Unhatched bovine blastocysts express all transcripts of the estrogen biosynthetic pathway, but steroid hormone synthesis could not yet be demonstrated.

Early embryos of rodent species and rabbits but also farm animals such as pigs, horses and cattle produce estrogens, which are considered important regulators of the implantation process. In cattle, the exact stage at which embryonic estrogen synthesis commences is yet unknown. However, this information is regarded as important to consider a possible role of embryonic estrogens in preimplantation development. Therefore, in this study, we first used quantitative reverse transcription PCR to examine the mRNA expression of the enzymes required for the conversion of cholesterol into free and sulfonated estrogens (CYP11A1, CYP17A1, HSD3B, CYP19A1, and SULT1E1), the cholesterol carrier protein STAR, and the estrogen receptors ESR1 and ESR2 in in vitro produced morulae and unhatched blastocysts (d 6-9). Only in the blastocysts, were the mRNAs of the entire estrogen biosynthesis chain and of both estrogen receptors clearly present, whereas mRNA specific to ESRs was already detectable in the morulae. We also examined the expression of the corresponding enzymes in blastocysts at the protein level. None of the enzymes were detectable by capillary-based western analysis. Immunofluorescence methods were established for the detection of CYP17A1, CYP19A1, and SULT1E1. CYP17A1 was observed in the inner cell mass and trophectoderm, whereas CYP19A1 and SULT1E1 were present only in trophectoderm. An attempt to detect estrogen sulfotransferase activity was unsuccessful. Despite clear evidence that some elements of the estrogen biosynthetic pathway are also present at the protein level, it remains to be clarified whether the enzyme cascade underlying estrogen production is already functional in unhatched blastocysts.

DNA methylation of placenta-specific Cyp19 promoters of cattle and sheep.

Locally produced estrogens are important paracrine regulators of placental growth and differentiation in cattle and sheep. The key enzyme of estrogen biosynthesis, aromatase cytochrome P450, is encoded by CYP19. Transcription of this gene in fetal cotyledons is the first regulatory step of placental estrogen production. The aim of the present study was to comparatively investigate if epigenetic mechanisms as tissue- and differentiation-specific DNA methylation might be involved in the regulation of placental CYP19 expression. Earlier investigations demonstrated that cattle and sheep use different promoters, P1.1 and P1.5 for placental CYP19 expression, respectively. During the present investigation, methylation of individual CpG dinucleotides within these promoter regions was analyzed with bisulfite direct sequencing in placental and luteal tissue. Transcript abundance in sheep was determined with qPCR. Both promoters contain only few CpGs and can therefore be classified as "low CpG densitiy regions". The average methylation levels of both promoter regions were significantly reduced in cotyledons compared to caruncles and corpus luteum of both species, which inversely coincides with high and low expression levels, respectively. It was evident however that even neighbouring CpGs can show very different, individual methylation levels. From the data we conclude that (1) CYP19 promoters are differentially methylated in ovine and bovine placental tissues of foetal and maternal origin; (2) DNA methylation is suggestively involved in the regulation of CYP19 expression; (3) the DNA methylation status on its own is not sufficient for the selection of tissue-and species-specific CYP19 promoters.

Cultured bovine trophoblast cells differentially express genes encoding key steroid synthesis enzymes.

Placental trophoblasts are an important source of endocrine, paracrine and autocrine acting hormones. The aim of the present study was to establish and evaluate a tissue culture model for bovine trophoblasts to study regulation of key genes of steroid hormone synthesis. Trophoblast cells were isolated from cotyledons by collagenase disaggregation and subsequent percoll density gradient centrifugation. The cells were seeded on collagen coated dishes and incubated for up to seven days. The cells were characterized for the presence of mesenchymal vimentin and epithelial cytokeratin filaments and for Dolichos biflorus agglutinin (DBA) binding, a marker for differentiated trophoblast giant cells. Transcripts of Hsd3b, Cyp17 and Cyp19 encoding 3beta-HSD, P450c17 and P450arom, the key enzymes of progesterone, androgen, and oestrogen biosynthesis, respectively, and of Csh1 encoding the trophoblast-specific hormone placental lactogen (PL) were measured by qPCR. Uninucleate cotyledonary epithelial cells and bi- and trinucleate trophoblast giant cells efficiently formed a dense cell layer on the collagen coated dishes within 24 h. Bi- and trinucleate cells showed DBA binding and weak or undetectable cytokeratin immunoreactivity. Vimentin-positive, fibroblast-like cells were found on top of this cell layer. Cyp19 transcripts were found in freshly dissociated but not in cultured cells. Cyp17 expression continuously increased, Hsd3b transcripts largely and rapidly increased during the first days in culture, followed by a decline after three days, whereas Csh1 decreased towards day seven. Serum free culture conditions significantly enhanced Cyp17 and Csh1 but not Hsd3b expression. The data indicate that collagen is a favourable substrate for cultured binucleate trophoblast giant cells. The cells represent an in vitro model to study the regulation of key genes of placental progesterone and androgen but not of oestrogen biosynthesis.

Promoter-2-derived Cyp19 expression in bovine granulosa cells coincides with gene-specific DNA hypo-methylation.

Cyp19, the key gene of oestrogen biosynthesis, is expressed at very different concentrations and from different promoters in bovine granulosa cells (GCs) and in pregnant corpora lutea (CL), respectively. The present study was aimed to investigate if DNA methylation and thus epigenetic mechanisms might play a potential role in the regulation of Cyp19 expression and promoter-specific activity in GCs of cycling versus CL of pregnant cows. It was demonstrated that GCs express high concentrations of promoter-2-derived Cyp19 transcripts whereas CL samples isolated before and after implantation, and at the end of the first trimester, showed very low Cyp19 transcript concentrations, all of them derived from promoter 1.1. Two genomic regions including promoter 1.1 and promoter 2 were largely unmethylated in GCs but methylated in all CL samples. The data suggest that promoter-2-derived high-level expression but not promoter-1.1-derived low-level expression of Cyp19 might be controlled by cell-type-specific DNA methylation.

Maternal treatment with somatotropin during early gestation affects basic events of myogenesis in pigs.

The effects of maternal treatment with somatotropin during early gestation on fetal muscle development were determined. Crossbred gilts received daily injections of either 3 ml of a placebo ( n=31) or of 6 mg porcine somatotropin ( n=31) from day (d) 10 to 27 of gestation and samples were collected from d 28 embryos, d 37 and 62 fetuses, and from neonates. Administration of somatotropin increased the total number of fibres (primary and secondary fibres) in neonatal semitendinosus muscle of middle- and low-weight littermates, whilst no increase was observed in psoas major muscle. Somatotropin induced increases in muscular protein concentration, creatine kinase activity, muscle fibre girth, as well as type II to type I fibre conversion which revealed an advanced degree of differentiation at birth. Treatment effects on prenatal development preceded these changes. Increased DNA concentrations at d 28 of gestation indicate stimulation of cellular proliferation during the embryonic stages. Thereafter, the withdrawal of somatotropin caused a transient delay of differentiation as indicated by lower protein concentrations and creatine kinase activity compared with controls at d 37 of gestation. This was compensated again at d 62, and the number of semitendinosus primary fibres was increased in middle-weight fetuses, whereas secondary or total fibre number did not yet differ. However, enhanced expression of Myf5 and MyoD indicates higher numbers of initially determined, proliferating myoblasts that may have contributed to increased formation of secondary fibres. In conclusion, maternal somatotropin is an influential factor in early pregnancy capable of affecting the basic events of myogenesis.

Chromatin structure of the bovine Cyp19 promoter 1.1. DNaseI hypersensitive sites and DNA hypomethylation correlate with placental expression.

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.

Expression of the aromatase cytochrome P450 encoding gene in cattle and sheep.

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.

Cis-acting elements regulating the placenta-specific promoter of the bovine Cyp19 gene.

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.

Placenta-specific transcripts of the aromatase encoding gene include different untranslated first exons in sheep and cattle.

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.

Genomic organization of the bovine aromatase encoding gene and a homologous pseudogene as revealed by DNA fiber FISH.

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.

Tissue-specific expression of the bovine aromatase-encoding gene uses multiple transcriptional start sites and alternative first exons.

Here we report on the genomic structure of the bovine aromatase cytochrome P450-encoding gene (Cyp19) and its tissue-specific transcript variants. The gene comprises at least 14 exons (1.1, 1.2a, 1.2b, 1.3,1.4, and 2-10) spanning more than 56 kilobases of genomic DNA. The coding area is confined to exons 2-10. Transcriptional start sites of Cyp19 were examined in granulosa cells, placenta, testis, adrenal gland, and brain, employing 5'-RACE (rapid amplification of complementary DNA ends) and primer extension. The analysis of 5'-RACE clones revealed six Cyp19 transcript variants that were different within their 5'-untranslated regions (5'-UTR). Yet, the coding region was identical in all clones. Although two of these 5'-UTR (the first 152 nucleotides of exon 2 and exon 1.4) are conserved among different species, four others (exons 1.1, 1.2a, 1.2b, and 1.3) did not show sequence homology to any other species. Transcription from exons 1.1 and 2 starts at several adjacent sites. In granulosa cells and placenta, but not in brain, a fraction of transcripts starting with exon 1.2a contains an additional untranslated exon, 1.2b, due to alternative splicing. Transcript variants comprising exon 1.1, 1.2a, 1.2b, or 1.3 were mainly found in the placenta, those with the 5'-UTR of exon 2 were predominant in granulosa cells, and transcripts with exon 1.4 prevailed in the brain. Estimates of Cyp19 transcript concentrations in six different tissues revealed high levels in granulosa cells and placenta, intermediate levels in testis and brain, and low levels in adrenal gland and liver. Our experiments demonstrate that six transcript variants of the bovine Cyp19 gene, including 9-11 exons, are expressed with tissue-specific preferences. These transcripts are presumably generated using five different promoter regions and tissue-specific alternative splicing.

Novel aromatase transcripts from bovine placenta contain repeated sequence motifs.

Aromatase cytochrome P-450 (Aro) is the major enzyme of estrogen biosynthesis. The aim of the present investigation was the isolation and comparative sequence analysis of the bovine aromatase cytochrome P-450 transcript (bCyp19) from a placental lambda gt10 cDNA library. From three overlapping clones, a total sequence of 5180 bp could be derived, including two polyadenylation sites and signals located next to each other. As found in other species, the open reading frame (ORF) comprises 1509 bp and shows 87, 78 and 78% sequence homology to the coding areas of the human, rat and mouse genes, respectively. The 3'-untranslated region (UTR) of the bovine transcript is about 2-kb longer than that of the human gene (hCYP19). It contains homologous retroposon elements of the bovidae dimer family (BDF) at two different positions, and ends with a sequence motif which also occurs repeatedly within the bovine genome. The 5'-UTR isolated from placenta includes a new sequence upstream from exon II that was not found in cattle or other species so far. We conclude from our data that (i) as found in other species, bCyp19 is likely to be transcribed into different mRNA species, (ii) the bovine 3'-UTR was the target for multiple insertions of repeated sequence motifs, (iii) the unusual length of the bCyp19 transcript is mainly due to the long 3'-UTR, (iv) it includes sequences which are found in humans only on the genomic level, conceivably due to mutational inactivation of a primordial polyadenylation signal (PAS) and (v) the recently used, functional PAS is contributed by a downstream bovine repeat element.

An aromatase pseudogene is transcribed in the bovine placenta.

The present investigation is focused on a new, truncated bovine aromatase cytochrome P-450-encoding transcript (bCyp19) which was repeatedly isolated from a placental lambda gt10 cDNA library. Different cDNA probes derived from bCyp19 were used during screenings. The deviant transcript contains areas of considerable sequence homology (89-98%) corresponding to exons II, III, V, VIII and IX of bCyp19. Exons VI and VII are missing. Exon IV is replaced by a bovine repeat sequence motif. Numerous translation stop codons occur within all reading frames, thus suggesting that the transcript does not encode a functional protein. The gene was detected by PCR in the genomes of all animals investigated (n = 38). The experiments demonstrate that the bovine genome contains a non-functional copy (pseudogene, Cyp19 phi) of bCyp19 that is transcribed together with the bCyp19 messenger in the bovine post-parturition placenta.

Mutant analysis of interaction of the Bacillus subtilis transcription regulator AbrB with the antibiotic biosynthesis gene tycA.

The AbrB protein of B. subtilis represses the transcription of various postexponentially expressed genes, such as the antibiotic biosynthesis gene tycA. Recently, we have shown that ArbB binds to the tycA promoter region at two A + T-rich sites; the 'promoter site' (-60 to -35) and the 'leader site' (+169 to +231). In this study we demonstrate that a Ptyc-lacZ fusion missing the leader region is constitutively expressed in wild-type B. subtilis cells and in B. subtilis cells carrying spoOA or abrB mutations. We also show that substitution mutations within the recently reported potential helix-turn-helix DNA binding motif of AbrB did not affect its specific DNA binding ability.

Interaction of AbrB, a transcriptional regulator from Bacillus subtilis with the promoters of the transition state-activated genes tycA and spoVG.

In Bacillus subtilis the abrB gene product negatively affects the transcription of some genes activated during the transition from vegetative to stationary phase of growth. Interaction of AbrB with the promoters of two such genes, spoVG, a sporulation gene, and tycA, an antibiotic biosynthesis gene, was studied by DNase I and hydroxyl radical footprinting. Two binding areas within the leader and promoter regions of tycA were identified. In spoVG the binding site is located at the A + T-rich region upstream of the promoter. Hydroxyl radical footprinting revealed that the AbrB-protected regions, in both the tycA and spoVG promoters, are short A + T-rich regions that are separated by one helical turn, indicating that AbrB binds to one face of the helix. To examine the role of spoOA in the expression of abrB-controlled genes, the levels of AbrB protein in Spo + and in spoOA cells were determined by Western blot analysis. In wild-type cells AbrB was detected only during vegetative growth, whereas in spoOA cells a high level of AbrB was detected during both the vegetative and stationary phases of growth. These findings support a model in which (i) spoOA negatively affects abrB expression, and (ii) the repression of the transition state-activated genes tycA and spoVG in spoOA cells is due to constitutive expression of AbrB, which acts as a repressor.

Cellular oncogenes in human teratocarcinoma cell lines.

We have analysed, by Northern blots, the expression of 14 cellular oncogenes in nine cell lines established from human teratocarcinomas. All lines expressed considerable amounts of p53, c-Ki-ras2, c-Ha-ras1, c-raf1, N-myc, and c-fos. Low level expression of c-myc was detected in some lines. Southern blot experiments revealed no amplification or rearrangement of the c-Ki-ras2, N-myc or c-fos genes. Using a rapid dot-blot screening procedure, based on a combination of in-vitro amplification of ras-specific sequences and oligonucleotide hybridization, we could detect no activation of Ha-ras or Ki-ras or any unexpressed N-ras sequences secondary to a point mutation at codons 12, 13, or 61.