Differentiation of trophoblast lineage is associated with DNA methylation and demethylation.

Our previous study has shown that the placenta and kidney had different genomic methylation patterns regarding CpG island loci detected by restriction landmark genomic scanning (RLGS). To investigate whether differentiation involves changes in DNA methylation, we analyzed the rat Rcho-1 cell line, which retains trophoblast cell features and differentiates from stem cells into trophoblast giant cells in vitro. By RLGS, a total of 1,232 spots were identified in the Rcho-1 stem and differentiated giant cells. Four spots (0.3%) were detected only in giant cells, implying that the loci were originally methylated, but became demethylated during differentiation. Another four spots (0.3%) were detected only in stem cells, implying that these loci, originally unmethylated, became methylated during differentiation. DNAs from three loci that became methylated during differentiation were cloned and sequenced. All showed high homologies with expressed sequence tags (ESTs) or with genomic DNA of other species, suggesting that these loci are biologically important. Thus, the eight differentially methylated loci should be good tools to study epigenetic modification specific to differentiation of trophoblast giant cells.     

Interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta

Mammalian embryos have an intimate relationship with their mothers, particularly with the placental vasculature from which embryos obtain nutrients essential for growth. It is an interesting vascular bed because maternal vessel number and diameter change dramatically during gestation and, in rodents and primates, the terminal blood space becomes lined by placental trophoblast cells rather than endothelial cells. Molecular genetic studies in mice aimed at identifying potential regulators of these processes have been hampered by lack of understanding of the anatomy of the vascular spaces in the placenta and the general nature of maternal-fetal vascular interactions. To address this problem, we examined the anatomy of the mouse placenta by preparing plastic vascular casts and serial histological sections of implantation sites from embryonic day (E) 10.5 to term. We found that each radial artery carrying maternal blood into the uterus branched into 5-10 dilated spiral arteries located within the metrial triangle, populated by uterine natural killer (uNK) cells, and the decidua basalis. The endothelial-lined spiral arteries converged together at the trophoblast giant cell layer and emptied into a few straight, trophoblast-lined "canals" that carried maternal blood to the base of the placenta. Maternal blood then percolated back through the intervillous space of the labyrinth toward the maternal side of the placenta in a direction that is countercurrent to the direction of the fetal capillary blood flow. Trophoblast cells were found invading the uterus in two patterns. Large cells that expressed the trophoblast giant cell-specific gene Plf (encoding Proliferin) invaded during the early postimplantation period in a pattern tightly associated with spiral arteries. These peri/endovascular trophoblast were detected only approximately 150-300 microm upstream of the main giant cell layer. A second type of widespread interstitial invasion in the decidua basalis by glycogen trophoblast cells was detected after E12.5. These cells did not express Plf, but rather expressed the spongiotrophoblast-specific gene Tpbp. Dilation of the spiral arteries was obvious between E10.5 and E14.5 and was associated with a lack of elastic lamina and smooth muscle cells. These features were apparent even in the metrial triangle, a site far away from the invading trophoblast cells. By contrast, the transition from endothelium-lined artery to trophoblast-lined (hemochorial) blood space was associated with trophoblast giant cells. Moreover, the shaping of the maternal blood spaces within the labyrinth was dependent on chorioallantoic morphogenesis and therefore disrupted in Gcm1 mutants. These studies provide important insights into how the fetoplacental unit interacts with the maternal intrauterine vascular system during pregnancy in mice.     

Expression of vascular endothelial growth factor and its receptors in the rhesus monkey (Macaca mulatta) endometrium and placenta during early pregnancy

Vascular endothelial growth factor (VEGF) is fundamental for development and maintenance of endometrial and placental vascular function during pregnancy. While there are a number of studies on VEGF in the human placenta, they are mostly restricted to late pregnancy. To further understand the role of VEGF in mediating angiogenesis during human early pregnancy, we employed a rhesus monkey early pregnancy model to study the temporal and spatial expression of VEGF and its receptors, fms-like tyrosine kinase (Flt)-1, and kinase-insert domain-containing receptor (KDR) mRNAs and proteins in the uteri on day 12, 18, and 26 of pregnancy using in situ hybridization, RT-PCR, and immunohistochemistry. VEGF mRNA had been identified in the luminal epithelium on day 12, in the glandular epithelium on day 12 and 18, and the highest expression was detected in the walls of some spiral arterioles adjacent to the implantation site on day 18, in the placental villi and in the fetal-maternal border on day 18 and 26. Besides, immunostaining of VEGF was detected in the placental villi and endometrial compartments including spiral arteries walls and the glandular epithelium. The localization of VEGF in the endothelium correlates with the presence of Flt-1 and KDR receptors on vascular structure. All the results above suggest that VEGF-VEGFR pairs were involved in the process of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development during the rhesus monkey early pregnancy. Expression of VEGF, Flt-1, and KDR in the epithelial cells also hints some additionally functional roles of VEGF during early pregnancy.     

Characterization of gene expression profiles in early bovine pregnancy using a custom cDNA microarray

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism (265), cell cycle and apoptosis (26), regulation of gene expression (113), expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placental-specific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.     

Effects of cadmium exposure on morphological aspects of pancreas,weights of fetus and placenta in streptozotocin-induced diabetic pregnant rats

This study was designed to evaluate the effects of Cd exposure on morphological aspects of β-cell and weights of fetus and placenta in streptozotocin (STZ)-induced diabetic pregnant rats. Ninety-nine virgin female Wistar rats (200–220 g) were mated with 33 males for at least 12 h. From the onset of pregnancy, the rats were divided into four experimental groups (control, Cd treated, STZ treated, and Cd+STZ treated). The Cd-treated group was injected subcutaneously daily with CdCl₂ dissolved in isotonic NaCl, starting at the onset of pregnancy throughout the experiment. Diabetes was induced on the 13th d of pregnancy by a single intraperitoneal injection of STZ in STZ-treated group. In addition to the daily injection of Cd, a single intraperitoneal injection of STZ was also given on the 13th d of pregnancy in the Cd+STZ-treated group. The rats received the last injection 24 h before being sacrificed and 10 randomly selected rats in each group were sacrificed on the 15th and 20th d of pregnancy. Blood samples were taken for the determination of the serum glucose and insulin levels. Maternal pancreases, fetuses, and placentas of sacrificed rats in all groups were harvested (fetal pancreas was also harvested only on the 20th d of pregnancy) for morphological and immunohistochemical examinations. Cd exposure alone caused a degeneration, necrosis, and weak degranulation, but Cd exposure with STZ caused a severe degeneration, necrosis, and degranulation in the β-cells of the pancreatic islets. No morphological or immunohistochemical differences were found in β-cells of fetal pancreatic islets of control or other treatment groups. Cd exposure alone also decreased the fetal and placental weights. The administration of STZ alone, on the other hand, increased the placental weight. Cd, STZ, and Cd+STZ administration increased the glucose and decreased the insulin level. The increase in glucose and decrease in insulin levels were higher when Cd and STZ were given together. All of these changes were more severe on the 20th d than those on the 15th d of the pregnancy. It is concluded that Cd exposure during pregnancy may reduce the birth and placental weights and produce necrosis, degeneration, and degranulation in β-cells of pancreatic islets, causing an increase in the serum glucose level. These changes might be severe in diabetic pregnant mothers.     

Localization of glycogen in the placenta and fetal and maternal livers of cadmium-exposed diabetic pregnant rats

This study was designed to investigate the effects of Cd exposure on the glycogen localization in the placenta and in fetal and maternal livers in streptozotocin (STZ)-induced-diabetic pregnant rats. Ninety-nine virgin female Wistar rats (200–220 g) were mated with 33 males for at least 12 h. From the onset of pregnancy, the rats were divided into four experimental groups (control, Cd treated, STZ treated, and Cd+STZ treated). The Cd-treated group was injected subcutaneously daily with CdCl₂ dissolved in isotonic NaCl, starting at the onset of pregnancy throughout the experiment. Diabetes was induced on d 13 of pregnancy by a single intraperitoneal injection of STZ in the STZ-treated group. In addition to the daily injection of Cd, a single intraperitoneal injection of STZ was also given on d 13 of pregnancy in the Cd+STZ-treated group. The rats received the last injection 24 h before being sacrificed and 10 randomly selected rats in each group were sacrificed on d 15 and d 20 of pregnancy. Blood samples were taken for determination of the serum glucose and insulin levels. Fetal and maternal livers of sacrificed rats in all groups were harvested on d 15 and d 20 of pregnancy, whereas placentas were harvested only on d 20 of pregnancy for histochemical examination. Although both Cd and STZ caused hyperglycemia and decreased insulin secretion, Cd-alone treatment increased the glycogen content only in the placental labyrinth, whereas STZ-alone treatment increased the glycogen content only in the maternal part of the placenta. Increased glycogen localization was observed in both the placental labyrinth and the maternal part of placenta when Cd and STZ were given together. Fetal and meternal livers of control and other treatment groups were not different regarding the glycogen content on d 15 or d 20 of pregnancy. It was concluded that Cd exposure during pregnancy might produce a glycogen localization in the placenta of diabetic rats. However, the function and the mechanisms of increased glycogen contents in the placenta of Cd-exposed pregnant diabetic rats remain unclear and further studies are needed.     

Estrogenic endocrine disruptive components interfere with calcium handling and differentiation of human trophoblast cells

During development, calcium (Ca) is actively transported by placental trophoblasts to meet fetal nutritional and the skeletal mineralization needs. Maternal exposure to estrogenic pesticides, such as 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) and methoxychlor (MTC), has been shown to result in reproductive disorders and/or abnormal fetal development. In this study, we have examined the effects of exposure of trophoblastic cells to MTC and DTT, in comparison to 17beta-estradiol (E2) and diethylstilbestrol (DES), to test the hypothesis that cellular Ca handling is a target for these endocrine disruptive components. Treatment with DDT, MTC, DES, or E2 increased cellular Ca uptake, and the expression of trophoblast-specific human Ca binding protein (HCaBP) was down-regulated by both MTC and DDT. Treatment with MTC, DDT, and DES inhibited cell proliferation, induced apoptosis, and suppressed expression of several trophoblast differentiation marker genes. These effects were reversed by overexpression of metallothionein IIa, a gene highly responsive to cadmium and other metals. These results strongly suggest that trophoblast Ca handling functions are endocrinally modulated, and that their alteration by candidate endocrine disruptors, such as MTC and DDT, constitutes a possible pathway of the harmful effects of these components on fetal development.     

AMP-deaminase from human term placenta

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S_0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S_0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that liver type of AMP-deaminase is an enzyme form present in human term placenta.     

Placental improvement and reduced distal limb defects by maternal interferon-gamma injection in methylnitrosourea-exposed mice

BACKGROUND: Methylnitrosourea (MNU), an alkylating agent derived from creatinine metabolism, is cytotoxic, genotoxic, and mutagenic. Mid-gestational exposure to MNU leads to distal limb defects in mice. Previous studies have shown that nonspecific maternal immune stimulation protects against MNU-induced teratogenesis. A role for immune-mediated placental improvement in this effect remains uncertain. METHODS: The immune system of timed-pregnant C57BL/6N and CD-1 mice was stimulated by GD 7 intraperitoneal (IP) injection with the cytokine interferon-gamma (IFN-gamma). A teratogenic dose of MNU was then administered by IP injection on the morning of GD 9 to disrupt distal limb formation. Fetal limb length, body length, digital deformities, and placental integrity were evaluated on GD 14. RESULTS: The incidence of syndactyly, polydactyly, and interdigital webbing in MNU-exposed mice was decreased by maternal IFN-gamma treatment. In C57BL/6N mice, these defects were reduced by 47, 100, and 63%, respectively, as compared to previous reports on CD-1 mice, by 39, 71, and 20%, respectively. Administration of IFN-gamma significantly diminished MNU-induced endothelial and trophoblast placental damage in both strains of mice. CONCLUSIONS: These findings support a possible link between maternal immunity, placental integrity, and fetal distal limb development. Further, these results suggest that IFN-gamma might act through placental improvement to indirectly protect against MNU-induced fetal limb malformations.