Mechanism Of Dysfunction Of Spermatogenesis In Mice Caused By CdSe/ZnS Quantum Dots

BackgroundFluorescent quantum dots(QDs)are highly promising nanomaterials for various biological and biomedical applications because of their unique optical properties,such as robust photostability,strong photoluminescence and size-tunable fluorescence.Several studies have reported the in-vivo toxicity of QDs,but their effects on the male reproduction system have not been examined.It is well known that autophagy,a cellular stress response to degrade damaged components,can be activated by many nano-particles.Increased levels of autophagy can cause apoptosis,nuclear degeneration and impaired DNA damage repair.DNA double-strand breaks(DSBs)are a highly toxic and dangerous kind of lesion.They have been shown to cause genomic instability and cell death.The main pathway responsible for DSBs repair in meiosis is homologous recombination(HR).Spermatogenesis refers to the process in which the cells undergo a series of divisional proliferation,differentiation and deformation,and finally form intact sperm in the seminiferous spermatogonium.This progress can be divided into three periods: mitosis,meiosis and sperm formation.The spermatogonia proliferate through mitosis and some of the cells differentiate into primary spermatocytes,completing the first meiosis to form secondary spermatocytes.Then the second meiosis is completed to form sperm cells.Then the sperm cells are deformed to form mature sperm.In the male reproductive system,primary spermatocytes are susceptible to external stimuli.When the meiosis process is blocked,it will directly cause male spermatogenic dysfunction.ObjectiveOur aim is to explore the effects of different concentrations of novel nanomaterials CdSe/ZnS quantum dots(QDs)on the physiological state,reproductive system structure and spermatogenesis of mice and related molecular mechanisms,which provided a reliable theoretical basis for the safety of clinical application.Methods1.Drug detection: Physical tests were used to detect physical properties of QDs,such as particle morphology,diameter,emission wave length and zeta potential distribution.Electron microscopy was used to observe the entry of drugs into mouse testis tissue.Cellular immunofluorescence and flow cytometry were used to observe whether QDs enter the cell and quantize the amount of QDs.2.Animal experiment: 60 healthy and sexually mature male CD1 mice were selected and bred in the environment of 18-25 degrees centigrade and 40%-50% humidity.The circadian rhythm was set to alternate for 12 hours.According to the dose measurement of tail vein injection,the mice were randomly divided into 3 groups: high dose group(2 nM QDs/ PCS),low dose group(0.2 nM QDs/ PCS)and blank control group(equal amount of normal saline).20 mice in each group were given the tail vein injection of 200 ul QDs for 5 days.The mice were weighed on days 1,14,30,60 and 90,respectively.Serum sex hormones(T,FSH,LH)were determined from peripheral blood.The content and distribution of QDs were detected in testicular tissue with Cd as the representative.The ratio of testis to epididymis was calculated by weighing testis and epididymis.Epididymis was taken for sperm count.TUNEL staining of paraffin tissue was used to detect apoptosis.Immunofluorescence was used to detect the expression of γ-H2 AX protein in spermatocytes in the convoluted tubule.The injury of testicular tissue was observed by HE staining.Western blot was used to analyze the expression of protein related to autophagy,apoptosis and DNA repair.RT-PCR technology was used to detect the expression of genes related to HR pathway.Spermatocytes were counted and classificated by testicular spreading and immunofluorescence staining.The proportion of spermatocytes in sub-stages of the meiosis I and the proportion of spermatocytes which stopped in the pachytene stage for DNA damage repair were counted.In addition,20 healthy and sexually mature male CD1 mice were introduced.After purchase,they were reared in the environment of 18-25 degrees centigrade and 40%-50% humidity.The circadian rhythm was set to alternate for 12 hours.A self-controlled model was established.10 mM autophagy inhibitor(40 ul)6-amino-3-methyladenine(3-MA)was injected into the right testicle in situ,and normal saline was injected into the left testicle in situ.Western blot was used to analyze of the expression of autophagy related protein.RT-PCR was used to detect the expression of genes related to HR pathway.The relationship between autophagy and DSB repair pathway was further analyzed by using the testis spread to analysize the proportion of spermatocytes in sub-stages of meosis I and the proportion of spermatocytes in pachytene stage for delayed DNA damage repairing.3.Cell experiment: A suitable number of GC-2 cells were inoculated in a six-well plate,divided into high-dose group(2 nM/well),low-dose group(0.2 nM/well),blank control group(added the same amount of normal saline).The cells were treated with 50 ul corresponding concentration gradient of CdSe/ZnS.After 6 hours,the drug was observed in the cells,and the expression of autophagy related protein and RAD51 was determined by Western blot.A DNA damage model was established by radiation.The PI3 K inhibitor 3-MA and non-homologous terminal junction pathway inhibitor,DNA protein kinase inhibitors(PKi),were added into the cell culture medium and used to detect the expression of RAD51,and γ-H2 AX by immunofluorescence technique after 1 hour and 8 hours,respectively.RT-PCR was used to detect the expression of genes involved in HR pathway.Results1.The content of quantum dots in mouse testis was time-dose-dependent.QDs had no significant effect on body weight of mice.On the 14 th and 30 th day,QDs reduced the quality of testicular and epididymis and decreased serum T levels,which had a time-dose-dependent effect.2.On the 14 th day,the high dose of QD damaged the testicular structure and made the sperm concentration on day 60 decreased.With the metabolism of QDs after 90 days in the epididymis,the effect gradually disappeared.On the 14 th day,the number of TUNEL-positive spermatocytes in the seminiferous tubules increased.3.On the 14 th day,and the number of γ-H2AX-positive cells increased.On the 14 th and 30 th day,the high dose QDs increased the proportion of spermatocytes arrested in the meiotic phase I in the testis tissue,and the number of DSB-positive cells increased in the pachytene stage,which showed that the meiosis process was disturbed.4.On the 14 th and 30 th day,the expression of genes related to the HR pathway in the testis tissue and GC-2 cells,such as Rad51,was down-regulated.After the 60 th day,the difference above was not detected.5.On days 14 and 30,QDs increased the expression of autophagy-associated protein LC3 II and ATG7 in testis tissue,and decreased the expression of P62,suggesting an increase in autophagy level.On the 60 th and 90 th days,no difference between groups was detected.Elevated levels of autophagy were also observed in GC-2 cells dosed for 12 hours.6.PI3 K inhibitor 3-MA can significantly improve the HR pathway by inhibiting the increased autophagy level induced by QDs,which can restore the ability of DSB repair and recover the spermatogenetic function.ConclusionHigh-dose QDs affect testicular and epididymis quality,destroy testicular tissue structure,decrease sperm concentration and increase apoptosis and DNA damage in spermatocyte with a time-dose-dependent effect.QDs can reduce the expression of some genes in HR pathway such as Rad51 by increasing the level of autophagy,which impairs the main pathway of DSB repair and then primary spermatocytes are largely arrested in the pachytene stage of meiosis I,affecting the overall meiosis process.Inhibition of autophagy can significantly improve the inhibition of homologous recombination pathway,allowing DSB repair to proceed smoothly.Therefore,the damage to the reproductive system of QDs is time-and-dose-dependent and can be significantly improved by autophagy inhibitor 3-MA.