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Functions and clinical significance of cold shock proteins in the testis

睾丸冷休克蛋白的功能及临床意义

基本信息

批准号：
11470335
负责人：
FUJITA Jun
金额：
$ 6.59万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Spermatogenesis is a complex process ; a diploid spermatogonium undergoes mitosis and develops into primary spermatocytes. As a result of two meiotic divisions and spermiogenesis, spermatozoa are produced. These processes are controlled by the germ cell - Sertoli cell interactions, and the tightly regulated expression of many genes. In addition to these, spermatogenesis has a unique feature. Unless the testis temperature is maintained to be lower than the body cavity temperature, germ cells will die. To elucidate the molecular mechanisms of spermatogenesis, we tried to isolate genes the expression of which are induced by the testis temperature (32 degree C) and analyze their involvement in male spermatogenesis. The major findings are as follows ; 1) We cloned the mouse cDNA of the cold shock protein, rbm3, and showed that it is expressed in the Sertoli cells of the testis and that its expression is decreased in the cryptorchid testis. 2) By using the yeast hybrid screening method, we tried to identify the RNAs that interact with the cold shock protein Cirp, but no success. 3) We determined the cold shock response element in the DNA sequence of the mouse cirp gene. By using yeast 1-hybrid screening method, we are isolating the protein that binds to this element. 4) Homozygous mutant mice that are deficient in cirp gene were produced. At present no abnormality in phenotypes is apparent. 5) We found that cirp is consticutively expressed the rat neuronal cells and that the expression decreases after transient forebrain ischemia or exposure to H2O2.6) Three proteins that interact with Cirp were isolated by the yeast 2-hybrid screening method. 7) We identified several genes whose expressions are increased in cultured cells by the cold stress.

精子发生是一个复杂的过程；二倍体精原细胞经历有丝分裂并发育成初级精母细胞。由于两次减数分裂和精子发生，产生了精子。这些过程受生殖细胞-支持细胞相互作用的控制，并受到许多基因表达的严格调控。除此之外，精子发生还有一个独特的特点。除非保持睾丸温度低于体腔温度，否则生殖细胞会死亡。为了阐明精子发生的分子机制，我们试图分离睾丸温度（32℃）诱导表达的基因，并分析其在雄性精子发生中的作用。主要研究结果如下：1)克隆小鼠冷休克蛋白rbm3的cDNA，发现该蛋白在睾丸Sertoli细胞中表达，在隐睾睾丸中表达减少。2)利用酵母杂交筛选方法，我们试图鉴定与冷休克蛋白Cirp相互作用的rna，但没有成功。3)我们确定了小鼠cirp基因DNA序列中的冷休克反应元件。通过酵母1-杂交筛选方法，我们分离出与该元件结合的蛋白。4)产生了cirp基因缺失的纯合突变小鼠。目前未见明显的表型异常。5)我们发现cirp在大鼠神经元细胞中呈连续表达，在短暂的前脑缺血或h2o2作用下，cirp的表达有所下降。2.6)通过酵母2杂交筛选法分离到3个与cirp相互作用的蛋白。我们在培养细胞中发现了几个在冷胁迫下表达增加的基因。