results showed that no matter during follicular development or follicular atresia,there was a statistically significant separation between healthy follicular fluid with large size group(LH group)and the healthy follicular fluid with medium size group(MH group),as well as between atresia follicular fluid with medium size group(MA group)and the healthy follicular fluid with medium size group(MH group).Metabolite analysis showed that the concentrations of endogenous metabolites differed due to follicular development and atresia.Compared with MH group,even through the concentration of glucose,oxoglutaric acid,proline and glucose 1-phosphoric acid was significantly higher(P<0.05),the concentration of the great majority of metabolites in LH group was significantly lower(P<0.05),including citric acid and so on.The concentration of vast majority of metabolites in MA group was significantly higher(P<0.05)compared with MH group,including glucose,amino acids,cholesterol and stearic acid and so forth,except that the concentration of glycerol 3-phosphate,arachidonic acid,D-galactose and L-carnitine et al was significantly reduced(P<0.05).In a word,the metabolic activity during follicular development is significantly higher than that during follicular atresia.Most of the metabolic substances in follicular fluid are used to improve the developmental potential of oocytes during the follicular development.The decrease of the concentration of saturated fatty acids in the follicular fluid also protects the oocyte from damage to some extent;the metabolic level of follicle cells during follicular atresia was significantly reduced and most of the metabolic substances were retained in follicular fluid.Elevating concentration of saturated fatty acids may accelerate the process of apoptosis of granulosa cells and oocytes.Experiment 2:The difference of expression of genes involving glycolipid metabolism in granulosa cell and anti-oxidative index of follicular fluid during follicular development and atresia.The expression of genes involving glycolipid metabolism in granulosa cell and anti-oxidative index of follicular fluid were investigated,combined with the distribution of glycogen,physicochemical indexes and molecular biological methods.The results of PAS staining showed that glycogen mainly concentrated in the follicular fluid.The distribution of glycogen in healthy follicle granulosa cells is less than that in atresia follicles.The results of genes showed that while the expression of GLUT1 mRNA in granulosa cells was highly significantly increased(P<0.01)in LH group compared with MH group,the expression of GLUT3 and GLUT8 mRNA was highly significantly decreased(P<0.01)in LH group,compared with MH group,the expression of GLUT8 mRNA in the granulosa cells was extremely significantly decreased(P<0.01)in MA group.In MH,LH and MA group,the genes(ACACA,CD36,SCD,FADS2,ELOVL2,ELOVL5,CPT1A,FABP5,PLIN2)related to lipid metabolism were all expressed in granulosa cells.Comparison with MH group,granulosa cells in LH group had a significantly increased(P<0.05)expression of AC AC A and CD36 mRNA and extremely significantly increased(P<0.01)expression of SCD,ELOVL2 and ELOVL5 mRNA.In MA group,the expression of ACACA,CPT1A and FADS2 mRNA in granulosa cells was extremely significantly decreased(P<0.01),while the expression of CD36,PLIN2,FABP5 mRNA was extremely significantly increased(P<0.01).In LH group,the content of malondialdehyde(MDA)of follicular fluid was significantly decreased(P<0.05),and the content of total antioxidant capacity(T-AOC)and glutathione peroxidase(GSH-PX)was significantly enhanced(P<0.05)compared with MH group.But the content of the total superoxide dismutase(T-SOD)was significantly decreased(P<0.05).In MA group,the level of MDA of follicular fluid was extremely significantly increased(P<0.01)and T-AOC extremely significantly decreased(P<0.01)compared with MH group(P<0.01),but the content of CAT and T-SOD enzyme was extremely significantly increased(P<0.01).All in all,the expression of SCD mRNA in granulosa cells during follicular development are significantly up-regulated,which may protect the oocytes from damage which saturated fatty acids caused in some way.During follicular atresia,the down-regulated expression of CPT1A mRNA and the up-regulated expression of CD36 mRNA in granulosa cells may increase the concentration of intracellular fatty acid and attribute granulosa cells to suffering lipid toxicity and apoptosis.During follicular development,the content of T-AOC in follicular fluid was increased,making oocytes free from free radical damage;the decrease of T-AOC during follicular atresia will accelerate the apoptosis and degradation of oocyte. |
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