The Effects And Mechanism Of M~6A Demethylase Of FTO On Regulation Of GABA_AR On Mn-induced Precocious Puberty Of Female Rat

Objective:Manganese?Mn?as an abundant environmental trace element that is essential for normal biological systems,both deficiency and excess of this mineral may cause adverse health effects.In addition to neurotoxicity,the lack or excess of Mn may also affect the normal function of mammalian reproductive system.Epidemiological studies show school-age children near the manganese-iron mine who overexposure to Mn might trigger hormone disorder and have a trend of precocious puberty.Previous studies on laboratory animals have indicated the endocrine-disruptive and puberty-advancing effects of Mn,however,the mechanism remains unclear.One of the performance of neuroendocrine behavior of puberty onset is that increased secretory of GnRH neurons in the preoptic area-anterior hypothalamus?POA-AH?transforms into LH pulse secretion,which causes the functional and morphological development of sexual organs.For mammals,GnRH neurons are highly active at birth,and then transforms into resting state within a few hours after birth.To initiate puberty,the inhibitory signal must be removed.Gamma-amino butyric acid?GABA?as the dominant inhibitory neurotransmitter in brain that plays a critical role in regulating pubertal development,GABA produces inhibition function in GnRH secretion by acting on GABA_A receptor?GABA_AR?in the hypothalamus.GABA_AR as a kind of ligand dependent ion channel include complex subunit,it reports that GABAA??1,?2,?3and?2?subunits expressed in GnRH neurons.Nitric oxide?NO?has a wide range of biological activities,studies demonstrate that NO is responsible for the release of GnRH.Studies have shown that GABAAR play an regulation role in the synthesis of nitric oxide synthetase?NOS?which is responsible for NO produce.At present,there are three different subtypes of NOS have been found,NOS1 is important in nervous system.N6-methyladenosine?m6A?is currently one of the most abundant and conservative internal modifications in eukaryotic mRNA,it exists in nearly all eukaryotic RNAs.Dynamic and reversible methylation of mRNA has opened up a new field for study of post transcriptional gene regulation in eukaryotes.Fat mass and obesity associated protein?FTO?,as the first recognized m⁶A demethylase,which is widely expressed in mammalian brain.As the"reader"protein of m6A,YTHDF1,YTHDF2,and YTHDF3can bind to the m⁶A site of target transcripts to promote its degradation or translation.M6A has been shown to regulate mRNA metabolism and translation,so that different mRNA will go to different fate.The first aim of this study is to observe the effect of Mn on puberty onset and the secretion of GnRH for juvenile female rats,then analyze the role of GABAAR on it,finally clarify the role and mechanism of FTO in Mn-induced early puberty initiation through GABAAR,providing a new theoretical and experimental basis for study the mechanism of Mn-induced reproductive toxicity.Methods:1.The day after weaning?PND21,50±5g?,female dams were randomly assigned to four groups according to weight?N=12 per group?.Rats in the first to fourth groups were administrated with 0.9%saline,2.5,5,and 10 mg/kg MnCl2?0.2 mL/25g BW/day?by oral gavage.All rats were administrated during PND 21-32.The weight of rats and the vaginal opening?VO?,the first estrus day?E1?,and the first diestrus day?D1?were examined daily.All animals would be sacrificed on D1 by decapitation,the trunk blood was collected,the uterus,ovary,and hypothalamus were removed and recorded.GABAAR protein subunits,GnRH mRNA,GnRH peptide and NO content were detected.Two rats in each group were perfusion with 4%paraformaldehyde for hematoxylin-eosin?HE?analysis nissl staining and immunofluorescence staining analysis.The levels of GnRH in hypothalamus,LH,FSH and INH-B in serum were detected by ELISA.The content of Mn in hypothalamus,ovary and uterus was detected by ICP-MS.In vitro:the dose of Mn in GT1-7 cells was determined by CCK-8,GT1-7 cells were divided into four groups,which treated with 0,50,100 and 200?M MnCl₂ respectively,and the cells were collected after 24 h after Mn exposure,and the fluorescence expression level of GABA_AR was detected.2?Rats?PND21?as mentioned above were divided into the control,Mn-only,isog-only,and isog+Mn group?N=12 per group?.Rats in control and Mn-only groups were both subcutaneous injection?s.c.?treated with 0.9%saline;rats in isog-only and isog+Mn groups were both s.c.administered of 1 mg/kg?1 mg/mL?isog.Two hours later,rats in control and isog-only groups were oral administration with 0.9%saline;rats in Mn-only and isog+Mn groups were oral administration with 10 mg/kg MnCl2.Griess Reagent colorimetric method was used to determine NO content,and western blotting was used to determine NOS1 protein content.Other procedures were similar to above.ACSF,10?M BIC,5?M isog and 400?M MnCl₂ were perfused into brain slices containing POA-AH of female rats?PND21?.The raw spike would be recorded and analysis by MED64.The electrical signal characteristics of MnCl₂ in the POA-AH were detected.3?After Mn treatment,dot blot assay was employed to detect total RNA m⁶A level of GT1-7 cells,MeRIP-qPCR assay was employed to determine mRNA m⁶A level of GABA_A?2,and western blot assay was used to detect the protein expression of m⁶A“eraser”enzyme and"reader"enzyme.Determining which siRNA is efficient and selecting a appropriate dose and transfection time.siNC cells were divided into the control and Mn-treatment group,siFTO cells were also divided into that two groups,the protein levels of GABA_A?2,mRNA m⁶A levels of GABA_A?2,GnRH mRNA levels were determined.Female SD rats?PND21,50±5?were divided into 5 groups according to body weight?n=10/each?.They are?1?control group,?2?10 mg/kg Mn group,?3?MA2+Mn group,?4?isog+Mn group,?5?MA2+isog+Mn group.MA2 is a highly selective inhibitor of FTO.Rats in groups?1?,?2?and?4?received stereotactic injection of1%DMSO into the third ventricle on PND21,and rats in groups?3?and?5?received stereotactic injection of MA2.After two days of recovery,rats in groups?1?,?2?and?3?received subcutaneous injection?S.C.?equal dose of 0.9%saline,while rats in groups?4?and?5?received S.C.of 1 mg/kg isog.Two hours later,rats in group?1?were given the same dose of 0.9%saline by gavage,and rats in groups?2?,?3?,?4?and?5?were given 10mg/kg MnCl2 by gavage for 10 days?PND23-32?.The follow-up detection refer to the first part of animal experiments.Results:With the increase of Mn,the VO and E1 were significantly earlier than controls.The body weight in 5 mg/kg and 10 mg/kg Mn group were significantly lower than these in control group in PND 35 and D1.The organ coefficient of uterus was significant higher than these in control group.There was no significant effect on the uterine morphology,endometrial thickness index and myometrial thickness index between these groups.With increase dose of Mn,many small antral follicles along with some unhealthy corpora lutea were observed in ovaries histopathological,but there was no significant effect on the number of corpus luteum.GnRH in the hypothalamus,LH,FSH and INH-B in serum were increased with the increase of Mn.The content of Mn in hypothalamus also increased with Mn increase.There was no significant difference of Mn content in uterus and ovary.He staining and Nissl staining showed that the pathological damage of hypothalamus was serious in 10 mg/kg Mn group.The expression of GnRH mRNA in hypothalamus increased with the increase of Mn.CCK-8results showed that with the increase of Mn and the prolongation of time,GT1-7 cell activity decreased continuously,with a dose-time response relationship.According to the OD,the dose of 200?M was selected as the maximum Mn group and 24 hours as the exposure time.The relative expression level of GnRH mRNA in GT1-7 cells increased with the increase of Mn concentration.2?There was no significant effect on the mRNA expression of GABA_AR??1,?2,?2,and?3?in GT1-7 cells and hypothalamus after Mn exposure,however,the GABAAR protein level and the fluorescence intensity of GABAA?2 were decreased with the increase of Mn exposure.The electrical signal from the POA-AH of acute brain slice?PND21?detected by MED64 found that electrical signal of GnRH neuron showed a distinctive pattern with a transient activation?about 1 s?followed by a gradual relative quiet period?with just a few small spikes?that lasted until the record finished.Pre-treatment of GABAAR agonist isog delayed Mn-induced VO,E1,and D1,increased body weight,and reduced organ coefficient of uterus and ovary.Compared with the control group,the GnRH,LH,FSH and INH-B hormones in the Mn group were significantly higher,and the GnRH,LH and INH-B levels in the isog+Mn group were significantly lower than those in the Mn group.Compared with the control group,the GnRH mRNA level,NOS1 protein level and NO concentration in the hypothalamus of Mn group increased significantly,while compared with Mn group,these in isog+Mn group were decreased significantly.3?With the increase of Mn dose,the total level of m⁶A,and m⁶A in GABA_A?2mRNA of GT1-7 cells both decreased significantly,and the expression of FTO protein increased significantly.However,there was no significant difference in ALKBH5,YTHDF1,YTHDF2,and YTHDF3 between each group.FTO protein in the hypothalamus of rats exposed to Mn was also increased significantly.Compared with the control group of si NC cells,the level of GABA_A?2 protein and GABA_A?2 mRNA m⁶A in Mn-treatment group were decreased significantly,but there was no significant difference between the Mn-treatment group and control group of siFTO cells.Compared with control group of siNC,the above indexs were significantly higher in control group of siFTO.Compared with the control group of siNC group,GnRH mRNA in the Mn-treatment group of siNC cells were increased significantly,however,there is no significant difference between the Mn-treatment group and control group of siFTO cells,but it was decrease in Mn-treatment group of siFTO cells than Mn-treatment group of siNC cells.Compared with the control group,the VO,E1 and D1 were significantly earlier in 10 mg/kg Mn group,but these were delayed in the MA2+Mn,isog+Mn,and MA2+isog+Mn groups,especially in MA2+isog+Mn group that was obvious delayed.Conclusion:1 Excessive overexposure to MnCl2 can lead to early initiation of sexual development in female rats.2 Mn stimulated the production of NO and increased the production and release of GnRH hormone by reducing the expression of GABAAR protein or interfering with its signal function in the POA-AH of female.3 Mn can down regulate the expression of GABA_AR by reducing m⁶A site of GABA_AR mRNA through FTO.It was found that Mn could regulate the initiation of sexual development in female rats by reducing the level of m⁶A of GABA_AR mRNA through increasing the expression of FTO,consequently increase NOS/NO by decrease the expression of GABAAR protein in the POA-AH,finally increase the production and release of GnRH hormone.