Non-placental Tissues Research Articles

The X-linked splicing regulator MBNL3 has been co-opted to restrict placental growth in eutherians

Understanding the regulatory interactions that control gene expression during the development of novel tissues is a key goal of evolutionary developmental biology. Here, we show that Mbnl3 has undergone a striking process of evolutionary specialization in eutherian mammals resulting in the emergence of a novel placental function for the gene. Mbnl3 belongs to a family of RNA-binding proteins whose members regulate multiple aspects of RNA metabolism. We find that, in eutherians, while both Mbnl3 and its paralog Mbnl2 are strongly expressed in placenta, Mbnl3 expression has been lost from nonplacental tissues in association with the evolution of a novel promoter. Moreover, Mbnl3 has undergone accelerated protein sequence evolution leading to changes in its RNA-binding specificities and cellular localization. While Mbnl2 and Mbnl3 share partially redundant roles in regulating alternative splicing, polyadenylation site usage and, in turn, placenta maturation, Mbnl3 has also acquired novel biological functions. Specifically, Mbnl3 knockout (M3KO) alone results in increased placental growth associated with higher Myc expression. Furthermore, Mbnl3 loss increases fetal resource allocation during limiting conditions, suggesting that location of Mbnl3 on the X chromosome has led to its role in limiting placental growth, favoring the maternal side of the parental genetic conflict.

Scavenger receptor class B, type 1 facilitates cellular fatty acid uptake

SR-B1 belongs to the class B scavenger receptor, or CD36 super family. SR-B1 and CD36 share an affinity for a wide array of ligands. Although they exhibit similar ligand binding specificity, SR-B1 and CD36 have some very specific lipid transport functions. Whereas SR-B1 primarily facilitates the selective delivery of cholesteryl esters (CEs) and cholesterol from HDL particles to the liver and non-placental steroidogenic tissues, as well as participating in cholesterol efflux from cells, CD36 primarily mediates the uptake of long-chain fatty acids in high fatty acid-requiring organs such as the heart, skeletal muscle and adipose tissue. However, CD36 also mediates cholesterol efflux and facilitates selective lipoprotein-CE delivery, although less efficiently than SR-B1. Interestingly, the ability or efficiency of SR-B1 to mediate fatty acid uptake has not been reported. In this paper, using overexpression and siRNA-mediated knockdown of SR-B1, we show that SR-B1 possesses the ability to facilitate fatty acid uptake. Moreover, this function is not blocked by BLT-1, a specific chemical inhibitor of HDL-CE uptake activity of SR-B1, nor by sulfo-N-succinimidyl oleate, which inhibits fatty acid uptake by CD36. Attenuated fatty acid uptake was also observed in primary adipocytes isolated from SR-B1 knockout mice. In conclusion, facilitation of fatty acid uptake is an additional function that is mediated by SR-B1.

Chromosomal architecture and placental expression of the human growth hormone gene family are targeted by pre-pregnancy maternal obesity.

The human (h) placental lactogenic hormone chorionic somatomammotropin (CS) is highly produced during pregnancy and acts as a metabolic adaptor in response to maternal insulin resistance. Maternal obesity can exacerbate this "resistance", and a >75% decrease in CS RNA levels was observed in term placentas from obese vs. lean women. The genes coding for hCS ( hCS-A and hCS-B) and placental growth hormone ( hGH-V) as well as the hCS-L pseudogene and pituitary growth hormone (GH) gene ( hGH-N) are located at a single locus on chromosome 17. Three remote hypersensitive sites (HS III-V) located >28 kb upstream of hGH-N as well as local hCS gene promoter and enhancer regions are implicated in hCS gene expression. A placenta-specific chromosomal architecture, including interaction between HS III-V and hCS but not hGH gene promoters, was detected in placentas from lean women (BMI <25 kg/m2) by using the chromosome conformation capture assay. This architecture was disrupted by pre-pregnancy maternal obesity (BMI >35 kg/m2), resulting in a predominant interaction between HS III and the hGH-N promoter, which was also observed in nonplacental tissues. This was accompanied by a decrease in hCS levels, which was consistent with reduced RNA polymerase II and CCAAT/enhancer-binding protein-β association with individual hCS promoter and enhancer sequences, respectively. Thus, pre-pregnancy maternal obesity disrupts the placental hGH/CS gene locus chromosomal architecture. However, based on data from obese women who develop GDM, insulin treatment partially recapitulates the chromosomal architecture seen in lean women and positively affects hCS production, presumably facilitating prolactin receptor-related signaling by hCS.

Methylation Status of the Chromosome Arm 19q MicroRNA Cluster in Sporadic and Androgenetic-Biparental Mosaicism–Associated Hepatic Mesenchymal Hamartoma

The C19MC gene on chromosome band 19q13.4 encodes a cluster of 46 microRNAs; those microRNAs are normally only expressed from the paternal allele and in the placenta. Placental expression correlates with selective demethylation of the paternal C19MC promoter, in contrast to methylation of both maternal and paternal alleles in nonplacental tissues. Prior investigations demonstrated "ectopic" activation of this gene in most hepatic mesenchymal hamartomas, including sporadic tumors and others with androgenetic-biparental mosaicism (subset of cells are diploid, but contain only paternally derived chromosomes). In the present investigation of C19MC promoter methylation status in a series of 14 mesenchymal hamartomas, a demethylated allele was identified in 6 tumors, including all 4 with androgenetic-biparental mosaicism. Conversely, only methylated alleles were cloned from sporadic hamartomas, including 3 tumors with chromosomal rearrangements thought likely to activate C19MC expression independent of the native promoter. In conjunction with published data, the findings suggest multiple molecular mechanisms for C19MC activation in hepatic mesenchymal hamartoma, including the existence of a normal placental imprinting pattern in mesenchymal cells in a subset of cases. Some or all of the latter hamartomas may result from placental "grafting," a hypothesis supported by endothelial expression of the placental vascular marker, glucose transporter-1, in 1 of the 6 cases with a demethylated allele.

Hypomethylation and Activation of Syncytin-1 Gene in Endometriotic Tissue

Syncytin-1 plays a critical role in the maintenance of normal pregnancy by mediating the formation of syncytiotrophoblasts through a fosugenic action. Encoded by the human endogenous retrovirus envelope gene HERV-W, syncytin-1 trophoblast-specific expression is controlled by epigenetic mechanisms. In non-placental tissues, the syncytin-1 gene is suppressed by hypermethylation in the LTR promoter region. Hypomethylated and activated syncytin-1 gene is found in placental trophoblast lineages and malignant cells. We here demonstrate that while syncytin-1 gene remains silenced in the eutopic endometrium from endometriotic patients, syncytin-1 mRNA and protein are detected in ectopic, endometriotic lesions; particularly the endometrioid glandular endothelial cells. LINE-1 COBRA assay and immunohistochemistry using the 5-MC-specific antibody did not detect any changes in global DNA methylation in the endometriotic tissues. However, results from COBRA and bisulfite sequencing indicated that the LTR region of the syncytin-1 promoter is hypomethylated in endometriotic tissues, highlighting the significance of DNA demethylation in syncytin-1 gene activation. Analysis of DNA methyltransferase 3B (DNMT3B) mRNA levels revealed that DNMT3B3, an isoform carrying methyltransferase activity, is downregulated; whereas DNMT3B7, the isoform without enzymatic activity, is upregulated in the endometriotic tissues, pointing to positive and negative regulatory functions, respectively, of these isoforms on syncytin-1 methylation. These results have provided the first evidence supporting the involvement of epigenetic mechanisms for syncytin-1 upregulation in endometriotic tissues. Considering recent findings on the nonfusogenic activity of syncytin-1, its expression in endometriotic tissues suggests that this multifunctional protein may be implicated in the pathogenesis and/or progression of endometriosis.

Transcriptional Derepression of the ERVWE1 Locus following Influenza A Virus Infection

Syncytin-1, a fusogenic protein encoded by a human endogenous retrovirus of the W family (HERV-W) element (ERVWE1), is expressed in the syncytiotrophoblast layer of the placenta. This locus is transcriptionally repressed in adult tissues through promoter CpG methylation and suppressive histone modifications. Whereas syncytin-1 appears to be crucial for the development and functioning of the human placenta, its ectopic expression has been associated with pathological conditions, such as multiple sclerosis and schizophrenia. We previously reported on the transactivation of HERV-W elements, including ERVWE1, during influenza A/WSN/33 virus infection in a range of human cell lines. Here we report the results of quantitative PCR analyses of transcripts encoding syncytin-1 in both cell lines and primary fibroblast cells. We observed that spliced ERVWE1 transcripts and those encoding the transcription factor glial cells missing 1 (GCM1), acting as an enhancer element upstream of ERVWE1, are prominently upregulated in response to influenza A/WSN/33 virus infection in nonplacental cells. Knockdown of GCM1 by small interfering RNA followed by infection suppressed the transactivation of ERVWE1. While the infection had no influence on CpG methylation in the ERVWE1 promoter, chromatin immunoprecipitation assays detected decreased H3K9 trimethylation (H3K9me3) and histone methyltransferase SETDB1 levels along with influenza virus proteins associated with ERVWE1 and other HERV-W loci in infected CCF-STTG1 cells. The present findings suggest that an exogenous influenza virus infection can transactivate ERVWE1 by increasing transcription of GCM1 and reducing H3K9me3 in this region and in other regions harboring HERV-W elements. Syncytin-1, a protein encoded by the env gene in the HERV-W locus ERVWE1, appears to be crucial for the development and functioning of the human placenta and is transcriptionally repressed in nonplacental tissues. Nevertheless, its ectopic expression has been associated with pathological conditions, such as multiple sclerosis and schizophrenia. In the present paper, we report findings suggesting that an exogenous influenza A virus infection can transactivate ERVWE1 by increasing the transcription of GCM1 and reducing the repressive histone mark H3K9me3 in this region and in other regions harboring HERV-W elements. These observations have implications of potential relevance for viral pathogenesis and for conditions associated with the aberrant transcription of HERV-W loci.

Maternal obesity alters feto-placental cytochrome P4501A1 activity

Cytochrome P4501A1 (CYP1A1), an important drug metabolizing enzyme, is expressed in human placenta throughout gestation as well as in fetal liver. Obesity, a chronic inflammatory condition, is known to alter CYP enzyme expression in non-placental tissues. In the present study, we test the hypothesis that maternal obesity alters the distribution of CYP1A1 activity in feto-placental unit. Placentas were collected from non-obese (BMI < 30) and obese (BMI > 30) women at term. Livers were collected from gestation day 130 fetuses of non-human primates fed either control diet or high-fat diet (HFD). Cytosol and microsomes were collected using differential centrifugation, and incubated with 7-ethoxyresorufin. The CYP1A1 specific activity (pmoles of resorufin formed/min/mg of protein) was measured at excitation/emission wavelength of 530/590 nm. Placentas of obese women had significantly reduced microsomal CYP1A1 activity compared to non-obese women (0.046 vs. 0.082; p < 0.05); however no such effect was observed on cytosolic activity. Similarly, fetal liver from HFD fed mothers had significantly reduced microsomal CYP1A1 activity (0.44 ± 0.04 vs. 0.20 ± 0.10; p < 0.05), with no significant difference in cytosolic CYP1A1 activity (control, 1.23 ± 0.20; HFD, 0.80 ± 0.40). Interestingly, multiple linear regression analyses of placental efficiency indicate cytosolic CYP1A1 activity is a main effect (5.67 ± 2.32 (β ± SEM); p = 0.022) along with BMI (−0.57 ± 0.26; p = 0.037), fetal gender (1.07 ± 0.26; p < 0.001), and maternal age (0.07 ± 0.03; p = 0.011). In summary, while maternal obesity affects microsomal CYP1A1 activity alone, cytosolic activity along with maternal BMI is an important determinant of placental efficiency. Together, these data suggest that maternal lifestyle could have a significant impact on CYP1A1 activity, and hints at a possible role for CYP1A1 in feto-placental growth and thereby well-being of fetus.

The epigenetic control of HERV loci encoding for fusogenic envelope glycoproteins in trophoblast

Background Up to 8% of the human genome is of retroviral origin, comprising numerous human endogenous retroviruses (HERVs), remnants of germ line transmission of exogenous retroviruses during primate and human evolution. Some endogenous retroviruses within the human genome remain intact open reading frames with translational potential. Gag and Env genes are often expressed in various human tissues and provide superantigens or restriction factors interfering with exogenous infections. Several Env products display fusogenic properties and it is temting to implicite them in various human pathologies like cancer, multiple sclerosis, and muscular dystrophies. Of particular importace are the fusogenic Env proteins expressed in placenta, syncytin-1 and 2, encoded by ERVWE1 and ERV-FRD proviral loci, respectively. These fusogenic Envs contribute to the differentiation of multinucleated syncytiotrophoblast in placental chorionic villi. In non-placental tissues, however, expression of these fusogenic proteins has to be suppressed because of high risk for tissue integrity and cancer development. We have previously shown that the 5' long terminal repeat, the regulatory region of syncytin-1 is epigenetically suppressed by DNA methylation in non-placental tissues.

Androgenetic/Biparental Mosaicism in an Infant with Hepatic Mesenchymal Hamartoma and Placental Mesenchymal Dysplasia

Androgenetic/biparental mosaicism, in which a subset of cells has complete paternal uniparental disomy, is associated with placental mesenchymal dysplasia (PMD), which is compatible with fetal development, indicating that fetal organs could also have androgenetic/biparental mosaicism, but few cases of somatic mosaicism have been described. A hepatic mesenchymal hamartoma (HMH) was resected from an otherwise healthy, nondysmorphic, 11-month-old girl, whose prenatal development was complicated by PMD. Placenta, HMH, histologically normal liver, and other tissues were examined for androgenetic/biparental mosaicism by analysis of (1) polymorphic DNA microsatellite markers, (2) the methylation status of an imprinted gene, SNRPN, and (3) immunohistochemically detectable protein products of the imprinted genes p57KIP2 and PHLDA2. The patient's liver, HMH, and 1 placental sample demonstrated an increased ratio of paternal to maternal alleles, indicating androgenetic/biparental mosaicism. The androgenetic component comprised 26% to 60% of the cells. Other tissues, including a 2nd placental sample, white blood cells, umbilical cord, and abdominal fascia, had no detectable androgenetic component. Methylation analysis confirmed a relative excess of the paternally imprinted SNRPN homolog in the normal liver, HMH, and placenta. Placental p57KIP2 immunoreactivity was consistent with androgenetic/biparental mosaicism, but neither p57KIP2 nor PHLDA2 immunohistochemistry were informative for HMH, because neither antigen was detected in control liver samples. We report androgenetic/biparental mosaicism in nonplacental tissues of an infant with PMD and provide the 1st description of genome-wide paternal uniparental disomy in HMH. Androgenetic/biparental mosaicism appears to play a role in the pathogenesis of HMH and other somatic lesions, particularly those associated with PMD.

Fine-scale quantification of HCG beta gene transcription in human trophoblastic and non-malignant non-trophoblastic tissues

Human chorionic gonadotropin (HCG) is produced by syncytiotrophoblast of placenta. It delays the apoptosis of corpus luteum and functions in implantation. Its possible role in male reproduction has been raised. HCG beta subunit is encoded by CGB, CGB5, CGB7 and CGB8 genes located at 19q13.3 in a common genome cluster with beta subunit non-coding CGB1 and CGB2. We conducted a sensitive quantification and comparison of CGB gene expression in human trophoblastic (blastocysts, n = 6; normal/failed pregnancy, n = 51) and non-malignant non-trophoblastic tissues (15 different tissue types, samples n = 241), by real-time RT-PCR. We showed a wide transcriptional window of CGB genes in normal pregnancy, a significant reduction in recurrent miscarriages, and a high expression (especially CGB1/CGB2) in ectopic and molar pregnancies. Expression was several orders of magnitude lower in the non-placental tissues, with the highest CGB levels being seen in testis, prostate, thymus, skeletal muscle and lung samples. The contribution of CGB1/CGB2 to the summarized expression of six CGB genes was not proportional to their gene dosage: 1/1000 to 1/10,000. An interesting exception was the testis exhibiting a much higher CGB1/CGB2 to total CGB mRNA ratio of approximately one-third, corresponding to gene dosage. In conclusion, the expressional profile of CGB genes, activated already in blastocyst stage, is associated with the status of pregnancy. The presence of CGB transcripts in testes, and in particular CGB1/CGB2 transcripts, may indicate a role in male reproductive tract.

CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues

Syncytin-1 is a captive envelope glycoprotein encoded by one of human endogenous retroviruses W. It is expressed exclusively in the placental trophoblast where it participates in cell-to-cell fusion during differentiation of syncytiotrophobast. In other tissues, however, syncytin-1 expression must be kept in check because inadvertent cell fusion might be dangerous for tissue organization and integrity. We describe here an inverse correlation between CpG methylation of syncytin-1 5′ long terminal repeat and its expression. Hypomethylation of the syncytin-1 5′ long terminal repeat in the placenta and in the choriocarcinoma-derived cell line BeWo was detected. However, other analyzed primary cells and cell lines non-expressing syncytin-1 contain proviruses heavily methylated in this sequence. CpG methylation of syncytin-1 is resistant to the effect of the demethylating agent 5-azacytidine. The inhibitory role of CpG methylation is further confirmed by transient transfection of in-vitro-methylated syncytin-1 promoter-driven reporter construct. Altogether, we conclude that CpG methylation plays a principal role in the transcriptional suppression of syncytin-1 in non-placental tissues, and, in contrast, demethylation of the syncytin-1 promoter in trophoblast is a prerequisite for its expression and differentiation of multinucleated syncytiotrophoblast.

Purinergic receptors in human placenta: evidence for functionally active P2X4, P2X7, P2Y2, and P2Y6

Appropriate regulation of ion transport by the human placental syncytiotrophoblast is important for fetal growth throughout pregnancy. In nonplacental tissues, ion transport can be modulated by extracellular nucleotides that raise intracellular calcium ([Ca2+]i) via activation of purinergic receptors. We tested the hypothesis that purinergic receptors are expressed by human placental cytotrophoblast cells and that their activation by extracellular nucleotides modulates ion (K+) efflux and [Ca2+]i. P2X/P2Y receptor agonists 5-bromouridine 5'-triphosphate (5-BrUTP), ADP, ATP, 2',3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP), and UTP stimulated 86Rb (K+ tracer) efflux from cultured cytotrophoblast cells at early (mononuclear) or later (multinucleate syncytiotrophoblast-like) stages of differentiation, with ATP and UTP particularly potent. 2-Methylthioadenosine 5'-triphosphate (2-MeS-ATP), and UDP elevated 86Rb efflux only from multinucleated cells. All agonists caused a significant peak and plateau increase in [Ca2+]i, although the magnitude of responses was variable. The effect of BzATP, UTP, and UDP in multinucleated cells was unaffected, and that of ATP partially inhibited, by removal of extracellular Ca2+, implicating P2Y receptor activation. mRNA encoding P2X1, P2X2, P2X4, and P2X7 and P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 were identified in mono- and multinucleated cells, whereas P2X3 and P2X5 mRNA were absent from all samples. Western blot analysis revealed P2X4, P2X7, P2Y2, and P2Y6 protein in cytotrophoblast cells, but P2Y4 was not detected. On the basis of published agonist selectivity, the data indicate the presence of functionally active P2X4, P2X7, P2Y2, and P2Y6 receptors in cytotrophoblast cells. We propose that activation of these receptors, and subsequent elevation of [Ca2+]i, modulates syncytiotrophoblast homeostasis and/or maternofetal ion exchange in response to extracellular nucleotides.

Imprinting of PEG1/ MEST Isoform 2 in Human Placenta

The PEG1 gene (a.k.a. MEST) is expressed in human placental trophoblast and endothelium, and data from knockout mice show that this gene regulates placental and fetal growth. Isoform 1 of PEG1 mRNA initiates from exon 1c and produces the long form of the MEST protein. This isoform is imprinted, with expression only from the paternal allele in many human and mouse organs, including placenta. In contrast, PEG1 isoform 2, initiating from exon 1a and producing the short form of MEST protein, is biallelically expressed (non-imprinted) in several non-placental organs. Here we show that PEG1 isoform 2 is in fact imprinted in a large subset of human placentae. A CpG island overlapping PEG1 exon 1a is unmethylated in various fetal and adult non-placental tissues, but is often substantially methylated in the placenta, with the extent of methylation in a large series approximating a normal distribution. Bisulfite conversion/sequencing indicates that the inter-individual differences reflect the relative representation of heavily methylated vs. unmethylated alleles, and RT-PCR/RFLP analysis shows strongly biased allelic expression of PEG1 isoform 2 mRNA in a majority of placentae with a high proportion of methylated alleles. These data highlight PEG1 isoform 2 as a marker for future studies of inter-individual epigenetic variation and its relation to placental and fetal growth in humans.

The Product of the Imprinted Gene IPL Marks Human Villous Cytotrophoblast and is Lost in Complete Hydatidiform Mole

The IPL/TSSC3 gene is expressed nearly exclusively from the maternal allele, and its protein product acts to limit placental growth in mice. This protein specifically marks Type II trophoblast in the labyrinthine layer of the mouse placenta. To investigate mouse–human homologies, we carried out immunohistochemistry with antibodies against human IPL. There was strong expression of IPL in villous cytotrophoblast of the human placenta, contrasting with complete lack of expression in syncytiotrophoblast. Staining for IPL was weak in cells of the villous mesenchyme and extravillous trophoblast, including the cytotrophoblast columns in the basal plate and the intervillous trophoblast islands. The IPL and p57 KIP2 /CDKN1C genes are closely linked and coordinately imprinted, and immunostaining showed that their protein products are co-expressed in villous cytotrophoblast. However, other cell types, including extravillous cytotrophoblast and cells in various non-placental tissues, expressed p57 KIP2, but not IPL. IPL protein was absent in both of two cases of androgenetic complete hydatidiform mole examined by immunostaining, and IPL mRNA was absent in an additional three cases of this neoplasm examined by northern blotting. In the mouse, Ipl-expressing cells disappear at mid- to late-gestation when placental growth ceases, but persistent IPL mRNA and protein expression was observed throughout human gestation, correlating with the continuous growth of the human placenta. These findings highlight dosage regulation of human IPL by imprinting and, more generally, suggest homology between Type II labyrinthine trophoblast in the mouse and villous cytotrophoblast in humans, both of which are proliferative stem cell-like compartments.

Baboon placentas express soluble and membrane-bound Paan-AG proteins encoded by alternatively spliced transcripts of the class Ib major histocompatibility complex gene, Paan-AG.

The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta contains messages encoded by a novel MHC gene, Paan-AG, which is evolutionarily related to the HLA-A locus, but shares unique characteristics with HLA-G. In this study, we show that the Paan-AG message is alternatively spliced to generate at least seven transcripts. One of these transcripts retains intron 4 and encodes a soluble glycoprotein with three external domains and a unique 21-amino-acid sequence at the carboxyl terminus, similar to soluble HLA-G1. This glycoprotein was detected in first trimester placental villous cytotrophoblast and syncytiotrophoblast, and in extravillous cytotrophoblast cells in the basal plate in term placenta. Four of the transcripts ( Paan-AG1, Paan-AG2, Paan-AG3, Paan-AG4) encode membrane-bound class Ib MHC glycoprotein isoforms. Paan-AG1 protein expression was similar to that of sPaan-AG, while Paan-AG2 protein was not detected in these tissues. The other two transcripts ( Paan-AGx and Paan-AGxi) contain a truncated exon 3 and multiple stop codons. Paan-AG1 and Paan-AGx transcripts were detected in a number of non-placental tissues, but these transcripts contained multiple stop codons. Because of the structural similarities and common features of organ-specific expression and splicing of the message, studies on Paan-AG may be of value in dissecting the functions of the class Ib proteins in human pregnancy.

DAX-1 represses the high-density lipoprotein receptor through interaction with positive regulators sterol regulatory element-binding protein-1a and steroidogenic factor-1.

The high-density lipoprotein receptor (HDL-R) mediates the selective uptake of high-density lipoprotein cholesterol in nonplacental steroidogenic tissues. We have previously demonstrated that sterol regulatory element-binding protein-1a (SREBP-1a) and steroidogenic factor-1 (SF-1) positively regulate HDL-R gene transcription. In the present study, we examined whether DAX-1 (dosage-sensitive sex adrenal hypoplasia congenital critical region on the X chromosome, gene-1) could influence the expression of the HDL-R gene. Cotransfection studies demonstrated that DAX-1 was able to repress SREBP-1a and SF-1-dependent activation of the HDL-R promoter. Mammalian two-hybrid assays demonstrated that DAX-1 could interact with SREBP-1a. In addition, electrophoretic mobility shift assays demonstrated that initial incubation of DAX-1 with SREBP-1a protein in the absence of DNA prevented subsequent binding of SREBP-1a to the HDL-R sterol regulatory elements in a dose-dependent manner, whereas, in the case of SF-1, DAX-1 formed a complex with SF-1 protein on the DNA. These data suggest that DAX-1 inhibits SREBP-1a- and SF-1-dependent activation of the HDL-R promoter through different mechanisms. This investigation confirms that DAX-1 has an important role in regulating steroidogenesis by interfering with SREBP-1a and SF-1 induction of a gene involved in the transport of cholesterol, thereby limiting the amount of substrate available for steroid hormone production.

Dose-related differences in the distribution of cocaine in the maternal–fetoplacental compartments in rats

Our goals were to examine whether a high dose of cocaine to causing CNS toxic manifestations in the pregnant rats influences the delivery of cocaine to the fetus, and whether the non-placental compartments have a significant role in the distribution of cocaine to the fetal tissues. Either a low or high dose of cocaine was infused intravenously to near-term pregnant rats. Arterial blood pressure and heart rate were monitored. Cardiac output and uterine and placental blood flows were measured by using radiolabeled microspheres. Plasma and tissue samples were obtained from the mother, placenta, and fetus and analyzed for cocaine and its metabolites via capillary gas chromatography/mass spectrometry. A high dose of cocaine induced convulsions that were accompanied by increased arterial blood pressure and decreased uteroplacental blood flow. However, the distribution pattern of cocaine and metabolites in the mother and fetus were similar between the high and low dose groups. Considerable amounts of cocaine and its metabolites were in the placenta. Previously ignored non-placental tissues, such as the amnion and myometrium appear to be a significant source for cocaine accumulation in the fetus.

Expression of non-classical MHC class I MAMU-AG transcripts in the rhesus monkey placenta and non-placental tissues

Expression of non-classical MHC class I MAMU-AG transcripts in the rhesus monkey placenta and non-placental tissues

The expression of the human chorionic gonadotropin beta subunit gene depends on negative control.

Transcription of the human chorionic gonadotropin (hCG) genes begins in the very early embryo stage and decreases or even disappears in nonplacental tissues. We have studied the regulation of hCG-beta genes by cell fusion and by the reverse transcription-polymerase chain reaction (RT-PCR). The choriocarcinoma cell lines, JAR and JEG-3, express high levels of the hCG-beta subunit while HeLa cells express extremely low levels of it. Most HeLa x JAR and HeLa x JEG-3 fusion clones expressed only a trace of the hCG-beta subunit mRNA, while JAR x JEG-3 fusion clones still expressed high levels of the hCG-beta subunit. Most transcripts of the hCG-beta subunit genes in JAR and JEG-3 came from the hCG-beta 5 subunit. Even the trace amount of hCG-beta transcripts from fusion clones came mainly from the beta-5 gene. The results suggest that the expression of the hCG-beta subunit genes depends on negative control. Probably when embryonic cells differentiate to form nonplacental tissues, specific inhibitors may appear and inhibit the expression of the hCG-beta subunit genes.

The Expression of the Human Chorionic Gonadotropin Beta Subunit Gene Depends on Negative Control

Transcription of the human chorionic gonadotropin (hCG) genes begins in the very early embryo stage and decreases or even disappears in nonplacental tissues. We have studied the regulation of hCG-beta genes by cell fusion and by the reverse transcription-polymerase chain reaction (RT-PCR). The choriocarcinoma cell lines, JAR and JEG-3, express high levels of the hCG-beta subunit while HeLa cells express extremely low levels of it. Most HeLa x JAR and HeLa x JEG-3 fusion clones expressed only a trace of the hCG-beta subunit mRNA, while JAR x JEG-3 fusion clones still expressed high levels of the hCG-beta subunit. Most transcripts of the hCG-beta subunit genes in JAR and JEG-3 came from the hCG-beta 5 subunit. Even the trace amount of hCG-beta transcripts from fusion clones came mainly from the beta-5 gene. The results suggest that the expression of the hCG-beta subunit genes depends on negative control. Probably when embryonic cells differentiate to form nonplacental tissues, specific inhibitors may appear and inhibit the expression of the hCG-beta subunit genes.