The Establishment Of Male Reproductive Toxicity Testing System Based On Three Dimentional Rat Testicular Cells Co-culture Model

Objectives Reproductive and developmental dysfunction is one of the major public health problems that seriously affect human health.However,the current reproductive and developmental toxicity evaluation methods are not consistent with the "3R" principle and can't meet the needs of safety or risk assessment.It is a trend to establish a better method which can integrate modern biological technology,provide more toxic mechanism information and be transformed to benefit human.Testis is the most sensitive organ of male reproductive toxicity which play the main role of male reproduction system such as spermatogenesis and production of sex hormones.Therefore,the testis has become the most important organ when consider establishing an in vitro male reproductive toxicity testing method.Now various in vitro testicular cells toxicity test methods have been established and the main problems include: the model included one type cell can only be used to study the specific toxicity and mechanism on this cell and difficult to be an testicular toxicity evaluation method;the effect of compounds on differentiation of spermatogenic cells or spermatogenesis can't be observed.Therefore,the establishment of the model includes major types of testicular cells such as Sertoli cells,Leydig cells and spermatogenic cells which can observe spermatogonial cell differentiation in recent years has become a research hotspot.In recent years,two significant advances in the field of testicular cell biology had been made,one is the extracellular matrix was successfully used as a scaffold in testicular cell short time culture which can maintain cell function better;the other is KSR(knock Out serum replacement)can promote the proliferation of embryonic stem cells and spermatogonial cells and differentiation from the primary cells to primary spermatocyte.Based on these,we speculated that if integrated the two results and the optimized culture conditions,it is possible to establish a stable culture systen which can not only observe the testicular cell function but also spermatogenic cell differentiation.The present idea is to establish an improved three-dimensional(3D)rat testicular cell co-culture model including Sertoli cells,Leydig cells and spermatogenic cells which used the extracellular matrix as a 3D scaffold,KSR as a serum substitute followed by selection of culture methods,extension of culture period and optimization of culture conditions based on the existed 3D rat testicular cell co-culture model.The application of the model as an in vitro testicular toxicity testing system will be validated that can lay the foundation for the establishment of an in vitro testicular toxicity testing system.The testing system based on the improved 3D rat testicular cell co-culture model can be used to evaluate the effects of various reproductive toxic chemicals on the function of the main somatic cells as well as spermatogonial cell differentiation and even spermatogenesis and to investigate the specific toxicant mechanisms in testes.The purpose of the present study was to lay the foundation for the establishment of an in vitro testicular toxicity test system in rats which can be used to monitor the function of the main somatic cells as well as spermatogonial cell differentiation and provide more information of the toxic mechanism.Methods1.The establishment of the 3D rat testis cells co-culture model: the testes from 6-day-old newborn male pups were decapsulated and sequential enzymatic digestion.a single-cell suspension was yielded that was subsequently filtered through a 100-?m screen mesh and re-suspended in DMEM/F12 culture medium containing 10% knockout serum replacement(KSR)(as a substitute for conventional fetal bovine serum(FBS))and then plated into 35-mm cell culture-treated dishes at a 105 cells/ml density in medium.An extracellular matrix(ECM)overlay was applied directly to the medium 30 min after seeding,with a final concentration of 200 ?g/ml medium.Leydig cell function(testosterone level and Star expression),Sertoli cell function(lactate levels and Abp expression),and spermatogonial cell meiosis-specific genes Stra8 and Sycp3 expression were selected as the endpoints of establishment of the improved 3D model.Secondly,the culture conditions were optimized by selecting different concentrations of ECM and human chorionic gonadotrophin(HCG),different densities of cells,different age of the suckling rats,different period of co-culture cycle.Finally,the advantages of the model under the condition of KSR and ECM were studied through comparing the effects of KSR+ECM with FBS+ECM(3D culture method)and KSR-ECM(three-dimensional(2D)culture method)on the endpoints used in the tests.2.Preliminary validation of the 3D rat testis cells co-cultured model as an in vitro testicular toxicity testing system: in order to validate the application of the model to be used as an in vitro testicular toxicity testing system for identification testicular toxicants and providing insight into the underlying mechanisms of testicular toxicity,two male reproductive toxicants known as bisphenol A(BPA)and dibutyl phthalate(DBP),which are recommended by the European Union Reference Laboratory for Alternatives to Animal Testing(ECVAM)as the references,were selected as the positive drugs,appropriate exposure time(8,16 and 24 days)and exposure dose(BPA: 0,17.85,37.5and 75?M;DBP: 0,6.25,12.5 and 25?M)were chosen.3.Subchronic toxicities of HZ1006 in rodents,Beagle dogs and testicular toxicity study based on 3D rat testis cells co-cultured model: in the present studies,the repeated dosage toxicity of HZ1006 in Beagle dogs and Sprague Dawley(SD)rats was identified.Dogs and rats received HZ1006 orally(0–80 and 0–120 mg/kg/day,respectively)on a continuous daily dosing agenda for 28 days following a 28-day and 14-day dosage-free period rspectively.The in vitro male reproductive toxic effects of HZ1006 and the mechanism of toxicity were clarified based on the 3D rat testis cells co-cultured model.According to the characteristics of HZ1006,the appropriate exposure duration(8,16 and24 days),dosage(0?3.125?6.25?12.5 and 25?M)and suitable for detection toxic endpoints were selected.Results1.The establishment of the 3D rat testis cells co-culture model: the results showed that the survival rate of testis cells was above 95%.The results of lactate level,testosterone level and spermatogonial cells meiosis-specific genes expression showed that lactate level and testosterone level were stable during 30 days and meiosis-specific genes were detected firstly from d24 to d26,which indicted the 3 main testicular cells(Sertoli cells,Leydig cells and spermatogenic cells)grew well in the established model and keep a good cell function.It was also found that 200?g/ml ECM,5×105/ml cells density,10IU/ml HCG,6-day-old newborn male rats and 26 days culture period could be used as the suitable experimental conditions for the experiment.The results indicated that the viability and proliferation of cells were better,testosterone and lactate levels were significantly increased cultured in KSR+ECM than that in FBS+ECM or KSR-ECM.The co-cultured cells grew well,and intercellular junctions were successfully formed in KSR+ECM.In addition,meiosis-specific markers(the Sycp3 and Stra8 genes and proteins)were detectable firstly from d24 to d26 in the co-culture system with KSR+ECM but not with FBS+ECM or KSR-ECM.2.Preliminary validation of the 3D rat testis cells co-cultured model as a of reproductive toxicity test system: The main findings of the present study were the significant changes in the detected endpoints of the model when exposed to BPA and DBP at the concentrations used in vitro,and these changes were dose and time dependent.The endpoints used in the validation tests included cell viability,Leydig cell function(testosterone level and Star expression),Sertoli cell function(lactate levels and Abp expression)and spermatogonial cell meiosis-specific genes expression.The results also showed that the most sensitive target cells of BPA and DBP in the 3D rat testis cells co-cultured model were Sertoli cells and Leydig cells which indicated that Sertoli cells and Leydig cells were first affected when the model exposed to BPA and DBP respectively followed by the dysfunction of other testis cells.3.Subchronic toxicities of HZ1006 in rodents,Beagle dogs and testicular toxicity study based on 3D rat testis cells co-cultured model: the results of HZ1006 in vivo showed that a certain dose range(40-80mg/kg)of the compound in vivo after a period of time can cause male reproductive toxicity in dogs,such as the testis,epididymis and prostate pathological changes.The testicular pathological changes included: the numbers of seminiferous tubules decreased in dogs belong to 80 and 40 mg/kg dose groups meanwhile tubule lumen became smaller and spermatogenic cells reduced in numbers and types when compared with the control group.Only spermatogonia,some primary and secondary spermatocytes were observed and there were no mature sperms compared with the control group.There were no obvious male reproductive toxicity-related changes in the rats in the subchronic toxicity study.In vitro tests showed that the IC50 values of HZ1006 after 24 h,48 h and 72 h exposure was 325.19,277.17 and 149.64?M respectively.The in vitro results showed inhibition of cell proliferation,reduced testosterone level and decreased spermatogonial cell meiosis-specific genes expression when the in vitro 3D co-culture rat testicular cell model was exposed to HZ1006 for some time.The effects of HZ1006 on cell proliferation and testosterone secretion were dose and time dependent,and spermatogonial cell meiosis-specific genes expression were also dose dependent.Decrease of Ar protein level and Ar target gene PSA expression accompanied by inhibition of Hdac6 expression were also found after HZ1006 exposure to the in vitro model.Conclusions1.We established of an improved 3D co-culture rat testicular cell model using KSR and ECM which can monitor the function of the main somatic cells as well as spermatogonial cell differentiation and even spermatogenesis that can be used as a 3D co-culture model to research the main biological function of rat testis.The in vitro 3D co-culture rat testicular cell model was reported firstly.2.The results of validation tests indicated that the 3D co-culture rat testicular cell model can be used to detect various toxic effects of BPA and DBP on testicular cells,determine the target cells more accurately and afford more information for mechanism research which validated the improved 3D co-culture rat testicular cell model can be as a male reproductive test system used for research.3.The results of HZ1006 in vivo showed that a certain dose range(40-80mg/kg)of the compound in vivo after a period of time can cause male reproductive toxicity in dogs,such as the testis,epididymis and prostate pathological changes.But there were no obvious male reproductive toxicity-related changes in the rats in the subchronic toxicity study.HZ1006 can induce testicular toxicity in vitro which was consistent with the observed toxicity changes in vivo,and Leydig cells maybe its target cells,spermatogenesis dysfunction maybe followed by the inhibition of testosterone.Further mechanism studies suggested that the reproductive toxicity of HZ1006 may be induced by the inhibition of Hdac6 which inhibited protein expression of Ar and its target gene Psa.The results of this study provide a new experimental basis for the toxicity evaluation and mechanism study of HZ1006,also confirmed the applicability of the proposed model as a male reproductive toxicity test system.4.The present study showed that the 3D co-culture rat testicular cell model has the potential to become a new testicular toxicity test system to be used to test toxic characters and mechanism of new compounds and it has a good application prospect,meanwhile it is necessary to do more research on the model.