Effect Of IGFBP-5 On The Puberty And The Litter Performance In Female Rat

Objective:The objective of this study is to investigate the expression of insulin-like growth factor-binding protein-5?IGFBP-5?protein and mRNA on the hypothalamus-pituitary-ovary?HPO?axis of female rat during the development stages,and the effects of IGFBP-5 with intracerebroventricular?ICV?injection on the onset of puberty,the expression of reproductive related genes,reproductive hormone and litter performance of female rats,which provide a scientific theory basis for the further study on the regulation of puberty in animals.Methods:Firstly,Real-time quantitative PCR and immunofluorescence were utilized to determine the changes of of Igfbp-5 mRNA and its on the hypothalamus-pituitary-ovary?HPO?axis of female rats at different developmental stages?infant,prepuberty,puberty and adult?,respectively.25d SD rats?n=18?were divided into three groups,that is,control group,1?g/kg and 2?g/kg group.Then the exogenous IGFBP-5?1?g/kg,n=6;2?g/kg,n=6?was intracerebroventricularly?ICV?injected into the hypothalamus of female rats,and the control group was treated with the same volumn saline?n=6?.The hypothalamus,pituitary,ovary and serum were collected at vaginal opening?VO?or the age of rats with 2?g/kg IGFBP-5 matched control group.RT-qPCR was used to determine the change of reproductive-related genes transcriptional level.ELISA was used to analyze the concentratsions of reproductive hormone in serum,and histological section was adopted to observe the microstructure of ovary and counted the number of follicle and corpus luteum.The changes of the time of VO and estrous cycle were recorded.Litter size,alive litter size and birth weight of female rats were counted after mating during the oestrus of adult rats.Finally,primary hypothalamic cells was added with IGFBP-5?25ng/m L,50ng/m L?to determine the transcriptional level of reproductive-related genes.Western Blot?WB?was used to analyze the expression of Akt-mTOR protein.Results:?1?Igfbp-5 mRNA was expressed in the hypothalamus,pituitary and ovary of female rats at different developmental stages.The Igfbp-5 mRNA transcriptional level in the hypothalamus were highest at infant?P<0.05?,and significantly lower?P<0.05?at prepuberty?28d?than that of puberty and adult stages.In the pituitary,no significant differences were found from infant to adult?P>0.05?.In the ovary,Igfbp-5mRNA level was the highest?P<0.01?at infant,and significantly lower at prepuberty?21d?and adult compared to prepuberty?28d?and puberty?P<0.01?.IGFBP-5 immunoreactivity was detected in the retrosplenial agranular cortex?RSA?,primary motor cortex?M1?,secondary motor cortex?M2?,and primary somatosensory cortex?S1?.The IGFBP-5 fluorescence intensity in the RSA,M1 and M2 was were highest at infant?P<0.01?,and significantly lower at puberty?P<0.05?.IGFBP-5 was distributed in the arcuate nucleus?ARC?,median eminence?ME?,supraoptic nucleus retrochiasmatic part&supraoptic decussation?SOR&SOX?,anterior pituitary,ovarian stroma,granulosa cells,and theca cells.The fluorescence intensity of IGFBP-5 was significantly decreased in ARC and SOR&SOX of pubertal rats?P<0.05?;The fluorescence intensity of IGFBP-5 was significantly lower in the anterior pituitary of pubertal rats than that of infancy rat?P<0.01?,but was the highest at adult?P<0.01?;The fluorescence intensity of IGFBP-5 in the ovary of prepubertal?28d?rats was significantly increased compared to the infant?P<0.01?,and significantly decreased at puberty?P<0.01?.?2?The time of VO was significantly delayed in prepubertal rats with at the dose of 1?g/kg?P<0.05?and 2?g/kg IGFBP-5?P<0.01?.?3?After ICV injecting IGFBP-5 into the hypothalamus of the rats,when the vagina of rats were opened,compared with the control group,1?g/kg IGFBP-5 reduced the transcriptional levels of Gnrh and Igf-1 mRNA in the hypothalamus?P<0.01?and Fsh?mRNA levels in the pituitary gland?P<0.01?,and the level of Fsh?mRNA also decreased at 2?g/kg group?P<0.05?.The transcriptional levels of Igf-1 and Igf-1r mRNA of experimental groups were not different compared with control?P>0.05?.No significant differences were observed among the three groups in the level of Igf-1,Igfbp-5 and Igf-1r genes in the pituitary gland and ovary?P>0.05?.The concentratsions of E₂,FSH,and LH in serum were decreased at the 1?g/kg and 2?g/kg groups compared to control?P<0.05?.The concentrations of IGF-1?P<0.01?and P₄?P<0.05?in the serum of the 2?g/kg group were lower than that of control group.The results of ovarian morphological showed that the ovarian weight was significantly decreased at the dose of 1?g/kg group?P<0.05?,and transverse diameter,longitudinal diameter,transverse perimeter and longitudinal perimeter of the ovary were not significantly different among the three groups?P>0.05?.No significant difference was found in ovarian weight and size between the 2?g/kg group and the control group?P>0.05?.The primordial follicle,primary follicle,secondary follicle and corpus luteum were observed in all animals,but experimental groups were mostly primary follicles?P<0.05?.Compared with the control group,the number of corpus luteum were decreased in the1?g/kg group?P<0.05?and 2?g/kg group?P=0.052?.No differecnce was found among the three groups in secondary follicle?P>0.05?.?4?After ICV injecting 2?g/kg IGFBP-5 into the hypothalamus of the rats,when the vagina of female rats were opened in control group and the age of 2?g/kg IGFBP-5group matched control group,the transcriptional level of Igf-1r and Gnrh mRNA in hypothalamus at the dose of 2?g/kg group was significantly lower than that in control group?P<0.05?.The level of Tgf-?mRNA was reduced?P=0.054?.However,no significant differences were observed in the level of Igf-1,Kiss-1,Rfrp-3,Akt-1,and Mtor genes between the two groups?P>0.05?.Compared with the control group,Igf-1mRNA level in pituitary gland of 2?g/kg group was significantly down-regulated?P<0.05?,Igf-1,Kiss-1,Rfrp-3,Akt-1,and Mtor mRNA levels were not different?P>0.05?.No differences were found in the levels of Igf-1r,Fsh?and Lh?mRNA in pituitary and Igfbp-5,Igf-1 and Igf-1r mRNA in ovary?P>0.05?.The concentration of P₄in the serum of the female rats with 2?g/kg IGFBP-5 was lower than that of the control group?P<0.05?,and the concentratsions of FSH,LH and IGF-1 in serum were decreased in the 2?g/kg group compared to control?P<0.01?.In contrast,no difference was observed in the concentratsion of E₂ in serum after 2?g/kg IGFBP-5 ICV injection?P>0.05?.There was no significant difference in transverse diameter,longitudinal diameter,transverse perimeter and longitudinal perimeter of the ovary between the two groups?P>0.05?.Ovarian tissue sections showed that primordial follicle,primary follicle andsecondary follicles were observed between the two groups.2?g/kg group was mostly primary and secondary follicles?P<0.05?,and the corpus luteum was almost absent?P<0.01?.However,litter performance and estrus cycle was not affected?P>0.05?.?5?The levels of Kiss-1 and Igf-1 mRNA were decreased after adding different concentratsions of IGFBP-5 to primary hypothalamic cells?P<0.05?.However,no significant differences were observed among the three groups in the levels of Gnrh,Igf-1r,Rfrp-3,Akt-1,and Mtor mRNA?P>0.05?.But IGFBP-5 caused a significant increase of AKT and mTOR protein?P<0.01?.Conclusion:?1?IGFBP-5 is distributed in the hypothalamus,pituitary and ovary of female rats at different developmental stages,and the transcriptal level of Igfbp-5mRNA in the hypothalamus and ovary varies with developmental process.?2?IGFBP-5delays the onset of puberty in female rats,and changes the level of Igf-1 mRNA,Gnrh mRNA in hypothalamus and Fsh?mRNA in pituitary;IGFBP-5 protein decreased the concentratsions of E₂,P₄,FSH,LH and IGF-1 in serum,and delays the developmental process of follicle in pubertal rats,but does not affect estrus cycle and the litter performance.?3?IGFBP-5 decreases the transcriptal level of Kiss-1 and Igf-1 mRNA in hypothalamus cell,and promotes the expression of AKT and mTOR,suggesting that IGFBP-5 may affect puberty in rats through AKT-mTOR pathway.