Rcho-1 Cells Research Articles

Expression profiles of zinc transporters in rodent placental models

Zinc is a vital metal that is a structural and functional component of many proteins. The precise mechanism of zinc transport in the placenta remains unclear. In this study, we investigated the expression of zinc transporters (ZnTs) in the mouse placenta and in two rat trophoblast cell lines, TR-TBT cells, which are syncytiotrophoblast cells of the labyrinth zone, and Rcho-1 cells, which retain trophoblast cell features and differentiate into trophoblast giant cells of the junctional zone. All of the ZnTs that have been identified in mice (ZnT1–7) were detected in the mouse placenta by RT-PCR. The expression profiles of ZnTs in the placenta during pregnancy were different. The mRNA levels of ZnTs, with the exception of ZnT7, did not change during pregnancy. The ZnT7 mRNA level in placenta was elevated during pregnancy. In TR-TBT cells, ZnT1, ZnT3 and ZnT4 were detected by RT-PCR analysis. In Rcho-1 cells, all of the ZnTs that have been identified in rats (ZnT1–4) were detected by RT-PCR analysis. There were no differences between the mRNA expression levels of ZnT family members in undifferentiated Rcho-1 cells and differentiated Rcho-1 cells. This is the first report of expression profiles of ZnTs during differentiation of the placenta in the mouse placenta and rat placental cell models.

Expression of CYP4A isoforms in developing rat placental tissue and rat trophoblastic cell models

Maintaining fatty acid homeostasis during pregnancy is critical for normal fetal development. As an organ that controls nutrient supply from the mother to the fetus, the placenta plays a significant role in guiding fatty acid transfer to the developing fetus. The cytochrome P450 4A (CYP4A) subfamily of metabolizing enzymes is a group of structurally and functionally conserved proteins that are specialized in the ω/ω−1 hydroxylation of saturated and unsaturated fatty acids and their derivatives. To understand the function of the CYP4A system in the placenta and its significance in maintaining fetal fatty acid homeostasis, information about the placental expression of individual CYP4A isoforms is required. In the present study, we have elucidated the temporal and spatial patterns of expression of the four known rat CYP4A isoforms (CYP4A1, CYP4A2, CYP4A3, and CYP4A8) in the junctional and labyrinthine zones of the developing rat chorioallantoic placenta as well as two rat trophoblastic cell lines, HRP-1 and Rcho-1, using semi-quantitative RT-PCR and immunohistochemical analyses. The mRNA from the four rat CYP4A isoforms was detected in the developing rat placenta with CYP4A1 exhibiting the strongest expression (4A1 > 4A2 ≫ 4A3 ≈ 4A8). CYP4A1 was also detected by immunohistochemical staining in the developing rat placenta. We also observed CYP4A1 in both HRP-1 and Rcho-1 cells by RT-PCR, suggesting the utility of these cells as in vitro tools to study the effects of xenobiotics on placental fatty acid metabolism. Establishing the expression of CYP4A isoforms in these tissues and cell models provides a framework for further investigation of their functional and physiological significance in guiding proper fetal development.

Androgen Secretion by Rcho-1 Cells is Independent of Extracellular Glutamate Concentration

In primates, progesterone, secreted by the placenta, is important for the maintenance of pregnancy. Androstenedione, a progesterone metabolite, fulfils a similar role in the rodent. Prior work has suggested that glutamate sufficiency and subsequent oxidation is important to placental androgen synthesis, presumably because of the production of NADPH (Trophoblast Res (1993) 7, 77). Rcho-1 cells possess a phenotype similar to that of rat placental giant cells, and secrete androstenedione and progesterone when in the differentiated state (J Endocrinol (1996) 150, 161). Our objective was to determine whether extracellular glutamate concentrations impact hormone synthesis in Rcho-1 cells. Rcho-1 cells were kept in culture under differentiating conditions. Extracellular glutamate concentrations were varied from 0–5 m m, and hormone concentrations assayed by ELISA. Rcho-1 cells secreted both progesterone and androstenedione. There was no direct correlation between the extracellular concentration of glutamate and the secretion of either hormone. Inhibition of transaminases (aminooxyacetic acid) or of glutaminase (6-diazo-5-oxo- l-norleucine) did not alter hormone production. Therefore, extracellular glutamate concentrations did not impact progesterone or androstenedione secretion. These findings may relate to the central position of glutamate in a variety of metabolic pathways, making intracellular depletion of this amino acid difficult to accomplish, or may represent a species specific difference in regulation.

Puralpha, a single-stranded deoxyribonucleic acid binding protein, augments placental lactogen gene transcription.

Placental lactogen (PL) is thought to alter maternal metabolism to increase the pool of nutrients available for the fetus and to stimulate fetal nutrient uptake. The ovine (o) PL gene is expressed in chorionic binucleate cells (oBNC) and cis-elements located within the proximal promoter (-124 to +16 bp) are capable of trophoblast-specific expression in human (BeWo) and rat (Rcho-1) choriocarcinoma cells. Protein-DNA interactions were identified with oBNC nuclear extracts, and mutational analysis of these regions revealed a previously undefined cis-element from -102/-123 bp that enhances promoter activity in BeWo cells but not Rcho-1 cells. Characterization of this region identified the nucleotide sequence CCAGCA (-105/-110; o110) as the responsible cis-acting element. Southwestern analysis with this element identified a binding protein with an apparent M(r) of approximately 41,000. Expression screening of an ovine placental cDNA library identified six homologous cDNAs, which shared identity with human (97%) and mouse (95%) Pur alpha, a single-stranded DNA binding protein. The Pur alpha-o110 interaction was confirmed by electrophoretic mobility-supershift assays with oBNC and BeWo extracts but was absent with Rcho-1 extracts. Furthermore, overexpression of ovine Pur alpha enhanced transactivation of the oPL gene proximal promoter in both choriocarcinoma cell lines through this novel cis-element. This study identified a previously undefined cis-element, which interacts with Pur alpha to augment PL gene transcription.

Transferrin Expression by Placental Trophoblastic Cells

Transferrin (TF), a 76–80 kDa glycoprotein, is responsible for the transport of iron to cells within both the fetal and maternal systems, but it does not cross the multiple cell layer barrier of the placenta. Recent findings that both rat and human placental cells produce TF indicated that placental TF may function in some manner to transport or regulate iron passage across this barrier. However, placental production of TF was brought into question because the cell preparations used to identify TF were obtained using dispersed tissue and may have contained non-placental contaminating elements. In this study, cultures of phenotypically distinct cell types containing only placental cells were used to firmly establish whether or not TF is expressed, and if so to begin to identify the cell(s) associated with its synthesis. Utilizing RT–PCR, in situ hybridization, and Western blot analysis, we identified TF mRNA and protein in three trophoblast cell types, HRP-1 (rat), Rcho-1 (rat), and BeWo (human) cells. Additionally, TF mRNA and protein were found in Giant cells, the differentiated form of Rcho-1 cells. When taken together, these results demonstrate clearly that TF is expressed by both differentiated and non-differentiated placental cells, and when viewed in light of previous findings, strengthen the possibility that placental TF may be central to the passage of iron from the mother to the fetus during development.

Glutamate transport by Rcho-1 cells derived from rat placenta.

Marginal giant cells within the rodent placenta are important sources of androgens, critical to maintenance of pregnancy. Androgen synthesis requires NADPH, a by-product of glutamate oxidation. We examined the uptake of glutamate into rat choriocarcinoma cells, which have been shown to maintain many of the characteristics of marginal giant cells in culture. Na+-dependent, d-aspartate inhibitable uptake consistent with System XAG- mediated transport was present, as were proteins capable of System XAG- activity, EAAC1, GLAST1, and GLT1. Glutamate uptake in rat choriocarcinoma cells was up-regulated by amino acid deprivation-a response that was not reversed by the addition of glutamate to the media. Inhibition data suggested up-regulation of transport activity mediated by either EAAC1 or GLAST1 at 6 h, whereas at 24 and 48 h, up-regulation of GLT1 plays an increasing role. Levels of EAAC1 immunoreactive protein increased with time under amino acid depleted conditions, whereas those of GLAST1 and GLT1 remained stable or declined but not significantly.

Effect of high concentrations of glucose on differentiation of rat trophoblast cells in vitro.

Previous studies have shown that diabetic placentas are characterized by structural and biochemical anomalies, including defects in the differentiation of trophoblasts. In this study, the Rcho-1 cell line was used to investigate the impact of high glucose concentrations on different markers of differentiation of rat trophoblast cells in giant cells (endoreduplication, invasive phenotype and endocrine phenotype). Rcho-1 cells were incubated for 12 days in medium supplemented with different concentrations of glucose and 10% horse serum to optimize differentiation. The cells were examined for the proportion of nuclei showing signs of apoptosis. The effect of high glucose was investigated on the endoreduplication process, on invasive phenotype (secretion of gelatinase B) and on endocrine phenotype (expression of placental lactogen I (PL-I) and II (PL-II) and progesterone secretion). Apoptosis was not induced by high glucose in Rcho-1. The number of cells was higher in the cultures exposed to high glucose (p<0.05) and their nuclei contained more DNA compared with control cells (p<0.001), while their nuclear size was smaller (p<0.001). Gelatinase B secretion increased during differentiation but no difference was found when gelatinase B secretion from trophoblasts exposed to high glucose was compared with the control cells. Rcho-1 cell cultures showed an increase in PL-I and PL-II mRNA expressions during differentiation and which was not affected by high glucose. Progesterone secretion increased during differentiation in control cultures. However, this increase was abolished when trophoblasts were cultured in high glucose. Our data suggest that high glucose influences the endoreduplication process and the steroidogenesis during differentiation of rattrophoblasts.

Lead enters Rcho-1 trophoblastic cells by calcium transport mechanisms and complexes with cytosolic calcium-binding proteins

Within the placenta, a specialized Ca 2+ transport pathway develops in trophoblasts to promote growth of the fetus and hypothetically to enhance fetal uptake of Pb 2+. This hypothesis could not be tested until a method to monitor Pb 2+ influx by indo-1 fluorescence quench became available. We have applied this new method to cultured undifferentiated and differentiated Rcho-1 trophoblastic cells. Pb 2+ concentrations of 1 and 10 μM are equivalent to blood levels of 20 and 200 μg/dl in pregnant women. Over this range, Pb 2+ uptake increased with time and concentration in medium containing 1 mM Ca 2+ but was greater in Ca 2+-omitted solutions. Activation of capacitative Ca 2+ entry (CCE) with thapsigargin, an endoplasmic reticulum (ER) Ca 2+ pump inhibitor, increased Pb 2+ uptake, while inhibition of CCE by La 3+ decreased influx. Parathyroid hormone-related peptide (PTHrP) stimulates the synthesis of Ca 2+-binding proteins (CaBPs), as well as Ca 2+ transporters, during trophoblastic differentiation. Pretreatment for 72 h with PTHrP increased Pb 2+ uptake by undifferentiated Rcho-1 cells but had little effect on the quench in differentiated cells, probably due to their greater content of CaBPs which competed for Pb 2+-binding with indo-1. This competition was most evident in differentiated cells when 1 μM Pb 2+ caused an initial quench, followed by a rise in fluorescence. This rise was not inhibited by thapsigargin, thereby ruling out sequestration into the ER and leaving complexation of Pb 2+ by CaBPs as the most plausible interpretation. We conclude that trophoblasts have the ability to clear Pb 2+ from the maternal circulation and deliver it to the fetus.

Characterization of the Expression of the Retinoid-related, Testis-associated Receptor (RTR) in Trophoblasts

Previous studies have provided evidence indicating that the nuclear orphan receptor RTR plays an important role during embryonic development and in spermatogenesis. In this study, we examine the expression of RTR in murine placenta and several human placental choriocarcinoma cell lines. Northern blot analysis showed high expression of RTR mRNA in placental tissue. In contrast to murine testis, which contains 7.4 and 2.3kb transcripts, placental tissue expressed only the larger transcript. Examination of RTR expression in murine placental tissue by immunohistochemistry demonstrated the presence of RTR protein in the nuclei of giant trophoblasts and spongiotrophoblasts. RTR mRNA was also expressed in rat choriocarcinoma Rcho-1 cells and in the human placental choriocarcinoma cell lines BeWo, JAR, and JEG-3. In trophoblasts, RTR was co-expressed with the estrogen-related receptors ERRα and ERRβ. Giant trophoblast differentiation in Rcho-1 cells, characterized by induction of placental lactogen I (PL-I), was accompanied by a steady decrease in the expression of RTR mRNA and down-regulation of ERRβ expression while levels of ERRα mRNA did not change significantly. RTR was able to inhibit ERRα-mediated transactivation through the consensus RTR-response element (RTRE) likely by competing with ERRα for binding to the RTRE. These results suggest the possibility of cross-talk between RTR and ERRα receptor signalling pathways in trophoblasts.

Involvement of Nuclear Transcription Factor Sp1 in Regulating Glucose Transporter-1 Gene Expression during Rat Trophoblast Differentiation

Glucose transporter-1 (GLUT1) is important in placental glucose transport. However, the mechanism of regulation of placental GLUT1 expression remains to be elucidated. We show here that the level of GLUT1 protein in rat choriocarcinoma cells (Rcho-1) decreased during differentiation. To analyze the regulatory mechanism of rat GLUT1 (rGLUT1) gene expression, we transfected rGLUT1 promoter–chloramphenicol acetyltransferase constructs into Rcho-1 cells. Deletion analysis of the rGLUT1 promoter suggested that the region −76/−53 bp was essential for basal transcriptional activity. Electrophoretic mobility shift assays showed that transcription factors Sp1 and Sp3 bound two GC boxes in the region −99/−33 bp of the rGLUT1 promoter. Mutation analysis of the Sp1 binding sites revealed that the promoter-proximal site located between −76 and −53 bp was essential for basal rGLUT1 promoter activity. Furthermore, the decreased level of GLUT1 may result from a decreased level of Sp1 during differentiation. These findings suggest that Sp1 is involved in the regulation of rGLUT1 gene expression during rat trophoblast differentiation.

PAL31 Expression in Rat Trophoblast Giant Cells

PAL31 is a proliferation-related acidic nuclear protein that belongs to the leucine-rich protein family and is expressed cell-cycle-dependently. Trophoblasts differentiate into the trophoblast giant cells (TGCs) through the unusual type of cell cycle, namely endoreduplication. In the present study, we investigated the spatiotemporal pattern of PAL31 expression in rat placenta and Rcho-1 cell line. The PAL31 mRNA concentration varied in different areas of the placenta, and was barely detectable in the TGC layer. In Rcho-1 cells, although the level of PAL31 mRNA decreased dramatically during differentiation, PAL31 was detected even after differentiation. The site of intranuclear localization of PAL31 mostly overlapped with that of PCNA in the undifferentiated Rcho-1 cells, while they were not overlapped in differentiated cells. Thus, the subcellular localization of PAL31 in Rcho-1 cells significantly changed, and loss of cell cycle dependency after differentiation was noted. PAL31 is suggested to play a role in the endoreduplication distinct from the usual DNA duplication.

Progesterone inhibits transcriptional activation of human chorionic gonadotropin-α gene through protein kinase A pathway in trophoblast cells

The purpose of this study was to analyze the mechanism of transcriptional inhibition of human chorionic gonadotropin-α (hCGα) gene by progesterone in trophoblast cells. We stably transfected −290 bp hCGα promoter-CAT constructs (−290hαCAT) into Rcho-1 cells and monitored the promoter activities. Differentiation-dependent activation of −290 bp hCGα promoter containing a tandem repeat of cAMP response element (CRE) was inhibited by progesterone in a dose-dependent manner. To further analyze the mechanism of the progesterone action, Rcho-1 cells stably transfected with −290hαCAT were treated with forskolin in the presence of progesterone. Progesterone inhibited forskolin-induced transcriptional activation of hCGα gene. Moreover, progesterone inhibited forskolin-induced transcriptional activation of CRE-CRE-tk-CAT. These results suggest that progesterone may inhibit cAMP-induced transcriptional activation of hCGα gene through CRE. Although progesterone did not alter the amount of CRE-binding protein (CREB), which is a main transcriptional factor bound to CRE(s) on hCGα promoter, progesterone abolished forskolin-induced CREB phosphorylation. In addition, pretreatment with progesterone abolished forskolin-induced activation of nuclear protein kinase A (PKA). In conclusion, progesterone inhibits hCGα gene transcription, at least in part, via the CRE region by inhibiting CREB phosphorylation through PKA pathway in trophoblast cells.

Stage-specific modification of G protein beta subunits in rat placenta

We previously analysed the plasma membrane proteins of rat placenta and prepared a database of 150 plasma membrane proteins, expressed in a stage-specific manner, utilizing two-dimensional gel electrophoresis (2D/E) [Mol. Cell. Endocrinol. 115(1995)149]. In this study, we focused on the proteins, tentatively named psL-I (MW 36.2 kDa, p I 5.3) and psL-II (35.9 kDa, 5.3), which were expressed mainly in late pregnancy. Close to psL-I and psL-II on 2D/E gels, we also recognized more abundant proteins [psC-I (36.2 kDa, 5.4) and psC-II (35.9 kDa, 5.4), respectively] arranged side by side with the same MW but different p I. Expression of psL-I and psL-II was detected only in junctional zone of placenta, whereas psC-I and psC-II were expressed in both labyrinth and junctional zones. In addition, psL-I and psL-II began to increase on day 16 of pregnancy and peaked at term, whereas expression of psC-I and psC-II was relatively constant. The analysis of these four proteins (psL-I, psL-II, psC-I and psC-II) by preparative 2D/E, peptide mapping, amino acid sequence and mass spectrometry (MALDI-TOF-MS) revealed that psC-I was a G protein β1 subunit, and psC-II was a β2 subunit, and showed that psL-I and psL-II were molecular modified forms of psC-I and psC-II, respectively. Expression of these G protein β subunits (psL-I, psL-II, psC-I and psC-II) was also observed in rat choriocarcinoma cells, Rcho-1 cells. Expression of psC-I and psC-II was much higher than those of psL-I and psL-II, and their level was relatively constant regardless of the stage of differentiation in vitro. Interestingly, expression of psL-I and psL-II gradually increased in association with the differentiation. Since the expression of β1 and β2 subunit proteins and their mRNAs was constant during the process of differentiation in Rcho-1 cells, the expression of these lower p I forms of G protein subunits (psL-I and psL-II) was thought to be post-translationally regulated. In conclusion, there are modified forms of G protein β1 and β2 subunits, in the placenta and Rcho-1 cells, which are expressed in a pregnancy-stage or differentiation stage specific manner.

Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cbeta.

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55-66% amino acid identity with mouse PLP-Calpha and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cbeta for consistency. We have also isolated rat PLP-Cbeta from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cbeta were isolated: one mRNA (rPLP-Cbeta) encodes a 241-amino acid product, but another mRNA (rPLP-Cbetadelta39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Calpha, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cbeta form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cbeta mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cbeta expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cbeta mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cbeta expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cbeta clearly indicate that PLP-Cbeta is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cbeta during pregnancy.

Characterization of palladin, a novel protein localized to stress fibers and cell adhesions.

Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with alpha-actinin in the stress fibers, focal adhesions, cell-cell junctions, and embryonic Z-lines. Palladin is expressed as a 90-92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with alpha-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH(2)-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions.

Functional Analysis of Placental 57-kDa Ca2+-Binding Protein: Overexpression and Downregulation in a Trophoblastic Cell Line

The placental trophoblastic epithelium functions to transport nutrients needed by the fetus, including calcium, which is required in the greatest amounts during the last third of pregnancy when the majority of fetal skeletal mineralization occurs. The mechanism of placental calcium transport and the developmental changes in the trophoblast that facilitate this process are currently incompletely understood. We have previously identified a 57-kDa, Ca2+-binding protein (CaBP) functionally implicated in placental calcium transport and trophoblast differentiation. In this study we have directly examined the role of CaBP in these processes by (1) recombinantly overexpressing CaBP in an inducible manner and (2) downregulating CaBP expression using antisense technology, using the rat choriocarcinoma cell line Rcho-1 as a trophoblastic cell model system. Our results show that overexpression of CaBP stimulates both cellular calcium uptake and vectorial calcium transport activities in Rcho-1 cells. Those cells stably expressing CaBP also exhibit higher levels of steady-state intracellular calcium and enhanced calcium-buffering ability. In addition, prolonged overexpression of CaBP in Rcho-1 cultures promotes trophoblast differentiation. Conversely, downregulation of CaBP expression had a negative effect on calcium uptake, calcium transport, and trophoblast differentiation in Rcho-1 cells. These data indicate that CaBP plays a direct role in placental calcium transport, functioning both as an intracellular calcium buffer and as a shuttle. These results also support a more direct role for CaBP in the trophoblast differentiation pathway.

Psx homeobox gene is X-linked and specifically expressed in trophoblast cells of mouse placenta.

We previously isolated a cDNA clone for a homeobox-containing gene with its expression restricted to the extraembryonic tissues. In this study, Psx gene expression was further examined using in situ hybridization to determine the cellular distribution of Psx transcripts during embryo development. Psx expression was first detected at embryonic day 8.5 only in trophoblast giant cells and chorionic ectoderm. At E 9.5 and E 13.5, the expression was restricted to the giant cells and the labyrinthine trophoblast layer. In addition, the gene expression was detected in differentiated Rcho-1 trophoblast cells in vitro, which is typical of trophoblast giant cells in vivo, but not in proliferating Rcho-1 cells and HRP-1 cells. Interestingly, rat Psx homologue mRNA is about 200 bp shorter than mouse Psx, suggesting that there is a high degree of sequence divergence between the mouse and rat Psx homologues. The sequence divergence, perhaps as a result of rapid evolution, is further supported by the zoo blot analysis because the Psx gene was detectable only in mouse and rat but not in other vertebrate species tested. Psx is localized to the murine X chromosome. Taken together, our results suggest that Psx gene plays a unique role in the function of differentiated trophoblast cells and also serves as a useful model for studying trophoblast cell lineages and the rapid evolution of homeobox genes.

Rhythmicity of β-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat

During the first half of gestation in the rat, prolactin (PRL) from the anterior pituitary gland exerts its luteotropic function on the ovary to stimulate progesterone secretion. During this period, β-endorphin stimulates PRL secretion by regulation of dopaminergic neurons in the hypothalamus. During the second half, placental lactogens (PLs) take the place of PRL in maintenance of pregnancy, and initiate a negative feedback to suppress PRL secretion. However, the effect of PLs on β-endorphinergic neurons is not known. The aim of this study was to examine the possibility that PLs suppress PRL secretion by inhibiting β-endorphinergic neuronal activity. To accomplish this aim, we examined the changes in the neuronal activity of β-endorphinergic neurons in the mediobasal hypothalamus, as measured by Fos immunoreactivity, after manipulating the levels of PRL and PLs during pregnancy. On day 4 of pregnancy, animals received either Rcho-1 cells in the lateral ventricle that secrete PLs or HRP-1 cells as controls. In a separate experiment on day 12, hysterectomy was performed to remove the intrinsic source of PLs. These rats received Rcho-1 cells, HRP-1 cells, or nothing. Intracerebroventricular (i.c.v.) injection of Rcho-1 into hysterectomized rats was done to examine the effect of PL replacement. Sham-hysterectomy was also performed as a control. Animals were sacrificed 2 days after each treatment at 0200 h, 1400 h, and 1800 h. Brains were used for dual immunocytochemistry of Fos/β-endorphin. The neuronal activity of β-endorphinergic neurons of HRP-1 i.c.v. injected animals showed a daily rhythm, with high levels at 0200 h and 1800 h, and a low level at 1400 h. These animals also exhibited two surges of PRL secretion on day 6 of pregnancy. This rhythmicity of β-endorphinergic neurons was also observed in Rcho-1 i.c.v. injected animals, which showed very low and unchanging PRL levels. However, the magnitude of neuronal activity was reduced. On day 14 of pregnancy, all four experimental groups showed diurnal rhythms of β-endorphinergic neurons. This rhythmicity occurred even though PRL was elevated at all three time points in the hysterectomized rats and very low in the Rcho-1 i.c.v. injected hysterectomized and sham-hysterectomized rats. Our results demonstrate that there is a diurnal rhythm of β-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat. PLs might reduce the neuronal activity of β-endorphinergic neurons, but only during the first half of pregnancy, partially explaining the suppression of PRL surges.

Feedback effects of placental lactogens on prolactin levels and Fos-related antigen immunoreactivity of tuberoinfundibular dopaminergic neurons in the arcuate nucleus during pregnancy in the rat.

PRL in the rat exerts its luteotropic action during the first half of pregnancy. After midpregnancy, placental lactogens (PLs) take the place of PRL to stimulate progesterone secretion from the corpus luteum. Simultaneously, PLs trigger a negative feedback on PRL secretion. However, the brain mechanisms for the negative feedback induced by PLs are not fully understood. Here, we report changes in plasma PRL levels, tuberoinfundibular dopaminergic (TIDA) neuronal activity as measured by Fos-related antigen (FRA)/tyrosine hydroxylase (TH) immunoreactivity, and TH catalytic activity as measured by dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME) after experimental manipulation of PL levels. On day 4 of pregnancy, animals received Rcho-1 cells intracerebroventricularly (i.c.v.) to increase the level of PLs in the brain or HRP-1 cells as controls. On day 12 of pregnancy, hysterectomy alone or i.c.v. HRP-1 injection plus hysterectomy were performed to remove the source of PLs. Rcho-1 i.c.v. injection plus hysterectomy were performed to examine the effect of replacement of the PL source. Sham-hysterectomized animals were used as a control group. Animals were killed 2 days after each treatment at 0200 and 1800 h, which represent the peak times of PRL surges, and at 1400 h, which represents the intersurge time, by either transcardial perfusion for FRA/TH immunocytochemistry or decapitation 30 min after NSD 1015 injection to assess DOPA accumulation with HPLC-electrochemical detection. Rcho-1 cells completely abolished PRL surges on day 6 of pregnancy and increased the percentage of FRA/TH immunoreactivity in the dorsomedial, ventrolateral, and caudal subdivisions of the arcuate nucleus. This change in neuronal activity reflected the amount of DOPA accumulation in the SME, which was high at all time points. On day 14 of pregnancy, removal of the PL source by hysterectomy resulted in increased PRL levels and decreased neuronal activity of TIDA neurons at all three time points. Similar profiles were observed in animals that received i.c.v. HRP-1 injection plus hysterectomy. Replacement of the source of PL with Rcho-1 cells in hysterectomized rats resulted in low PRL secretion, high neuronal activity of TIDA neurons, and high TH catalytic activity. These patterns were the same as those in sham-operated animals. Our results demonstrate that PLs induce an increase in the neuronal activity of dopaminergic neurons, as measured by FRA/TH immunoreactivity and TH catalytic activity in the SME. Removal of the PL source elevates plasma PRL levels at all times during the second half of pregnancy and does not restore PRL surges.

Expression of Rat DNA (cytosine-5) Methyltransferase (DNA MTase) in Rodent Trophoblast Giant Cells: Molecular Cloning and Characterization of Rat DNA MTase

Methylation of genomic DNA is involved in the basic methanism of gene inactivation, chromatin organization, X chromosome inactivation and genomic imprinting. A pattern of DNA methylation is maintained in mitotic cells by DNA (cytosine-5) methytransferase (DNA MTase). The DNA MTase has been shown to be also expressed in postmitotic cells such as neurons. In the present report, as an approch to analyzing mechanisms underlying regulation of DNA MTase expression, we first isolated rat DNA MTase cDNA. The isolated cDNA encoded a protein of 1,622 amino acid residues showing 88.3% and 64.2% of homology with mouse and human DNA MTase, respectively. Northern blot analysis showed that DNA MTase mRNA was highly expressed in placenta during mid- to late- pregnancy. We then analyzed the expression of DNA MTase in Rcho-1 cells, a rat choriocarcinoma-derived cell line, which cease cell division but keep replicating genomic DNA when differentiated in vitro. We found that the expression of DNA MTase protein was decreased in terminally differentiated Rcho-1 cells whereas DNA MTase mRNA was consistently expressed. This result suggested posttranscriptional regulation of DNA MTase activity in Rcho-1 cells. The Rcho-1 cells would be a valuable model for studying the regulation of gene expression and function of DNA MTase in postmitotic, differentiated cells.