Effect Of GABA-T On Puberty And Reproduction Performance In Female Rat

Objective:?-aminobutyric acid transaminase?GABA-T?participates in?-aminobutyricacid?GABA?catabolism,the paper mainly explored the expression of GABA-T and its genes in the reproductive axis of female rats at different developmental stage,and the effect of inhibiting GABA-T on the puberty and reproductive performance of Sprague-Dawley female rat,which provides a scientific evidence for the regulation of reproduction in female animal.Methods:Female rats at different developmental stage?infancy,10 d?,prepuberty?21 dand 28 d?,puberty?33.67±0.49 d?and adult in diestrus?64.16±0.79 d?)were used to detect the expression of GABA-T in the hypothalamus-pituitary-ovary axis?HPOA?with an immunofluorescence technique and a quantitative real-time PCR?RT-q PCR?method,respectively.Hypothalamic cell was cultured and GABA-T inhibitor?vigabatrin?was added to evaluate the effect on the expression of reproduction-related genes by RT-q PCR.Vigabatrin was intracerebroventricularly?ICV?injected into the hypothalamus of rats at prepuberty?25 d,n=36?and adult in diestrus?approximately 65 d,n=32?,then the change of the vulvar opening?VO?time of 24 rats at prepuberty were observed,and sacrificed when the vulvar of rats were open,12 rats at prepuberty and 12 animals at adult were killed after injection with vigabatrin for 4 h,blood was collected to separate serum,and hypothalamus,pituitary,the left ovary were collected and stored at-80°C,serum hormone levels were measured by enzyme-linked immunosorbent assay?ELISA?,and reproductive-related gene transcription levels of HPOA were measured using RT-q PCR,the right ovary were weighted and observed histological structure.The change of estrous cycle and mating time of the remaining 20 adult rats in diestrus were observed through vaginal smear,and the litter performance and lactation performance of the rats were analyzed.Results:?1?In the hypothalamus,GABA-T was mainly distributed in the arcuate nucleus?ARC?,paraventricular nucleus?PVN?and periventricular nucleus?Pe N?,and the fluorescence intensity of GABA-T was highest in the puberty?P<0.01?.Compared with adulthood,Abat m RNA transcription levels were significantly reduced at infancy,prepuberty?28 d?and puberty?P<0.01?,and significantly decreased at prepuberty?21 d??P<0.05?;Abat m RNA transcript levels at infancy and prepuberty?28 d?were significantly lower than those of at prepuberty?21 d?and puberty?P<0.01?.In the pituitary,GABA-T was mainly located in the adenohypophysis,and the fluorescence intensity of GABA-T was the lowest at puberty?P<0.01?and was the highest at prepuberty?21 d??P<0.01?;Abat m RNA transcription level at the prepuberty?28 d?was the lowest,which is significantly lower than those of at infancy,prepuberty?21 d?,puberty and adult?P<0.01?;there was no significant difference in Abat m RNA transcription level between infancy and puberty?P>0.05?;Compared with infancy and puberty,the transcription level of Abat m RNA was significantly reduced at prepuberty?21 d?and adult?P<0.01?.In the ovary,GABA-T was mainly presented in oocytes and granulocytes,and the fluorescence intensity of GABA-T was the strongest at adult?P<0.05?and weakest at prepuberty?21 d??P<0.05?;but Abat m RNA transcription level at prepuberty?21 d?was the highest?P<0.01?;the Abat m RNA transcript level at infancy was significantly higher than that at prepuberty?28 d??P<0.05?,puberty and adult?P<0.01?,there were no significant difference in the transcription level of Abat m RNA among puberty,adult and prepuberty?28 d??P>0.05?.?2?After vigabatrin was added to hypothalamus cells,in the 25?g/m L and 50?g/m Lgroups,the transcription level of Abat m RNA and Rfrp-3 m RNA were significantly reduced?P<0.01?,and the Gnrh m RNA was increased?P<0.01?.The transcription level of Kiss1 m RNA in the group at the dose of 50?g/m L was significantly increased?P<0.05?,and the transcription level of Gabbr1 m RNA was reduced?P<0.05?.No differences were found in the Kiss1 m RNA and Gabbr1 m RNA in the group at the dose of 25?g/m L?P>0.05?.?3?Prepubertal?25 d?rats injected with vigabatrin?1?g/g and 2?g/g?were significantly delayed the onset of puberty?P<0.05?;compared with the control group,1?g/g and 2?g/g vigabatrin reduced the transcription level of Abat m RNA?P<0.05?in hypothalamus and the concentration of GABA-T?P<0.01?,LH?P<0.01?and P₄?P<0.05?in serum,and Rfrp-3 m RNA transcription level in hypothalamus was increased?P<0.01?;however,Kiss1 m RNA?P<0.05?and Gnrh m RNA?P<0.01?transcription levels reduced in the group at the dose of 1?g/g;and Kiss1 m RNA transcription level in 2?g/g group was significantly reduced?P<0.01?.Ovarian histological sturcture showed that corpus luteum and secondary follicles were both present in the vigabatrin group and the control group,in the 1?g/g group,there were more secondary follicles,and the ovary index group was significantly lower?P<0.05?than that in the control group;the corpus luteum was larger and there were more primary follicles in the 2?g/g group,but no significant change was found in ovarian index?P>0.05?.?4?The estrous cycle of adult rats was disturbed after ICV injected with vigabatrin,there was no change in mating time,gestation period and litter performance,but the weight of the pups decreased?P<0.05?.?5?After 4h ICV injected with vigabatrin,there was no significant difference in thetranscription level of Abat,Rfrp-3,Kiss1,Gnrh and Gabbr1 m RNA in the reproductive axis o at adult and prepubertal?25 d?rats?P>0.05?;but the serum hormone concentration were changed,vigabatrin reduced the concentrations of GABA-T?P<0.01?and LH?P<0.05?,extremely increased the level of FSH?P<0.01?at prepubertal?25 d?of rats.vigabatrin reduced the concentrations of GABA-T and LH extremely at adult animals,?P<0.01?.Conclusion:?1?GABA-T is expressed in the hypothalamus,pituitary and ovary atdifferent developmental stage in female rat;the transcription level of Abat m RNA are changed with the development of the rat.?2?Inhibition of GABA-T in vivo and in vitro both changed the transcription levels of Rfrp-3,Gnrh and Kiss1 m RNA,but the results are opposite between in vivo and in vitro.Inhibition of GABA-T in vivo delays the onset of puberty and changs the concentrations of GABA-T,LH and P₄ in serum.?3?Inhibition of GABA-T results in disordered estrous cycle and and has an effect on breastfeeding performance in adult female rats.?4?Short-term inhibition of GABA-T changes the concentrations of GABA-T,LH,and FSH in the serum but does not affect the transcription levels of Abat,Rfrp-3,Kiss1,Gnrh and Gabbr1 m RNA in HPOA.