Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.     

Substrate-dependent angiotensin II formation in the peripheral circulation.

An alternative angiotensin II-forming system distinct from the vascular renin-angiotensin system was demonstrated using a rat hindlimb perfusion system and synthetic substrates. This pathway was resistant to captopril and aprotinin, but was highly sensitive to chymostatin. Moreover, angiotensin II formation was substrate-dependent, i.e. angiotensin II formation from tridecapeptide human renin substrate in the presence of captopril was more than twice than that from an equimolar amount of angiotensin I. Both pathways may play a role in regulating the peripheral circulation.     

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.     

Enhanced endocytotic and transcytotic activity in the rat endometrium prior to embryo implantation

Background Understanding the mechanisms by which fluid absorption and secretion occur in the endometrium is clinically important since conditions that deregulate this process reduce fertility. It has been suggested that luminal epithelial cells induce a crucial step in the process of embryo implantation called uterine closure via endocytotic fluid uptake. Uterine lumen closure is a key step in the process of embryo implantation and is absent in some infertile strains of mice. Methods To investigate the process of uterine closure a ferritin-based tracer, used as a marker of endocytosis, was injected into the uterine lumen on day 5 of pregnancy when closure occurs. Results Unexpectedly, luminal epithelial uptake of tracer was minimal on day 5 of pregnancy discrediting endocytosis as the induction method of uterine closure. In contrast, ferritin was found deep in the stromal portion of the endometrium in pre-pregnant animals. Conclusions We have shown for the first time that uterine closure is not induced by luminal epithelial cell driven endocytosis. Another novel finding of this study was the passage of the tracer ferritin up to 15 cells deep into the endometrium suggesting an as yet unstudied mechanism by which information can be transported from the uterine lumen to the underlying stroma.     

Mercury in human maternal and cord blood, placenta, and milk.

Total mercury concentration was measured by atomic absorption spectrophotometry in 100 maternal-umbilical cord blood pairs, 39 placentae, and 32 breast milk samples, all from patients at the University of Iowa Hospitals. The mean maternal and cord blood levels were 1.01 and 1.24 parts per billion (ppb), respectively. Though the difference between maternal and cord values was not statistically significant, the two were significantly correlated with each other, and maternal blood level increased significantly with maternal age. Mean placental and milk concentrations were 2.28 and 0.93 ppb, respectively. The mercury levels in this study were lower than those reported from elsewhere, probably reflecting a combination of methodological refinement and relatively low mercury exposure in a rural population whose diet is low in fish consumption. These low levels suggest that the risk of perinatal mercury damage is small in such a population.     

The ability of the murine placenta to absorb monoclonal anti-fetal H-2K antibody from the maternal circulation.

In previous work we have found that partially purified 125I-labeled anti H-2 antibody is localized in the placenta when injected i.v. into females pregnant by males bearing the target haplotype. This led to the concept that the placenta is an H-2 antigen-bearing immunoabsorbent barrier between mother and fetus. In this report we have used an anti-H-2Kk monoclonal antibody of the IgG2a subclass, also labeled with 125I, to verify this concept, as well as to improve the genetic definition of the immunoabsorbent antigen. In addition we have prepared F(ab')2 fragments of this antibody, and these also show the immunoabsorbent effect. This indicates that transport into fetally derived tissues via Fc binding is not a prerequisite for reaction of the antibody with paternal strain H-2 K antigens.     

Serotonin-induced disruption of implantation in the rat: II. Suppression of decidualization

Subcutaneous injection of serotonin (20 mg/kg) on Day 5 of pregnancy disrupts implantation in the rat as indicated by the reduction in number of live fetuses/cornu present on Day 19 (0.9 vs. 6.1, treated vs. control). Such disruption of implantation possibly results from impaired decidualization. To test for suppression of decidualization, serotonin was administered to pseudopregnant rats on the day before, on (Day 4) or after artificial induction of the decidual cell reaction. Relative to saline-treated controls (C), serotonin (S) reduced decidualization when injected either before [C: 1987 +/- 130 vs. S: 1085 +/- 155 mg (Day 3); P less than 0.005] or after [C: 1987 +/- 130 vs. S: 173 +/- 8 mg (Day 5); P less than 0.001] administration of the deciduogenic stimulus. In addition, serotonin markedly decreased uterine blood flow (C: 0.47 +/- 0.05 vs. S: 0.25 +/- 0.06 ml/min per g; P less than 0.01) during pseudopregnancy. However, serotonin altered neither the duration of luteal function in pseudopregnant rats (C: 15.3 vs. S: 14.3 days) nor serum progesterone levels (C: 74-91 vs. S: 53-82 ng/ml) in pregnant animals. It is concluded that serotonin may disrupt implantation, in part, by suppression of decidualization. The loss of endometrial competence to undergo decidualization appears to be a consequence of serotonin-induced uterine ischemia rather than impaired corpus luteum activity.     

Newman's chamber models in the study of blood circulation through the liver and heart

Models based on Newman's chamber theory are proposed to describe, evaluate and eventually interpret blood circulation processes. Application to circulation through the liver and heart is made, with emphasis on the analysis of radiohepatogram and radiocardiogram.     

Development of the implantation chamber in the pregnant bitch.

Uteri taken from 25 bitches at various times during the early stages of pregnancy were studies cytologically to determine how the implantation chamber developed and how fetal-maternal relations were established. On day 13 after the end of estrus, knobs of trophoblastic syncytium formed and became wedged between cells of the uterine luminal epithelium. The syncytium quickly spread along the uterine lumen and into the mouths of the glands, dislodging and surrounding maternal cells. As invasion continued trophoblastic villi, consisting of cores of cytotrophoblast covered by a continuous layer of syncytium, penetrated deeper into the endometrium. The syncytium spread to surround maternal vessels and decidual cells. By day 26 the trophoblast had extended down to the large lacunae. Here syncytial trophoblast covering tips of the villi degenerated, leaving cytotrophoblast exposed to the necrotic zone. These cells possessed characteristics of absorbing cells. Hematomas were formed by focal necrosis of fetal and endometrial tissue at the poles of the implantation sites. Large pools of extravasated blood accumulated and red blood cells were phagocytized by surrounding trophoblastic cells. Therefore, the endotheliochorial relationship in the canine placenta appeared to be established by syncytial trophoblast invading a cellular endometrium. In the necrotic zone and hematomas, cellular trophoblast may have lost its syncytial covering, but elsewhere maternal vessels and decidual cells in the placenta were in direct contact only with syncytial trophoblast.     

Developmental origin of the rat adenohypophysis prior to the formation of Rathke's pouch

In amphibians, it has already been shown that the adenohypophysis originates from the anterior neural ridge. During the migration and morphogenesis of this organ, the anterior neural ridge transiently forms a Rathke's pouch-like structure by attaching itself to the rostral tip of the foregut, and finally gives rise to the adenohypophysis by detaching from the foregut and becoming connected to the infundibulum of the hypothalamus. In order to identify the origin of the adenohypophyseal cells in mammalian embryos prior to the formation of Rathke's pouch (RP), we labeled the rostral end of the neural plate and the adjacent area focally with DiI at the open neurula stage (9.5 dpc). After a 48-hours culture of the whole embryos, strongly labeled cells were detected in the RP only when DiI was applied to a small area situated just anterior to the rostral end of the neural plate. By explanting the labeled RP for a further 7 days, we confirmed immunohistochemically that the labeled cells developed into the secretory cells of the adenohypophysis. The developmental origin of the adenohypophysis is identified for the first time in the early mammalian embryo before the formation of RP.     

Extracorporeal circulation in the isolated in situ lamb placenta: hemodynamic characteristics.

Decreased placental perfusion and respiratory gas exchange have been observed after experimental fetal cardiopulmonary bypass (CPB). To better characterize placental hemodynamics during CPB, seven isolated in situ lamb placentas were placed on a CPB circuit by use of umbilical arterial and venous cannulation. Measures were taken to simulate normal placental hemodynamics. Perfusion flow rates were varied from 15 to 300 ml.min-1.kg fetal wt-1 during normothermia and hypothermia. Placental vascular resistance (PVR) remained constant when perfusion pressure and flow were varied above 40 mmHg and 150 ml.min-1.kg-1, respectively. Below these values, PVR varied inversely. This increase in PVR was more marked when CPB was performed with hypothermia than with normothermia. The clinical implication is that decreased placental flow and pressure on CPB may lead to a vicious cycle, resulting in further impairment of placental perfusion and respiratory gas exchange. Hypothermia promotes this impairment.     

Effects of maternal blood loss on embryonic and placental development in the rat.

The effects of acute loss of maternal blood on embryonic and placental development was examined in 50 rats on Days 8 or 9 of gestation. Blood was withdrawn from conscious, cannulated rats over a 1-min period at 1-0 or 2-0 ml/100 g body weight. These degrees of blood loss were expected to produce a mild (about 50%) and severe (about 80%) reduction in uterine blood flow, respectively, for at least 15 min. There was no evidence that loss of blood affected either fetal survival and malformation rates or fetal weights and sex ratios. The anaemia resulting from haemorrhage lasted no longer than 6 days. Placental weights were 11% higher in rats losing 2-0 ml blood/100 g than in controls (P less than 0-05). It appears that the 8- or 9- day rat embryo is highly resistant to the partial reduction in uterine blood flow, maternal anaemia and other possible challenges induced by maternal loss of blood at levels sufficient to affect the mothers.     

Studies on implantation traces in rats. II. Staining of cleared uteri, formation and distribution of implantation traces

We made an investigation of implantation traces in delivered rats. 1. Non-fixed rat uteri were immersed in 2%-NaOH solution for over one hour. The uteri were then cleared and the implantation traces were seen to be stained yellowish-brown. This staining method was convenient for observation of the implantation traces. 2. Stillbirth was induced by stabbing or crushing, either in some embryos on one day between the 6th and 12th day of gestation, or in all embryos after the 13th day of gestation. Also, abortion was induced by stabbing or crushing all embryos before the 10th day of gestation. 3. When live embryos existed in the uterine horns, abortion traces were not detected. 4. In cleared uteri stained with 2%-NaOH solution, abortion traces were observed as small globes, reddish-brown. Normal delivery traces were observed as large globes, yellowish-brown, covered with yellowish-white of agglomerate cells, while stillbirth traces appeared as middle-sized, orange or yellowish-brown masses. 5. Though the implantation traces in rats which had been delivered four times were arranged like beads, they were recognizable by NaOH stain as either old or new traces, the former appearing smaller without agglomerate cells and a little more brown in comparison with the latter. 6. As the implantation traces increased in the uterine horns they became distributed from the central region to the cervical end, and the mean space between them narrowed. The implantation traces occupying the ovarian end or central region of the uterine horns trendes smallest size or largest size. 7. The implantation traces were composed of cicatrical tissue, and the area they occupied did not show adhesion of the placenta. On the other hand, placenta adhering to the uterine wall proliferated, and formed new implantation traces which did not overlap the old traces after decollement of the placenta.     

Serotonin-induced disruption of implantation in the rat: I. Serum progesterone, implantation site blood flow, and intrauterine pO2

Serotonin, administered on the day after the initiation of implantation, promptly terminates pregnancy in the rat. Consequently, the effects of serotonin on serum progesterone levels, implantation site blood flow, and intrauterine oxygen tension were determined to see whether the disruption of implantation is related to altered corpus luteum and/or uterine vascular function. Animals received a subcutaneous injection of physiological saline (C: control) or serotonin (S: 20 mg/kg) on Day 5 of pregnancy. Serotonin did not alter the number of blastocysts implanting (C: 6.02 +/- 0.52 vs. S: 6.29 +/- 0.46, sites/cornu) but did cause subsequent implantation site resorption (C: 0.08 +/- 0.07 vs. S: 5.46 +/- 0.44/cornu; P less than 0.001). Progesterone levels in serotonin-treated rats did not differ from those of controls at 6 h postinjection or on Days 6 through 10 of pregnancy. Implantation site blood flow was reduced at 30 min (C: 0.76 +/- 0.12 vs. S: 0.25 +/- 0.02 ml/min per g; P less than 0.01) and remained suppressed at 2 h after serotonin injection. A prompt and sustained reduction in intrauterine oxygen tension (C: 48.9 +/- 3.7 vs. S: 25.9 +/- 4.5 mmHg; P less than 0.005; 120 min) accompanied the reduced uterine perfusion. Thus, disruption of implantation is not a result of impaired corpus luteum function but is associated with marked and protracted reductions in uterine blood flow and intraluminal oxygen availability.     

Selenium levels in related biological samples: human placenta, maternal and umbilical cord blood, hair and nails.

A study on selenium levels has been carried out in human placenta, maternal and umbilical cord blood, hair and nails of a group of 50 mothers and in the hair of the newborns. The determinations were perfomed by electrothermal atomic absorption spectrometry. The selenium concentration obtained for each sample type was as follows: For the human placenta the values obtained were between 0.56 and 1.06 microg/g (mean +/- standard deviation: 0.81 +/- 0.02 microg/g). The levels for the umbilical cord blood were 51.1-104.2 microg/l (76.3 +/- 6.5 microg/l). For the maternal blood the values measured were between 57.3 and 117.9 microg/l (90.0 +/- 15.2 microg/l), and for hair and nails were 0.22-1.5 microg/g (0.60 +/- 0.37 microg/g) and 0.46-1.57 microg/g (0.90 +/- 0.27 microg/g), respectively. For the hair of the newborns the values obtained were between 0.40 and 2.53 microg/g (1.04 +/- 0.48 microg/g). The effect of different variables as age, habitat, nutritional index or gestation age of the mothers on the selenium concentration in the samples was studied. The influence of the habitat is significant with a confidence level of 95% for the selenium concentration in maternal blood and umbilical cord blood samples. The influence of the mothers' age is significant with a confidence level of 95% for the selenium concentration in the umbilical cord blood samples. For the placenta samples, the effect of the nutritional index is significant with a confidence level of 95%. There is a positive correlation between samples of umbilical cord blood and the newborns' hair, between placenta and umbilical cord, and between cord blood and maternal blood.