| Object: Most sheep are seasonal estrus animals(eg,Kazakh sheep),their reproductive system function is mainly controlled by various hormones secreted by the hypothalamus pituitary-gonadal axis(HPG axis).The GnRH synthesized and secreted by the hypothalamus is the key to regulating fertility,and it’s not only regulated by the photoperiod,but also has a certain regulation effect on the estrus of sheep.Our previous studies found that GNAQ may play an important role in nutrient regulation of sheep oestrus,but the mechanism of GNAQ in nutrient regulation of sheep oestrus remains to be studied.Foreign studies have shown that GNAQ is a downstream gene of the kisspeptin/GPR54 system in the photoperiodic regulatory pathway and has the function of regulating the expression and secretion of GnRH in the hypothalamus.Therefore,whether the GNAQ serves as a link between the nutritionally regulated sheep oestrus pathway and kisspeptin/GPR54 system in the light cycle regulatory pathway remains to be studied.Methods:(1)The hypothalamus is the key to the secretion of GnRH,thereby sheep hypothalamic nerve cells were isolated and cultured,then used as the research model.(2)GNAQ expression vectors and interference vectors were constructed and transfected into the hypothalamic cells of Kazak fetal sheep.qRT-PCR,Western Blot and ELASA methods were used to analyze the regulation of GnRH expression in hypothalamic cells.(3)The expression of GNAQ gene in sheep tissues was detected by qRT-PCR.(4)Different levels of folic acid were treated with the hypothalamic cells,0,4,40,and 80 mg/mL were the the final concentrations of folic acid,respectively.Following that,the methylation levels of GNAQ promoter region were detected by BSP in each group.The effects of folic acid on GNAQ expression and GnRH secretion in sheep hypothalamus were analyzed by qRT-PCR and ELASA method.Results:(1)Sheep hypothalamic nerve cells were successfully cultured.The purity of MAP2 positive cells reached 95.6% through immunofluorescence identification.(2)When GNAQ expression was down-regulated,its upstream genes KISS1,GPR54,and ER were up-regulated,and downstream genes PLCB1,PRKCB,and GnRH were also up-regulated,meanwhile GnRH secretion was up-regulated as well.(3)The GNAQ was expressed in differently degrees in various tissues of Kazakh ewe and in pituitary,hypothalamus,brain and uterus had higher expression level.(4)The relative expression of DNMT1 in 40 mg/ml folic acid group was significantly higher than that in other groups(P<0.05),while the relative expression of GNAQ was significantly lower than other groups(P<0.05),and as the level of folic acid were increased.The expression of GNAQ followed the trend of decreasing first and then increasing.BSP detected methylation of one CpG site in the GNAQ promoter region in the 40 mg/mL folic acid group,and no methylation of the CpG site in the other groups.In addition,the GnRH level was significantly higher in the 40 mg/mL folic acid group than in the other three groups(P<0.05).The high folic acid concentration affected GnRH secretion.Conclusion:(1)GNAQ regulates the downstream PLCβ in hypothalamic cells and directly regulates GnRH expression and secretion through calcium signal pathway and PKC signaling pathway.Also GNAQ can indirectly regulate the expression and secretion of GnRH by regulating the expression of kisspeptin in the kisspeptin/GPR54 signaling pathway.(2)The appropriate folic acid level can increase the expression level of DNMT1,promote the methylation level of GNAQ promoter,then affect the GNAQ expression level,and indirectly regulate the secretion of GnRH.(3)Based on GNAQ,the relationship between nutrient regulation pathway and photoperiod regulation pathway was established. |
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