Introduction

Top Abstract Introduction Genomic studies of human... Pattern of cyp expression... Possible function for p45014dm... Future directions References

The cytochrome P450 (P450, or CYP)¹ superfamily of genes includes more than 1000 genes, being found in species of all of the kingdoms of biology (Nelson, 1998). Only one of these genes, however, is found in prokaryotes and eukaryotesCYP51, encoding sterol 14-demethylase (P45014DM) (Yoshida et al., 1997). Other P450-dependent activities, such as -hydroxylation of fatty acids, are also found in different phyla, but different gene families have evolved in different species to serve this function. The conservation of CYP51 is probably due to the complex chemical reaction required for 14-demethylation, combined with the essential role of the synthesis of membrane sterols. The methyl group is first converted to an alcohol, then to an aldehyde, and then removed as formic acid, each step requiring one molecule of molecular oxygen and one molecule of NADPH (fig 1). Thus human and rat CYP51 share 93% amino acid identity. Even the Mycobacterium tuberculosis CYP51-like gene, which is the most distant from the human enzyme, is still clearly recognizable, showing greater than 30% identity to other CYP51 genes.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Three-step process in the cholesterol biosynthetic pathway converting lanosterol to FF-MAS. All steps require lanosterol 14-demethylase (CYP51) and P450 reductase.

In yeast and fungi, CYP51 plays an essential role in ergosterol biosynthesis. It is involved in phytosterol biosynthesis in plants, and in animals it is involved in cholesterol biosynthesis. The CYP51-like gene in M. tuberculosis has recently been shown to catalyze sterol 14-demethylase activity, although its function in vivo is unknown (A. Bellamine, unpublished results, 1998). In animals, CYP51 is the only P450 in the cholesterol biosynthetic pathway. It is localized in the endoplasmic reticulum of all nucleated cells, being the only P450 involved in a housekeeping metabolic pathway (Rozman et al., 1996b). Upon cloning human CYP51, we found by Northern-blot analysis that it is expressed in all cells studied as expected (Stromstedt et al., 1996). However, we were surprised to find that the highest level of expression was in the testes. Also, two different-sized transcripts were observed in the testes, one being 3.8 kb, the transcript size found in all human tissues, and the other being 2.0 kb, a testes-specific shorter transcript (Stromstedt et al., 1996). This minireview summarizes the studies undertaken to try to explain the unusually high-level expression of CYP51 in the testis.

	Genomic Studies of Human CYP51

Top Abstract Introduction Genomic studies of human... Pattern of cyp expression... Possible function for p45014dm... Future directions References

Restriction endonuclease analysis of human DNA suggested that the CYP51 gene might be quite large, compared with many other P450 genes (Rozman et al., 1996b; Rozman et al., 1996a). However, localization of the CYP51 gene, using human/mouse and human/hamster cell hybrids, revealed that there are three CYP51-related genes in the human genome (Rozman et al., 1996b). These genes are localized on chromosome 3, chromosome 7, and chromosome 13. The genes on chromosomes 3 and 13 were found to be processed pseudogenes. Processed pseudogenes arise from reverse transcription of mRNA in germ cells followed by illegitimate recombination of the resulting cDNA into the genome. They have no introns since they arise from mRNA. Most often, processed pseudogenes are found for housekeeping genes. The CYP51 processed pseudogenes are the only intronless genes in the CYP superfamily. The functional gene on chromosome 7 is a typical P450 gene, being approximately 22 kb in length and having 10 exons (Rozman et al., 1996a). The presence of processed pseudogenes and a testes-specific transcript suggests that CYP51 is highly expressed in germ cells.

	Pattern of CYP Expression in Testes

Top Abstract Introduction Genomic studies of human... Pattern of cyp expression... Possible function for p45014dm... Future directions References

To study CYP51 expression in testes, it was necessary to use an animal model. The rat has proven particularly useful for such studies. As in humans, the highest level of CYP51 expression in rats is found in the testes. In situ hybridization of rat testes (fig. 2) showed high-level expression of CYP51 in certain seminiferous tubules, moderate or low levels of expression in other seminiferous tubules, and no expression in other seminiferous tubules (Stromstedt et al., 1998). This indicated two things. First, CYP51 was expressed at high levels in developing germ cells. Second, it indicated that CYP51 expression in germ cells follows a stage-specific pattern. This refers to the fact that spermatogenesis takes more than 40 days to develop mature sperm in the rat, going through many different stages during this differentiation process (Russel et al., 1990). Stages include both premeiotic (diploid) and postmeiotic (haploid) germ cells. In order to understand why such high levels of CYP51 expression occur in the testes, it was necessary to determine the temporal pattern of expression of CYP51 during spermatogenesis. Only with this information would it be possible to elucidate the function of high-level CYP51 expression in germ cells. Northern-blot analysis of mRNA from different populations of rat germ cells was carried out. As in humans, rat testes contain a shortened, testes-specific mRNA (Stromstedt et al., 1998). However, rat testes contain three somatic cell transcripts, rather than just one as in humans. These four transcripts in rat testes (fig. 3) result from the presence of four different polyadenylation sites in the 3'-untranslated region of the rat gene (J. DeLeon, unpublished results, 1998). The shortened, germ-cell-specific transcript uses the most upstream (5') polyadenylation site. The reason for the existence of one somatic transcript in humans and three in rats is not apparent. Northern-blot analysis of isolated populations of rat germ cells suggested that CYP51 is expressed in postmeiotic, haploid germ cells (Stromstedt et al., 1998). When staged segments of rat seminiferous tubules (Parvinen and Vanha-Perttula, 1972; Parvinen and Ruokonen, 1982) were used, it was found that CYP51 is, in fact, overexpressed in round and elongating spermatids, both of which are postmeiotic haploid germ cells. Thus, while somatic-like CYP51 transcripts are present in both premeiotic and postmeiotic germ cells at low levels, the germ-cell-specific transcript is found at high levels in round and elongating spermatids. Furthermore, when CYP51 enzymatic activity was measured, a significant increase in activity (table 1) was found in samples containing postmeiotic germ cells, compared with samples having only premeiotic germ cells (Stromstedt et al., 1998). Thus the increase in CYP51 mRNA levels observed in postmeiotic germ cells, compared with premeiotic germ cells, leads to increased P45014DM activity.

View larger version (130K): [in this window] [in a new window]   Fig. 2.   In situ hybridization of CYP51 in rat testes, using antisense probe. This darkfield image shows the expression in seminiferous tubules.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Northern-blot analysis of CYP51 mRNA transcripts in rat livers (somatic) and testes. Note that the three largest transcripts are in both RNA samples, while the shortest (1.9 kb), which is germ-cell-specific, is found only in the testes.

	Possible Function for P45014DM Activity in Postmeiotic Germ Cells

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After completion of meiosis, spermatids undergo major morphological changes (from round to elongating spermatids), which must include the remodeling of cellular membranes. These processes surely could reflect the need for increased cholesterol biosynthesis, and, thus, one reason for increased CYP51 expression could be a requirement for increased de novo cholesterol biosynthesis. However, it is also possible that increased P45014DM activity in postmeiotic germ cells is important for the production of signaling sterols. Byskov and colleagues purified sterols from human follicular fluid (FF-MAS) (fig. 1) and bull testes (T-MAS), which enhance the activation of meiosis of mouse oocytes when added in vitro (Byskov et al., 1995): thus the designation MAS, meiosis-activating substance. In bull testes, the predominant sterol is T-MAS, which is the product of the 14-reductase, the enzyme immediately following P45014DM in the cholesterol biosynthetic pathway (Yoshida et al., 1996). FF-MAS is a minor product in the bull testes. The accumulation of these intermediates in testes suggests that they might have a signaling function during spermatogenesis. The mammalian 14-reductase has not been cloned, and it is unknown whether it also is elevated in postmeiotic germ cells. Nevertheless, the elevated levels of P45014DM could indicate its role in signaling sterol production. What could the function of these sterols be during spermatogenesis? Since they seem to accumulate in postmeiotic germ cells, it is perhaps unlikely that they serve a meiosis-activating role in male germ cells. The complexity of spermatogenesis makes it difficult to speculate on the role of MAS sterols, and it must remain an intriguing question as to why there is an elevated level of expression of CYP51 in male haploid germ cells.

	Future Directions

Top Abstract Introduction Genomic studies of human... Pattern of cyp expression... Possible function for p45014dm... Future directions References

Elucidation of the role of high-level expression of CYP51 in postmeiotic germ cells cannot be directly addressed by classic knockout technology because CYP51 is required for the housekeeping function of cholesterol biosynthesis during embryogenesis. Instead, a conditional knockout experiment that focuses on blocking the expression of CYP51 only in the postmeiotic germ cells of adult animals will be necessary to determine what function MAS sterols serve during spermatogenesis.