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:Background: Inhalatory anesthetics may impact spermatogenesis and sexual behavior. Comprehensive evaluation should be conducted to screen the effect of inhalatory anesthetics on the sperm and semen quality. This experimental research was organized to assess the impacts of sevoflurane during the period of neonatal spermatogenesis. Materials and methods: Twenty-one pregnant mice were obtained from the Pasteur Institute. After birth, neonates were categorized based on exposure to Monitored Anesthesia Care (MAC) of sevoflurane into three groups: experimental 1, experimental 2 and control. In order to investigate the testicular condition, a histological evaluation, including apoptosis study and immunohistochemistry, was performed. Not only apoptotic target genes such as Bax and Bcl-2, but also microRNA17-92, were investigated in testicular samples via real-time polymerase chain reaction (real-time PCR). Results: The outcomes of this work indicated the effects of sevoflurane on spermatogonial and germ cells in testicular tissue via stimulating apoptotic target genes and microRNA-17-92. The proportion of Bax/Bcl-2 in the experimental group was 8.318699 ± 1.093, and the proportion of Bax/Bcl-2 in the control group was 2.631 ± 0.079. There was a significant (p ≤ 0.002) difference among the control group and both experimental groups. Conclusion: Sequential sevoflurane exposure during the neonatal period may create testicular dysfunction due to the high level of apoptosis in spermatogonial cells. Also, sevoflurane may affect spermatogenesis by influencing other biomarkers, such as microRNA.
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Project description:In mammals, females undergo reproductive cessation with age, whereas male fertility gradually declines but persists almost throughout life. However, the detailed effects of ageing on germ cells during and after spermatogenesis, in the testis and epididymis, respectively, remain unclear. Here we comprehensively examined the in vivo male fertility and the overall organization of the testis and epididymis with age, focusing on spermatogenesis, and sperm function and fertility, in mice. We first found that in vivo male fertility decreased with age, which is independent of mating behaviors and testosterone levels. Second, overall sperm production in aged testes was decreased; about 20% of seminiferous tubules showed abnormalities such as germ cell depletion, sperm release failure, and perturbed germ cell associations, and the remaining 80% of tubules contained lower number of germ cells because of decreased proliferation of spermatogonia. Further, the spermatozoa in aged epididymides exhibited decreased total cell numbers, abnormal morphology/structure, decreased motility, and DNA damage, resulting in low fertilizing and developmental rates. We conclude that these multiple ageing effects on germ cells lead to decreased in vivo male fertility. Our present findings are useful to better understand the basic mechanism behind the ageing effect on male fertility in mammals including humans.
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Project description:Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6-8 weeks post-transplant, with donor derived structures including epithelial, endothelial, and mesenchymal cells. Epithelial cells within these donor-derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about threefold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Our study provides proof of concept for lung regeneration by adult lung cells after preconditioning to vacate the pulmonary niche. Stem Cells Translational Medicine 2018;7:68-77.
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Project description:There are various autoimmunogenic antigens (AIs) in testicular germ cells (TGCs) recognized as foreign by the body's immune system. However, there is little information of TGC-specific AIs being available. The aim of this study is to identify TGC-specific AIs. We have previously established that immunization using viable syngeneic TGC can also induce murine experimental autoimmune orchitis (EAO) without using any adjuvant. This study is to identify TGC-specific AIs by TGC liquid chromatography-tandem mass spectrometry analysis, followed by two-dimensional gel electrophoresis that reacted with serum IgG from EAO mice. In this study, we identified 11 TGC-specific AIs that reacted with serum from EAO mice. Real-time RT-PCR analysis showed that the mRNA expressions of seven TGC-specific AIs were significantly higher in only mature testis compared to other organs. Moreover, the recombinant proteins of identified 10 (except unnamed protein) TGC-specific AIs were created by using human embryonic kidney 293 (HEK293) cells and these antigencities were reconfirmed by Western blot using EAO serum reaction. These results indicated Atp6v1a, Hsc70t, Fbp1 and Dazap1 were candidates for TGC-specific AIs. Identification of these AIs will facilitate new approaches for understanding infertility and cancer pathogenesis and may provide a basis for the development of novel therapies.
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Project description:An essential step in the success of germ cell transplantation is the preparation of the recipient's testicular environment to increase the availability of stem cell niches. However, most methods for this purpose in birds face serious limitations such as partial germ cell depletion, high toxicity and mortality, or the need to use expensive technologies. Here, we validated a simple and practical technique of transferring quail testicular cells into chicken testes depleted of endogenous spermatozoa by fractioned chemotherapy (20 mg/kg/week busulfan for 5 weeks). This protocol resulted in a very low mortality of the treated day-old chicks and, despite maintenance of androgenic activity, sperm production was decreased by 84.3% at 25 weeks of age. NANOG immunostaining revealed that very few to no germ cells were present following treatment with 20 and 40 mg/kg, respectively. RT-qPCR data also showed that c-MYC and NANOG expression declined in these treatments, but GRFα1 and BID expressions remained unaltered among groups. After xenotransplantation, quail germ cells were immunodetected in chicken testes using a species-specific antibody (QCPN), and quail ovalbumin DNA was found in seminal samples collected from chicken recipients. Together, these data confirm that fractionated administration of busulfan in hatchlings is a practical, effective, and safe protocol to prepare recipient male birds capable of supporting xenogeneic spermatogenesis.
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