Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

The transcription factor PLA-1/SKN-1A is expressed in human placenta and regulates the placental lactogen-3 gene expression.

Here we report the selective expression of two POU transcription factor genes, PLA-1 and OCT-1, in human placenta and choriocarcinoma cell lines JAR, JEG-3 and BeWo. Pla-1 protein binds to a POU-consensus DNA sequence in the human placental lactogen-3 (PL-3) promoter and it is capable of trans-activating its transcription up to 18-fold. Other tissue-specific or ubiquitous POU transcription factors such as Pit-1/GHF-1 or Oct-1 showed none or low levels of trans-activation of the PL-3 promoter. In addition, we identified an unique and highly charged region in the N-terminal portion of Pla-1 protein required for full trans-activation of the PL-3 promoter.

Differential display RT-PCR analysis of human choriocarcinoma cell lines and normal term trophoblast cells: identification of new genes expressed in placenta.

In this study, we performed the differential display technique to identify genes specifically expressed in human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and normal placental term cells. Few differences were found among the expression profiles of the three choriocarcinoma cell lines and most of the differentially expressed genes were detected in normal term placenta. A total of 36 cDNA fragments were isolated and analysed. Of these, 19 sequences corresponded to regions in the human genome coding for potential novel genes. We confirmed by RT-PCR, the placental mRNA expression of three selected new human genes, on chromosomes 16q12, 9q32 and 6q22. The other 17 sequences showed high similarity to known human genes (like PSG3, FN1, PAI-2). Interestingly, the functions of five known proteins (from genes IK, TRA-1, HERPUD1, UBA-2, and TRAP240) have not yet been well characterized in placenta tissue. In addition, new alternative spliced mRNAs were detected for IK, TRAP240 and PLAC3 genes. The differential expression of the PAI-2 gene among the choriocarcinoma cell lines was also confirmed. The genes identified in this analysis will be of interest for future studies regarding both a better understanding of the biology of the trophoblast cell and the formation of placental tumors.

Molecular characterisation of sea bream (Sparus aurata) transforming growth factor beta1.

A transforming growth factor beta1 (TGF beta1) full length cDNA was characterised and sequenced from the head kidney of sea bream (Sparus aurata) previously challenged with a nodavirus. The cloned cDNA of 1778bp contains a predicted open reading frame of 379 amino acids, which includes the mature peptide region of 112 amino acids. The regulating region of the peptide possesses four potential N-linked glycosylation sites (N-X-T/S), as well as an RGD integrin binding site, an RKKR tetrabasic cut site and nine conserved cysteines all characteristic of the TGF beta superfamily. Compared to other teleost TGF beta1 genes, the sea bream TGF beta1 is most closely related to hybrid striped bass (Moronesaxatilis xM. chrysops) TGF beta1 (80% amino acid identity). The genomic organisation of TGF beta1 was determined through the generation of contiguous PCR clones. The sea bream TGF beta1 gene is approximately 3.6kb in length and consists of five coding regions. Two introns are absent in comparison to the genomic organisation of rainbow trout Oncorhynchus mykiss TGF beta1, whilst an additional intron not present in other sequenced TGF beta genes, but present in the trout TGF beta1 gene, is conserved in sea bream.A reverse transcription polymerase chain reaction (RT-PCR) assay was developed to study TGF beta expression in different sea bream tissues. Constitutive TGF beta1 expression was detected in the liver, brain, muscle, kidney, heart, gills and spleen of sea bream, as well as in head kidney macrophages and blood leucocytes.

Anterior pituitary development and Pit-1/GHF-1 transcription factor.

Anterior pituitary differentiation is a well-established paradigm in mammalian organogenesis. PIT1/GHF1, a homoeotic gene, plays a key role in terminal pituitary differentiation. Recently, a new set of transcription factors involved in early pituitary differentiation have been identified: Lhx-3, Lhx-4, P-OTX and Prop1. A pituitary-specific transcriptional cascade regulating a developmental programme leads to the determination of the mature cell types.

Nuclear factor 1 regulates the distal silencer of the human PIT1/GHF1 gene.

Here we report the characterization of 12 kb genomic DNA upstream of the human PIT1/GHF1 promoter. Different regions involved in the modulation of human PIT1/GHF1 gene expression were defined by transient transfection studies. Two regions, one proximal (-7.1/-2. 3) and one distal (-11.8/-10.9), presented an enhancer activity in pituitary cells when placed upstream of the SV40 promoter. The 0.9 kb distal region was analysed further and found to decrease the basal transcriptional activity of the human PIT1/GHF1 minimal promoter, indicating that this region behaves as a silencer for its own promoter. Three Pit-1/GHF-1-binding sites and two ubiquitous nuclear factor 1 (NF-1)-binding sites were identified by DNase I footprinting in the distal regulatory region. Deletion analysis indicated that NF-1 or NF-1-related protein(s) participate in the down-regulation of human PIT1/GHF1 gene expression by interacting with an NF-1-binding site within the distal regulatory region.

The transcription factor GHF-1, but not the splice variant GHF-2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression.

The rat growth hormone (GH) promoter was significantly activated in non-pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand-dependent activation was found in the presence of the pituitary-specific transcription factor GHF-1. When compared with GHF-1, the splice variant GHF-2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF-2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over-expression of GHF-2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF-1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.

Molecular cloning of gilthead seabream (Sparus aurata) pituitary transcription factor GHF-1/Pit-1.

We report here the complete nucleotide sequence of a cDNA clone encoding Sparus aurata GHF-1/Pit-1 isolated from an expression library prepared from gilthead seabream pituitary gland poly(A)+ RNA. The cDNA sequence (saGHF-1/Pit-1) encodes a protein of 371 amino acids (aa) containing a POU domain (aa 194-343) and a transactivation, STA domain (aa 1-128). Northern blot hybridization of pituitary RNA detected a single 3.0 kb band and a rat GHF-1/Pit-1 antiserum was found to immunoreact with pituitary protein species of 42 kDa by Western blot analysis. When compared with mammalian GHF-1/Pit-1 aa sequence, the POU and STA domains of saGHF-1/Pit-1 protein show 83% and 48% aa identity, respectively. In spite of the low homology of the transactivation domain, saGHF-1/Pit-1 is able to activate the transcription of the human growth hormone promoter.

AP-1 and Oct-1 transcription factors down-regulate the expression of the human PIT1/GHF1 gene.

The pituitary-specific transcription factor Pit-1/GHF-1 is a member of the POU domain family of regulatory proteins. It is involved in the commitment and expansion of the somatotropic cell lineage and activates the transcription of a set of anterior pituitary genes. We have cloned the human PIT1/GHF1 gene and characterized the regulatory mechanisms controlling its promoter activation and regulation. A minimal promoter region (-102 to +15) contains the cis-acting elements that confer to the human PIT1/GHF1 gene a high basal transcriptional activity, the tissue-specific expression, and the autoregulation by Pit-1/GHF-1 protein. The upstream promoter region contains a multiplicity of Pit-1/GHF-1 binding sites that do not show any synergistic interaction with the minimal promoter. The transcriptional activity is negatively regulated by Oct-1 and mediated by an octamer-binding site (OTF). In addition, we have also identified a 12-O-tetradecanoylphorbol-13-acetate-responsive element, which overlaps with a Pit-1/GHF-1 binding site. A mutually exclusive binding of the activator protein-1 (AP-1) and Pit-1/GHF-1 has been observed on this composite site, and AP-1 was shown to down-regulate PIT1/GHF1 transcription.

Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.

Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture.

Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel pituitary transcription factor is produced by alternative splicing of the human GHF-1/PIT-1 gene.

An alternative splice acceptor site in intron 1 of the human GHF-1/PIT-1 gene was sequenced. The use of this splice site is responsible for a 78-bp in-frame insertion upstream from exon 2 and leads to the hGHF-2/PIT-2 cDNA detected in normal human pituitary.

Pit-1 and Pit-2 role in growth hormone gene regulation.

The role of the POU domain transcription factor Pit-1 (GHF-1) in differentiation and proliferation of the somatotrophic lineage is well known. In our study of differential splicing of the PIT1 gene we found a new protein, Pit-2, in which 26 amino acids are inserted into the transactivation domain. Pit-2 can activate the growth hormone promoter but has lost the ability to activate the prolactin promoter or PIT1 promoter itself.

Differential splicing of the GHF1 primary transcript gives rise to two functionally distinct homeodomain proteins.

The POU domain protein GHF-1 has a critical role in generation, proliferation and phenotypic expression of three pituitary cell types. GHF-1 functions in part by binding to and transactivating the promoters of both the growth hormone (GH) and prolactin (PRL) genes and that of the GHF1 gene itself. We describe a naturally occurring isoform of GHF-1, GHF-2, in which an additional 26 amino acids are inserted into the activation domain of the protein as a result of alternative splicing. GHF-2 retains the DNA binding activity of GHF-1 and can activate the GH promoter but has lost the ability to activate the PRL and GHF1 promoters. These results suggest that GHF-2 may function in differential target gene activation during differentiation of the somatotrophic lineage. Both GHF-1 and GHF-2 transcripts are specifically expressed in the anterior pituitary. Analysis of the genomic GHF1 gene shows that most of the distinct functional domains of GHF-1 (and GHF-2) are encoded by separate exons. Gene segment duplication and exon shuffling may have contributed to the evolution of this cell type-specific transcriptional regulatory gene.

Function of the homeodomain protein GHF1 in pituitary cell proliferation.

Mutations that cause pituitary dwarfism in the mouse reside in the gene encoding the transcription factor growth hormone factor 1 (GHF1 or pit1). These dwarf mice (dw and dwJ) are deficient in growth hormone (GH) and prolactin (PRL) synthesis and exhibit pituitary hypoplasia, suggesting a stem cell defect. With antisense oligonucleotide technology, a cell culture model of this genetic defect was developed. Specific inhibition of GHF1 synthesis by complementary oligonucleotides led to a marked decrease in GH and PRL expression and to a marked decrease in proliferation of somatotrophic cell lines. These results provide direct evidence that the homeodomain protein GHF1 is required not only for the establishment and maintenance of the differentiated phenotype but for cell proliferation as well.

Growth hormone gene regulation: a paradigm for cell-type-specific gene activation.

The growth hormone (GH) gene is specifically expressed within specialized cells of the anterior pituitary. The central role in GH gene activation is played by GHF-1, a homeodomain protein that is itself specifically expressed in the anterior pituitary.

Expression of GHF-1 protein in mouse pituitaries correlates both temporally and spatially with the onset of growth hormone gene activity.

The relationship between expression of the pituitary-specific transcription factor, GHF-1, and activation of the growth hormone and prolactin genes during mouse anterior pituitary development was investigated. While GHF-1 transcripts were detected within 24 hr of the first observable events in anterior pituitary differentiation, no GHF-1 protein could be detected until about 3 days later. The appearance of GHF-1 protein showed good temporal and spatial correlation with activation of the growth hormone gene. Prolactin gene expression, on the other hand, was observed transiently during embryonic day 16 in two different populations of cells, of which the major one does not contain GHF-1 or growth hormone. These results suggest that expression of GHF-1 is controlled both transcriptionally and posttranscriptionally. The spatial and temporal correlation between the appearance of GHF-1 protein and growth hormone gene activation suggests that GHF-1 is responsible for this very last step in the specialization of somatotrophic cells.

Dissection of functional domains of the pituitary-specific transcription factor GHF-1.

The specific expression of growth hormone (GH) in the somatotrophic cells of the anterior pituitary is largely attributable to a short promoter in the 5' flanking region of the GH gene. This promoter contains two binding sites for the transcription factor GHF-1, the expression of which is also specific to cells of the somatotrophic lineage and correlates with activation of the GH gene in the developing mouse pituitary. Various studies indicate that GHF-1 is the main determinant of cell type-specific expression of the GH gene. GHF-1 is a member of the POU-domain class of proteins that each contain two highly conserved sequence motifs, the homoeodomain and the POU-specific domain. Here we report that the GHF-1 homoeodomain is sufficient for sequence-specific DNA binding, although its activity is stimulated by the POU-specific domain, which does not interact directly with the DNA. Transcriptional activation is mediated by a separate domain rich in hydroxylated amino-acid residues. Similar sequences are present in other cell type-specific transcription factors.