Rat Placental Tissue Research Articles

MicroRNA-195-5p Attenuates Pregnancy-Induced Hypertension by Inhibiting Oxidative Stress via OTX1/MAPK Signaling Pathway.

Pregnancy-induced hypertension (PIH) is a hypertensive disorder during pregnancy and can induce perinatal death of human infants. MicroRNA (miR)-195-5p was validated to display low expression in severe preeclampsia placentas, but the role of miR-195-5p in pregnancy-induced hypertension (PIH) has not been investigated. The study emphasized on the functions and mechanism of miR-195-5p in PIH. A reduced uterine perfusion pressure (RUPP) rat model was established to mimic PIH in vivo. Adenovirus (Ad)-miR-195-5p agomir and/or Ad-OTX1 were further injected into some model rats. RT-qPCR was conducted to assess the expression of miR-195-5p and orthodenticle homeobox 1 (OTX1) in rat placental tissues, the isolated aortic endothelial cells (AECs), and in serum samples of PIH patients. Western blot analysis was implemented to measure the protein levels of OTX1, VEGFA, and key factors involved in the MAPK signaling pathway. The concentrations of oxidative stress markers (superoxide dismutase, catalase, and lipid hydroperoxide) in AECs and placental tissues of RUPP rats were measured by corresponding kits. The binding relation between miR-195-5p and OTX1 was verified using the dual-luciferase reporter assay. Hematoxylin-eosin staining was conducted to evaluate the pathological features of rat placental tissues. MiR-195-5p was downregulated, while OTX1 was upregulated in rat placental tissues and human serum samples of PIH patients. MiR-195-5p could target OTX1 and inversely regulate OTX1 expression in AECs and rat placental tissues. In addition, miR-195-5p can negatively regulate VEGFA level. Furthermore, miR-195-5p inactivates oxidative stress and the MAPK signaling by downregulating OTX1 in AECs. In vivo experiments revealed that OTX1 overexpression reversed the protective effect of miR-195-5p overexpression on placental damage and oxidative stress. MiR-195-5p alleviates PIH by inhibiting oxidative stress via targeting OTX1 and inactivating MAPK signaling.

MiR-218-5p promotes trophoblast infiltration and inhibits endoplasmic reticulum/oxidative stress by reducing UBE3A-mediated degradation of SATB1.

This research evaluated the effects of miR-218-5p on trophoblast infiltration and endoplasmic reticulum/oxidative stress during preeclampsia (PE). The expression of miR-218-5p and special AT-rich sequence binding protein 1 (SATB1) in placental tissues from 25 patients with PE and 25 normal pregnant subjects was determined using qRT-PCR and western blotting. Cell invasion and cell migration were detected by performing Transwell assays and scratch assays, respectively. MMP-2/9, TIMP1/2, HIF-1α, p-eIF2α, and ATF4 expression in cells was assessed through western blotting. Intracellular reactive oxygen species were detected using 2,7-dichlorodihydrofluorescein diacetate, and intracellular malondialdehyde and superoxide dismutase activities were determined with kits. Dual-luciferase and RNA pull-down assays were performed to verify the interaction between miR-218-5p and UBE3A. Co-immunoprecipitation and western blotting were used to detect the ubiquitination levels of SATB1. A rat model of PE was established, and an miR-218-5p agomir was injected into rat placental tissues. The pathological characteristics of placental tissues were detected via HE staining, and MMP-2/9, TIMP1/2, p-eIF2α, and ATF4 expression in rat placental tissues was determined through western blotting. MiR-218-5p and SATB1 were expressed at low levels, while UBE3A was highly expressed in the placental tissues of patients with PE. The transfection of an miR-218-5p mimic, UBE3A shRNA, or an SATB1 overexpression vector into HTR-8/SVneo cells promoted trophoblast infiltration and inhibited endoplasmic reticulum/oxidative stress. It was determined that UBE3A is a target of miR-218-5p; UBE3A induces ubiquitin-mediated degradation of SATB1. In PE model rats, miR-218-5p alleviated pathological features, promoted trophoblast infiltration, and inhibited endoplasmic reticulum/oxidative stress. MiR-218-5p targeted and negatively regulated UBE3A expression to inhibit ubiquitin-mediated SATB1 degradation, promote trophoblast infiltration, and inhibit endoplasmic reticulum/oxidative stress.

Кількісний та якісний склад кріоекстрактів плаценти щурів до та після ліофілізації

The purpose of the study was to investigate a peptide composition of cryoextracts from rat placental tissue using a gel-penetrating chromatography prior to and after low temperature storage and lyophilization. Materials and methods. Cryoextracts were harvested from tissue homogenates of the rat placenta with a method of freezing (-20oC, 1 day), subsequent thawing and centrifugation. The following types of cryoextracts were analyzed: cryoextracts-1 – immediately after preparation; cryoextracts-2 – frozen down to -196oC and stored for 1 month at this temperature; cryoextracts-3 – subjected to cryosublimation after storage at -196oC and thawing. Separate fractions of cryoextracts were obtained with a method of gel chromatography using columns of 27x2 cm with a Sephadex G-100. Protein load was measured with a method of spectrophotometry. The quantitative and qualitative evaluation of a peptide content in the substances with molecular masses from 100 to 12,000 Da was done with the help of gel-penetrating chromatography with a column of 400x16 mm, filled with a polyvinyl gel. Results and discussion. The analysis of gel chromatograms showed the volume ratio of proteins with molecular masses from 20 to 150 kDa in placental cryoextracts to be 80.28%, while total concentration of protein equaled 23.81 ± 1.03 mg/ml. A portion of low molecular fractions with molecular masses from 12 to 4 kDa comprised 19.60%. The data, obtained by gel-penetrating chromatography, demonstrated the extracts to contain from 7 to 10–12 protein-peptide fractions with molecular masses from 447 to ≥12,000 kDa. The basic points were the peaks Pr with molecular masses ≥12000 Da, and also in the following groups: A (molecular masses from 7,500 to 2,000 Da), B (molecular masses from 1,413 Da), C (molecular masses from 888 to 949 Da), D (molecular masses from 706 to 694 Da), E (molecular masses of 846 Da), F (molecular masses of 556 Da) and H (molecular masses of 447 Da). The cryoextracts-1 were represented mainly by 5 fractions of the group A, while comprised 7.59% from the total volume of a cryoextract. The volume ratio of low molecular peptides with molecular masses from 1,249 to 706 Da was 20.82%. In cryoextracts, stored at -196oC (cryoextracts-2), the volume ratio of the group A fractions comprised 7.76%, while concentration of low molecular fraction was as high as 25.15%. In cryoextracts, subjected to sublimation (cryoextracts-3), 5 high molecular peptide fractions of the group A were missing. The total volume ratio of low molecular fraction was significant and comprised 24.97%. It contained 6 low molecular peptide fractions – peaks B, C, E, F, H with molecular masses from 1,873 to 447 Da. Conclusion. Cryoextracts, subjected to freeze-drying, demonstrated an increase in the volume ratio of both high molecular fractions and peptides with molecular masses up to 1,000 Da, which may be due to coagulation of proteins as well as their destruction in the process of evaporative drying. Freezing down to -196oC and lyophilization of cryoextracts resulted in a strong tendency to increase the content of low molecular compounds of a peptide nature

Sexually concordant and dimorphic transcriptional responses to maternal trichloroethylene and/or N-acetyl cysteine exposure in Wistar rat placental tissue

Numerous Superfund sites are contaminated with the volatile organic chemical trichloroethylene (TCE). In women, exposure to TCE in pregnancy is associated with reduced birth weight. Our previous study reported that TCE exposure in pregnant rats decreased fetal weight and elevated oxidative stress biomarkers in placentae, suggesting placental injury as a potential mechanism of TCE-induced adverse birth outcomes. In this study, we investigated if co-exposure with the antioxidant N-acetylcysteine (NAC) attenuates TCE exposure effects on RNA expression. Timed-pregnant Wistar rats were exposed orally to 480 mg TCE/kg/day on gestation days 6–16. Exposure of 200 mg NAC/kg/day alone or as a pre/co-exposure with TCE occurred on gestation days 5–16 to stimulate antioxidant genes prior to TCE exposure. Tissue was collected on gestation day 16. In male and female placentae, we evaluated TCE- and/or NAC-induced changes to gene expression and pathway enrichment analyses using false discovery rate (FDR) and fold-change criteria. In female placentae, exposure to TCE caused significant differential expression 129 genes while the TCE+NAC altered 125 genes, compared with controls (FDR< 0.05 + fold-change >1). In contrast, in male placentae TCE exposure differentially expressed 9 genes and TCE+NAC differentially expressed 35 genes, compared with controls (FDR< 0.05 + fold-change >1). NAC alone did not significantly alter gene expression in either sex. Differentially expressed genes observed with TCE exposure were enriched in mitochondrial biogenesis and oxidative phosphorylation pathways in females whereas immune system pathways and endoplasmic reticulum stress pathways were differentially expressed in both sexes (FDR<0.05). TCE treatment was differentially enriched for genes regulated by the transcription factors ATF6 (both sexes) and ATF4 (males only), indicating a cellular condition triggered by misfolded proteins during endoplasmic reticulum stress. This study demonstrates novel genes and pathways involved in TCE-induced placental injury and showed antioxidant co-treatment largely did not attenuate TCE exposure effects.

Ferulic acid confers protection on islet β cells and placental tissues of rats with gestational diabetes mellitus

Gestational diabetes mellitus (GDM) refers to glucose intolerance of variable degree with onset or first recognition during pregnancy. It causes polyhydramnios, ketoacidosis, fetal macrosomia, and neonatal respiratory distress syndrome. The incidence of GDM has greatly increased, hence the search for new interventions that can effectively treat it. The present study investigated the protective effect of ferulic acid on islet β cells and placental tissues of rats with GDM. Female Sprague Dawley rats (n = 30) were used in this study. The rats were randomly assigned to three groups of 10 rats each: control group, GDM group and ferulic acid group. The rats were fed high-fat diet consecutively for 12 weeks, except those in control group. In addition, ferulic acid group rats received ferulic acid at a dose of 20 mg/kg body weight (bwt) intragastrically daily for 12 weeks. The expressions of insulin signal transduction proteins and inflammatory factors were determined in rat placental tissue. Apoptosis and levels of expression of apoptosis-related proteins were assessed in isolated islet β cells. The results showed that the expressions of p-IRS1, p-IRS2, p-PI3K, GLUT1, GLUT3, and GLUT4 were significantly reduced by GDM, but were significantly upregulated after treatment with ferulic acid (p &lt; 0.05). The levels of expression of NF-kB, ICAM-1, TNF-α and IL-β in placental tissues were significantly higher in GDM group than in control group, but were significantly reduced by ferulic acid treatment (p &lt; 0.05). However, the protein expression of visfatin was significantly reduced by GDM, but was significantly increased by ferulic acid treatment (p &lt; 0.05). Apoptosis was significantly promoted by GDM in islet β cells, but was significantly and concentration-dependently reduced after treatment with ferulic acid (p &lt; 0.05). Gestational diabetes mellitus (GDM) also significantly down-regulated the expression of bcl-2, but markedly upregulated the expressions of bax and caspase-3 (p &lt; 0.05). However, treatment with ferulic acid significantly and concentration-dependently upregulated the expression of bcl-2, but down-regulated bax and caspase-3 protein expressions (p &lt; 0.05). These results indicate that ferulic acid protects β cells from GDM-induced apoptosis and improves insulin signaling in placenta of female rats with GDM.

PP053. The role of peroxisome proliferator-activated receptor gamma co-activator 1-alpha in pregnancy

PGC-1α is a transcriptional co-activator that drives mitochondrial biogenesis in many tissues, including cardiac. PGC-1α is a powerful regulator of angiogenesis, a process of fundamental importance to the development of a healthy placenta in normal pregnancy. While the angiogenic role of PGC-1α has previously been described in the heart, its function and expression in the placenta is unknown. It may be possible that PGC-1α plays a key role in the imbalance of angiogenic factors observed in the pregnancy specific condition of pre-eclampsia. To determine if PGC-1α is expressed in placental tissue and its implications for pathophysiological disorders of pregnancy. Human placental samples were obtained from normal healthy pregnant women for processing and immunohistochemical analysis using an anti-PGC-1α rabbit polyclonal antibody. In addition, PGC-1α expression was examined in placental tissue from normal pregnant and reduced uterine perfusion pressure (RUPP) rats. Positive staining for PGC-1α was noted in both human and rat placental tissue with varying levels of expression. Staining appeared to be localised to the vascular endothelial cells, trophoblasts and syncytiotrophoblasts, with some minor staining in the maternal decidua and stroma. Moreover, intensity of staining for PGC-1α appeared reduced in placental tissue from RUPP rats. This study indicates that PGC-1α strongly localised to endothelial cells in both human and rat placental tissue. Furthermore, our data may implicate PGC-1α in the pathogenesis of pre-eclampsia, but further research is needed. Future studies could suggest the use of PGC-1α as a future drug target for treatment of complicated pregnancies.

Effect of paternal folate deficiency on placental folate content and folate receptor α expression in rats

We investigated the effect of paternal folate status on folate content and expression of the folate transporter folate receptor α (FRα) in rat placental tissues. Rats were mated after males were fed a diet containing 0 mg of folic acid/kg of diet (paternal folate-deficient, PD) or 8 mg folic acid/kg of diet (paternal folate-supplemented, PS) for 4 weeks. At 20 days of gestation, the litter size, placental weight, and fetal weight were measured, and placental folate content (n = 8/group) and expression of FRα (n = 10/group) were analyzed by microbiological assay and Western blot analysis, respectively. Although there was no difference observed in litter size or fetal weight, but significant reduction (10%) in the weight of the placenta was observed in the PD group compared to that in the PS group. In the PD group, placental folate content was significantly lower (by 35%), whereas FRα expression was higher (by 130%) compared to the PS group. Our results suggest that paternal folate status plays a critical role in regulating placental folate metabolism and transport.

Factors Affecting the Accuracy of Bisphenol A and Bisphenol A-Monoglucuronide Estimates in Mammalian Tissues and Urine Samples

ABSTRACTBisphenol A (BPA) (CAS Number 80–05-7; EINECS Number 201–245-8) is used in the production of plastics having food contact applications. Some biomonitoring studies have reported free BPA in blood or urine of humans. Since complete first-pass metabolism of orally administered BPA to BPA-monoglucuronide (BPA-G) occurs in humans, the presence of free BPA in human specimens raises questions as to the origin and/or possible sources of the free BPA. We hypothesized that BPA-G instability during specimen collection and analysis contributes to the presence of free BPA in the biological samples. Investigation of the in vitro hydrolysis of BPA-G in blood plasma, tissue homogenates, and diluted urine from laboratory rats and in aqueous/organic solutions commonly used for extraction in BPA analyses lent support to the hypothesis of BPA-G instability as a possible source of free BPA determinations in the biological specimens. Hydrolysis of BPA-G occurred at neutral pH and room temperature in diluted urine and in rat placental or fetal tissue homogenates at room temperature. Hydrolysis of BPA-G in aqueous/organic solutions began within minutes at pH 2 and 80°C. BPA-G was degraded to an unidentified compound in a urine/water mixture or when stored in a 25/75 mixture of urine/acetonitrile at pH 9 at either 22 or 80°C. Based upon these experiments, it was concluded that methods demonstrating BPA-G stability or accounting for its instability during analysis are warranted in studies designed to measure free BPA in biological specimens.

Synthesis of Oxytocin Analogues with Replacement of Sulfur by Carbon Gives Potent Antagonists with Increased Stability

[Chemical reaction: See text] The neuropeptide oxytocin 1 controls mammary and uterine smooth muscle contraction. Atosiban 2, an oxytocin antagonist, is used for prevention of preterm labor and premature birth. However, the metabolic lifetimes of such peptide drugs are short because of in vivo degradation. Facile production of oxytocin analogues with varying ring sizes wherein sulfur is replaced by carbon (methylene or methine) could be achieved by standard solid-phase peptide synthesis using olefin-bearing amino acids followed by on-resin ring-closing metathesis (RCM). These were tested for agonistic and antagonistic uteronic activity using myometrial strips taken from nonpregnant female rats. Peptide 8 showed agonistic activity in vitro (EC50= 1.4 x 10(3) +/- 4.4 x 10(2) nM) as compared to 1 (EC50= 7.0 +/- 2.1 nM). Atosiban analogues 17 (pA2= 7.8 +/- 0.1) and 18 (pA2= 8.0 +/- 0.1) showed substantial activity compared to the parent oxytocin antagonist 2 (pA2= 9.9 +/- 0.3). Carba analogue 35 (pA2= 6.1 +/- 0.1) had an agonistic activity over 2 orders of magnitude less than its parent 3 (8.8 +/- 0.5). A comparison of biological stabilities of 1,6-carba analogues of both an agonist 8 and antagonist 18 versus parent peptides 1 and 2 was conducted. The half-lives of peptides 8 and 18 in rat placental tissue were shown (Table 2) to be greatly improved versus their parents oxytocin 1 and atosiban 2, respectively. These results suggest that peptides 8 and 18 and analogues thereof may be important leads into the development of a long-lasting, commercially available therapeutic for initiation of parturition and treatment of preterm labor.

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.

Laminin 2 (merosin), but not laminin 1 (EHS-laminin), may be a major laminin isoform present in normal and fibrotic rat liver

Recently several laminin isoforms were newly identified and found to distribute in a tissue and cell type-specific manner. We have studied the expression of several laminin isoforms, including α1(A), α2(M), β1(B1), β2(S), and γ1(B2) subunits, in normal and CCl 4-induced fibrotic rat liver by RT-PCR and performed immunohistochemical study with newly developed monoclonal antibody to merosin (laminin-2). RT-PCR study for laminin subunits revealed that α2(M) subunit mRNA transcript was dominant chain present in normal rat liver tissue, but α1(A) subunit mRNA was not. Laminin β1(B1) and γ1(B2) subunit mRNA were detected strongly and β2(S) subunit mRNA was detected weakly. During the CCl 4-induced rat hepatic fibrosis, α2, β1, β2, and γ1 subunit mRNA showed increased expression, but α1 transcript could not be detected during fibrotic process even in the condition where α1 subunit mRNA was detectable in rat placental tissue. Immunohistochemical studies with a novel monoclonal antibody to merosin also revealed that deposition of merosin increased during CCl 4-induced rat fibrotic liver. These data strongly suggest that laminin isoform containing α2 subunit (merosin) may be a major laminin present in normal and fibrotic rat liver.

Study on melatonin in human and rat placental tissue

Human and rat placental tissues contain high levels of melatonin even during daytime. The alteration of placental melatonin suggests that the role of melatonin in the placenta differs from that in the pineal gland. Expression of the gene encoding AA-NAT strongly suggests that placental melatonin was synthesized in the placenta. However, the precise mechanism by which placental melatonin is regulated remains to be elucidated.

Study on melatonin like substance in both human and rat placental tissue

Study on melatonin like substance in both human and rat placental tissue

Concentrations of leptin in the serum of pregnant, lactating, and cycling rats and of leptin messenger ribonucleic acid in rat placental tissue

Leptin concentrations were measured in the serum of cycling, pregnant, and lactating Sprague-Dawley rats. Serum leptin concentrations did not vary significantly during the estrous cycle. In contrast, as gestation advanced, serum leptin concentrations increased significantly, p < 0.0001. Following delivery, leptin concentrations declined and remained stable during lactation. Leptin messenger ribonucleic acid (mRNA) was identified in the visceral adipose tissue and placenta of rats sacrificed on days 14 and 21 of pregnancy. The relative abundance of placental leptin mRNA increased ≈ 4 to 5 fold from day 14 to 21 of gestation. The pattern of elevated leptin concentrations in the serum of late pregnant rats is similar to that reported in pregnant women, therefore the rat may be a useful model for the study of leptin during pregnancy. The increase in leptin in the serum of late pregnant rats, as well as an increase in placental mRNA, raises the possibility that leptin may serve a physiological role for the late parturient rat and/or its young.

Vascular Endothelial Growth Factor Expression in the Rat Uterus and Placenta throughout Pregnancy.

To elucidate the physiological roles during pregnancy of vascular endothelial growth factor (VEGF), we examined the temporal and spatial expression of VEGF protein and mRNA in the rat uterine and placental tissues throughout pregnancy. The uterine tissue of nonpregnant rats, pregnant rats, and the rat placental tissue were examined by Northern blotting, in situ hybridization and immunohistochemistry. Northern analysis revealed the existence of VEGF mRNA in the uterus and placenta regardless of the stage of estrous cycle or pregnancy. The results of in situ hybridization and histochemistry for nonpregnant rat specimens showed that VEGF mRNA and protein accumulated in myometrial smooth muscle cells, glandular epithelial cells, stromal cells, and endometrial epithelial cells during each stage of the estrous cycle. The level of accumulation increased in all but the myometrial smooth muscle cells on estrus, and further increased during pregnancy. Furthermore fetal compartments such as trophoblasts, trophoblast giant cells, vitelline epithelial cells, and amnion were labeled more strongly than the maternal compartment.In conclusion, the spatial expression of VEGF was relatively constant in rat maternal uterus during the estrous cycle and during pregnancy; however, there was a temporal change of VEGF expression.

Gestational and smoking effects on peptidase activity in the placenta

Angiotensin converting enzyme (ACE) and leucine aminopeptidase (LAP) regulate fetally and maternally generated peptides in the placenta. In this study, ACE-like activity was found to be decreased and LAP-like activity increased with increasing days of gestation in rat placental tissues forming the fetal:maternal interface. Membrane-associated ACE-like and LAP-like activities in the placenta of smokers were also found to be significantly higher than their respective activities in placenta of nonsmokers. Our collective findings suggest that gestational and environmentally-induced changes in placental peptidase activities may account for variable peptide hormone and/or therapeutic peptide metabolism in the placenta.

Prolactin-like protein-B: heterologous expression and characterization of placental and decidual species.

Prolactin-like protein-B (PLP-B) is a member of a family of proteins expressed by the rat placenta and/or decidua with characteristics similar to prolactin (PRL). In this report, we present the heterologous expression and characterization of PLP-B. Recombinant PLP-B heterologously expressed in Chinese hamster ovary cells exhibited similar immunoreactive and electrophoretic characteristics with PLP-B produced by rat placental and decidual tissues. N-terminal sequencing verified the identity and purity of the recombinant PLP-B species and the site of cleavage of the signal peptide from the mature secreted PLP-B species. Polyclonal antibodies were generated to the recombinant PLP-B and used for Western blot and immunocytochemical analyses. Recombinant and native PLP-B migrated as a doublet at 30-31 kDa in SDS-PAGE under reducing conditions. Treatment of recombinant and native PLP-B with N-glycanase accelerated their electrophoretic mobility, indicative of their glycoprotein nature. PLP-B was localized exclusively to decidual cells in the developing deciduum and spongiotrophoblast cells in the placental junctional zone. The level of PLP-B protein expression dramatically declined prior to parturition. Potential PRL-like biological actions of PLP-B were also investigated. PLP-B bound weakly to ovarian and liver PRL receptors and did not stimulate the proliferation of lactogen-dependent Nb2 lymphoma cells. In conclusion, recombinant PLP-B possesses characteristics similar to native decidual and placental PLP-B and may represent a hormone/cytokine that has important modulatory actions during the establishment of pregnancy and the initiation of parturition.

Transferrin in cultured human term cytotrophoblast cells: synthesis and heterogeneity.

Transferrin (Tf) mRNA was recently demonstrated in rat and mouse placental tissue. Rat placental cells were shown to secrete transferrin. The cell type with which Tf mRNA was associated was not investigated. We therefore studied the ability of immunopurified human term cytotrophoblast cells in culture to synthesize Tf, by means of pulse-label experiments with 35S-methionine and report that these cells do synthesize Tf. Tf mRNA was demonstrated in the cell lysates by means of RT-PCR. Tf isolated from cytotrophoblast and syncytiotrophoblast cells was shown to be different from both maternal and fetal serum Tf with respect to the distribution of isoforms as demonstrated by means of iso-electric focusing. The iso-electric points were found at lower pH values (pH 5.0-5.4), compared to the iso-electric points of maternal and fetal serum Tf, suggesting a higher degree of sialylation and glycan chain complexity.

Lectin histochemistry of rat placental tissues

Lectin binding of rat trophoblast subpopulations was examined at days I0, 12 and I5 of gestation. Biotinylated lectins, specific for a range of carbohydrates, were applied to paraffin wax embedded tissues. At day I0 of pregnancy, a wide and similar range of lectins reacted with ectoplacental cone and giant cells: chorionic lamina was relatively unreactive except with wheat germ agglutinin. At days 12 and I5, the range of lectins reacting with the placental labyrinth was relatively restricted. Spongiotrophoblast at day 12 showed lectin reactivity similar to that of ectoplacental cone cells but at day I5 the reaction pattern indicated loss of N-acetylgalactosamine residues in the large, glycogen-rich cells. Trophoblastic giant cells also showed wide lectin reactivity: some reacted only with cytoplasmic components whilst others showed pericellular reactivity. Varying lectin reactivity of giant cells in different regions of the placenta suggests functional differences. There was reduced lectin reactivity of endovascular trophoblast at day I5, compared with day I2 of pregnancy, which may reflect altered invasive potential associated with loss of sialic acid and N-acetylgalactosamine residues. The pattern of lectin reactivity suggests that ectoplacental cells, as well as giving rise to giant cells, may also contribute to spongiotrophoblast and to endovascular trophoblast.

Rat Placental Hepatocyte Growth Factor/Scatter Factor: Purification, Characterization, and Developmental Regulation

In view of significant species-specific sequence differences between human and rat placental hepatocyte growth factor (HGF)/scatter factor (SF), the rat placental HGF/SF (rpSF) was purified, and its properties compared with human placental HGF/SF (hpSF). Like hpSF, rpSF scattered Madin-Darby canine kidney cells at 1-2 ng/ml and is composed of two subunits of 60 kDa and 30 kDa. Higher amounts (> 50%) of uncleaved 90-kDa form was present in the HGF/SF preparations from both human and rat placentas. Rat placental SF reacts with antibodies raised against hpSF in rabbits and chickens. The SF activity when expressed per gram rat placental tissue rises rapidly up to 9 days and then levels off. When expressed per milligram tissue protein it also increases rapidly up to 9 days and then declines. The expression of HGF/SF mRNA during development parallels that of HGF/SF activity. The specific activity of HGF/SF receptor (c-met) mRNA also appears to peak at 6 days. These findings suggest that (i) in spite of significant (> 10%) sequence differences between rpSF and hpSF, they exhibit similar structural, biologic, and immunologic characteristics and (ii) HGF/SF and its receptor are expressed in high amounts on Day 6 and then decline in developing placenta.