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Expression of sterol responsive element binding proteins in mouse germ cell maturation

INTRODUCTION Sterol responsive element binding proteins (SREBPs) are membrane-bound transcription factors which control the homeostasis of cholesterol and fatty acids in animal cells1. Cholesterol level/SREBP-dependent pathway is responsible for regulation of cholesterol homeostasis in liver and other somatic tissues. However, cholesterol/SREBP-independent regulation of cholesterogenic genes occurs in certain physiologic/pathophysiologic conditions. Although our previous RT-PCR results clearly established the expression of SREBP-1c and SREBP-2 transcripts in the whole mouse testis and in specific testis cell types (interstitial and germ cells)2, the role of SREBPs in germ cells is not yet clear. AIM The aim of this study was: (1) to determine the expression of SREBP-2 protein in nuclear extracts of mouse liver and testis, and (2) to localize SREBPs proteins in mouse testis at the ultrastructural level. METHODS (1) Expression of SREBP-2 protein in mouse liver and testis was determined by Western blotting. Nuclear proteins were prepared from liver and testis of normally fed and starved prepubertal and adult male mice. Nuclear extracts were separated on 8% SDS-polyacrylamide gels, and immunoblotting was performed using anti-SREBP-2 monoclonal antibody. Protein visualization was done by chemiluminiscence Western blotting detection system. (2) The cellular localization of SREBPs in mouse tissue was analyzed by immunogold electron microscopy using primary antibodies that recognize SREBP-1 and SREBP-2, and secondary antibodies bearing 10 nm gold particles, which were applied to ultrathin sections of mouse testis. RESULTS Western blot analysis showed the presence of mature SREBP-2 protein, migrating on SDS gels as 66-68 kDa, in liver and testis nuclear extracts. Also, three SREBP-2 immunoreactive proteins (72, 63 and 55 kDa), which are not present in mouse liver nuclei, were detected in testis nuclei of prepubertal and adult mice2. Short 55 kDa isoform corresponds to recently described germ cell specific SREBP-2 isoform (SREBP-2gc)3. Preliminary immuno-electron microscopy data showed the presence of SREBPs in nuclei of primary spermatocytes, in nuclei of round and elongated spermatides, and in nuclei of interstitial cells. CONCLUSION Although the presence of SREBPs isoforms in mouse germ cells has been shown on the level of mRNA and protein expression, and cellular localization, the role of SREBPs in male germ cell maturation remains unclear. Interestingly, in testis nuclei we confirmed the presence of SREBP-2gc, which enables cholesterol level-independent/SREBP2gc-dependent regulation of cholesterogenic genes. In conclusion, previously described discordant activation of cholesterogenic genes4 in the testis is cholesterol level-independent but it may involve cholesterol level-independent SREBPs isoforms. REFERENCES 1. M.S. Brown, J.L. Goldstein. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell, 89 1997 331-40 2. K. F. Tacer, S. Kalanj Bognar, M. R. Waterman, D. Rozman. Lanosterol Metabolism and SREBP Expression in Male Germ Cell Maturation, J Ster Biochem Mol Biol, 2003 (in press) 3. H. Wang, F. Liu, C.F. Millette, D.L. Kilpatrick. Expression of a novel, sterol-insensitive form of sterol regulatory element binding protein 2 (SREBP2) in male germ cells suggests important cell- and stage-specific functions for SREBP targets during spermatogenesis, Mol. Cell Biol., 22 (24) 2002 8478-90 4. K. F. Tacer, T.B. Haugen, M. Baltsen, N. Debeljak, D. Rozman. Tissue-specific transcriptional regulation of the cholesterol biosynthetic pathway leads to accumulation of testis meiosis-activating sterol (T-MAS), J. Lipid Res., 43 (1) 2002 82-9.

sterol responsive element binding proteins (SREBPs); transcriptional regulation of cholesterol homeostasis; germ cell maturation

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