Molecular mchanisms of stress tolerance observed at mildly low temperature environment and the clinical relevance of their abnormality in the testis

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In the mammalian testes, germ cells and somatic colic undergo active cell proliferation and differentiation at mildly low temperatures (32-34℃). To elucidate how testicular cells adapt to the lower temperature and become more resistant to various stressors, we have analyzed the molecular mechanism underlying the cold shock response, especially in relation to the cold shock protein alp (cold-inducible RNA-binding protein).1. We failed to identify the transcription factor that binds to the mild-cold response element (MCRE) and activates transcription at lower temperatures by the yeast 1-hybrid screening, but successfully identified several proteins by mass spectrometry that associate with MORE in vitro and in vivo. Most of them seemed to modify activity of the elusive transcription factor.2. When over-expressed in cultured rolls, several of the identified "MCRE-binding proteins" enhanced the expression of Cap as well as the reporter gene driven by MCRE and CMV promoter One of the genes was transiently over-expressed in cultured cells and the induced genes similarly to Cirp were identified by using a DNA microarray.3. MCRE was found to be ineffective as a cold-responsive enhancer when it is integrated into the chromosome at a cm all copy numbers. We optimized the sequence and copy numbers for better induction at 32℃.4. We produced the dip knockout mice and demonstrated that Cirp is involved in wound healing process.5. By using the yeast 2-hybrid screening, we identified proteins, including a kinase, that interact with Cirp and potentially endow resistance to apoptotic stresses.