The Effect And Mechanism Of Ptprn-2 Gene On The Puberty In Female Rat

The present study aims to evaluate the expression of PTPRN-2 and its genes in the reproductive axis of female rats at before and after puberty,and the effect and mechanism of knockdown on Ptprn-2 gene on puberty in female rat,to provide scientific theoretical basis for the gene regulation network of puberty in female animal.Firstly,immunofluorescence and RT-q PCR technology was utilized to study the expression change of PTPRN-2 on the hypothalamus-pituitary-ovary axis of female rats at before and after puberty,that is,infancy,prepuberty,puberty(21d and 28d)and adult;Then the knockdown Ptprn-2 lentiviral vector(sh RNA-Ptprn-2)was constructed to package lentivirus and infect hypothalamic neurons,RT-q PCR was used to analyze the knockdown efficiency of the lentivirus and the transcription level of puberty-related genes.The lentiviral vector(sh RNA-Ptprn-2 group,n=12,and negative control group,n=12)and dd H2O(blank control group,n=12)was injected into the hypothalamus of prepuberty female rats(25d)using the intracerebroventricular injection(ICV injection),respectively.The time of vaginal opening(VO)was recorded.The rats were sacrificed after recording the body weight when the vulva was opened for all animals,the hypothalamus,pituitary,ovary and serum were collected.the transcriptional level of puberty-related genes was determined with RT-q PCR,the serum concentratsions of reproductive hormone were detected by ELISA,the size and microstructure of ovaries was observed using the morphological method;Again the animal experiment,when the control group animals vulva was opened,and the sh RNA-Ptprn-2 group was matched the same age with the controls,ditto for testing items,add the number of dense core vesicles(DCV)of adenohypophysial gonadotropin cells were observed by transmission electron microscopy.The results were as follows:(1)The observation of immunofluorescence showed that PTPRN-2 was mainly distributed in arcuate(ARC),periventricular nucleus(Pe N),and paraventricular nucleus(PVN),adenohypophysis and ovarian stromal cells,granule cells and corpus luteum at female rats at before and after puberty.The hypothalamic transcriptional level of Ptprn-2m RNA was significantly lower from infancy to prepuberty(28d)(P<0.05),the level was the lowest in the puberty,and significantly higher in adult(P<0.05).In addition,the levels of Ptprn-2 m RNA at prepuberty(28d)and puberty were significantly lower than those of at infant,prepuberty(21d)and adult(P<0.05).There was no significant difference in the transcriptional level of Ptprn-2 m RNA in the pituitary of rats from infant to prepuberty(28d)(P>0.05),significantly increase(P<0.01)was found at puberty and adult.However,we could not detect the level of Ptprn-2 m RNA in the ovary.(2)Knockdown of Ptprn-2gene in hypothalamic neurons,RT-q PCR results showed that Ptprn-2 m RNA decreased by80%,the expression of Rfrp-3 m RNA significantly lower(P<0.05)and Gnrh、Kiss-1、Ca N m RNA were significantly increased(P<0.05).(3)Prepubertal(25d)rats injected with sh RNA-Ptprn-2 were significantly delayed the onset of puberty(P<0.01),the transcriptional level of Ptprn-2,Gnrh,Tac-3,Kiss-1,Ca N m RNA in the hypothalamus significance reduced(P<0.05),but the level of Rfrp-3,Igf-1and Cpe m RNA increased(P<0.05).In the pituitary and ovary,the transcriptional levels of Ptprn-2 and Gnrh R m RNA were not different compared with the control(P>0.05),the levels of Fshβ and Lhr m RNA were significantly reduced(P<0.01),Lhβ and Fshr m RNA was significantly increased(P<0.01).The results of ovary morphology showed that the number of corpus luteum,the ovary transverse and longitudinal circumference,and the body weight of the rats in sh RNA-Ptprn-2 group were significantly increased(P<0.05),however,there was no significant difference in transverse diameter,longitudinal diameter and ovarian index(P>0.05).The concentratsions of FSH(P<0.05)and P4(P<0.05)in serum were reduced compared to controls,the concentrations of LH and E2 in the serum was not significantly difference(P>0.05).(4)After injection of sh RNA-Ptprn-2 into the lateral ventricle,when the vulva of the blank control group or the negative control group was opened,it was matched with sh RNA-Ptprn-2 rats of the same age.The transcriptional levels of Ptprn-2,Gnrh m RNA in the hypothalamus significance reduced(P<0.01),the level of Rfrp-3 and Igf-1 m RNA in the hypothalamus was significantly increased(P<0.01),and the ERα、Insr m RNA were significance descrease(P<0.05).The transcriptional levels of pituitary Gnrh R m RNA(P<0.05)、ovarian Fshr m RNA(P<0.01)were significantly increased,pituitary Fshβ m RNA reduced(P<0.05),but Ptprn-2、Lshβ and Lhr m RNA were no difference(P>0.05).The number of follicles increased in the sh RNA-Ptprn-2 group,longitudinal diameter and transverse perimeter of ovary were significantly reduced(P<0.05),but the ovary index significantly increased(P<0.05),the transverse diameter,longitudinal perimeter and weight were all no significant differences(P>0.05).The concentration of serum FSH was significantly reduced(P<0.05),and E2 was significantly increased(P<0.05),no differences were found in the concentrations of serum LH and P4(P>0.05)compared to control group.The number of the big DCV in adenohypophysial gonadotropin cells was significantly reduced(P<0.05),but the small DCV was no difference(P>0.05).The results showed that:(1)PTPRN-2 is expressed in the hypothalamus,pituitary and ovary in female rats at before and after puberty,and the transcriptional level of Ptprn-2m RNA in the hypothalamus and pituitary is changed with the development of rat.(2)Knockdown of Ptprn-2 gene delays the onset of puberty and affects the transcriptional levels of puberty-related genes in reproductive axis on vivo 、 vitro and the serum concentrations of reproductive hormones.(3)Knockdown of Ptprn-2 gene changes the number of big DCV in adenohypophysial gonadotropin cells.