Glucose transport across cellular membranes is mediated by integral membrane proteins (glucose transporters). GLUT1, an isoform of facilitated-diffusion glucose transporters, is abundant in human and rat placentae. In the human placenta, syncytiotrophoblast serves as the structural basis of the placental barrier. GLUT1 is present along the luminal and basal plasma membranes of this cell. GLUT1 seems to play an important role in the transfer of glucose through a syncytiotrophoblast layer in the human placenta. In the rat placenta, two layers of syncytiotrophoblasts (namely syncytiotrophoblasts I and II from the maternal blood side to the fetal side) serve as a barrier. GLUT1 is abundant in these syncytiotrophoblasts. It is concentrated along the plasma membrane of the maternal side in syncytiotrophoblast I and also along the plasma membrane of the fetal side in the adjacent syncytiotrophoblast II. These two syncytiotrophoblast layers are connected by numerous gap junctions. Connexin 26 is present in these gap junctions. GLUT1 in conjunction with connexin 26 in the gap junction seems to serve as the machinery for trans-placental transfer of glucose in the rat placental barrier.

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Class I membrane glycoproteins encoded by major histocompatibility complex (MHC) genes are not expressed during early stages of development. This regulation is thought to play an important role in maternal tolerance of the fetal allograft. To elucidate the significance of developmental regulation of MHC class I genes, we produced transgenic mice expressing transgenes encoding the class I L^d heavy chain or the light chain, β₂-microglobulin, in the developing mouse embryo. These transgenes were driven by the chiken β-actin promoter, which is very active in the developing mouse embryo. The heavy chain and light chain transgenic mice were mated, and the resultant double transgenic offspring expressed L^d antigen in all the tissues examined by immunostaining and Northern blot analysis. The L^d antigen was also detected by immunostaining in the placenta of the double transgenic fetus. The double transgenic fetus was not rejected by the immunocompetent nontransgenic mother. These results suggest that expression of MHC class I antigens in embryos is not sufficient to provoke a maternal immune response.

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We identified the TTK-1 cell line to be a human large granular lymphocyte (LGL)/natural killer (NK) cell line from bone marrow or lymphoid tissue. This cell line expressed laminin on the cell surface and secreted interleukin-3 (IL-3) into culture supernatant in addition to immunosuppressive factors. We also revealed that cytokines of the colony stimulating factor (CSF) family essential for the proliferation and differentiation of trophoblast cells were generated in the mixed lymphocyte reaction (MLR) and the trophoblast cell line could be established by adding the products of MLR to the culture. Furthermore, the therapeutic effects of cytokines of the recombinant human (rh) CSF family including IL-3, granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF) and granulocyte-CSF (G-CSF) on the spontaneous abortion prone mice were examined. The results demonstrated that each cytokine definitely responded to the increase in placental and fetal weight and the incidence of fetal resorption in various ways.

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During the early stages of pregnancy in the mouse, part of the trophectodermal cells undergo endoreplication and are transformed into trophoblastic giant cells (TGCs) that contain up to several hundred times the haploid amount of DNA per nucleus. TGCs, which position at the border between fetal and maternal tissues throughout mouse pregnancy, not only form the cell layer between fetal and maternal tissues, but they also secrete progesterone and several hormonal proteins structurally similar to prolactin and growth hormone. Thus, these cells play an important role in fetal-maternal interaction and tissue remodeling and function as endocrine cells essential for the maintenance of pregnancy. In this paper, we review current knowledge on the mechanisms of the TGC transformation of trophoblast cells and the biological function of TGCs in mouse placentation.

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The placenta plays an essential role in fetal growth and the maintenance of pregnancy. Successful development and maturation of the embryo is totally dependent on placental function. The main endocrine participation of the placenta is attributed to placental lactogens (PLs). Progesterone is essential for pregnancy in all mammals and is secreted by the ovary and placenta, depending on the animal species. In the rat, the main source of progesterone throughout pregnancy is the ovary, and 20α-hydroxysteroid dehydrogenase (20α-HSD) is a key enzyme for ovarian progesterone secretion. The primary action of prolactin (PRL) in the maintenance of ovarian progesterone secretion is suppression of the activity of ovarian 20α-HSD. In this review, the sequence homologies between cDNAs for PLs and PRL and the intimate functional relationship between the ovary and placenta are discussed. We postulate the possibility of co-evolution of PLs and ovarian 20α-HSD.

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Existence of an endothelio-endothelial placenta is controversial. Insectivores may have a placenta of this type or the endothelio-chorial type; thus, studies of the placentae of Suncus murinus, the laboratory shrew, were undertaken. Cellular components of the interhemal membrane included maternal endothelium, intermediate trophoblast (syncytiotrophoblast) and fetal endothelium. Up to day 20 of gestation, the intermediate trophoblast clearly intervened between the hypertrophied maternal endothelium and the fetal endothelium throughout the labyrinthine zone. From day 20 to 24, the intermediate trophoblast was sieve-like in appearance with extensive infoldings at both surfaces. Basal laminae of both the maternal endothelium and the intermediate trophoblast were broad, irregular and continuous. Therefore, at this stage the placenta was endothelio-chorial in type. After day 24 of gestation, the intermediate trophoblast could scarcely be recognized as an obvious and continuous layer. Only cytoplasmic processes of the intermediate trophoblast were present between the maternal and the fetal endothelium. Finally, projections of both the maternal and the fetal endothelium contacted each other. We conclude that the intermediate trophoblast does not play a central role within the placental barrier throughout the last week of gestation. Hence, the placenta of Suncus murinus is endothelio-endothelial in type.

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Prostaglandin biosynthesis in umbilical vessels was investigated in patients of normal pregnancy, pregnancy induced hypertension, diabetes mellitus and intrauterine growth retardation of unknown cause. To the homogenates of umbilical vessels, isotope labeled arachidonic acid was added and incubated for 30 min. Then the biosynthetic products were extracted and identified by silicic acid column chromatography, thin layer chromatography and HPLC. In normal pregnancy, PG biosynthesis was examined in both arteries and veins. Biosynthetic products in arteries and veins were 6keto PGF_1α, PGF_2α, PGE₂, PGD₂, 15HETE and 8, 15 diHETE. In the umbilical veins, conversion rates to 15HETE and 8, 15 diHETE were higher than those in the umbilical arteries. Conversion rates to 6keto PGF_1α were lower in PIH and IUGR patients than in the normal control. This means that PGI₂ synthesis in those patients was reduced. On the other hand, hydroxyacid biosynthesis in patients with PIH, DM and IUGR was increased. Therefore in pathological conditions, bioconversion of arachidonic acid to PGI₂ is reduced and it may be said that blood circulation in the umbilical vessels in those patients is also reduced.

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To investigate endocrine responses of isolated premature goat fetuses during long-term extrauterine incubation with umbilical arteriovenous extracorporeal membrane oxygenation (A-V ECMO), we conducted experiments in seven goat fetuses (95-134 days gestation). The fetuses were cannulated from the umbilical vessels, and their blood-gas exchange was totally supported by A-V ECMO, while they were maintained in an isothermal incubator containing artificial amniotic fluid. The survival period was between 84 and 190 h. At 24-h intervals, fetal blood samples were collected, and plasma concentrations of catecholamines, ACTH, and cortisol were determined. After 24 h of incubation, fetal circulatory and respiratory variables remained stable, until evident circulatory failure occurred before their deaths. A similar pattern was observed in temporal changes in plasma concentrations of catecholamines, ACTH, and cortisol. Plasma levels were high during the initial 24 h of incubation, subsequently decreased, and then increased before death. Hormone levels during stable periods were equivalent to or slightly higher than values for fetuses in utero. These results suggest that conditions during the stable period of long-term extracorporeal fetal incubation are not highly stressful for the isolated fetuses.

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Steroids in fetal circulation are known to exist in a sulfoconjugated form. To investigate the contribution of human fetal liver to the sulfoconjugation of steroids, and the properties of steroid sulfotransferase, an attempt was made to separate the specific sulfotransferases toward estrone (E-ST) and 3β-hydroxysteroid (3β-ST). For the purification procedure, successive chromatography was used:(1) DEAE-Sepharose CL-6B chromatography (2) Chromatofocusing and (3) Adenosine 3', 5'-diphosphateagarose affinity chromatography. Enzymatic activity was obtained with ³H-estrone (E₁), ³H-pregnenolone (P) and ³H-dehydroepiandrosterone (DHA) as substrates. Two active fractions of steroid sulfotransferase were collected from the eluate of a CL-6B column. Fractions 88-120 found to be specific for E₁, were purified approximately 1, 800 fold by further chromatographic procedures. The affinity column condition used was set at pH 7.2. The apparent Km and Vmax values were 1.66 μM and 35.6 nmol/mg protein/min, respectively. Fractions 130-162 specific for P were further purified approximately 144 fold. The affinity column condition used was set at pH 6.0. The apparent Km and Vmax values were 0.25 μM and 4.36 nmol/mg protein/min, respectively. Both enzymes were found to be a single band and had almost the same molecular weight of approximately 36 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both enzymes equally catalyzed DHA to form DHA-sulfate. In the present study, E-ST and 3β-ST were separated and purified in human fetal liver for the first time, with kinetic analysis for both enzymes.

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Placenta as a physiological source for oxytocic factor was implicated in our previous report, where we investigated the serum concentration of oxytocin (OT) and neurophysins during pregnancy, labor and lactation. The existence of immunoreactive (ir)-OT in placenta was suggested first by our study of the serum concentration in the umbilical vein, artery, and maternal circulation. The concentration of ir-OT in the placenta in the second trimester (20.8 ng/g-tissue) was 12-fold of that in the first trimester (1.6 ng/g-tissue). An immunohistochemical study on term placenta with the antiserum to OT revealed that ir-OT was mainly localized in the layer of syncytiotrophoblast. In vitro culture of trophoblast indicated that cycloheximide and prostaglandins (PGs) modulated the synthesis and/or release of ir-OT in the placenta. Further, the placental extract showed sings of mimicry of synthetic OT both in the elution profile through a carboxymethyl cellulose column and in bioactivity assayed by isometric tension of uterine muscle. The bioactivity and immunoreactivity of the extract were compared in the fractionated eluent of the extract on a Sephadex G-25 column. The results indicated that the bioactivity could be parallel with the immunoreactivity assayed by our RIA system, which was consistent with the fact that our antiserum neutralized the bioactivity of OT. We recently obtained a cDNA clone by immunoscreening from a human placental library, and the clone size was approximately 900 nucleotides. The clone was transfected into a Chinese hamster ovary cell line, the cultured media of which contained the recombinant ir-OT proved by assays of both immunoreactivity and bioactivity. A search for the major contractile factor in the initiation of labor is in progress.

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We found 9 proteases in human placenta. Our studies showed that these placental proteasesmetabolize vasoactive and immunomodulating peptides, possibly derived from the fetus, and protect the exchange of peptide hormones across the placenta in order to maintain feto-placental homeostasis. We clarified the pregnancy serum oxytocinase discovered by Fekete in 1930 and angiotensinase by Page in 1947, respectively. In addition we showed the pregnancy serum bradykininase. Changes in maternal serum protease activities were useful for monitoring of pre-elcampsia and predicting the onset of labor. In addition, the ratio of peak systolic over least diastolic pressure of uterine or umbilical artery assessed by the Doppler technique was closely correlated with the levels of maternal serum proteases in preeclampsia, which suggested that placental proteases might control uteroplacental circulation via the regulation of concentrations of vasoactive peptides in uteroplacental circulation. The degradation of immunomodulating peptides by placental protease also suggests the possible involvement of placental protease in the immunological aspect of pregnancy.

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With a culture system of human endometrial stromal cells, the effects of cytokines of interleukin (IL)-1, tumor necrosis factor (TNF)α, interferon (IFN)β, and IFNγ were examined. In a dose-dependent manner, IL-1 and TNFα inhibited progesterone-induced in vitro decidualization indicated by PRL production and cellular transformation with no cytotoxicity. IFNβ had no effect, but IFNγ inhibited PRL production caused by cytotoxicity. By Northern blot analysis and quantitative reverse transcription-polymerase chain reaction, human endometrium was shown to express the genes of macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF) and leukemia inhibitory factor (LIF). During stromal cell culture with progesterone, mRNA expression of both M-CSF and its receptor, c-fms, was significantly increased in a dose- and time-dependent manner. There was no difference in the expression of SCF mRNA between proliferative and secretory phase endometrium, but higher expression of SCF mRNA was observed in decidua of early pregnancy. LIF mRNA was more strongly expressed in secretory phase endometria than in proliferative ones. In culture experiments, SCF gene expression was higher in stromal cells, on the other hand, LIF mRNA was more strongly expressed in glandular epithelial cells. But no significant changes in SCF or LIF mRNAs were identified in our culture system for in vitro decidualization. These findings suggested an important regulatory role of cytokines in endometrial differentiation, and, in addition, endometrial stromal cells and glandular cells may play independent roles in producing cytokines essential for local immune cell proliferation and/or differentiation. Thus an immune-endocrine network exists in the human uterus to regulate the endometrial function.

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Endothelin (ET) was originally isolated from culture medium of porcine aortic endothelial cells as a potent vasoconstrictor peptide.Subsequently, ET was also reported to be produced by nonvascular tissues and to be involved in various biological phenomena in these tissues.Recently, a high concentration of ET-1-like immunoreactivity (ET-1-LI) was found in human amniotic fluid.Amnion tissue also contained a large amount of ET-1-LI, and cultured amnion cells secreted large amounts of ET-1-LI.The major component of ET-1-LI in these samples was ET-1.Moreover, the expression of prepro-ET-1 mRNA was detected in both amnion tissue and cultured amnion cells, indicating that ET-1 in the amniotic fluid originated from amnion cells.In cultured amnion cells, ET-1 secretion was stimulated by EGF, TGF-β, IL-1α, β and TNF-a.In addition, specific receptors for ET, ET-AR and ET-BR were detected in myometrium, decidua vera, chorion laeve, and placenta at term by both ligand binding and Northern blot analysis.These findings suggest that ET-1 secreted from amnion cells plays a physiological role in human pregnancy.In this paper, the regulation of production of ET-1 and expression of receptors for ET-1 by avascular human amnion tissue are reviewed.The possible importance of amniotic ET in human pregnancy is also discussed.

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Previous studies have shown that human placental trophoblast is the site of the reception of thyroid hormone and epidermal growth factor (EGF) and that cellular levels of thyroid hormone receptor and EGF receptor are notably higher in early placenta than those in term placenta. On the other hand, it is now evident that a close similarity exists between thyroid hormone receptor and erb A protein and between EGF receptor and erb B protein. In the present study, we have therefore analyzed staged human placentas by Northern blot and In situ hybridization with c-erb A and c-erb B cDNA probes.
In situ hybridization revealed that c-erb B mRNA is localized in the syncytiotrophoblast, consistent with our previous results, which showed the cytological localization of EGF receptor in developing human placenta. Northern blot analysis demonstrated that placental RNA contains c-erb A transcripts of 5.7-kb and c-erb B transcripts of 10.0-kb and 5.6-kb and that the amounts of the c-erb A and c-erb B transcripts vary remarkably during the course of gestation, being most abundant in early placenta, more abundant in midterm placenta, and least abundant in term placenta. It is therefore likely that the increased expression of c-erb A and c-erb B gene in early placental cells indicates an increase in cellular levels of thyroid hormone receptor and EGF receptor in early placental trophoblasts. These findings suggest an important role for the c-erb A and c-erb B oncogenes in the induction of trophoblast proliferation and differentiation in early gestation.

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Twenty-five placentae between 24 and 41 gestational weeks showing signs of intrauterine infection were studied pathologically. The placentae with chorioamnionitis at less than 33 gestational weeks had thick amniotic layers due to infiltration by polymorphonuclear leucocytes (PMNL) and fibrin deposits. Umbilical cords also showed signs of infiltration and occasional calcification. The main cause of chorioamnionitis in these cases is ascending bacterial intrauterine infection had villitis with cholangiosis and dysmature villi in 6 placentae. The clarification of these infectious processes is difficult, and they are mostly diagnosed as villitis of unknown etiology (VUE). Autopsy data are useful for the clarification of infectious pathology in stillborn cases.

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Peculiar mononuclear cells existing in the mesenchymal stroma of human chorionic villi, termed Hofbauer cells, are macrophages, and they are characterized by many vacuoles of various sizes and small granules in the cytoplasm. Their functional properties are still a matter of controversy. The current study was designed to determine the biological association between these macrophages and human chorionic gonadotropin (hCG), mainly using immunohistochemistry and immunoelectron microscopy. By light microscopic immunohistochemistry, these macrophages showed a weak positive reaction for the hCG-alpha subunit and a marked positive reaction for hCG-beta subunit (hCG-β). They were observed throughout gestation, and the reactivity was unrelated to the fetal sex. However, immunoreactivity for hCG-β C-terminal peptide was negative. Immunoreaction products of hCG-β were localized in coated pits, coated vesicles, multivesicular bodies, and also within the cytoplasmic vacuoles and granules but not in the intracytoplasmic spaces, organelles, or nucleus. In addition, acid phosphatase activity was observed in these vacuoles and granules. The hCG/LH receptor was not defined in the cell suspensions of these macrophages by hCG radioreceptor assay. The present study provides evidence that these macrophages ingest hCG secreted from the syncytiotrophoblasts without a receptormediated endocytotic mechanism, and they may participate in the control of hCG. Both cell types are not mutually exclusive in the developing chorionic villi.

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