| Project/Area Number |
17591693
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | Nagoya City University |
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Principal Investigator |
IKEUCHI Takahito Nagoya City University, Graduate School of Medical Sciences, Researcher (90315888)
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| Co-Investigator(Kenkyū-buntansha) |
KOJIMA Yoshiyuki Nagoya City University, Graduate School of Medical Sciences, Assistant Professor (60305539)
KUBOTA Hiroki Nagoya City University, Graduate School of Medical Sciences, Researcher (10347403)
HAYASHI Yutaro Nagoya City University, Graduate School of Medical Sciences, Associate Professor (40238134)
KOHRI Kenjiro Nagoya City University, Graduate School of Medical Sciences, Professor (30122047)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,610,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥210,000)
Fiscal Year 2007: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | transcription factors / DAX-1 / gene therapy / 遺伝子治療 / 造精機能 / 男性不妊症 / Sertoli細胞 |
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| Research Abstract |
Background: Ad4BP/SF-1 and DAX-1 are orphan members of the nuclear hormone receptor superfamily of transcription factors. In order to obtain better understandings of human testicular steroidogenesis and spermatogenesis, we examined the expression levels of both factors in human normal and idiopathic azoospermic testes and investigated their physical meaning. In addition, we identify the most appreciate gene transfer method to testis for future clinical application.
Methods : (1)We examined the expression level of Ad4BP/SF-1 and DAX-1 by quantitative RT-PCR, immunohistochemistry and western blotting analysis using 8 normal human testicular tissues from infants to adults. Next, we performed quantitiative RT-PCR using testicular biopsy samples obtained from 22 idiopathic azoospermic patients to examine the expression of Ad4BP/SF-1 and DAX-1, and analyzed the correlation between the expression levels of both factors and the serum hormone levels or histological evaluation to study their pote
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ntial correlation with steroidogenesis and spermatogenesis on idiopathic azoospermia. (2) We directly injected DNA into mouse testes in vivo using three methods, liposome, electroporation and adenovirus vector, to transfect testicular cells using the same exogenous DNA, and compared the three methods for transfection efficiency and effects of gene transfer on spermatogenesis to identify the most appreciate method for future clinical application.
Results : (1)The expression levels of both factors in the normal testes increased with testicular development. Ad4BP/SF-1 was abundantly expressed in Leydig cell, whereas DAX-1 was expressed in Sertoli cell. The expression level of Ad4BP/SF-1 in idiopathic azoospermic patients testes positively correlated with serum testosterone (p<0.05). The average expression levels of DAX-1 mRNA for patients with maturation arrest (0.39+/-0.19)and Sertoli cell-only syndrome (0.13+/-0.08) were lower than that with hypospermatogenesis (1.60+/-1.32) and normal spermatogenesis (1.30+/-1.41). (2)Although liposome and electroporation methods were simple, they had low efficiency, short duration of the physiological effect and high spermatogenic damage in comparison with adenovirus-mediated gene transfer. The disadvantage of the liposome and electroporation methods for clinical application was that they showed potential for gene expression in germ cells and strong adverse effects on spermatogenesis. Our results suggest that adenovirus-mediated gene transfer may be a more effective and superior method for transfecting testicular cells among these three methods and may be more applicable for in vivo gene therapy for male infertility in the future.
Conclusion : (1)Ad4BP/SF-1 is essential for the maintenance of steroidogenesis in the human testis. DAX-1 plays a critical role in spermatogenesis in the human testis, and Sertoli cell-only syndrome and maturation arrest may result from abnormal Sertoli cell function that disrupts the normal progression of spermatogenesis. (2)Although more investigations on the mechanism of spermatogenesis and male infertility and the establishment of techniques for more efficient and safer gene transfer to the sperm and testis will be needed, gene therapy will enable a revolutionary advance for reproductive treatment and provide great benefit for patient with male infertility in the future. Less
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