Effect Of Grid1 Gene On The Puberty And Litter Performance In Female Rats

The purpose of this study is to investigate the expression of Grid1 and its protein on the reproductive axis of female rats during the development of puberty,and the effect of knockdown Gridl on the puberty and litter performance of female rats,which provides a theoretical basis for the regulation of animal puberty.Firstly,RT-qPCR and immunofluorescence were utilized to analyze the changes of Gridl mRNA and its protein on the reproductive axis of female rats at different physiological stages(infant,prepuberty,puberty and adult),respectively.Secondly,Gridl Ientiviral vector(LV-Gridl)was constructed,and added to hypothalamic neurons in vitro,RT-qPCR and fluorescent cell counting were used to detect the infection efficiency,knockdown efficiency and the change of reproductive-related gene transcription.Lastly,21d SD rats were divided into three groups,that is,LV-Gridl group,control group and LV-(-)group.LV-Gridl was intracerebroventricularly(ICV)injected into hypothalamus,hypothalamus,pituitary,ovary and serum were collected on 7d and the day of vaginal opening(VO).RT-qPCR was used to analyze knockdown efficiency and the change of reproductive-related gene transcription.ELISA and histological methods were used to evaluate reproductive hormone levels in serum and ovarian structure,respectively.Mating in oestrus,litter size,alive litter size and birth weight were counted.The main results were as follows:(1)From infant to prepuberty,Gridl mRNA transcription level in hypothalamus of rat was significantly decreased(P<0.05),and increased in puberty and adult(P<0.05),and it was significantly lower at puberty and adulthood than that of infancy rat(P<0.05),no significant difference between puberty and adult was found(P>0.05);GluD1 distributed in the arcuate nucleus(ARC),paraventricular nucleus(PVN)and periventricular nucleus(PeN)in hypothalamic of female rats.However,the fluorescence intensity of ARC was highest at infant rat(P<0.05).The fluorescence intensity of PVN increased with the development of rats,it was significantly higher at adult rat compared to infant animal(P<0.05).The immunofluorescence intensity of PeN was the weakest at infant(P<0.05),and then showed an upward trend and there was no significant difference between the prepuberty,puberty and adult(P>0.05).In the pituitary of female rats,the expression level of Gridl mRNA decreased gradually,it was significantly higher at infant than prepuberty,puberty and adult(P<0.05),and puberty was significantly higher than that of adult(/P<0,05);the immunofluorescence results showed that GluD1 mainly distributed in adenohypophysis.The expression level of Grid1 mRNA in ovary was extremely low,but GluD1 was distributed in corpus luteum and theca cells.(2)LV-Gridl infected hypothalamic neurons,Gridl mRNA expression level was significantly decreased(P<0.05),Gnrh mRNA increased(P<0,05),Rfrp-3 mRNA decreased(P<0.05),but Gludl mRNA was not change(P>0.05).(3)After ICV injection with LV-Gridl into hypothalamus of rat,the time of VO in LV-Grid1 group rats was significantly advanced(P<0.05).However,litter performance was not affected(P>0.05).(4)ICV injection with LV-Gridl into hypothalamus of rat after 7 days,the Gridl mRNA in the hypothalamus decreased about 46%(JP<0.05),the changes of Gnrfh,Rfrp-3 and Glud1 mRNA were consistent with the above results(2);The concentrations of GluD1,E2,P4,FSH and LH in serum were not significantly different among LV-Gridl,control and LV-(-)group(P>0.05).Ovarian tissue sections showed that the follicles in the LV-Gridl group developed rapidly and were mostly tertiary follicles,while the control and LV-(-)group were mostly secondary follicles.However,the weight,transverse diameter,longitudinal diameter,transverse perimeter and longitudinal perimeter of the ovary did not vary among the three groups(P>0.05).(5)After ICV injection with LV-Grid1 into hypothalamus of rat,when all rats vaginal were opened,Gridl mRNA expression level in hypothalamus of LV-Gridl group was significantly down-regulated(P<0.05),Gnrh,Rfrp-3 and Gludl mRNA were not different(P>0.05)compared with control and LV-(-)group.The concentration of GluD1 in serum increased(P<0.05),P4 decreased(P<0.05),and there was no change in E2,FSH and LH(P>0.05).Ovarian tissue sections showed that corpus luteum were found in three group,but LV-Grid1 group was mostly primary and secondary follicles.There was no difference between the three groups in weight,transverse diameter,longitudinal diameter,transverse perimeter and longitudinal perimeter(P>0.05).(6)After ICV injection with LV-Gridl into hypothalamus of rat,when the vaginal of rats in LV-Gridl group was opened,the age of control and LV-(-)group were matched with LV-Gridl,the transcription levels of Gridl,Gnrh,Rfrp-3 and Gludl mRNA in hypothalamus,and concentrations of GluD1 and FSH in serum were not significantly different(P>0.05).The concentration E2,P4 and LH were significantly higher than those of control and LV-(-)groups(P<0.05).Ovarian histological observation showed that corpus luteum,primary follicle and secondary follicle appeared in LV-Gridl group,but there were no corpus Iuteum was found in control and LV-(-)group,and control and LV-(-)group was mostly secondary and tertiary follicles.The ovary weight,transverse diameter,longitudinal diameter and longitudinal perimeter of LV-Gridl group were significantly greater than control and LV-(-)group(P<0.05),no significant difference in transverse perimeter(P>0.05).The results showed that:(1)During the developmental of puberty in female rats,the expression level of Gridl mRNA in the hypothalamus and pituitary varies with age;GluD1 is distributed in the Hypothalamic-Pituitary-Gonadal axis(HPGA),and the distribution of GluD1 in hypothalamic different nuclei is different.(2)Knockdown Gridl promotes the onset of puberty in rats,but does not affect the litter performance of female rats.(3)Knockdown of Gridl affects the transcription level of Gnrh mRNA and Rfrp-3 mRNA in hypothalamus and the concentration of reproductive hormone in serum.