Norbert Walther

The role of sex steroids in the oxytocin hormone system

The sex steroids and the peptide hormone oxytocin are both ancient modulators of the reproductive system of most metazoan species responsible for tissue differentiation and acute events respectively. In vivo experimentation implies estrogenic control of both the oxytocin (OT) gene and that for its receptor (OTR). Yet neither gene promoter appears able to bind classic estrogen-dependent nuclear receptors (ER) in vitro. The literature is confused by some transfected cell culture experiments which suggest that the human and rat OT gene promoter can be regulated by both ER alpha and ER beta through a major hormone response element at -160 bp upstream of the transcription start site. These findings depended, however, upon the presence of a high molar excess of the nuclear estrogen receptor. The current consensus suggests that the sex steroids are acting indirectly on both the OT and OTR genes, possibly involving intermediate transcription factors or cofactors. They may also act upon the OTR at the cell membrane, though more study is needed before the few current observations can be generalized. Due to the OT system being so ancient and fundamental to all aspects of reproduction, it is likely that the mechanisms by which the sex steroids influence this system are going to be of general importance to many other basic aspects of reproductive control.

The Affinity and Activity of the Multiple Hormone Response Element in the Proximal Promoter of the Human Oxytocin Gene

In vivo there appears to be a marked association between oestrogen levels and the expression of the oxytocin (OT) gene in most tissues. Transfection and DNA‐protein binding experiments using high levels of either oestrogen receptor (ER)α or ERβ imply a direct interaction of these transcription factors with the multiple hormone response element (HRE) at approximately −160 from the transcription start site of the OT gene in most species. In an extensive set of experiments, we show, using both transfection and protein‐DNA binding, that low to moderate amounts of either oestrogen receptor, while being able to interact directly with a classic oestrogen response element (ERE) fail to interact with the human OT −160 HRE. Instead, this element, similar to its bovine counterpart, has a high affinity for the orphan receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. Second, the human and bovine OT promoter can be made artificially responsive towards oestrogen in a cotransfection system over‐expressing ERα or ERβ, but not in cells expressing natural levels of these steroid receptors. Interestingly, nuclear extracts from both ERα‐positive MCF7 cells and ERα‐negative MDA‐MB231 cells both contain a transcription factor which binds specifically to both the hOT‐HRE element and to a classic ERE, and which has orphan receptor‐like binding properties rather than those of an oestrogen receptor. Together, these and other results suggest that oestrogen action in vivo on the OT gene in all species is more likely to involve a DNA‐independent mechanism than classic direct interactions with dimeric oestrogen receptors.

Transcriptional activation of the oxytocin promoter by oestrogens uses a novel non-classical mechanism of oestrogen receptor action.

Transcriptional activation of the gene coding for the neuropeptide hormone oxytocin by oestrogens does not follow the classical model of oestrogen receptor action. The oxytocin promoter does not contain an oestrogen response element (ERE), but instead a high‐affinity binding site for nuclear orphan receptors. In the present study, the oestrogen‐dependent up‐regulation of the bovine oxytocin promoter is investigated in MDA‐MB 231 cells. Control by oestrogen is shown to be dependent on the integrity of the nuclear orphan receptor binding site and the presence of ligand‐activated oestrogen receptor, but independent of oestrogen receptor binding to DNA. Partial agonists tamoxifen and raloxifen and the pure antagonist ICI 182 780 all show agonistic activities on transcription, while exhibiting normal binding affinities to oestrogen receptor (ER)α. Nuclear orphan receptors oestrogen receptor‐related receptor α (ERRα) and germ cell nuclear factor (GCNF) are expressed to significant levels in MDA‐MB 231 cells. Binding of ERRα to the oxytocin promoter binding site can be demonstrated, suggesting the involvement of this nuclear orphan receptor in oestrogen‐dependent up‐regulation. The oestrogenic stimulation of the oxytocin promoter apparently is dependent on the stimulation of the transcriptional activity of this nuclear orphan receptor by ERK‐1/ERK‐2 mitogen‐activated protein kinases (MAP kinases). This novel nonclassical mechanism of oestrogen action most probably is not restricted to the regulation of neuropeptide hormone expression, but may further contribute to the multitude of tissue‐specific effects of oestrogenic substances.

Cloning of bovine estrogen receptor beta (ERβ): 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.

SPEER—A New Family of Testis-Specific Genes from the Mouse

Abstract Differential cloning revealed a partial mRNA sequence expressed in the mouse testis, which on further molecular characterization proved to be a member of a new family of 14 transcribed genes. Six of the genes appear to be expressed pseudogenes. The remainder indicate an open reading frame of approximately 200–220 amino acids encoding proteins with a very high proportion of alpha helical secondary structure, comprising approximately 15% glutamate residues. Because of this property, the family has been named SPErm-associated glutamate (E)-Rich protein (SPEER). Three members were chosen for more detailed characterization: SPEER-1 (pseudogene), SPEER-2, and SPEER-4D. All three are expressed tissue specifically in the testis of mice, with only very weak expression evident in the rat testis but in no other species tested. Using reverse transcription-polymerase chain reaction (RT-PCR), all three transcripts can be detected also in the epididymis, presumably due to the presence of spermatozoa. All three transcripts are expressed to high levels in haploid germ cells at the spermatocyte-spermatid transition. SPEER-1 mRNA is present in the cytoplasm as a sense transcript, SPEER-2 appears to be made mostly as an antisense transcript, whereas SPEER-4D appears to be localized within a subcellular compartment as a conventional sense transcript. Codon usage analysis suggests that all but the pseudogenes can be expressed as protein, confirmed for SPEER-2 and SPEER-4D by in vitro transcription/translation. An antibody raised against a peptide region of SPEER-4D, which probably cross-reacts with other SPEER members, immunohistochemically stains the nuclei of early round spermatids. While there are no true homologies to other proteins in the genome databases, some motifs are present that suggest a relationship to nuclear matrix proteins, implying that the SPEER family is a new group of haploid sperm-specific nuclear factors.

Mapping of the Bovine Oxytocin Gene Control Region: Identification of Binding Sites for Luteal Nuclear Proteins in the 5′ Non‐Coding Region of the Gene

In view of the small number of hormone‐producing cells, the factors regulating oxytocin gene expression in the classic site of synthesis, in the magnocellular neurons of the hypothalamus, have not yet been characterized. In the early bovine corpus luteum there is a tissue‐specific oxytocin expression involving many more cells. This tissue therefore was chosen as a experimental system to identify deoxyribonucleic acid elements and nuclear proteins involved in the regulation of oxytocin gene expression. 3.2 kb from the 5’non‐coding region of the bovine oxytocin gene have been sequenced and subcloned fragments used as probes for gel retardation and footprinting experiments. Binding sites for luteal as well as more ubiquitous proteins were detected in the oxytocin promoter region and in an artiodactyl‐specific dispersed repeated deoxyribonucleic acid element. A binding site in the promoter region with a superficial similarity to an estrogen‐responsive element (‐159 to ‐152) was shown not to bind this steroid hormone receptor but to bind two nuclear proteins alternatively. One is a luteal protein, the other a more general transcription factor belonging to the steroid hormone receptor superfamily and similar, if not identical to the COUP protein. This alternative binding of a tissue‐ and phase‐specifically expressed protein or an ubiquitous factor to the same site in the oxytocin promoter suggests a role for these two proteins in the transient up‐regulation and subsequent down‐regulation of the oxytocin gene during the differentiation of the bovine corpus luteum.

The COUP transcription factor (COUP-TF) is directly involved in the regulation of oxytocin gene expression in luteinizing bovine granulosa cells

Competition with specific oligonucleotides in DNA-binding experiments, polyacrylamide gel electrophoresis, and recognition by specific antibodies have identified the ubiquitous transcription factor COUP as one of the nuclear proteins binding to the promoter region of the bovine oxytocin gene in endogenously expressing bovine granulosa cells. PCR cloning of partial cDNA sequences for bovine COUP-TF I and II and development of RNase protection assays demonstrated the up-regulation of COUP-TF in bovine granulosa cells and corpus luteum under conditions where the oxytocin gene is switched off. These experimental results from in vitro and in vivo studies point to the direct involvement of COUP-TF in oxytocin gene down-regulation during luteinization of bovine granulosa cells.

The Orphan Receptor SF-1 Binds to the COUP-Like Element in the Promoter of the Actively Transcribed Oxytocin Gene

The factors regulating oxytocin expression have not yet been characterized in detail. Although direct control by ligand‐dependent binding of nuclear hormone receptors to the oxytocin promoter has been suggested, the presence of these receptors in the tissues expressing oxytocin has not been shown consistently. We have analyzed nuclear proteins from preovulatory bovine granulosa cells and corpus luteum, tissues actively expressing the oxytocin gene, and describe here the characterization of a tissuespecific factor binding to the conserved element in the oxytocin promoter that has been implicated in the control of this gene. This factor is the bovine homologue of SF‐1, an orphan receptor expressed specifically in steroidogenic tissues. It is suggested that SF‐1 binds to the oxytocin promoter in vivo and is involved in control of oxytocin gene expression possibly by interaction with other factors.

The Molecular Basis of Oxytocin and Oxytocin Receptor Gene Expression in Reproductive Tissues

Transcriptional regulation of the oxytocin and oxytocin receptor genes underly to a large degree the highly specific and often transient physiologies associated with this peptide hormone system. Using a variety of homologous transcription assays we have endeavoured to identify and characterize the cis and trans elements responsible for the regulation in vivo of the oxytocin peptide gene and the gene for the oxytocin receptor. The bovine ovarian granulosa cell model is a primary culture system where under stimulation by insulin or IGF-I and LH the endogenous oxytocin gene is massively upregulated. We have identified a proximal response element at -160, which in vivo binds the competing nuclear receptors, SF1 and COUP-TF. Additionally ovarian specific transcription factors bind at two additional sites in the distal promoter region. For the bovine oxytocin receptor gene, we have taken advantage of the high endogenous expression of the receptor in the endometrium of the estrous cycle. Using a combination of primary cell culture techniques and in vitro binding of nuclear protein extracts from tissues expressing the receptor in vivo, we have shown there to be a combination of constitutive and inhibitory elements controlling oxytocin receptor gene expression. Similar results were obtained for the human oxytocin receptor gene. At birth there may additionally be a specific stimulatory effect on transcription in the myometrium.

Progressive inactivation of the haploid expressed gene for the sperm-specific endozepine-like peptide (ELP) through primate evolution.

The endozepine-like peptide (ELP) is a novel intracellular molecule which is expressed in high amounts at both mRNA and protein levels very specifically in late haploid male germ cells. It is closely related to the ubiquitous acyl-CoA binding protein, is highly conserved, shares a similar ability to bind mid-long chain acyl-CoA, and is thus likely to be involved in mature sperm metabolism. While it has been characterized from diverse mammals, it has so far not been possible to identify an equivalent molecule in the primate testis. Using a PCR approach, combined with cDNA cloning and Northern hybridization, testicular transcripts and/or genomic DNA were analysed for different primate species, including human. In the marmoset and cynomolgus macaque normally structured transcripts appear to be expressed, though at a low level. In the human testis, two rare transcripts were characterized, hELP1 and hELP2, the products of independent duplicated genes. Both transcripts were longer than in non-mammalian species, included frame-shift mutations and substantial sequence insertions, preventing the translation of a sensible protein. Genomic PCR analysis of three anthropoid species, chimpanzee, gorilla and orangutan, showed the presence of a similarly mutated hELP1 gene. Only in the gorilla was a hELP2 gene identified, apparently lacking the frame-shift mutation, and thus potentially able to give rise to a functional ELP protein. Taken together, these results show that during primate evolution there has been a progressive inactivation of the ELP gene, initially with a down-regulation in lower primates, and subsequently with inactivating mutations in the open reading frame. At some time during simian evolution prior to these mutations there has been a gene duplication, though this second gene has also become inactivated in humans. In its pattern of evolution the ELP gene shows similarities with the MDC/fertilin family, whose members are also considered essential components of haploid sperm in non-primates, but which are progressively inactivated in anthropoids and humans. We should like to speculate that the established subfertility of the human male may not be a recent event, but the consequence of a longer evolutionary process whereby primates have traded off absolute fertility against social or sexual advantages.