THE ROLE OF COLD SHOCK PROTEIN IN SPERMATOGENESIS

我们已经克隆并鉴定了两个冷休克蛋白，CIRP和RBM3。这两个蛋白质都属于由一个氨基端共有序列RNA结合结构域（CS-RBD）和一个羧基端富含甘氨酸结构域组成的蛋白质家族。在小鼠睾丸中，CIRP仅在初级精母细胞中表达，而RBM3仅在支持细胞中表达。在实验性隐睾中，CIRP和RBM3的表达减少。精索静脉曲张组生殖细胞CIRP蛋白表达较正常组明显减少。CIRP和RBM3表达在睾丸中的升高的温度下下调。对这些冷休克蛋白的分析可能有助于阐明精索静脉曲张患者男性不育的分子机制。为了分析这些蛋白在精子发生中的作用，我们尝试将这些基因导入生精细胞。利用小鼠生精细胞系 ...更多信息 、GC-1 spg和GC-2 spd（ts）作为靶细胞，试图定义将有效地逆转录基因的逆转录病毒载体的设计。以氯霉素乙酰转移酶（CAT）为报告基因，检测了不同逆转录病毒长末端重复序列（LTRs）的启动子活性。在两种细胞系中，脾病灶形成病毒（SFFVp）的U3区的增强子活性均高于Molony鼠白血病病毒（MoMuLV）。骨髓增生性肉瘤病毒（MPSV）的U3区仅在GC-1 spg细胞中表现出较高的活性。表达受到抑制的引物结合位点（PBS）的抑制元件的莫洛尼相关病毒。比较了鼠胚胎干细胞病毒（MESV）PBS和SFFVp或MPSV LTR组成的混合载体与MoMuLV载体对多药耐药基因（mdr-1）的转导效率。罗丹明外排试验和秋水仙素抗性克隆形成试验表明，杂交载体的基因表达更高。两性和亲性受体被发现在两种细胞系中表达和功能。因此，利用逆转录病毒载体将目的基因导入GC-1 spg和GC-2 spd（ts）生精细胞系，并在体外分析其对生精细胞的影响是可能的。由于GC-2 spd（ts）细胞在体外可分化为单倍体细胞，因此可以在体外分析所转移的基因对减数分裂过程的影响。代替通过电穿孔引入质粒DNA（Yamazaki等人，1998），将逆转录病毒载体注射到体内睾丸的生精小管中也可以分析转移的基因对体内精子发生过程的影响。少

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