Optimal dose of busulfan for depleting testicular germ cells of recipient mice before spermatogonial transplantation.
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ABSTRACT:
SUBMITTER: Wang DZ
PROVIDER: S-EPMC3739084 | biostudies-other | 2010 Mar
REPOSITORIES: biostudies-other
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Project description:Spermatogonial transplantation has been used as a standard assay for spermatogonial stem cells (SSCs). After transplantation into the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) between Sertoli cells and settle in a niche. Unlike in the repair of other self-renewing systems, SSC transplantation is generally performed after complete destruction of endogenous spermatogenesis. Here, we examined the impacts of recipient conditioning on SSC homing. Germ cell ablation downregulated the expression of glial cell line-derived neurotrophic factor, which has been shown to attract SSCs to niches, implying that nonablated niches would attract SSCs more efficiently. As expected, SSCs colonized nonablated testes when transplanted into recipients with the same genetic background. Moreover, although spermatogenesis was arrested at the spermatocyte stage in Cldn11-deficient mice without a BTB, transplantation not only enhanced donor colonization but also restored normal spermatogenesis. The results show promise for the development of a new transplantation strategy to overcome male infertility.
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Project description:Spermatogonial stem cell (SSC) transplantation is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5 -/- mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5 -/- mice. At 12 weeks of age, the tubules of Etv5 -/- mice lacked almost all spermatogenic cells with a Sertoli cell-only phenotype, and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3- (immature) or 7-week-old (mature) Etv5 -/- mice. Partial restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5 -/- mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5 -/- mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.
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Project description:In vitro differentiation of spermatogonial stem cells (SSCs) promotes the understanding of the mechanism of spermatogenesis. The purpose of this study was to isolate spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinoic acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of SSCs and somatic cells. Cell colonies were present after culture in M1-medium for 3 days. Through alkaline phosphatase, RT-PCR and indirect immunofluorescence cell analysis, cell colonies were shown to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for 2 days. Then the cell colonies of SSCs were again cultured in M1-medium for 6-8 days, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. It could be demonstrated that 10(-7) mol l(-1) of RA effectively induced the SSCs into haploid male germ cells in vitro.
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Project description:BACKGROUND:Spermatogonial 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. METHODS:A 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). RESULTS:The 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. CONCLUSION:Co-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.
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Project description:Adult male fertility depends on spermatogonial stem cells (SSCs) which undergo either self-renewal or differentiation in response to microenvironmental signals. Activin A acts on Sertoli and Leydig cells to regulate key aspects of testis development and function throughout life, including steroid production. Recognising that activin A levels are elevated in many pathophysiological conditions, this study investigates effects of this growth factor on the niche that determines spermatogonial fate. Although activin A can promote differentiation of isolated spermatogonia in vitro, its impacts on SSC and spermatogonial function in vivo are unknown. To assess this, we examined testes of Inha KO mice, which feature elevated activin A levels and bioactivity, and develop gonadal stromal cell tumours as adults. The GFRA1+ SSC-enriched population was more abundant and proliferative in Inha KO compared to wildtype controls, suggesting that chronic elevation of activin A promotes a niche which supports SSC self-renewal. Intriguingly, clusters of GFRA1+/EOMES+/LIN28A- cells, resembling a primitive SSC subset, were frequently observed in tubules adjacent to tumour regions. Transcriptional analyses of Inha KO tumours, tubules adjacent to tumours, and tubules distant from tumour regions revealed disrupted gene expression in each KO group increased in parallel with tumour proximity. Modest transcriptional changes were documented in Inha KO tubules with complete spermatogenesis. Importantly, tumours displaying upregulation of activin responsive genes were also enriched for factors that promote SSC self-renewal, including Gdnf, Igf1, and Fgf2, indicating the tumours generate a supportive microenvironment for SSCs. Tumour cells featured some characteristics of adult Sertoli cells but lacked consistent SOX9 expression and exhibited an enhanced steroidogenic phenotype, which could arise from maintenance or acquisition of a fetal cell identity or acquisition of another somatic phenotype. Tumour regions were also heavily infiltrated with endothelial, peritubular myoid and immune cells, which may contribute to adjacent SSC support. Our data show for the first time that chronically elevated activin A affects SSC fate in vivo. The discovery that testis stromal tumours in the Inha KO mouse create a microenvironment that supports SSC self-renewal but not differentiation offers a strategy for identifying pathways that improve spermatogonial propagation in vitro.
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Project description:High-dose regimes containing etoposide, carboplatin and an oxazaphospharine can salvage 30-40% of patients with relapsed or refractory male germ cell tumours (GCTs). The additional benefit of paclitaxel in such high-dose therapy has not been tested. Between March 1995 and November 2002, 36 male GCT patients were treated with Carbop-EC-T (paclitaxel 75 mg x m(-2), etoposide 450 mg x m(-2), carboplatin AUC 10 on days -7, -5 and -3 and cyclophosphamide 60 mg x kg(-1) on days -5 and -3) followed by peripheral blood stem cell infusion (day 0). The 1-year overall survival rate for all patients is 67% (median follow-up 29 months). For the 24 patients with cisplatin-sensitive disease, the 1-year overall and event-free survivals are 88 and 64%, respectively. For those with cisplatin refractory or absolutely refractory disease, the 1-year overall survival is 25%. In all, 12 patients relapsed at a median duration of 5 months, 11 of whom have died. There were also six treatment-related deaths, five associated with pneumonitis. Pulmonary toxicity has been reported with paclitaxel in other high-dose regimes. Since altering our protocol so that paclitaxel is infused over 24 h with steroid prophylaxis, only one of 18 patients (13 testicular GCTs and five other tumour types) has had a treatment-related death. Our results suggest that Carbop-EC-T may enable a greater proportion of patients with relapsed and refractory GCTs to enter long-term remission.
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Project description:Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19(MOLF/Ei)), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.
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Project description:Germ cell tumor development in humans has been proposed to be part of testicular dysgenesis syndrome (TDS), which manifests as undescended testes, sterility, hypospadias, and, in extreme cases, as germ cell tumors. Males of the Ter mouse strain show interesting parallels to TDS because they either lack germ cells and are sterile or develop testicular germ cell tumors. We found that these defects in Ter mice are due to mutational inactivation of the Dead-end (Dnd1) gene. Here we report that chromosome X modulates germ cell tumor development in Ter mice. We tested whether the X or the Y chromosome influences tumor incidence. We used chromosome substitution strains to generate two new mouse strains: 129-Ter/Ter that carry either a C57BL/6J (B6)-derived chromosome (Chr) X or Y. We found that Ter/Ter males with B6-Chr X, but not B6-Chr Y, showed a significant shift in propensity from testicular tumor development to sterile testes phenotype. Thus, our studies provide unambiguous evidence that genetic factors from Chr X modulate the incidence of germ cell tumors in mice with inactivated Dnd1.
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Project description:The endocrine disruptor bisphenol A (BPA) is well known for its adverse effect on male fertility. Growing evidence suggests that BPA may interact with testicular germ cells and cause infertility as a result of its estrogenic activity. Objective of current in vitro study was to investigate the proliferation, survivability and stemness properties of mouse testicular germ cells exposed to BPA, and to evaluate possible expression of cellular proteome. Our results showed that germ cell viability and proliferation were not affected by low concentrations (0.01, 0.1, 1, and 10?µM) although significant reduction observed at 100?µM BPA. Germ cell self-renewal and differentiation related marker proteins expression found unchanged at those concentrations. When BPA-exposed germ cells were transplanted into recipient testes, we observed fewer colonies at higher concentrations (10 and 100?µM). Additionally, a significant frequency of recombination failure during meiosis was observed in 10?µM BPA-exposed germ cell transplanted recipient. Moreover, experiment on continuous BPA-exposed and 100?µM BPA-recovered germ cells suggested that spermatogonial stem cells are more potential to survive in adverse environment. Finally, scrutinizing differentially expressed cellular proteins resulted from our proteomic analysis, we conclude that BPA exposure might be associated with several health risks and infertility.
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