Effect Of Lentivirus-mediated Silencing Of PNX On Reproductive Development In Female Mice

Mammalian reproduction is a complicated physiological process and coordinated by a series of reproductive factors.In recent years,PNX,a newly reprodctive peptide,has been proved to regulate the expression of key reproductive peptides such as Gn RH and kisspeptin,as a result,affect the reproductive process of animals.Nevertheless,it is still controversial whether PNX mainly regulates reproduction at the level of hypothalamus or pituitary.It is worth noting that the existing studies mainly focused on exploring the pharmacological effects of PNX peptide treatment,and whether endogenous PNX playing a decisive role in the reproductive development of mammal remains to be further studied.In addition,puberty is an important period for the development of the reproductive system.Presently,researches are only limited to exploring the effect of PNX on estrus in adult animals,but the effect of PNX on puberty has not yet been elucidated.In terms of this,the present study intends to use lentiviral-mediated RNAi technology to silence endogenous PNX,and investigate the effect of PNX silencing from the cellular and individual levels to further explain the regulatory mechanism of PNX on reproductive development.The main research contents and results of this study are as follows:(1)Construction and detection of lentivirus-mediated PNX silenced cell linesRNAi lentiviral vector targeting PNX and negative control lentiviral vector were constructed,and a three-plasmid packaging system was used to produce lentivirus.Subsequently,lentivirus was used to infect hypothalamus GT1-7 cells and pituitary L?T2 cells derived from mice and then screened by puromycin.The results showed that the RNAi lentiviral vector and negative control vector were successfully constructed.The average titer of RNAi lentivirus targeting PNX was 7.67×10~8 TU/m L and the average titer of negative control lentivirus was 8.66×10~8 TU/m L.The above two virus titers were qualified and there was no significant difference.After infecting cells with lentivirus,the hypothalamic PNX silenced cell line(GT1-7-sh PNX)and the pituitary PNX silenced cell line(L?T2-sh PNX)were successfully selected.Quantitative real-time PCR and Western blot were used to detect the expression of important reproductive genes in PNX silenced cell lines,and the"off-target effect"of RNA interference technology was eliminated by rescue experiments.The results showed that:1)PNX silenced cell lines GT1-7-sh PNX and L?T2-sh PNX had significantly decreased the protein and m RNA levels of PNX,which confirms the silencing effect of RNAi lentivirus targeting PNX;2)The m RNA levels of Gn RH and GPR54 genes of GT1-7-sh PNX were significantly down-regulated while there was no significant difference in the expression of reproductive genes such as LH and FSH of L?T2-sh PNX;3)The false positives caused by the"off-target effect"were eliminated by rescue experiments,which further confirmed that endogenous PNX could regulate the reproductive development process at the hypothalamus level.(2)Effects of lentivirus-mediated PNX silencing on puberty development in female micePrevious part of this research confirmed the inhibitory effect of endogenous PNX silencing on reproductive genes at the hypothalamic level.This study explored the effects of endogenous PNX silencing on the development of adolescence from the individual level.After injection of RNAi lentiviral particles targeting PNX in the lateral ventricle of prepubertal female mice,the hypothalamus reproductive gene expression,initiation time of puberty and uterine and ovarian development were measured.The results showed that:1)Post-injection of PNX-targeted RNAi lentivirus resulted in significant down-regulation of PNX protein and m RNA levels in the hypothalamus of mice,indicating that endogenous PNX was significantly silenced;2)PNX silencing significantly delayed the vaginal opening time and reduced the uterine and ovarian organ index in mice.At the same time,morphological analysis showed that PNX silencing significantly reduced the endometrial thickness and the number of Antral follicle in mice.This indicated that endogenous PNX silencing delayed the initiation of puberty and inhibited gonad development;3)PNX silencing significantly inhibited the m RNA levels of Gn RH,kiss1,GPR54 genes in the hypothalamus;4)PNX silencing caused a reduction in Gn RH hormone levels in the serum of mice.Meanwhile,the hormone levels of LH,FSH and estrogen were reduced.In summary,PNX silencing could inhibit the synthesis and secretion of Gn RH in hypothalamus,and then inhibit the synthesis and secretion of pituitary LH and FSH,and ultimately inhibit the pubertal development of female mice.This study confirmed that endogenous PNX silencing can inhibit the Gn RH synthesis and secretion in the hypothalamus,thereby interfering with the endocrine environment of the hypothalamic-pituitary-gonadal axis and ultimately inhibiting the initiation of puberty and reproductive development.This study indicated that maintaining physiological levels of endogenous PNX played an essential role on the reproductive development and also layed the foundation for further elucidating the reproductive regulation mechanism of PNX in mammals,which could help to further understand the human reproductive development process and provide theoretical basis for the treatment of reproductive diseases.