The Role Of YTHDC2 Regulation Of Notch1 In Spermatogenesis Abnormalities In Male Rats Induced By Prepubertal DEHP Exposure

Objective:Di-2-ethylhexyl phthalate(DEHP)is a plasticizer that is widely used in various consumer products,such as flooring and wall coverings,food containers,and medical devices.However,excessive exposure to DEHP can affect male fertility by causing degeneration of the testicular tissue and reducing sperm quality.The effects of excessive exposure in childhood may persist into adulthood.Recent studies have found that the N~6-methyladenosine(m~6A)modification of mRNA plays a very important role in the reproductive system.The m~6A modified recognition protein YTHDC2 is highly expressed in the testis and plays an important role in spermatogenesis.Therefore,this study aims to investigate the organic changes after exposure to DEHP in prepubertal male rats and the changes of sperm in adult male rats.We also explore the role of YTHDC2 in Notch signaling abnormalities and provide a theoretical basis for revealing the effects of DEHP exposure in prepubertal male rats on the organic lesions of testicular tissues in adolescence and sperm quality in adulthood.Methods:A total of 48 clean-grade male Sprague Dawley(SD)rats weaned on Postnatalday21(PND21)were selected.The rats were randomly divided into 4 groups according to body weight,with 12 rats in each group.The DEHP model was established by intragastric administration.The first group(the control group)was given corn oil.The second to fourth groups were low,medium and high dose DEHP stained groups,which were given DEHP dissolved in corn oil at 5,50 and 500 mg/kg,respectively.The body weight and anogenital distance(AGD)were recorded every three days.After 12 days of continuous exposure,rats in each group were divided into two parts.In the first part,a total of 4 rats were fed with regular feed until 60 days.In the second part,a total of 8animals were collected immediately after the poisoning.After the first part was raised to60 days,the rats were sacrificed and the epididymis tissue was isolated.The bilateral epididymis tissues were cut into three segments and placed in 37℃normal saline to dissociate the sperm by themselves.The sperm suspension was prepared for sperm count and morphology observation.After the second part of the exposure,6 rats were taken from each group,the testis and epididymis tissues were separated and weighed,and then the protein and mRNA were extracted.Two rats from each group were perfused with 4%paraformaldehyde.The testes was fixed and embedded.Then,the hematoxylin and eosin(HE)staining and glycogen staining were performed on the sections to observe the structural changes of the tissues.The mouse spermatocyte cell line(GC-2spd)was cultured in vitro.The spermatocyte cell line was treated with 0,100,200,400μmol/L of DEHP.The effects of DEHP on cell viability,morphology,mRNA and protein expression levels of YTHDC2 and Notch1,apoptosis and expression sites of YTHDC2 and Notch1 were observed.A cell model of YTHDC2 overexpression was constructed by plasmid transfection and its apoptosis,Notch1 protein expression and mRNA level changes were detected.The Notch signaling pathway was blocked by aγ-secretase inhibitor(DAPT)and apoptosis was detected again.Results:1.In vivo experiments showed that DEHP increased the body weight and AGD relatively slowly.When weighing on the 30th and 33rd days,the 500 mg/kg DEHP group was significantly different from the control group.ELISA results showed that serum testosterone level decreased gradually with the increase of exposure dose,but the difference was not statistically significant.2.The sperm count of rats in the 500 mg/kg DEHP group decreased significantly after adulthood and there were abnormal sperms,including headless,tailless,bicephaly and curled tail sperms.The testis and epididymal organ coefficients of rats decreased after the exposure.With the increase of the exposure dose,the testis organ coefficients of rats in the 500 mg/kg DEHP group were significantly different from those in the control group.The epididymal organ coefficients of rats in the 50 and 500 mg/kg DEHP groups were significantly different from those in the control group.HE and glycogen staining results showed that DEHP caused testicular tissue damage and reduced the number of germ cells.Compared with the control group,the seminiferous tubules of the testes of the rats in the medium-and the high-dose groups were atrophied and germ cell fragments and macrophages were shed.DEHP reduced YTHDC2 and Notch1 mRNA expression and protein levels in the testis.The levels of Notch1 protein in the 50 and 500 mg/kg DEHP groups were statistically significant compared with those in the control group.The expression of Notch1 mRNA,YTHDC2mRNA and protein levels in 500 mg/kg DEHP group decreased significantly compared with the control group.3.In the in vitro experiment,DEHP staining changed the morphology of GC-2spd cells.However,with the increase of the dose of DEHP,the number of spermatocytes decreases and the shape of spermatocytes became rounder.DEHP could increase the apoptosis level of GC-2spd cells.The apoptosis rate of 200μmcame round,gradually losing the original cell state.Flow cytometry results showed that 200μmol/L and 400μmol/L DEHP groups was statistically significant compared with the control group.Notch signaling inhibitor DAPT alone also induced apoptosis of GC-2spd cells,and the level of apoptosis was similar to that of 400μmol/L DEHP group.However,the level of apoptosis increased in the DAPT and DEHP combined intervention group.Immunofluorescence staining results showed that YTHDC2 and Notch1 were co-expressed in GC-2spd cells.DEHP also decreased the mRNA expression and protein levels of YTHDC2 and Notch1 in GC-2spd cells.YTHDC2 protein levels in the three doses of DEHP groups were significantly different from those in the control group.The level of Notch1 protein in 400μmol/L DEHP group was significantly different from that in control group.As for the mRNA expression levels of YTHDC2 and Notch1 in GC-2spd cells,the changes of 400μmol/L DEHP group were the most significant compared with the control group.4.In the GC-2spd cell model overexpressing YTHDC2,the experimental results showed that the expression of Notch1 in the OE-NC+DEHP group was significantly lower than that in the OE-NC group.The mRNA expression and protein content of Notch1 in OE-YTHDC2+DEHP group were increased compared with that in OE-NC+DEHP group,and the difference was statistically significant.Flow cytometry experiments were performed on the GC-2spd cell model overexpressing YTHDC2,and the results showed that the levels of apoptosis in the OE-NC group and OE-YTHDC2 group were similar.The apoptosis level of OE-YTHDC2+DEHP group was lower than that of OE-NC+DEHP group.The apoptosis level of OE-YTHDC2+DEHP+DAPT group was also decreased compared with that of OE-NC+DEHP+DAPT group.Conclusion:1.Prepubertal exposure to DEHP can lead to testicular tissue damage in rats and affect sperm quality in adulthood.2.DEHP can reduce the expression of YTHDC2and Notch1 in testicular and GC-2spd cells.3.DEHP can reduce the level of Notch1 by reducing the expression of YTHDC2 in testis,which leads to spermatocyte apoptosis and thus to sperm abnormalities.