THE ROLE OF COLD SHOCK PROTEIN IN SPERMATOGENESIS

我们已经克隆并鉴定了两种冷休克蛋白：CIRP和RBM3。它们都是由一个氨基末端共有序列核糖核酸结合域和一个羧基末端甘氨酸富含结构域组成的蛋白质家族，在体外32℃诱导小鼠体细胞表达。CIRP仅在初级精母细胞中表达，而RBM3仅在睾丸支持细胞中表达。在实验性隐睾症睾丸中，CIRP和RBM3的表达减少。精索静脉曲张患者睾丸生殖细胞表达的CIRP蛋白低于无精索静脉曲张患者。随着温度升高，睾丸组织中CIRP和RBM3的表达下调。在试验中分析这些冷休克蛋白可能有助于阐明精索静脉曲张患者男性不育的分子机制。为了分析这些蛋白质在精子发生中的作用，我们试图将这些基因引入生精细胞。利用小鼠生精细胞系…此外，以GC-1 spg和GC-2 spd(Ts)为靶细胞，试图定义一种能有效转导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，检测了不同类型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，脾肿瘤形成病毒(SFFVp)的U3区比Molony鼠白血病病毒(MoMuLV)的U3区具有更高的增强活性。骨髓增生性肉瘤病毒(MPSV)的U3区仅在GC-1 SPG细胞中有较高的活性。表达受Molony相关病毒的引物结合部位(PBS)抑制元件抑制。以多药耐药基因(MDR-1)为载体，以小鼠胚胎干细胞病毒(MESV)为载体，以SFFVp和MPSV的LTR为载体，比较了MDR-1基因的转导效率。罗丹明外排试验和秋水仙碱抗性克隆形成试验表明，杂合载体具有较高的基因表达水平。两性和亲生性受体在两种细胞系中都有表达和功能。因此，利用逆转录病毒载体将目的基因转移到GC-1 spg和GC-2 spd(Ts)生精细胞中，并在体外分析它们的作用效果是可能的。由于GC-2 Spd(Ts)细胞在体外可以分化为单倍体细胞，因此可以在体外分析转基因对减数分裂过程的影响。将逆转录病毒载体注射到体内睾丸生精小管中，代替通过电穿孔导入质粒DNA(Yamazaki等人，1998)，也可以分析转移的基因对体内精子发生过程的影响。较少

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