The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic aci...The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid(5-ALA).Furthermore,this study aimed to explore the underlying mechanism of the protective effects of 5-ALA.First,we collected and stored mouse testicular fragments in different media,including Hank’s balanced salt solution(HBSS;n=5),Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12(DMEM/F12;n=5),and alpha-minimum essential medium(αMEM;n=5).Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group(P<0.05)and theαMEM group(P<0.01).Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA(0[control],1 mmol l−1,2 mmol l−1,and 5 mmol l−1)to determine the most effective concentration of 5-ALA.The 2 mmol l−15-ALA group(n=3)presented the highest positive rate of spermatogonial stem cells compared with those in the control,1 mmol l−1,and 5 mmol l−15-ALA groups.Finally,the tissue fragments were preserved in HBSS with control(n=3)and 2 mmol l−15-ALA(n=3)under low-temperature conditions.A comparative analysis was performed against fresh testes(n=3)to elucidate the underlying mechanism of 5-ALA.Gene set enrichment analysis(GSEA)for WikiPathways revealed that the p38 mitogen-activated protein kinase(MAPK)signaling pathway was downregulated in the 2 mmol l−15-ALA group compared with that in the control group(normalized enrichment score[NES]=−1.57,false discovery rate[FDR]=0.229,and P=0.019).In conclusion,these data suggest that using 2 mmol l−15-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.展开更多
An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell(SSC)transplantation.Busulfan has been commonly used to develop such a model,but 30%–87%of mice die when adm...An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell(SSC)transplantation.Busulfan has been commonly used to develop such a model,but 30%–87%of mice die when administered an intraperitoneal injection of 40 mg kg^?1.In the present study,hematoxylin and eosin staining,Western blot,immunofluorescence,and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses(20,30,and 40 mg kg^?1)on day 36 or a dose of 40 mg kg^?1 at different time points(0,9,18,27,36,and 63 days).The survival rate of the mice was 100%.When the mice were treated with 40 mg kg^?1 busulfan,dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36.In addition,the gene expressions of glial cell line-derived neurotrophic factor(GDNF),fibroblast growth factor 2(FGF2),chemokine(C-X-C Motif)ligand 12(CXCL12),and colony-stimulating factor 1(CSF1)were moderately increased by day 36.A 63-day,long-term observation showed the rare restoration of endogenous germ cells in the testes,suggesting that the potential period for SSC transplantation was between day 36 and day 63.Our results demonstrate that the administration of two intraperitoneal injections of busulfan(40 mg kg^?1 in total)at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.展开更多
基金funded by the National Natural Science Foundation of China(No.81971759 and No.82171604)the Guangdong Basic and Applied Basic Research Foundation(2023B1515020108)+3 种基金the Science and Technology Program of Guangzhou(202206010089)the Excellent Talents Training Project of The Sixth Affiliated Hospital of Sun Yat-sen University(R20210217202601970)the Postdoctoral Fellowship Program of CPSF(GZC20233216)the Basic and Applied Basic Research Foundation of Guangdong Province(2021A1515111195).
文摘The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid(5-ALA).Furthermore,this study aimed to explore the underlying mechanism of the protective effects of 5-ALA.First,we collected and stored mouse testicular fragments in different media,including Hank’s balanced salt solution(HBSS;n=5),Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12(DMEM/F12;n=5),and alpha-minimum essential medium(αMEM;n=5).Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group(P<0.05)and theαMEM group(P<0.01).Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA(0[control],1 mmol l−1,2 mmol l−1,and 5 mmol l−1)to determine the most effective concentration of 5-ALA.The 2 mmol l−15-ALA group(n=3)presented the highest positive rate of spermatogonial stem cells compared with those in the control,1 mmol l−1,and 5 mmol l−15-ALA groups.Finally,the tissue fragments were preserved in HBSS with control(n=3)and 2 mmol l−15-ALA(n=3)under low-temperature conditions.A comparative analysis was performed against fresh testes(n=3)to elucidate the underlying mechanism of 5-ALA.Gene set enrichment analysis(GSEA)for WikiPathways revealed that the p38 mitogen-activated protein kinase(MAPK)signaling pathway was downregulated in the 2 mmol l−15-ALA group compared with that in the control group(normalized enrichment score[NES]=−1.57,false discovery rate[FDR]=0.229,and P=0.019).In conclusion,these data suggest that using 2 mmol l−15-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.
基金This study was supported by the National Natural Science Foundation of China(No.81571489,No.81671834,No.81671449 and No.81871110)the Frontier and Key Technology Innovation Special Foundation of Guangdong Province,China(No.2016B030230001)+3 种基金the Natural Science Foundation of Guangdong Province,China(No.2014A030310359,No.2016A030313229 and No.2015A030313013)the Science and Technology Planning Project of Guangdong Province,China(No.2016A020214004)the Health Care Collaborative Innovation Foundation Major Projects of Guangzhou City,Guangdong Province,China(No.201604020189)the Youth Teacher Training Project of Sun Yat-sen University(No.17ykpy68 and No.18ykpy09).
文摘An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell(SSC)transplantation.Busulfan has been commonly used to develop such a model,but 30%–87%of mice die when administered an intraperitoneal injection of 40 mg kg^?1.In the present study,hematoxylin and eosin staining,Western blot,immunofluorescence,and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses(20,30,and 40 mg kg^?1)on day 36 or a dose of 40 mg kg^?1 at different time points(0,9,18,27,36,and 63 days).The survival rate of the mice was 100%.When the mice were treated with 40 mg kg^?1 busulfan,dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36.In addition,the gene expressions of glial cell line-derived neurotrophic factor(GDNF),fibroblast growth factor 2(FGF2),chemokine(C-X-C Motif)ligand 12(CXCL12),and colony-stimulating factor 1(CSF1)were moderately increased by day 36.A 63-day,long-term observation showed the rare restoration of endogenous germ cells in the testes,suggesting that the potential period for SSC transplantation was between day 36 and day 63.Our results demonstrate that the administration of two intraperitoneal injections of busulfan(40 mg kg^?1 in total)at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
