| High-concentration diets can cause a decrease of rumen pH in ruminants and subacute ruminal acidosis(SARA),resulting in rumen abnormal metabolism as well as endocrine disruption.When SARA occurs in the body,the rumen will release a large amount of lipopolysaccharide(LPS).LPS may cause ovarian dysfunction by damaging the hypothalamic-pituitary-gonad axis,therefore leads to reproductive disorder.The current study includes in vitro and in vivo experiments,and techniques such as enzyme-linked immunosorbent assay(ELISA),real time-PCR,oil red O staining and western blotting(WB)were applied.Firstly,the effects of different forage to concentrate ratios on estrus,progesterone level and luteal PLINs gene expression were studied in Hu sheep.Secondly,in vitro culture of goat ovarian LGCs were used to study the effects of LPS on cell proliferation,lipid droplet accumulation,and steroidogenesis.The result will provide a scientific basis for the further study in the molecular mechanism of nutrient levels affecting animal reproductive function.The main contents are as follows:1.Effects of high concentrate diet on the estrous,progesterone level and mRNA expression of luteal PLINs in Hu EwesThe current study was conducted to investigate the effects of high concentrate diet on the estrous,reproductive hormones and mRNA expression of luteal PLIN family members of Hu Ewes.Twenty Hu Ewes aged 5 months were divided into low concentrate group(LC,2:8 ratio,n=10)and high concentrate group(HC,6:4 ratio,n=10).Estrous synchronization was carried out on day 20 of the formal period.On day 58 of the formal period,the ovaries were collected and corpora lutea were isolated.Blood was collected to measure the content of estrogen(E2),progesterone(P4)and lipopolysaccharide(LPS).The average daily gain(ADG)and dry matter intake(DMI)were measured during the experiment period.Real time-PCR were applied to investigate the mRNA expressions of luteal PLINs.The results showed that ADG of HC group was significantly higher than that in LC group(P<0.05),while DMI was significantly lower(P<0.05).The concentration of serum LPS in the HC group was significantly higher than that in LC group(P<0.05).The natural estrous rate of HC and LC groups were 30%and 70%,respectively(P=0.089).The P4/E2 value of non-estrus ewes were significantly higher than that of estrus ewes(P<0.05).There was no significant difference in the ovary weight,luteal number and serum P4 concentration in luteal phase between two groups(P>0.05).The mRNA of PLIN family members were expressed at different degrees in the corpus luteum,among which the expression level of PLIN2 mRNA was the highest,and PLIN2 mRNA expression level in the LC group was significantly higher than that in the HC group(P<0.05),while PLIN3,PLIN4,and PLIN5 levels were significantly lower than those in the HC group(P<0.05).The results suggest that high concentrate diet feeding can increase serum LPS,which might be detrimental to estrus in ewe.The differences in the expressions of luteal PLINs genes between HC and LC indicate that PLINs gene expression might be affected by LPS.2.Effects of LPS on proliferation,accumulation of lipid droplets and steroidogenesis in goat luteinized granulosa cellsThe purpose of this study was to investigate the effects of LPS on proliferation,accumulation of lipid droplets(LDs)and steroidogenesis in goat luteinized granulosa cells(LGCs).GCs isolated from the ovarian follicles were spontaneously luteinized under media with FBS,resulting in increased progesterone and shifted shape from spherical to star with multiple prolongations.Then,LGCs were treated with LPS(0-10μg·mL-1)for 0-48 h.Oil Red O staining was performed to observe LDs accumulation and commercial kit was applied to detect intracellular triglyceride(TG)content.The cell proliferation were detected by CCK-8 kit.Expressions of cell-cycle related genes were determined by real-time PCR.Estradiol(E2),progesterone(P4)and Proinflammatory cytokines from cell supernatants were determined by ELISA,and expressions of StAR,P450scc,3β-HSD,CYP19A1 and NF-κB were detected by Western blot.Results showed that LPS treatment significantly increased LDs accumulation after 24 h,and 5 μg·mL-1 LPS increased TG content(P<0.05).LPS treatment for 24 h stimulated the LGCs activities(P<0.05),which was confirmed by the increases in the expressions of PCNA,cyclinB1 and cyclinD1,while 48 h treatment had no effect.The expression of NF-κB protein increased with the increase of pro-inflammatory factors.LPS treatment suppressed E2 and P4 output of LGCs(P<0.05).Western blot results showed that 10μg·mL-1 LPS decreased the protein expression of 3β-HSD in LGCs(P<0.05).In conclusion,LPS increased LDs accumulation and cell proliferation,increased levels of pro-inflammatory cytokines and LPS-mediated P4 reduction could be attributed to the decreased 3β-HSD protein expression. |
