The Mechanisms Of The Effects On Endocrine Axes And Asthenospermia Caused By Aluminum Exposure

Aluminum as a kind of common environmental pollution metal,can damage the nervous and reproductive system.But most of researches focused on only single tissue or organ,without analyzing how their connectors,the endocrine axes are influenced by aluminum exposure.By using the golden hamster,a special photoperiod animal,this research designed a series of experiments to observe how circadian controlled endocrine axes is affected and further caused the damage of reproductive system by aluminum exposure.Methods: Firstly,at cultured mouse spermatogonium cell line(SPG-GC1)level,we performed the basic dose test of aluminum exposure.The CCK8 assay was conducted to evaluate the proliferation of the spermatogonium cell and the and wound healing assay was used for testing the migration ability of spermatogonium cell.Secondly,in the animal level,wild type male golden hamsters were used to established 3 pairs experimental groups in short day(SD: L8:D16,8 hours of light: 16 hours of darkness)and long day(LD: L16:D8,16 hours of light: 8 hours of darkness)condition: 1)the control group of short day responder(SDR)hamster in the short day condition(SDR-Ctr)and the aluminum exposure group of short day responder hamster in the short day condition(SDR-Al);After SD condition,some animals were switched back to long day condition,2)the control group of SDR hamster switched back to the long day condition(LDR-Ctr)and the aluminum exposure group of SDR hamster switched back to the long day condition(LDR-Al);3)the control group of SD nonresponder hamster switched back to the long day condition(LDNR-Ctr)and the aluminum exposure group of SD non-responder hamster switched back to the long day condition(LDNR-Al).After the completion of the modeling,the tissue samples of blood,testis and epididymis were collected.The sperm in hamster epididymis were observed under microscope for morphology check and the nucleus condensation in sperm of the Al exposure hamsters were noticed.Because the poly ADP-ribose polymerase,PARP was known to be closely related with the nucleus condensation,we further detected the level of Mono-ADP ribose(MAR)of intracellular proteins in cultured spermatogonia cell(through MAR fluorescent probe and Western-blot).We further detected the major hormone level in serum and testis,such as androgen,estrogen and progesterone,sugar/salt cortical hormone,and thyroid hormone,by high performance liquid chromatography-mass spectrum(HPLC-MS/MS).We also performed strand specific RNA-seq on testes of hamsters in each group followed bioinformatic analysis including the differential gene expression,gene ontology(GO),pathway,and weighted correlation network analysis.For parts of the differentially expressed genes,real-time PCR was conducted for the verification.Results: In cultured SPG-GC1 cells,aluminum exposure inhibits the proliferation and migration.With the increase of the aluminum chloride concentration,the inhibition became more obvious.By checking the morphology of the sperm,we found that aluminum exposure resulted in higher deformity rate and the nucleus condensation.In cultured mouse spermatogonia cell(SPG-GC1),the cellular MARylated proteins were increased and the condensation dots of MARylation in cells were also observed after Al exposure.The assay of hormone levels in hamster blood and testis demonstrated that aldosterone in serum and corticosterone in testis of SDR-Al group were significantly lower than those of SDR-Ctr group.The dihydrotestosterone,progesterone,and transandrosterone in testis of LDR-Al group were significantly lower than those in LDR-Ctr group.The estriol and estrone in testis of LDNR-Al group were significantly lower than those of LDNR-Ctr group,but trans-androsterone in serum of LDNR-Al group was significantly higher than that of LDNR-Ctr group.RNA-seq and Real-time PCR validation found that the Tfg gene was significantly down-regulated in testis of LDNR-Al group comparing to LDNR-Ctr group.WGCNA analysis indicated that a MEthistle2 transcript cluster module in testis was highly related with the significant changes(SDR-Al vs SDR-Ctr)of serum aldosterone and testis corticosterone;and a MEcoral transcript cluster module in testis was highly related with the significant changes(LDR-Al vs LDR-Ctr)of testis dihydrotestosterone and trans-androsterone.GO/Pathway analysis discovered that the genes in these cluster modules related with spermatogenesis,cellular protein modification,macro molecular synthesis,regulation of defense response,etc.In summary,aluminum exposure has the certain toxicity to reproductive system,which inhibited the proliferation and migration of spermatogonia cell,increased the sperm malformation,sperm nucleus condensation and protein MARylation level.It also affected the endocrine hormones and down-regulated Tfg gene expression.We also discovered that the significantly changes of aldosterone in serum,corticosterone,dihydrotestosterone,and trans-androsterone in testis were highly correlated with certain transcript cluster modules expressed in testis.These findings provided important clues for searching genes and key mechanism related to the disorder of endocrine caused by Al exposure.They also implied possible new targets for diagnosis or treatment of aluminum exposed caused the Asthenospermia.