| Project/Area Number |
10671503
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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 | KANSAI MEDICAL UNIVERSITY |
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Principal Investigator |
MATSUDA Tadashi KANSAI MEDICAL UNIVERSITY, FACULTY OF MEDICINE, PROFESSOR, 医学部, 教授 (20192338)
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| Co-Investigator(Kenkyū-buntansha) |
MUGURUMA Kouei KANSAI MEDICAL UNIVERSITY, FACULTY OF MEDICINE, RESEARCH ASSOCIATE, 医学部, 助手 (10239460)
KAWAKITA Mutsushi KANSAI MEDICAL UNIVERSITY, FACULTY OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (50252458)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | cold shock / RNA-binding protein / CIRP / spermatogenesis / infertility / spermatogenic cell lines / retroviral vector / 低温誘導性蛋白 / 減数分裂 / レトロウィルス / 男性不妊 / 精索静脈瘤 |
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| Research Abstract |
We had cloned and characterized two cold shock proteins, CIRP and RBM3. Both of them were belong to families of proteins consisting of one amino-terminal consensus sequence RNA-binding Domain (CS-RBD) and one carboxyl-terminal glycine-rich domain and induced at 32℃ in mouse somatic cells in vitro. CIRP was exclusively expressed in primary spermatocytes and RBM3 was expressed only in Sertoli cells in mouse testes. In experimental cryptorchid testes, expression of CIRP and RBM3 decreased. In human testis with varicocele, germ cell expressed less CIRP protein than those in the testis without varicocele testes. CIRP and RBM3 expression are down regulate at elevated temperature in the testis. Analysis of these cold shock proteins in the tests may help elucidate the molecular mechanism of male infertility in patients with varicocele testes. To analyze the role of these proteins in spermatogenesis, we try to introduce the genes to spermatogenic cells. Utilizing murine spermatogenic cell lines
… More
, GC-1 spg and GC-2 spd(ts), as target cells an attempt was made to define the design of a retroviral vector which would transduce genes efficiently. Promoter activities of different retroviral long terminal repeats (LTRs) were examined by using chloramphenicol acetyltransferase (CAT) as a reporter. The U3 region of spleen focus-forming virus (SFFVp) showed higher enhancer activity than that of Molony murine leukemia virus (MoMuLV) in both cell lines. The U3 region of myeloproliferative sarcoma virus (MPSV) showed a higher activity only in GC-1 spg cells. Expression was suppressed by the repressor element of the primer binding site (PBS) of the Molony-related virus. The transduction efficiency of multidrug-resistance gene (mdr-1) was compared by a MoMuLV-based vector with hybrid vectors consisting of murine embryonic stem cell virus (MESV) PBS and LTR of either SFFVp or MPSV. Rhodamine efflux assays and colchicine resistant colony-forming assays demonstrated higher gene expressions by the hybrid vectors. Amphotropic and ecotropic receptors were found to be expressed and functional in both cell lines. Thus, these hybrid vectors would provide a powerful tool to transfer into and analyze their effects in spermatogenic cells.
Thus, it is possible to transfer the genes of interest into spermatogenic cell lines of GC-1 spg and GC-2 spd(ts) by using retroviral vectors and to analyze the effects of them in vitro. Since GC-2 spd(ts) cells can be differentiated into haploid cells in vitro, effects of the transferred genes on the process of meiosis can be analyzed in vitro. Instead of introducing plasmid DNAs by electroporation (Yamazaki et al., 1998), injection of retroviral vectors into seminiferous tubules of the testis in vivo might also enable the analysis of the effects of transferred genes on the process of spermatogenesis in vivo. Less
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