Key regulatory transcription factors involved in placental trophoblast development--a review.

Specification of the trophoblast cell lineage comprising the outermost epithelial cell layer of the blastocyst occurs early in development and is a prerequisite for implantation of the embryo and subsequent formation of the placenta, a multifunctional organ which is indispensable for the proper development of the fetus. Trophoblast stem cells of the placenta give rise to distinct highly differentiated trophoblast subtypes which build the functional units of the organ. These specialized cells assure anchorage of the embryo to the mother, establishing a vascular connection transporting nutrients and gases and expression of hormones that are required for the successful progression of pregnancy. Developmental processes of the trophoblast occur in a spatially and temporally highly organized manner. Despite these facts, little is known on the key regulatory factors which commit and differentiate trophoblast cells in humans. Recent studies in mice, however, provided evidence that various cell-type specific transcription factors play crucial roles in the developmental programme of the trophoblast. In this review we will focus on the function of these major regulatory factors in murine trophoblast/placental development and discuss the potential role of their homologues in the human system.

Promoter elements and transcription factors involved in differentiation-dependent human chorionic gonadotrophin-alpha messenger ribonucleic acid expression of term villous trophoblasts.

Differentiation of primary villous cytotrophoblasts into syncytia is associated with increasing production of alpha and beta human CG subunits, which is predominantly governed at the level of messenger RNA expression. Here, we present a detailed study on the mechanisms involved in the differentiation-dependent regulation of the trophoblast-specific CGalpha gene promoter. Site-directed mutations in each of the five DNA-elements of the composite enhancer were performed to investigate the contribution of the individual regulatory sequences to the overall transcriptional activity of the promoter at two different stages of trophoblast in vitro differentiation. We show that deletion of one cyclic AMP response element (CRE) did not affect CGalpha promoter activity in cytotrophoblasts; however, it reduced transcription by 33% in differentiating cultures. Removal of both CREs almost abolished transcription at early and later stages of in vitro differentiation. Upon mutation the enhancer elements alphaACT, JRE, and CCAAT significantly decreased luciferase reporter transcription; however their contribution to the total promoter activity did not change during in vitro differentiation. Contrary to that, mutated TSE diminished promoter activity by 19% during 12 and 48 h of cultivation but reduced luciferase expression by 78% between 48 and 84 h of differentiation. In electrophoretic mobility shift assay, the TSE interacted with activating protein (AP)-2alpha in both primary trophoblasts and choriocarcinoma cells. While CRE-interacting proteins were detectable 12 h after isolation, the TSE-binding complex did not appear before 36 h of in vitro differentiation. During syncytium formation increasing protein expression of activating transcription factor (ATF)-1, cAMP response element-binding protein (CREB)-1, and AP-2alpha was observed on Western blots. Moreover, phosphorylated CREB-1 and ATF-1 accumulated between 24 and 78 h of trophoblast cultivation. By fluorescence immunohistochemistry, we show that CREB-1 was predominantly expressed in syncytiotrophoblasts, whereas ATF-1 and AP-2alpha localized to the syncytium and some cytototrophoblasts as well as to stromal and endothelial cells of the placental villus. Phosphorylated CREB-1/ATF-1 and the coactivator protein CBP were primarily detected in syncytial nuclei, suggesting the presence of functional, cAMP-dependent transcriptional complexes in the differentiated tissue. In agreement to the in vivo situation, phosphorylated CREB-1/ATF-1 were observed in nuclei of the differentiated trophoblast cultures. The activity of the CGalpha promoter as well as CREB-1/ATF-1 phosphorylation increased upon elevation of cAMP levels and overexpression of the catalytic subunit of protein kinase A. Additionally, we demonstrate that overproduction of the enzyme enhanced protein expression and binding of AP-2alpha to the TSE. We conclude that differentiation-dependent transcription of the CGalpha gene in villous trophoblasts is mainly governed by increasing expression of AP-2alpha and PKA-dependent phosphorylation of CREB-1 and ATF-1.

Molecular cloning of the human Hand1 gene/cDNA and its tissue-restricted expression in cytotrophoblastic cells and heart.

The basic helix-loop-helix (bHLH) factor Hand1 plays a role in the developing chicken heart and is required for trophoblast giant cell differentiation and cardiac looping of mouse embryonic development. Here, we report the cloning of the human Hand1 cDNA and gene from a heart-specific cDNA library and a genomic lambda-DNA library, respectively. We present the nucleotide sequence of a 1.75kb cDNA clone, encoding the presumptive 215 amino acid human Hand1 protein, and show homology comparison of the conserved bHLH region between different species. In vitro transcription-translation of Hand1 mRNA and analysis of protein size suggest that the Hand1 polypeptide is (post)translationally modified. By Southern blot analysis we demonstrate that the isolated genomic DNA clone harbours the entire Hand1 gene and describe molecular structure and sequences of the two 799 and 938bp exons and the single 1.56kb intron. The expression pattern of the mRNA in different human tissues revealed that Hand1 transcripts are restricted to the heart, suggesting that the protein could be required for cardiac-specific gene transcription and function in adults. Hand1 transcripts were undetectable in a non-tumorigenic villous trophoblast cell line, immunopurified cytotrophoblasts undergoing in vitro differentiation, and first trimester placental tissue, suggesting that the transcription factor is not involved in the development of villous and extravillous trophoblast cell lineages. Hand1 mRNA, however, was abundantly expressed in cytotrophoblastic Jeg-3 and BeWo cells, suggesting that Hand1 could be required for early trophoblast differentiation.

[Menstruation disorders following artificial interruption of pregnancy]. / Poruchy menstruacního cyklu po umelém prerusení tehotenství.

PIP: Information given by 263 women about their menstrual cycle within 1 year after interruption of their pregnancy was evaluated. 3 age groups were considered: up to 20 years, 21-30, and over 30. Greater regularity, length, and intensity of menstrual bleeding occurred in women without painful symptoms accompanying menstruation. The largest number of negative changes of the cycle after interruption of pregnancy occurred in women up to 20 and over 30 years of age. These age groups suffer the most from disorders of their menstrual cycles after the interruption of a pregnancy.^ieng