cDNA Cloning, Genomic Structure and Expression Analysis of the Bovine Lanosterol 14.ALPHA.-Demethylase (CYP51) in Gonads

Abstract

Sterol biosynthesis is an essential metabolic pathway in all eukaryotic organisms. Lanosterol 14a-demethylase (CYP51) is a cytochrome P450 enzyme of the sterol biosynthesis pathway belonging to the CYP51 gene family which is the most evolutionarily conserved member of the CYP superfamily and the only cytochrome P450 family present in all kingdoms of biology.1,2) CYP51 protein catalyzes the oxidative removal of the 14a-methyl group (C32) of lanosterol and 24-methylene-24,25-dihydrolanosterol in yeast and fungi, lanosterol and 24,25-dihydrolanosterol in mammals, and obtusifoliol in plants.In mammalian gonads, CYP51 enzyme is not only important to the synthesis of cholesterol, but also has a role in overproducing meiosis activating sterol (MAS), an intermediate of the postsqualene part of cholesterol biosynthesis, including follicular fluid meiosis activating sterol (FF-MAS) and testis meiosis activating sterol (T-MAS) accumulating mainly in ovaries and testis respectively. MAS was proved to have the ability to trigger resumption of oocytes meiosis, 3) which can promote not only the nuclear but also plasma maturation and preimplantation development of mouse oocytes and fertilized oocytes in vitro. 4,5) Our lab recently have proved MAS also can promote the formation of follicle in fetal mouse ovaries (Xie, unpublished, 2005). CYP51 protein is highly expressed in mammalian testis leydig cell and round, elongating spermatids.6,7) It resides in the endoplasmatic reticulum of most cells and also in acrosomal membranes of spermatids where transport through the Golgi apparatus is suggested.8) But the role of MAS in the spermatogenesis is not well known.The mammalian CYP51 cDNA and the gene have previously been characterized in mouse, 9) rat, 10) human, 11,12) and pig. 13) CYP51 mRNAs are widely expressed in those mammalian tissues with the highest level in haploid male germ cells spermatids. Two different types of CYP51 transcripts somatic transcripts and additional testis-specific transcripts are observed for differential polyadenylation site usage. CYP51 genes have several common characteristics: ten exons and nine introns, highly conserved exon/intron borders and proximal promoter structures.14) The promoter region is TATA-less and contains several highly conserved regulatory elements, such as GC-box, cAMP-responsive elements (CRE), sterol regulatory element (SRE), suggesting an evolutionary conserved mechanism of CYP51 transcriptional regulation in mammals. In somatic cells CYP51 expression is regulated by sterol regulatory element binding proteins (SREBPs), 14) while the germ-cell-specific cAMP/CREMtdependent upregulation of CYP51 expression might contribute to increase the production of MAS in testis. 14,15) Although T-MAS was first found and purified from bovine testis, the information of bovine CYP51 is little. In the present study, we characterized the bovine CYP51 gene and its expression and discussed the probable role of CYP51 and MAS in reproduction. MATERIALS AND METHODS RNA ExtractionBovine tissues were coll...

Mammalian cytochromes P450—Importance of tissue specificity

“…The CYP51 gene family is evolutionary very conserved and has orthologues in animals, fungi, plants and few prokaryotic groups [43]. It has been well characterized in five mammalian species, the human [44,45], mouse [46], rat [47,48], pig [49] and bull [15,50,51]. CYP51 is one of the enzyme in cholesterol biosynthesis, which is a tightly regulated houskeeping pathway composed of over 20 enzymatic steps.…”