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Gestagens and oestrogens are important regulators of pregnancy and parturition. The aim of the present study was the comparative quantification of steroidogenic transcripts in placenta and corpus luteum of cattle and sheep during pregnancy and post partum. Cyp19 transcript variants, derived from different promoters, as well as transcripts of Hsd3b, Cyp11A1, and Cyp17, encoding the steroidogenic enzymes P450arom, 3beta-HSD, P450SCC, and P450C17, respectively, were quantified by newly developed real-time PCR assays. All steroidogenic transcripts were detected in ovine and bovine corpus luteum and placenta during pregnancy, however at a very different concentration. In both species Cyp11A1 and especially Hsd3b transcripts predominated in corpus luteum, outnumbering transcripts of Cyp17 and Cyp19 by more than two and three orders of magnitude, respectively. Cyp19 transcript were found at high concentration in the placenta and at a very low concentration in corpus luteum. Cyp17 transcripts had a relatively low concentration in both, placenta and corpus luteum, however showed a peak of expression in the ovine and bovine term placenta. Tissue- and species-specific Cyp19 transcripts derived from different promoters were detected. In order to map all promoters, the bovine Cyp19 locus was reconstructed by in silico analysis. In the placenta, transcripts were primarily derived from the proximal promoter P1.5 in sheep, but from the distally located P1.1 in cattle. Corpora lutea of both species predominantly expressed P1.1 derived transcripts. Contrary to the bovine, the sheep corpus luteum also showed considerable P1.5 derived expression. This demonstrates that cattle and sheep use different promoters to direct Cyp19 expression during pregnancy.

Aromatase cytochrome P450, the key enzyme of estrogen biosynthesis, is encoded by Cyp19. To elucidate the complex regulation of this gene in mouse gonads (ovary and testis) and brain (thalamic/hypothalamic areas), Cyp19 transcripts were isolated using rapid amplification of cDNA 5' ends and transcript concentrations were estimated in juveniles at different postnatal days (P0, P7, and P14) and in adult animals by real time polymerase chain reaction (PCR). In addition, the murine Cyp19 locus including all known exons and promoters was reconstructed from a recently published sequence of a mouse bacterial artificial chromosome. From each of the tissues investigated, Cyp19 transcripts with a specific 5' untranslated region (5' UTR) were isolated: T(ov) from ovary, T(br) from brain, and T(tes) from testis. T(tes) included a novel 5' UTR that did not show sequence similarities to other Cyp19 transcripts. Real time PCR experiments revealed similar levels of Cyp19 transcript concentrations in neonatal gonads of both sexes. The majority of transcripts were T(ov) in ovaries and T(tes) in testes. During further postnatal development, testicular Cyp19 transcript concentrations transiently decreased, but the contributions of different transcript variants basically remained unchanged. However, ovarian Cyp19 transcript concentrations increased by about 100 times, and almost 100% of all Cyp19 transcripts were identified as T(ov) in adult ovaries. Brains of both sexes showed highest transcript concentrations at P0. However, concentrations in female brains were reduced to adult levels earlier than in male brains. In brains of both sexes, T(br) was found to predominate throughout postnatal life. The results suggest that the mouse Cyp19 gene includes three different promoters that specifically direct expression in ovary, testis, and brain.

During this report the tissue-specific expression and promoter usage of the aromatase cytochrome P450 encoding gene, Cyp19, are compared between cattle and sheep. In addition, data will be presented on the identification of cis-acting regulatory sequences located in the bovine placenta-specific promoter 1.1. In cattle and sheep Cyp19 is mainly expressed in the foetal placental layer and ovarian granulosa cells but also in other organs as brain or testis. Differently spliced transcripts of Cyp19 which include an invariable coding region but a variable 5'-untranslated region could be detected in tissues of both species. However, in contrast to ovary and brain which express homologous transcript variants, different transcripts are present in placentae suggesting that also different placenta-specific promoter regions are active in cattle and sheep. The analysis of the chromatin structure of the main placental promoter 1.1 in different bovine tissues revealed that hypomethylation and the occurrence of DNaseI hypersensitive sites (HS) within this region are associated with promoter activity. Active regulatory elements were identified in reporter gene studies in JEG-3 choriocarcinoma cells. The co-localisation of an E-box element within one of the placenta-specific HS suggests that this element is important for Cyp19 expression in the bovine placenta.

Expression of the Cyp19 gene, encoding aromatase cytochrome P450, is driven by several tissue-specific promoters. The underlying mechanisms of this complex regulation have not yet been elucidated in detail. In the present report we investigate a possible link between chromatin structure and tissue-specific regulation of the bovine Cyp19 gene. We analysed the DNA methylation status and mapped DNaseI hypersensitive sites in the region encompassing the Cyp19 promoter 1.1 (P1.1) which controls Cyp19 expression in the bovine placenta. We show that P1.1 is hypomethylated in placental cotyledons (foetal layer) whereas it is methylated in placental caruncles (maternal layer), testis and corpus luteum. Furthermore, two placenta-specific DNaseI hypersensitive sites, HS1 and HS2, were observed within P1.1. Both DNA hypomethylation and the presence of DNaseI hypersensitive sites correlate with transcriptional activity of P1.1. Sequence analysis of hypersensitive sites revealed potential cis-regulatory elements, an E-box in HS1 and a trophoblast-specific element-like sequence in HS2. It could be demonstrated by electrophoretic mobility shift assays that both sequence motifs are specific targets for placenta-derived nuclear factors. In conclusion, observed tissue-specific differences of the chromatin structure which correlate with tissue-specific promoter activity suggest that chromatin might be an important regulator of aromatase expression in cattle.

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

The aim of the present study was the characterization of the ovine aromatase cytochrome P450 encoding gene (Cyp19) and the analysis of its tissue-specific expression. Two loci with considerable sequence identity were found (Cyp19 and Cyp19b). From Cyp19, tissue-specific transcript variants with different untranslated first exons but identical coding regions could be identified. Cyp19b transcripts were not detected. In the sheep brain and ovarian granulosa cells transcript variants, starting with the untranslated exons 1.4 and 2, respectively, were preferentially found. Exons 1.2 and 1.3 which had been described in bovines could not be detected in sheep and the major 5' untranslated region of the bovine placental transcript, exon 1.1, was also not found to predominate in the sheep placenta. However this exon frequently was combined with a new untranslated exon (exon 1.1a) thus generating an alternative splice variant. The main placental transcripts in sheep had a different first exon (exon 1.5). Two alternatively spliced variants of this transcript were found with tissue-specific preference. From the present data it can be concluded: (a) that the ovine genome contains two copies of Cyp19 of which only one is transcribed and may encode a functional protein; and (b) that in spite of being closely related species, sheep and cattle have remarkable differences concerning tissue-specific transcript distribution and presumable promoter usage.

In cattle, the CYP19 locus comprises the aromatase cytochrome P450-encoding gene (CYP19) and a homologous pseudogene (CYP19P1). It has been assigned to chromosome region 10q26. Cloning of genomic DNA revealed that the CYP19 gene covers more than 56 kb. Its precise extent is still unknown because the DNA spanning the untranslated first exon 1.1 and the coding region (exons 2 to 10) have not been isolated. Furthermore, the chromosome arrangement of closely linked CYP19 and CYP19P1 was also not clear. To establish a high resolution physical map of the entire CYP19 locus, fluorescence in situ hybridization to extended bovine genomic DNA fibers (fiber FISH) was performed. The results demonstrate (1) that the clone containing exon 1.1 is located about 19 kb upstream from the CYP19 coding region. (2) Within the chromosome region 10q26 CYP19 and CYP19P1 are arranged "tail-to-head", being separated by a distance of about 24 kb between the labeled clones. (3) The physical size of the bovine CYP19 locus amounts to a minimum of 130 kb.

Focuses on the comparative investigation of Cyp19 gene and its transcript in cattle and sheep. Genomic organization of Cyp19; Transcript variants and promoters; Tissue-dictribution of Cyp19 transcript variants and tissue-specific promoter activity.