Jens Vanselow

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 placen...

Three Different Promoters Control Expression of the Aromatase Cytochrome P450 Gene (Cyp19) in Mouse Gonads and Brain

Abstract 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: Tov from ovary, Tbr from brain, and Ttes from testis. Ttes 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 Tov in ovaries and Ttes 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 Tov 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, Tbr 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.

Research resource: preovulatory LH surge effects on follicular theca and granulosa transcriptomes.

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.

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.

Down-regulation of genes encoding steroidogenic enzymes and hormone receptors in late preovulatory follicles of the cow coincides with an accumulation of intrafollicular steroids.

The transformation of the dominant follicle into a functional corpus luteum is accompanied by a profound molecular and morphological reorganization of somatic cell layers. Several studies have focused on gene expression during early processes of follicular differentiation as it relates to recruitment and selection of dominant follicles. However, little information exists on changes of gene expression profiles in late preovulatory follicles. This lack of information is addressed here to elucidate molecular mechanisms behind the LH-induced transition from the large dominant estrogen-active to the preovulatory follicle, an intermediate stage toward full luteinization. Transcripts encoding key molecules for the biosynthesis of steroid hormones and prostaglandins, as well as receptors for gonadotropic and growth hormones (Star, Cyp11a1, Hsd3b, Cyp17, Cyp19, Ptgs2, Fshr, Lhr, and Ghr), were quantified by real-time polymerase chain reaction (PCR) in the granulosa and theca of large dominant and late preovulatory follicles. The steroid hormones progesterone (P4) and estradiol-17beta (E2) were monitored to distinguish estrogen-active and estrogen-inactive follicles. We found that (1) independent of the follicular stage, the gene expression profile was very different in granulosa and theca; (2) the abundance of several key transcripts was lower in estrogen-inactive, compared with estrogen-active, dominant follicles; (3) in the granulosa of late preovulatory follicles, transcripts encoding steroidogenic enzymes and hormone receptors were largely down-regulated, whereas (4) progesterone and E2 were found at high concentrations in the follicular fluid. Collectively, our data show that late preovulatory follicles have a transient and unique gene expression profile and are clearly different from both the preceding and subsequent (follicular and luteal, respectively) stages.

DNA Methylation Is Not Involved in Preovulatory Down-Regulation of CYP11A1, HSD3B1, and CYP19A1 in Bovine Follicles but May Have a Role in Permanent Silencing of CYP19A1 in Large Granulosa Lutein Cells

Abstract The luteinizing hormone-induced morphological and physiological reorganization of the bovine follicle is preceded by a profound and well-orchestrated modulation of gene expression. In the present study, the cell type-specific methylation profiles of CYP11A1, HSD3B1, and CYP19A1, genes that encode key enzymes of steroid hormone biosynthesis, were analyzed to elucidate whether epigenetic parameters such as DNA methylation might be involved in gene regulation during luteinization. Transcript abundance and DNA methylation levels were determined in granulosa and theca of large dominant and late preovulatory follicles and in large granulosa lutein cells isolated from corpora lutea cyclica and graviditatis. Levels of the steroid hormones progesterone and estradiol-17beta were monitored to assess the physiological status of individual follicles. From our results, we conclude that (1) individual, even closely neighboring, CpG dinucleotides can show very different methylation levels; (2) proximal (<300 base pair [bp] from the respective transcription start sites) but not distal CpGs show cell type-specific methylation levels; (3) higher methylation levels suggestively preclude high levels of gene expression; (4) DNA methylation is not involved in the transient (HSD3B1 and CYP11A1) respectively permanent (CYP19A1) down-regulation of gene expression in late preovulatory follicles; and (5) DNA methylation may have a role in the permanent shutdown of promoter 2-directed CYP19A1 expression in large (granulosa derived) lutein cells.

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.

Increasing cell plating density mimics an early post-LH stage in cultured bovine granulosa cells.

Cultured ovarian granulosa cells are essential models to study molecular mechanisms of gene regulation during folliculogenesis. Here, we characterize primary tissue culture models for bovine granulosa cells by morphological and physiological parameters and by novel molecular luteinization markers, as transcript abundance and DNA methylation levels. The data show that: (1) collagen substrate increased the number of attached, viable cells; (2) the expression of the key transcripts of estrogen synthesis, CYP19A1, could be induced and maintained in granulosa cells from small to medium but not from large follicles, whereas (3) only granulosa cells from large but not from smaller follicles were responsive to LH; (4) serum supplementation unfavorably transformed the cellular phenotype, induced proliferation and PCNA expression, reduced or abolished the transcript abundance of steroidogenic key genes and of gonadotropin receptor genes, CYP11A1, CYP19A1, FSHR and LHCGR but, however, did not increase the abundance of the luteinization-specific marker transcripts PTGS2, PTX3, RGS2 and VNN2; but (5) by increasing the plating density, estradiol production and the abundance of CYP19A1 transcripts, in particular those derived from the main ovarian promoter P2, were decreased concurrently leaving P2-specific DNA methylation levels unchanged, whereas progesterone secretion was stimulated and the expression of both luteinization-specific marker transcripts, RGS2 and VNN2, was significantly induced. From these data, we conclude that increasing the plating density induces a different, partly complementary, physiological and gene expression profile in cultured bovine granulosa cells and drives the cells towards an early post-LH stage of luteinization, even in the absence of luteinizing agents.

Two high-fertility mouse lines show differences in component fertility traits after long-term selection

UNLABELLED Two selected high-fertility mouse lines, namely FL1 and FL2, and a non-selected control (Fzt:DU), all derived from the same genetic pool, were analysed as an animal model for polytocous species to elucidate the effects of long-term selection and to identify relevant component traits that may be responsible for fertility performance. The index trait used for breeding selection was largely increased by 104% and 142% in the FL1 and FL2 lines, respectively, resulting in an average litter size of 17.3 pups and 18.7 pups per litter in the FL1 and FL2 lines, respectively, compared with a litter size of 11.0 pups per litter in the control (Fzt:DU). In addition, different component fertility traits were analysed in females of all three lines at different stages of the oestrous cycle and pregnancy. IN CONCLUSION (1) early embryonic development was accelerated in the FL1 and FL2 lines compared with control; (2) plasma progesterone levels were not correlated with fertility performance; (3) a largely increased ovulation number (i.e. number of corpora lutea) was responsible for high prolificacy in both lines; however, (4) the number of ova shed, as well as the rate of loss of ova and pre- and postimplantation conceptuses, was very different in the FL1 and FL2 lines, suggesting that different genetic components may be responsible for the high prolificacy in both high-fertility lines.

Epigenetic mechanisms contribute to enhanced expression of immune response genes in the liver of cows after experimentally induced Escherichia coli mastitis.

Endotoxins, such as lipopolysaccharide (LPS), are released during infection with Gram-negative bacteria, which can result in excessive activation of toll-like receptor (TLR) signalling. The aim of the present study was to investigate whether epigenetic mechanisms are involved in controlling the onset and progression of the systemic inflammatory response. Using chromatin accessibility by real-time (CHART) PCR to assess livers from cows with experimentally induced Escherichia coli mastitis, this study demonstrated that the chromatin at the site of the promoters of the genes encoding TLR2, TLR4, lipopolysaccharide binding protein (LBP) and haptoglobin (HP) was opened up 24 h after infection, accompanied by enhanced mRNA expression by these genes. Such modulation did not occur in the same samples for the αS1-casein promoter, which served as a negative control. Demethylation of the TLR4 promoter accompanied opening up of chromatin. These data suggest that modulation of epigenetic factors might offer a novel approach to treating adverse systemic reactions elicited in cows with E. coli mastitis.