| Busulfan has a highly efficient destructive effect on male germ cells and is used as a recipient for the preparation of spermatogonial stem cell transplants.However,the safety risk due to its greater toxicity is a challenge in the production process,and the mechanism of toxicity is not yet clear.In order to reveal the mechanism of damage to testicular spermatogenic function by Busulfan treatment,improve the efficiency and safety of Busulfan production application and alleviate its damage to male reproductive function.In this study,mice were injected with Busulfan in the testis,and body weight,testicular diameter,testicular weight,epididymal weight,epididymal sperm concentration and sperm malformation rate were examined,and it was found that Busulfan significantly decreased testicular weight and epididymal weight,and significantly decreased sperm production and increased sperm malformation rate.In view of the effects of Busulfan on spermatogenesis and quality,this study observed testicular tissue sections after Busulfan treatment by H&E staining technique,and serum detection by ELASA,and found that Busulfan treatment led to a significant increase in the levels of inflammatory cytokines IL-6,IL-1β,and TNF-α,and broke through the physiological upper limit 3 d after Busulfan treatment,with cellular infiltration in the interstitial tissue of testis,disruption of the seminiferous tubule The lumen of the tubules became smaller,and the spermatogenic cells in the lumen were disorganized and shed,which proved that the treatment of Busulfan could induce testicular inflammation and damage spermatogenesis.Based on the above findings,we further detected that the immunofluorescence signal of blood testis barrier(BTB)related protein(N-Cadherin)and cytoskeleton of sertoli cells(Vimentin)were gradually weakened and inhomogeneous after the treatment with diazoxide,and we speculated that Busulfan damaged the BTB structure and endangered the environmental stability of spermatogenesis,and The damage of Vimentin led to the shrinkage of sertoli cells,which reduced the support and regulation of spermatogenic cells by sertoli cells and led to the shedding of spermatogenic cells;ELASA was used to examine the testicular homogenate of mice,and it was found that androgen receptors gradually decreased after the treatment of BTB and reached a significant level at 21 d.The decrease of androgen receptors reduced the regulation of spermatogenic cells by sertoli cells and affected spermatogenesis.In order to verify the above speculation,transmission electron microscopy ultrastructural observation was performed,and it was found that BTB gradually narrowed,blurred the structure of actin filament bundles,and vacuolated near the BTB after the treatment of Busulfan,which provided the first ultrastructural evidence to reveal that Busulfan damaged the sertoli cells and spermatogenic cells,and affected their mutual support and regulation.In addition,it was found that the mitochondria as a whole became smaller,and cristae disappeared and aggregated,indicating that the balance between mitochondrial aggregation and division may be disrupted by the treatment of Busulfan,which damaged the mitochondrial structure and function and affected the energy supply of spermatogenesis;the damaged mitochondria were also involved in the testicular inflammatory response and affected spermatogenesis and development.In summary,Busulfan induced testicular inflammation damaged BTB and the cytoskeleton of sertoli cells,and damage to BTB destroyed the stable niche environment of spermatogenesis;damage to skeletal proteins led to atrophy of sertoli cells,which affected the sertoli cells’ support and regulation of spermatogenesis,causing cell shedding and apoptosis;damage to sertoli cells also reduced androgen receptor levels,affecting androgen-androgen receptor signaling,which in turn weakened the regulatory function of Finally,based on the inflammatory inhibitory principle of melatonin,a melatonin treatment protocol was developed to alleviate the damage of testicular spermatogenesis caused by Busulfan.The present study revealed the mechanism of testicular spermatogenesis damage by Busulfan and explored the melatonin mitigation scheme,which provides an important reference for the establishment of efficient and safe Busulfan application scheme in the future. |
