Establishment of recipient model for spermatogonial stem cells transplantation in Kunming mice

The objective of this study was to establish a recipient model for spermatogonial stem cells (SSCs) transplantation in the Kunming mice after different doses busulfan treatment. The results showed that the most optimal dose of busulfan was 20 mg/kg and the most appropriate time for transplantation was 5–7 wk after busulfan treatment. Then, the cloned fragments existed in the testis of recipient mice after 20 mg/kg busulfan treatment and the offspring with enhanced green fluorescent protein (EGFP) were produced by the transplanting SSCs. Hence, we established the effective recipient model for donor-derived SSCs transplantation in Kunming mice.     

探索Sertoli细胞对去除精原细胞的动态反应

目的探索Sertoli细胞对去除小鼠精原细胞后睾丸的动态反应。方法采用15、30和44 mg/kg的白消安腹腔注射法建立不同程度去除精原细胞的动物模型,处理后5 d和28 d时对睾丸进行组织学检测,评价精子发生状态,并运用实时定量荧光PCR技术检测这两个时期睾丸GDNF、PLZF、Nanog和GFRα1基因mRNA的表达量。结果在白消安处理后第5天,GDNF出现显著升高,且呈剂量依赖趋势,而PLZF与GFRɑ1并无显著变化,睾丸组织学观察亦无明显变化。在白消安处理后28 d时,GDNF、PLZF、Nanog、GFRɑ1基因mRNA相对表达量均出现大幅度的升高,睾丸组织学切片观察显示随着给药剂量的增加,精子发生受到的损伤愈加严重。结论 Sertoli细胞早在白消安处理后第5天就对精原细胞的变化发生了反应,Sertoli细胞分泌GDNF的能力发生代偿性增加,进而刺激精原干细胞自我更新速度加快,体现在Nanog和PLZF水平提高,从而实现精子发生的重建。     

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67+/-0.29 to 16.5+/-3.8%) but also for alpha6-integrin, beta1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRalpha-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.     

Testicular Busulfan Injection in Mice to Prepare Recipients for Spermatogonial Stem Cell Transplantation Is Safe and Non-Toxic

Current methods of administering busulfan to remove the endogenous germ cells cause hematopoietic toxicity, require special instruments and a narrow transplantation time. We use a direct testicular injection of busulfan method for preparing recipients for SSC transplantation. Male ICR mice (recipients) were divided into four groups, and two experimental groups were treated with a bilateral testicular injection of 4 or 6 mg/kg/side busulfan (n = 60 per concentration group). Mice received an intraperitoneal injection (i.p.) of 40 mg/kg busulfan (n = 60, positive control) and bilateral testicular injections of 50% DMSO (n = 60, negative control). Donor SSCs from RFP-transgenic C57BL/6J mice were introduced into the seminiferous tubules of each recipient testis via efferent duct injection on day 16–17 after busulfan treatment. Recipient mice mated with mature female ICR mice and the number of progeny was recorded. The index detected at day 14, 21, 28, 35 and 70 after busulfan treatment. Blood analysis shows that the toxicity of busulfan treated groups was much lower than i.p. injection groups. Fertility was restored in mice treated with busulfan and donor-derived offspring were obtained after SSC transplantation. Our study indicated that intratesticular injection busulfan for the preparation of recipients in mice is safe and feasible.     

Efficient and safe recipient preparation for transplantation of mouse spermatogonial stem cells: pretreating testes with heat shock

Recipient preparation is of prime importance for the successful transplantation of spermatogonial stem cells (SSCs). Busulfan destroys endogenous germs cells and is commonly used for recipient preparation. However, busulfan produces significant side effects, including systemic toxicity, and it is lethal in certain species. The side effects associated with busulfan compromise the efficiency of SSC transplantation and threaten the safety of recipients. Here, we show that heat shock treatment of testes can be used as an alternative to busulfan treatment. Fourteen days after heat shock treatment, mice received a testicular injection of donor germ cells expressing enhanced green fluorescent protein (EGFP). Busulfan-treated mice were used as controls. Two months after transplantation, the number (12 ± 1 mm) and length (30.46 ± 5.23 mm) of EGFP-expressing testicular colonies in heat shock-treated recipients were not significantly different from those in busulfan-treated recipients. Furthermore, healthy EGFP-expressing offspring were obtained after intracytoplasmic injection of round spermatids recovered from heat shock-treated recipients. This result indicates that donor SSCs undergo complete spermatogenesis in the heat shock-treated testes of recipients. Our findings demonstrate the feasibility of using heat shock for the preparation of recipients before SSC transplantation in mice. Heat shock may prove to be useful for recipient preparation in mammalian species in which busulfan produces significant toxicity.     

Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations

The alkylating agent busulfan (Myleran) adversely affects spermatogenesis in mammals. We treated male mice with single doses of busulfan in order to quantitate its cytotoxic action on spermatogonial cells for comparison with effects of other chemotherapeutic agents, to determine its long-term effects on fertility, and to assess its possible mutagenic action. Both stem cell and differentiating spermatogonia were killed and, at doses above 13 mg/kg, stem cell killing was more complete than that of differentiating spermatogonia. Azoospermia at 56 days after treatment, which is a result of stem cell killing, was achieved at doses of over 30 mg/kg; this dose is below the LD50 for animal survival, which was over 40 mg/kg. Busulfan is the only antineoplastic agent studied thus far that produces such extensive damage to stem, as opposed to differentiating, spermatogonia. The duration of sterility following busulfan treatment depended on the level of stem cell killing and varied according to quantitative predictions based on stem cell killing by other cytotoxic agents. The return of fertility after a sterile period did not occur unless testicular sperm count reached 15% of control levels. Dominant lethal mutations, measured for assessment of possible genetic damage, were not increased, suggesting that stem cells surviving treatment did not propagate a significant number of chromosomal aberrations. Sperm head abnormalities remained significantly increased at 44 weeks after busulfan treatment, however, the genetic implications of this observation are not clear. Thus, we conclude that single doses of busulfan can permanently sterilize mice at nonlethal doses and cause long-term morphological damage to sperm produced by surviving stem spermatogonia.     

Toxicological review of busulfan (Myleran)

Busulfan is a bifunctional alkylating agent that appears to be cytotoxic to slowly proliferating or non-proliferating stem cell compartments, although its specific molecular and cellular mechanisms are unknown. It is the drug of preference in treatment of chronic myelogenous or granulocytic leukemia because its cytotoxic activity results in primary damage or destruction of hematopoietic cells. Additional effects resulting from the cytotoxicity of busulfan in hematological and other tissues, as documented by both human and animal model studies, include lethality, sterility, teratogenicity, and alteration of immune function. Busulfan has been shown to be mutagenic to microorganisms, mammalian cells in culture, Drosophila, and rodents. This agent is also considered potentially carcinogenic to humans. Various tissue hyperplasia and preneoplastic cells have been observed in animal model studies with busulfan, and case reports on human patients implicate busulfan as the causative agent in induction of secondary malignancies. Reports from human and animal studies of busulfan's cytotoxicity, teratogenicity, carcinogenicity, and mutagenicity have been reviewed. This information may be useful in a quantitative assessment of the effects of this agent and the identification of significant deficiencies in the data base. Demonstration that busulfan induces mutations in both somatic and germ cells suggests the need to assess its risk to humans.     

Suppression of spermatogenesis for cell transplantation in adult mice

Summary Spermatogenesis occurs within the testis of adult males by a complex and very well organized process. Breakthroughs in techniques such as cryopreservation and culture of spermatogenic cells and the maturation of these cells in exogenous testes after transplantation renewed the interest in this process. Transplantation of spermatogenic cells from a donor to a recipient animal needs a preparatory step that consists in the elimination of the endogenous population of spermatogenic cells. The most common method used to empty the seminiferous tubules is the treatment with busulfan (1,4-butanediol dimethanesulfonate). Busulfan partially eliminates stem cells because of its alkylating nature, but a residual component of stem cells survives the treatment and competes in the regeneration of the testis with transplanted cells. Estradiol has also been used as an agent that causes a delay in the process of spermatogenesis by altering its hormonal stimulation, although it does not affect the spermatogonia population. Therefore, we have tested different treatments with busulfan, estradiol benzoate, and also an agonist of the chorionic gonadotrophin-releasing hormone, leuprolide acetate, for the inhibition of endogenous spermatogenesis. We have found that a combination of estradiol, busulfan, and leuprolide can destroy the population of endogenous spermatogenic cells without altering Sertoli cells and maintains the optimal environment needed to allow the development of transplanted cells.     

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism >80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.     

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.     

Prediction of intravenous busulfan clearance by endogenous plasma biomarkers using global pharmacometabolomics

Introduction

High-dose busulfan (busulfan) is an integral part of the majority of hematopoietic cell transplantation conditioning regimens. Intravenous (IV) busulfan doses are personalized using pharmacokinetics (PK)-guided dosing where the patient’s IV busulfan clearance is calculated after the first dose and is used to personalize subsequent doses to a target plasma exposure. PK-guided dosing has improved patient outcomes and is clinically accepted but highly resource-intensive.

Objective

We sought to discover endogenous plasma biomarkers predictive of IV busulfan clearance using a global pharmacometabolomics-based approach

Methods

Using LC-QTOF, we analyzed 59 (discovery) and 88 (validation) plasma samples obtained before IV busulfan administration.

Results

In the discovery dataset, we evaluated the association of the relative abundance of 1885 ions with IV busulfan clearance and found 21 ions that were associated with IV busulfan clearance tertiles (r² ≥ 0.3). Identified compounds were deoxycholic acid and/or chenodeoxycholic acid, and linoleic acid. We used these 21 ions to develop a parsimonious seven-ion linear predictive model that accurately predicted IV busulfan clearance in 93 % (discovery) and 78 % (validation) of samples.

Conclusion

IV busulfan clearance was significantly correlated with the relative abundance of 21 ions, seven of which were included in a predictive model that accurately predicted IV busulfan clearance in the majority of the validation samples. These results reinforce the potential of pharmacometabolomics as a critical tool in personalized medicine, with the potential to improve the personalized dosing of drugs with a narrow therapeutic index such as busulfan.

     

Increase in erythroid colony formation in rabbits following the administration of testosterone.

Changes in the numbers of erythroid colonies formed in cultures from rabbits pretreated with either testosterone or busulfan plus testosterone were studied using a methyl cellulose gel system. The mean number of erythroid colonies in bone marrows from rabbits treated with testosterone in vivo was significantly higher than that of controls. However, this increase in erythroid colonies in cultures seen following testosterone treatment was completely blocked by the concurrent administration of busulfan as seen in the rabbits treated with busulfan orally and followed by testosterone injections. Busulfan has been postulated block the formation of new erythropoietin responsive cells (ERC) from hematopoietic stem cells (CFU). Thus, these findings suggest that testosterone may act directly on CFU to enhance their differentiation into the ERC compartment causing an increase in nucleated erythroid cells.     

Comparative effects of Yoshi-864 and busulfan on certain transplantable murine tumors.

Yoshi-864 extends markedly the survival times of mice bearing L1210 leukemia or Ehrlich ascites carcinoma. Busulfan, with methanesulfonate leaving groups identical with those of Yoshi-864, is without effect. Tumor cells from mice bearing the Ehrlich tumor and treated with Yoshi-864 have a persistent reduction in ability to synthesize DNA. Synthesis of DNA in cells from mice treated with busulfan is moderately suppresed at 48 hr after treatment, but returns virtually to the control value at 72 hr.     

The effect of prenatal treatment with busulfan on in vitro androgen production by testes from rats of various ages

Treatment of rats with busulfan in utero severely depletes the germ cell population of the seminiferous tubules. These studies have examined the in vitro capacity of testicular tissue and Leydig cells from such testes to secrete androgens. Leydig cells were identified by staining for 3 beta-hydroxy steroid dehydrogenase. Rats were studied at several ages to identify any developmental changes in the androgen-secreting capacity of control and treated gonads. At 30 days of age, no effect of treatment on serum androgen was found. At 60 and 90 days of age, treatment caused decreased androgen and increased LH content of the serum. At 12, 30, 60, and 90 days of age, the amount of androgen secreted per milligram of testicular tissue in response to LH was higher in busulfan-treated rats. Leydig cells from 60- and 90-day-old rats which had received busulfan were also hyperresponsive to LH. It was concluded that Leydig cells from testes essentially devoid of germ cells were hyperresponsive to LH. Serum androgen levels were decreased yet androgen production per Leydig cell was increased. A possible explanation of this apparent paradox is that busulfan treatment resulted in decreased numbers of Leydig cells in the gonads.     

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.     

Effect of prenatal treatment with busulfan on the hypothalamo-pituitary axis, genital tract and testicular histology of prepubertal male rats

Female Wistar rats were treated with busulfan or with solvent on Day 20 of pregnancy. Thirty male offspring of each group were killed at 38 days of age. In busulfan-treated rats, compared to controls, hypothalamic LH-RH content was decreased by 52%, whereas pituitary LH and FSH concentrations were increased by 60 and 43% respectively. Plasma LH and FSH were increased by 112 and 275% respectively. Prolactin concentrations were not changed, but plasma testosterone concentration was decreased by 48%. The total number of Leydig cells per testis was decreased by 52%, and LH binding sites per testis were decreased by 70%. The total number of Sertoli cells was decreased by 44%, while FSH binding sites per testis were decreased by 62%. Spermatogenesis was practically absent after prenatal exposure to busulfan. These data demonstrate that on Day 20 of pregnancy all the dividing cells in the fetal testes were depleted by an antimitotic treatment. The stimulation of the hypothalamo-pituitary axis could have been partly induced by the decrease in testosterone production, and by the aplasia of germ cells involving modifications of the remaining Sertoli and Leydig cells.     

Quantification of busulfan in plasma by liquid chromatography–ion spray mass spectrometry: Application to pharmacokinetic studies in children

Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. A new method was developed coupling liquid chromatography with mass spectrometry (LC–MS) and was validated for the determination of busulfan concentrations in plasma. Recovery was 86.7%, the limit of detection was 2.5 ng/ml and linearity ranged from 5 to 2500 ng/ml. The correlation between the busulfan concentrations measured by our previously published HPLC–UV method and the new HPLC–MS method was highly significant (P<0.0001). Sample volume was reduced and the method was rapid, sensitive and less expensive than the methods previously used in our laboratory. This method was used to determine the pharmacokinetic parameters of busulfan after the first administration of 1 mg/kg orally, in 13 children receiving the drug as part of the preparative regimen for bone marrow transplantation. Our results were similar to previously reported data. They showed that the apparent oral clearance of busulfan was 0.299±0.08 l/h/kg, and that it was significantly higher (P=0.02) in patients below the age of 5 years than in older children.     

Synthesis and biological effects of new hybrid compounds composed of benzylguanidines and the alkylating group of busulfan on neuroblastoma cells

¹³¹Iodine-labelled (meta-iodobenzyl)guanidine ([¹³¹I]-mIBG) and busulfan [butane-1,4-diylbis(methanesulfonate)] are well-established pharmaceuticals in neuroblastoma therapy. We report the design, synthesis, and testing of hybrid molecules—mBBG and pBBG—which combine key structural features of (meta-iodobenzyl)guanidine and busulfan: they contain a benzylguanidine moiety for accumulating in neuroblastoma cells via the noradrenaline transporter and, in the meta- or para-position, respectively, one of the two identical alkylating motives of busulfan for killing cells. Uptake and toxicity of hybrids mBBG and pBBG in human neuroblastoma cells compared favorably to their ancestors [¹³¹I]-mIBG and busulfan.     

精原干细胞移植及受体制备技术研究进展

精原干细胞移植为研究精子发生、雄性生殖能力及新型转基因技术奠定了基础.尽管已利用小鼠建立了较成熟的移植技术体系,白消安受体制备法和曲细精管及睾丸网移植法已得到广泛应用,但白消安可导致动物较高的死亡率,局部射线照射和无内源性精子发生受体动物的制备费用较昂贵,热处理受体制备法应用范围较窄且效果不稳定;三种移植方法均对操作有较高的技术要求,曲细精管、睾丸输出管移植需要显微注射装置,而睾丸网移植需要超声仪的辅助.而且,移植效果在不同实验间、物种间差异较大,移植效率有待提高,对移植排斥反应的认识也有待进一步深入.对睾丸结构和精原干细胞生物学特性的深入研究,将有助于建立更简单高效的受体制备和移植的方法.     

Signal One and Two Blockade Are Both Critical for Non-Myeloablative Murine HSCT across a Major Histocompatibility Complex Barrier

Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.