Evidence for a clathrin-mediated recycling of albumin in human term placenta.

During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 microM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-beta-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.

Colloidal effect of albumin on the placental lactogen and chorionic gonadotrophin releases from human term placental explants.

Albumin has been reported to stimulate the release of placental lactogen and chorionic gonadotrophin from human term placental explants within physiological concentrations. This study aimed at characterizing further its effect on the placental hormonal secretion. The placental lactogen and chorionic gonadotrophin secretory response of incubated explants to 5% albumin was reproduced by colloidal agents, i.e., dextran (4.5%) and polygelin (4%), indicating that a rise in colloidal osmotic pressure can elicit hormonal release from the syncytiotrophoblast. Their secretory effects were not modified by the absence of extracellular calcium or the presence of verapamil in the medium. The three agents also provoked a marked increase in (45)calcium outflow from preloaded and perifused explants that persisted in absence of extracellular calcium. These data indicate that the triggering effect of albumin on placental lactogen and chorionic gonadotrophin release can be partly reproduced by colloidal agents and is independent of extracellular calcium.

Physiological concentrations of albumin stimulate chorionic gonadotrophin and placental lactogen release from human term placental explants.

This study investigates whether albumin, a major plasma protein in direct contact with the trophoblast in vivo, can modulate human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) releases from placental explants. Incubating explants with a near physiological, i.e. 5%, concentration of human or bovine albumin during 30 min increased HCG and HPL release by at least 150%. This albumin effect was not mediated by any difference in hormone adsorption onto glass surfaces. In contrast to the sustained stimulation of hormone releases elicited by the addition of 10 mmol/l extracellular calcium, the albumin-mediated secretory responses were transient. However, the albumin- and calcium-stimulatory effects were abolished at 4 degrees C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 micromol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca(2+) or in the presence of 1 or 10 mmol/l Ca(2+) in the extracellular milieu. These data suggest that albumin is involved, at physiological concentration, in the secretion of HCG and HPL by human placenta. The cellular mechanism(s) underlying the albumin-mediated secretory responses may be partly different from those involved during the calcium-mediated stimulation.

Apoptosis in human term placenta is not increased during labor but can be massively induced in vitro.

Apoptosis in human placental villi is reported to increase until close to delivery. However, the involvement of the apoptotic process in the initiation of labor, and more particularly in relation to the decrease in placental perfusion during uterine contractions, remains unknown. The purpose of the study was to examine the reactivity of the apoptotic machinery in term placentae obtained before or after the onset of labor and after in vitro incubations. The incidence of apoptotic nuclei (< 1%) as evidenced by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, and the histological distribution of immunoreactive Bcl-2, Bax, and Bcl-x proteins, were similar in placentae collected after delivery and before the onset of labor and in placental explants maintained overnight at 4 degrees C in a minimal salt-Hepes medium. By contrast, 28% of nuclei contained fragmented DNA when placental explants were incubated overnight at 37 degrees C. This marked increase was associated with a decrease in the intensity of the Bcl-2 immunostaining and an increase in the intensity of Bax and Bcl-x immunostaining. In conclusion, the present study clearly evidences the presence of an active apoptotic machinery in term placental cells that is not involved in normal parturition.

Epitope mapping on intact, heated and reduced molecular variants of human chorionic gonadotrophin.

Monoclonal antibodies (MAbs) raised against purified human chorionic gonadotrophin (hCG) (n = 30) and synthetic peptides derived from hCG (n = 3) were able to recognize by Western blotting several hCG dimers (57-47 and 42 kDa), free beta-subunits (35-32, 26 and 16 kDa) and free alpha-subunit (21 kDa) which coexist in commercially available hCG preparations. According to differences in the immunoreactivity of hCG-related molecular forms observed under native or denaturing conditions such as boiling or reducing hCG samples before or after gel electrophoresis, nine classes of MAbs able to recognize different immunoreactive domains were determined. Three domains corresponded to continuous epitopes recognized by MAbs raised against hCG-related peptides. The six remaining domains, recognized by the other MAbs, contained discontinuous epitopes from which three were surface-oriented and three disguised in the holo-hormone. This solid-phase approach, combining native and denaturing conditions, represents a simple and powerful tool to screen the specificity of MAbs from varying sources and to investigate molecular variants of proteic hormone.

In-vivo and in-vitro assessment of the influence of ritodrine and oxytocin on the placental secretion of human chorionic gonadotrophin and placental lactogen.

The purpose of this study was to evaluate, in vivo and in vitro, the influence of ritodrine and oxytocin on the placental release of human chorionic gonadotrophin (HCG) and placental lactogen (HPL). The in-vivo study was performed on maternal sera collected before and 1 h after the onset of either ritodrine treatment (50 micrograms i.v./min; administered to 15 women at risk of premature labour) or oxytocin infusion (2 mU i.v./min; administered to 21 women for acceleration of slow labour). The in-vitro study was performed on human term placental explants incubated in the presence of 4-400 ng ritodrine/ml or 15-1500 microU oxytocin/ml. HCG and HPL were measured by radioimmunoassay on maternal sera and incubation media. Maternal circulating concentrations of HCG and HPL remained unaffected after 1 h of ritodrine or oxytocin treatment. The in-vitro release of HCG and HPL by placental explants was not modified when ritodrine or oxytocin was added to the incubation media. The lack of influence of ritodrine and oxytocin on the placental secretion of HCG and HPL suggests that beta 2-adrenergic and oxytocin receptors are not involved in the releasing process.

Simultaneous measurement of 45Ca outflow and human placental lactogen release from placental explants.

Kinetics of 45Ca outflow and human placental lactogen (hPL) release were characterized in human placental explants. Measurements of the rate of 45Ca and [3H]-sucrose (extracellular space marker) outflow from preloaded explants showed that, after a 40 min washout period, the 45Ca effluent radioactivity presumably originated from an intracellular compartment. This view was further supported by the La3+ and temperature sensitivity of the 45Ca outflow. Moreover, the addition of ionomycin as well as an increase in the extracellular Ca2+ or Ba2+ concentration provoked a dose-dependent rise in both the 45Ca outflow and hPL release. There were no systematic temporal analogies between the pattern of 45Ca outflow and hPL release. Taken together, these observations suggest that the stimulation of 45Ca outflow reflects an increased rate of 40Ca entry. The present data also extend previous observations indicating that hPL release can be stimulated by Ca2+ entry. Lastly, the 'in vitro' method described herein allows one to compare rapid changes in hPL release with associated ionic events and can be used to further document the relationships between placental secretory responses and cell calcium metabolism.

Gestational malaria: assessment of its consequences on fetal growth.

In a region of Africa (Nord-Kivu, Zaire) where malaria is endemic, circulating malaria parasites, malaria-associated placental lesions, and a low hemoglobin level (< 10 g/dl) were observed, either singly or in combination, in 73.1% of women (n = 461) delivering at the maternity hospital. These pathologic findings were associated with low birthweight in 18.1% of the newborns, whereas the prevalence of low birthweight was 6.4% among cases without these findings (P < 0.05). Parasitemia was observed in 17.4% of all mothers and was associated with a significant decrease in birthweight. Malaria-associated lesions were found in 52.5% of all placentas and were associated with a decrease in birthweight, head circumference, and ponderal index of the newborns. Such lesions were more frequently observed among primiparae (60.5%) than among multiparae (49.5%; P < 0.05). Lastly, a low hemoglobin level, found in 38.6% of the mothers, was associated with a decrease in birthweight, length, and head circumference. The differences in the physical effects associated with each of the pathologic conditions suggest that parasitemia, placental lesions, and anemia result in acute, subacute, and chronic impairment of fetal growth, respectively. Moreover, their deleterious effects may be cumulative, since the most dramatically affected physical patterns were found when the pathologic findings were associated in the same patient. Frequent antenatal monitoring of maternal hemoglobin and parasitemia, accompanied, when necessary, with curative treatments, may help to reduce the prevalence of intrauterine growth retardation and its procession of perinatal complications.

Serum unconjugated estriol after intravenous cortisol administration in late pregnancy.

Serum unconjugated estriol levels were measured in 44 pregnant women treated with corticoids for the prevention of respiratory distress syndrome (RDS) in premature infants. Intravenous administration of cortisol (1 g every 6 hours for 24 hours) decreased the level of circulating unconjugated estriol to 40% of the initial value after 6 hours and to 27% after 24 hours. During the 2 succeeding days, all the results remain below the pretreatment values. On the third day, the mean value is no longer different from the control value, but in some patients recovery takes more than 3 days.