Cloning of bovine estrogen receptor beta (ERbeta): expression of novel deleted isoforms in reproductive tissues.

cDNAs coding for bovine estrogen receptor beta (ERbeta) isoforms were cloned from bovine granulosa cells using a combination of several RT-PCR strategies. The cloned full-length receptor contains an open reading frame of 474 amino acids encoding a protein with high homology to the ERbeta sequences from other species. A second isoform nearly totally lacking the ligand binding domain was cloned that is expressed to relatively high levels in reproductive tissues. Expression of both ERbeta isoforms is down-regulated in corpus luteum and endometrium during the luteal phase of the female cycle. In addition, in granulosa cells several ERbeta isoforms carrying major internal deletions were detected by RT-PCR and cloned. Transient transfection studies expressing the two major bovine ERbeta isoforms together with an ERE reporter construct show estrogen-dependent transactivation by the full-length isoform, whereas the isoform lacking the ligand binding domain did not show any transactivation.

Bovine endometrial epithelial cells as a model system to study oxytocin receptor regulation.

Endometrial epithelial cell cultures were established from bovine uterine tissue collected during the oestrous cycle from commercially slaughtered animals. These cells were shown to express moderately high levels of oxytocin receptors (OTR) (up to 30000 per cell) after about one week in culture. These receptors have been characterized at the molecular, pharmacological and functional level and shown to be identical to those expressed in the bovine endometrium in vivo. Preliminary experiments to investigate the regulation of the OTR and its gene using this system, have shown that expression is to a large degree constitutive, the receptors being spontaneously upregulated during culture. Sex steroids at concentrations close to or above the serum limits observed in vivo appeared to have no effect, although the cells were shown to express mRNA for the specific steroid receptors throughout culture. Only the blastocyst product, interferon-tau, showed a significant effect, downregulating both OTR and their gene transcripts in the cultured endometrial epithelial cells. Although more extensive studies are necessary, these results support the view that the OTR gene is controlled in part at least by a combination of constitutive and inhibitory elements.

An autocrine progesterone positive feedback loop mediates oxytocin upregulation in bovine granulosa cells during luteinization.

During luteinization of bovine granulosa cells in vitro in the presence of insulin, or insulin plus forskolin, there is a massive upregulation not only of progesterone production, but also of the gene for the peptide hormone oxytocin, with secretion of the peptide into the medium. By using the progesterone receptor antagonists, RU486 or onapristone, we have shown that this upregulation of the oxytocin gene is mediated by an autocrine loop involving endogenous progesterone and the progesterone receptor in the granulosa cells. The inhibition of oxytocin gene expression by the anti-gestagens can be reversed by medroxyprogesterone acetate but not by dexamethasone, showing that the effect is due to the endogenous progesterone. Since oxytocin also appears to be involved in a positive feedback loop regulating steroid output, these results show that endogenous progesterone itself is a key feedback element in the cascade of events termed luteinization, and that possibly anti-gestagens can influence ovarian function in vivo by directly disrupting this process.

Analysis of the 170-kDa lectin gene promoter of Entamoeba histolytica.

The promoter region driving the gene for the 170-kDa heavy subunit of the Entamoeba histolytica galactose-inhibitable lectin was analysed by transient transfection using the chloramphenicol acetyltransferase gene as reporter. S1 mapping confirmed our previous notion that the promoter is located within a 1.35-kb intergenic sequence preceding the structural lectin gene. Transcripts derived from the chloramphenicol acetyltransferase gene of transfected trophozoites were found to be polyadenylated and the transcriptional start mapped to a position similar to that of the wild-type lectin gene. By deletion analysis the entire promoter was restricted to a fragment covering about 550 bp upstream from the start of transcription. On the other hand, residual promoter activity required a sequence of about 140 bp only, encompassing a newly identified CCAAT-box like element around position -100, as well as the amebic specific TATA-box. This 140-bp fragment as well as a stretch of 15 bp, which is located some 100 nt further upstream, were found to be conserved within the 5' noncoding region of a second E. histolytica lectin gene. Point-mutation analyses indicated that the 15-bp fragment, the likely CCAAT-box, as well as the TATA-box are required for full promoter activity.

Unusual gene organization in the protozoan parasite Entamoeba histolytica.

We have analyzed three independent genomic loci of the protozoan parasite Entamoeba histolytica that contain coding regions for the iron-containing superoxide dismutase, the pore-forming peptide, and the galactose-inhibitable lectin. All of the three structural genes were found to be closely linked unidirectionally to other coding sequences. The intergenic regions did not exceed 1,350 nucleotides. Nuclear run-on data demonstrated that at least the galactose-inhibitable lectin gene is transcribed in a monocistronic fashion. Comparison of the genomic sequences described here with several others reported previously for E. histolytica revealed a number of invariable peculiarities for the gene organization of this parasite: (i) Coding sequences are not interrupted by introns; (ii) 5' untranslated regions are rather short and transcription starts at the consensus sequences ATTCA or ATCA; (iii) an unusual TATA-motif is located about 30 nucleotides upstream of the start of transcription and comprises the sequence TATTTAAA, which reveals protein binding activity as determined by gel retardation assays; (iv) the conserved pentanucleotide motif TAA/TTT is found within the relatively short 3' untranslated regions and functions putatively as the transcription termination signal; and (v) a stretch of up to 12 pyrmidine residues is located at the end of transcribed sequences.