Lanosterol 14alfa-demethylase in biosynthesis of cholesterol and signalling sterols

Lanosterol 14a-demethylase (CYP51) is an enzyme converting lanosterol to FF-MAS (folicular fluid meiosis activating sterol). MAS are intermediates of cholesterol biosynthesis and signalling sterols accumulating in gonads. CYP51 expression is regulated mainly at the transcriptional level. Regulation by sterol regulatory element binding protein (SREBP)-dependent pathway leads to overexpression of CYP51 coordinately with other cholesterogenic genes, to increase production of cholesterol in the liver. In contrast to that, a cholesterol feedback-independent regulation of CYP51 exists in male germ cells and it seems that discordant regulation of the entire pathway leads to accumulation of MAS. The aims of this project are: 1.to study molecular mechanisms that lead to production of meiosis activating sterols and/or cholesterol, at the level of gene expression and regulation, protein expression, enzyme activity and intracellular traficking. We will characterize regulatory pathways involved in regulation of cholesterogenic genes in general, and CYP51 in particular. The cAMP-dependent mechanism leading to overexpression of CYP51 in male germ cells will be characterized in detail. Cholesterol biosynthesis intermediates will be measured in animal tissues and in cell cultures where different signaling pathways will be turned on. CYP51 enzyme activity will be measured in subcellular fractions of spermatids. A CYP51-GFP fusion protein will be produced and intracellular traficking monitored. 2. to develop a Cyp51 conditionally knockout mouse and address the ‘in vivo’ role of CYP51 and MAS in reproduction. Cre/loxP strategy will be used to delete CYP51 gene only in spermatids of sexually mature mice after crossing Cyp51loxP female with Sycp1-Cre male. Revealing tissue-specificity of cholesterol and MAS production will contribute to a better understanding of mechanisms involved in cardiovascular and reproductive diseases and may lead to development of new treatment strategies.