Intraperitoneal Injection Of Busulfan Research Articles

Intratesticular versus intraperitoneal Busulfan administration: a comparative study on spermatogenesis suppression in quails and chickens

Generation of transgenic birds can be achieved by temporal suppression of endogenous spermatogenesis in males prior to primordial germ cell implantation. One of many established methods to induce male sterility is the intraperitoneal injection of busulfan, an alkylating agent. Nevertheless, the use of busulfan injections, which may also affect hematopoietic stem cells, carries the risk of potential lethality in animals. Given their safety and non-toxic nature, it has been demonstrated that intratesticular busulfan injections in mammals are less effective than intraperitoneal injections. This study aimed to compare, for the first time, the sterility and toxicity effects of intraperitoneal vs. intratesticular busulfan injections in quail and chickens. Our experimental design involved a previously established single intraperitoneal busulfan injection of 40 mg/kg of body weight (BW). In quail, busulfan was then administered intratesticularly at 3 different concentrations (6, 12, and 20 mg/kg BW), while in chickens, the working concentration was 20 mg/kg BW. We found that a single intraperitoneal busulfan injection of 40 mg/kg of BW resulted in 100% mortality in the treated roosters. In quails, however, this concentration only caused a temporary suppression of fertility for a 15-d period. Moreover, we found that a higher dose of intratesticular injection of busulfan is required to suppress spermatogenesis in quail (20 mg/kg BW) compared to mammals (4 mg/kg BW). Following these findings, we further confirmed that intratesticular injection of 20 mg/kg BW busulfan into roosters did not affect their overall viability. However, it induced a temporary state of male sterility, consistent with the effects observed with intraperitoneal injections. Hence, our data demonstrate that quail and chicken respond differently to busulfan administration. Furthermore, the present study provides evidence that direct injection into the rooster testes causes less physiological stress than intraperitoneal injection.

Antioxidant and antiapoptotic paracrine effects of mesenchymal stem cells on spermatogenic arrest in oligospermia rat model

BackgroundOligospermia is one of the common causative factors of male infertility. Some medical and hormonal therapy for male infertility is typically with unsatisfactory outcome. Stem cell therapy has become a new therapeutic strategy for restoring function in addition to inducing proliferation and differentiation of malfunctioning germ cells. This work aims at investigating the potential ability of BM-MSCs to repair the spermatogenic arrest in oligospermic rat model. MethodsIn this work, a rat model of oligospermia was induced using two intraperitoneal injections of busulfan (15 mg/kg) with two weeks interval. Rats were divided into (i) donor group [source of the bone marrow mesenchymal stem cells (BM-MSCs) that were labelled and transfected with green fluorescent protein (GFP)] and (ii) experimental groups that were subdivided into: GpI (control), GpII (spermatogenic arrest model), GpIII (untreated rats), and GpIV (BM-MSCs treated rats). Estimation of the testicular weight, sperm count and motility % were performed. Histological and immunohistochemical staining for inducible nitric oxide synthase (iNOS) and caspase-3 (Cas-3) were conducted. Besides, the level of the testicular malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α) and testicular testosterone were estimated by ELISA. ResultsOligospermic rats illustrated hypospermatogenesis of the seminiferous tubule with spermatocyte and spermatid arrest, focal thickening of the basement membrane and significant increase in germ cells apoptosis and testicular oxidative stress. Compared with the control, MDA and TNF-α were markedly elevated with marked suppression of the testicular testosterone. Intra-testicular injection of BM-MSCs substantially ameliorated these changes and effectively improved the sperm count and motility %. ConclusionsBM-MSCs improved the induced-spermatogenic arrest in the rat model mainly through anti-apoptotic and antioxidant paracrine effects.

Three-Dimensional Analysis of Busulfan-Induced Spermatogenesis Disorder in Mice.

We examined if the distribution of impaired or normal spermatogenesis differs along the length of seminiferous tubules in disorders of spermatogenesis. For this purpose, three-dimensional (3D) reconstruction of seminiferous tubules was performed in mice with experimental spermatogenesis disorder induced by intraperitoneal injection of busulfan, and the areas of impaired and normal spermatogenesis were analyzed microscopically. The volume of the testis and length of seminiferous tubules decreased, and the proportion of tubule areas with impaired spermatogenesis increased depending on the dose of busulfan. With the highest dose of busulfan, although the proportion of impaired spermatogenesis was similar among individual seminiferous tubules, it was slightly but significantly higher in shorter tubules and in tubule areas near branching points. The tubule areas with impaired and normal spermatogenesis consisted of many segments of varying lengths. With increasing doses of busulfan, the markedly impaired segments increased in length without changing in number, whereas normal segments, although reduced in number and length, remained even with the highest dose of busulfan. Individual remaining normal segments consisted of several different stages, among which stage I and XII were found at higher frequencies, and stage VI at a lower frequency than expected in normal seminiferous tubules. We also examined if the distribution of impaired or normal spermatogenesis differs among different 3D positions in the testis without considering the course of seminiferous tubules. Although the proportions of impaired spermatogenesis with the minimum dose of busulfan and normal spermatogenesis with the highest dose of busulfan greatly varied by location within a single testis, there were no 3D positions with these specific proportions common to different testes, suggesting that the factors influencing the severity of busulfan-induced spermatogenesis disorder are not fixed in location among individual mice.

Inhibiting Necroptosis of Spermatogonial Stem Cell as a Novel Strategy for Male Fertility Preservation.

Fertility preservation is a common concern for male cancer survivors of reproductive age. However, except for testicular tissue cryopreservation, which is not very effective, there is no feasible and precise therapy capable of protecting spermatogenesis for prepubertal boys before or during gonadotoxic treatment. This study aims to investigate the effects of inhibiting necroptosis of spermatogonial stem cell (SSC) in fertility preservation. Male mice 12 weeks of age were used to establish gonadotoxicity with two intraperitoneal injections of busulfan at a total dose of 40 mg kg-1. The mouse model and the primary cultured mouse SSCs were used to characterize the relationship between necroptosis of SSC and gonadotoxicity. Meanwhile, the effects of an inhibitor of necroptosis pathway, RIPA-56, were observed on day 36 in the mouse model of busulfan-induced gonadotoxicity. We found that the number of SSCs was decreased, but the level of necroptosis was upregulated on day 18 after busulfan treatment in testes from gonadotoxic mice. Further experiments in primary cultured cells showed that the necroptosis caused cell death in busulfan-treated SSCs and could be inhibited by RIPA-56. After suppressing the necroptosis of SSCs, the busulfan-induced mice had a decreased loss of spermatogenic cells as shown by histology and an increased Johnsen's score. Moreover, the quantities of SSCs and epididymal spermatozoa were restored after intervention with RIPA-56, indicating a series of beneficial effects by targeting the necroptosis of SSCs in mice undergoing busulfan treatment. In conclusion, our findings reveal that the necroptosis of SSCs plays a critical role in busulfan-induced gonadotoxicity and may be a potential target for male fertility preservation.

Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection

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.

Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice

BackgroundSpermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment.MethodsA mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10).ResultsThe best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs.ConclusionCo-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

Behaviour of spermatogonia following recovery from busulfan treatment in the rat.

This study describes the morphological behaviour of spermatogonia following recovery from two doses of busulfan treatment in the rat. Twenty days after the second intraperitoneal injection of busulfan, the testes lost most of their spermatogenic cells and there were fewer dispersed singly surviving spermatogonia. These surviving cells were in close contact with the basal portions of adjacent Sertoli cells and the shrunken basal lamina, and were the source for repopulating the depleted seminiferous epithelium. During the initial stage of repopulation (48 days later), surviving spermatogonia underwent a phase of active proliferation: type A spermatogonia underwent symmetric and asymmetric divisions; type B spermatogonia underwent asynchronous differentiation. At day 96, normal spermatogenesis was fully recovered in many seminiferous tubules, represented by 80% of the rats regaining various degrees of fertility at day 120. These data provide an additional model for the study of self-renewal of stem spermatogonia and suggest that the asymmetric division of type A spermatogonia and their close contact with both the basal lamina and the Sertoli cells may be involved in regulating the number of stem spermatogonia and the delicate process of normal spermatogenesis.