Visceral Yolk Sac Placenta Research Articles

Spatial Distribution of Putative Growth Factors in the Guinea Pig Placenta and the Effects of These Factors, Plasma, and Bile on the Growth and Chemotaxis ofCampylobacter jejuni

The pregnant guinea pig is an effective model for studying abortifacient Campylobacter spp, and previous experiments have demonstrated that C. jejuni IA3902 has a marked predilection for the subplacenta while sparing the placental disc in this species. In the study described here, the growth and chemotaxis of IA3902 and a reference strain (NCTC 11168) are compared in the presence of subplacental and placental factors, as well as bile and plasma, from pregnant and nonpregnant guinea pigs. Both strains grew better in subplacental versus placental disc tissue extracts at 24 hours; however, only IA3902 maintained this enhancement at 48 hours. Histochemistry and lectin histochemistry were used to localize mucin, iron, and l-fucose within the placental unit. Mucin was most abundant in subplacental lacunae, the junctional zone, and visceral yolk sac placenta, while iron was most abundant in the placental disc, and L-fucose-containing surface glycans were limited to the visceral yolk sac placenta. These 3 individual factors, along with progesterone and estradiol, were evaluated for effects on growth and chemotaxis of C. jejuni. Mucin, iron, and L-fucose were growth promoting, while l-fucose was also chemoattractive for both strains. Progesterone, estradiol, and pregnant guinea pig plasma did not affect growth or chemotaxis, and no difference was observed when bile from pregnant and nonpregnant animals was compared. These findings demonstrate the presence of specific factors within the guinea pig placental unit that drive chemotaxis and enhance growth of C. jejuni, shedding light on potential mechanisms underlying the fetoplacental tropism observed with this strain.

Species‐specificity of ethylene glycol‐induced developmental toxicity: toxicokinetic and whole embryo culture studies in the rabbit

High-dose gavage exposure to ethylene glycol (EG) is teratogenic in rats, but not rabbits. To investigate the reason for this species difference, toxicokinetic and whole embryo culture (WEC) studies were conducted in gestation day 9 New Zealand White rabbits, and the data compared to very similar data previously generated in pregnant rats. In the toxicokinetic study, maximal levels of unchanged EG in rabbits were comparable to those reported for rats. However, maximal levels of EG's teratogenic metabolite, glycolic acid (GA), in rabbit maternal blood and embryo were only 46% and 10% of the respective levels in rats. The toxicokinetic profile suggested that the lower GA levels in rabbits were due to a slower rate of maternal metabolism of EG to GA, slow uptake of GA into the yolk sac cavity fluid which surrounds the embryo, and negligible transfer via the visceral yolk sac (VYS) placenta. In the WEC study, exposure of rabbit conceptuses to high concentrations (< or = 12.5 mM) of GA was without effect, which contrasts with reported effects in rat WEC at > or = 3 mM. Overall, these data implicate toxicokinetics as an important factor underlying the species difference, although intrinsic insensitivity of the rabbit embryo might also be involved. Integration of these findings with published human data suggest that the rabbit is the more relevant model for human EG exposure, based on the negligible role of the rabbit VYS in placental transfer (humans lack a VYS) and similar rates of EG metabolism and extraembryonic fluid turnover.

Chorioallantoic and Yolk Sac Placentation in the Dassie Rat Petromus typicus and its Significance for the Evolution of Hystricognath Rodents

Placental characters are most important in understanding the evolutionary history of hystricognath rodents of which some act as animal models for human pregnancy. The data available deal mostly with species native to South America, but the current paper presents novel findings on chorioallantoic and yolk sac placentation in an Old World hystricognath and discusses its significance for the evolution of the group. Several hystricognath stem species characters are verified for Petromus, such as the unique trophoblast growth pattern within the chorioallantoic placenta. Subsequently, a novel set of characters belonging to the visceral yolk sac is added to the stem species pattern of the group. The nourishment of the embryo is facilitated by an inverted visceral yolk sac placenta from early pregnancy onward, later complemented by the chorioallantoic placenta. About mid term, the visceral yolk sac becomes partly folded and attached to the parietal yolk sac cover of the chorioallantoic placenta, suggesting a functional shift to the transfer of substances between the two placental types. Thus, the chorioallantoic and yolk sac placenta collaborate in nurturing the embryo. This apparently represents an evolutionary transformation along the stem lineage of hystricognaths.

Mechanisms regulating toxicant disposition to the embryo during early pregnancy: An interspecies comparison

The dose of toxicant reaching the embryo is a critical determinant of developmental toxicity, and is likely to be a key factor responsible for interspecies variability in response to many test agents. This review compares the mechanisms regulating disposition of toxicants from the maternal circulation to the embryo during organogenesis in humans and the two species used predominantly in regulatory developmental toxicity testing, rats and rabbits. These three species utilize fundamentally different strategies for maternal-embryonic exchange during early pregnancy. Early postimplantation rat embryos rely on the inverted visceral yolk sac placenta, which is in intimate contact with the uterine epithelium and is equipped with an extensive repertoire of transport mechanisms, such as pinocytosis, endocytosis, and specific transporter proteins. Also, the rat yolk sac completely surrounds the embryo, such that the fluid-filled exocoelom survives through most of the period of organogenesis, and can concentrate compounds such as certain weak acids due to pH differences between maternal blood and exocelomic fluid. The early postimplantation rabbit conceptus differs from the rat in that the yolk sac is not closely apposed to the uterus during early organogenesis and does not completely enclose the embryo until relatively later in development (approximately GD13). This suggests that the early rabbit yolk sac might be a relatively inefficient transporter, a conclusion supported by limited data with ethylene glycol and one of its predominant metabolites, glycolic acid, given to GD9 rabbits. In humans, maternal-embryo exchange is thought to occur via the chorioallantoic placenta, although it has recently been conjectured that a supplemental route of transfer could occur via absorption into the yolk sac. Knowledge of the mechanisms underlying species-specific embryonic disposition, factored together with other pharmacokinetic characteristics of the test compound and knowledge of critical periods of susceptibility, can be used on a case-by-case basis to make more accurate extrapolations of test animal data to the human.

Morphofunctional changes in the mitochondrial subpopulations of conceptus tissues during the placentation process.

To establish the role of mitochondrial subpopulations in the mitochondrial maturation process, we studied morphological and functional changes in the mitochondria of different mammalian conceptus tissues during the organogenic and the placentation processes. Mitochondrial subpopulations of three different conceptus tissues, embryo and visceral yolk sac placenta on gestational days 11, 12 and 13 and placenta on days 12 and 13, were examined morphologically by transmission electron microscopy. Cytochrome oxidase activity and protein levels were also measured in each mitochondrial subpopulation. The results indicate two different mitochondrial subpopulation profiles: a homogeneous one, which corresponds to immature mitochondria, and a heterogeneous one, which represents the mature mitochondria. The three tissues studied show different morphologic and metabolic patterns of mitochondrial maturation during the placentation process, rendering them suitable as experimental models to establish the possible relationship between mitochondrial maturation and the mitochondrial subpopulations.

92-kDa gelatinase (matrix metalloproteinase-9) is induced in rat amnion immediately prior to parturition.

The fetal membranes undergo striking changes in structure before delivery that involve catabolism of the extracellular matrix. To investigate the role of specific enzymes in this process, we examined gelatinase activities in rat amnion, visceral yolk sac placenta, and placenta and amniotic fluid between Days 18-21 of pregnancy. Matrix metalloproteinase (MMP)-2 was present in amnion on all days, and its activity increased slightly on Day 21. The 92-kDa gelatinase, MMP-9, was not detected on Days 18-20 but appeared by the morning of Day 21. There was a marked increase in MMP-9 mRNA in the amnion on Day 20, preceding the appearance of MMP-9 activity. Western blotting confirmed an increase in MMP-9 protein in amnion on Day 21. MMP-2 and MMP-9 activities were detected in extracts of whole yolk sac placenta, placenta, and amniotic fluid, but there were no striking changes in these gelatinases between Days 18 and 21. However, the capsular regions of the visceral yolk sac placentae, which thin and rupture during labor, did show higher MMP-9 activity on Day 21 than on Days 18 and 20. We suggest that the striking increase in MMP-9 expression in amnion and possibly the capsular region of the visceral yolk sac placenta approximately 12 h prior to delivery is responsible, in part, for the alterations in the structure of these fetal membranes before parturition.

Differences in binding characteristics of rat and human transferrin by rat visceral yolk sac placenta

Previous work has shown that human serum supplemented with rat transferrin can support the normal growth of cultured rat conceptuses, but that supplementation with human transferrin has no such effect. Such results strongly suggest an hypothesis of species-specificity. This potential specificity was therefore investigated by comparing uptake, binding, competition and vectorial transport of both rat and human transferrin using two well-established systems, those of 17.5 day rat visceral yolk sacs and anembryonic yolk sacs in culture. The results of these investigations show that human transferrin displays a lower rate of uptake and lower binding affinity for the rat transferrin receptor than does rat transferrin. Human transferrin competes poorly with rat transferrin for receptor occupancy unless present in 20-fold excess. Both molecules are taken up by receptor-mediated endocytosis and are processed in a similar manner. Anembryonic yolk sac experiments show that a greater proportion of intact rat transferrin is transported to the exocoelomic fluid than is intact human transferrin. Binding analyses show a difference in binding affinities of the two molecules for the rat transferrin receptor and also that human transferrin exhibits negative cooperativity in its binding. This evidence strongly supports an hypothesis of species-specificity in the binding of transferrin to the transferrin receptor in the rat visceral yolk sac.

Effects of sustained dietary ethanol on the ultrastructure of the visceral yolk‐sac placenta of the rat

The present investigation reports ultrastructural alterations in endodermal epithelial cells of the rat visceral yolk-sac placenta that accompany alcohol-induced changes in intracellular trafficking of endocytosed maternal serum proteins. Fine structural changes include restructuring of mitochondrial cristae (foliate to vesicular and tubular forms), beading of cisterns of granular endoplasmic reticulum, hypertrophy and hyperplasia of Golgi elements, differentiation of the Golgi membranes to a GERL configuration, and a notable increase in numbers of primary lysosomes. Such changes in ultrastructure suggest that exposure of this developing maternofetal exchange system to high levels of ethanol in maternal blood increases the production of primary lysosomes near term for proteolysis of maternal serum proteins within the visceral yolk-sac epithelium. Near term, targeting of endocytosed protein to secondary lysosomes for proteolysis appears to be augmented; transcellular routing of maternal protein (e.g., IgG) for placental transport may be impeded. Thus significance of the observed changes in the fine structure of this placenta may relate to the mechanism(s) of action of maternal alcohol consumption on the acquisition of neonatal immunity, intrauterine growth retardation, and the production of congenital malformations.

Teratogenic antibody internalization by rat visceral yolk-sac endoderm in vitro: an ultrastructural colloidal gold tracer study.

Previous work from our laboratory has demonstrated that specific rabbit immunoglobulins G (IgG) against a glycoprotein antigen of rat kidney proximal tubule or a cross-reacting visceral yolk-sac endodermal cell antigen will induce abnormal embryonic development when they are injected into pregnant rats during the period of organogenesis. It has been proposed that these antibodies may induce embryopathy by interfering with functions of the visceral yolk-sac placenta, an important organ providing nutrients to the embryo at this stage of development. In order to gain some insight into the underlying pathogenic mechanism(s) in which specific teratogenic IgG may interfere with visceral yolk-sac functions, we examined the uptake of these teratogenic IgG by the visceral yolk-sac endodermal cells at the electron microscopic level. The results demonstrated that teratogenic rabbit IgG specifically localized on the fuzzy coat of the external apical cell membrane of the visceral yolk-sac endoderm at the intermicrovillous region. Within 5 min, the IgG were rapidly internalized via coated pits and micropinocytic vesicles. Within 30 min, an increasing proportion of gold particles appeared within uncoated vesicles or vacuoles of various sizes; most of the gold particles were in close proximity to the inner membranous lining of the vesicles. Similar findings were observed after 1- or 2-hr incubation. After 24- to 48-hr culture, however, the gold particles appeared to have dissociated from the inner surface of the vesicle membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of d- and dl-β-hydroxybutyrate by mouse embryos in vitro

Rates of 14CO 2 production by mouse conceptuses in vitro from d- and dl-[3- 14C]-β-hydroxybutyrate (βOHB) were determined during the period of organogenesis (days 9 through 12 of gestation) in the presence of 4 to 32 mmol/L dl-βOHB. During this time period the rates 14CO 2 production from d-βOHB metabolism are concentration-dependent, increase on each day of gestation, and the site of metabolism appears to shift from the visceral yolk sac placenta to the embryo proper. In contrast to fetal and neonatal tissues, the rates of 14CO 2 production from dl-βOHB oxidation is significantly greater than from d-βOHB suggesting that the utilization of the l-isomer may be equal to or greater than that of the d-form.

Effect of amphotericin B and gestational age on sodium transport across the rat visceral yolk sac placenta in vitro.

The transport properties of the rat visceral yolk sac placenta from Days 14.5 to 18.5 of gestation were studied in vitro. All tissues had a positive potential difference, fetal side relative to maternal side, and showed net Na transport towards the fetus. Basal short-circuit current and net Na flux increased rapidly with gestational age over the period studied. Amphotericin B applied to the maternal surface of the yolk sac stimulated current and net Na flux, indicating that the apical membrane Na permeability limited transport and revealing a reserve capacity for transport. Contrary to their basal values, current and Na flux following treatment with amphotericin were independent of gestational age.

Antiserum to rat visceral yolk sac endoderm induced abnormal embryonic development.

The induction of abnormal embryonic development by heterologous tissue antisera has been well established. The underlying mechanism whereby such teratogenesis occurs is not known. There were implications that visceral yolk sac endoderm might be involved. Endoderm was isolated from rat visceral yolk sac of 14th day of gestation using a nonenzymic procedure. The purity of the endoderm preparation was examined by electron microscopy. The preparation contained sheets of single layer of endodermal cells with no apparent contamination by the underlying mesenchyme or basal lamina. The specificity of the antiserum was examined by in vitro immunofluorescent localization studies. The antibodies against the endoderm localized only in the endodermal cells and some of the renal tubular cells. Intraperitoneal injection of the endoderm antiserum into 9-day pregnant rats resulted in congenital malformation, embryonic death, and fetal growth retardation. The effects of the antiserum were dose-dependent. The most frequently observed defects were anophthalmia and microphthalmia. Retarding effect of the antiserum on the growth of the embryo at the egg cylinder stage was also observed. In vivo immunofluorescent localization studies indicated that the endoderm antibodies localized only in the endodermal cells of the visceral yolk sac placenta; no localization was observed in the visceral yolk sac mesenchyme, basal lamina. Reichert's membrane, maternal kidney tissue or the embryo proper.

Visualization of Antibody Transport in Rat Visceral Yolk-Sac Placenta

A technique has been developed for visualizing antibody against horseradish peroxidase (HRP) in rat visceral yolk sac, the placental membrane across which passive immunity previously has been shown to be transferred from mother to young just prior to birth. Female rats were immunized by injecting both hind foot pads with 1 mg HRP emulsified in complete Freund's adjuvant. They were given a booster of 0.5mg HRP in 0.1 ml normal saline i.v. after one week, then bred and autopsied at selected stages of pregnancy, viz., 12, 1 7 and 22 days post coitum, receiving a second booster, injected as above, five days before autopsy. Yolk sacs were removed surgically and fixed immediately in 2% paraformaldehye, 1% glutaraldehye in 0.1 M phosphate buffer with 0.01% CaCl2 at pH 7.4, room temperature, for 3 hr, rinsed 3X in 0.1 M phosphate buffer plus 5% sucrose, then exposed to 1 mg HRP in 1 ml 0.1 M phosphate buffer at pH 7.4 for 1 hr. They were refixed in aldehydes, as above, for 1 5 min (to assure binding of antigen-antibody complex). Following buffer washes, the tissues were incubated in 3 mg diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M Tris-HCl buffer for 30 min. After brief buffer washes, they were postfixed in 2% OsO4. in phosphate buffer at pH 7.4, 4°C for 2 hr, dehydrated through a graded series of ethanols, and embedded in Durcupan. Thin sections were observed and photographed without contrast-enhancement with heavy metals. Cytochemical reaction product marked the site of HRP (i.e., antigen) which, in turn, was present only where it was bound with anti-HRP antibody.

Whole-body autoradiography of 204Tl in embryos, fetuses and placentas of mice

Whole-body autoradiography was used to study thallium uptake and retention in mice during gestation. Fifteen minutes after an intraperitoneal injection of 50 μCi 204Tl 2So 4 into a 15-day pregnant mouse, thallium could be seen within the fetuses. Maximum fetal accumulation occurred 2–4 h after injection, and minimum at the last observation 4 days after injection. The fetal concentration of 204tl was constantly lower than the placental. Uptake of 204Tl in embryos, fetuses and placentas of mice with gestation ages varying from 5 days to 16 days indicated that thallium crosses the placental barriers throughout gestation. Thallium was retained by the visceral yolk sac placenta during early gestation, by the visceral yolk sac, chorioallantoic placenta and amnion during late gestation. Over a period of time placental transfer and embryonic/fetal retention of small amounts of thallium may be hazardous to the offspring due to the high cumulative toxicity of this metal.

Freeze-fracture observations on the visceral yolk sac placenta of rats, mice and hamsters. With special reference to endodermal cell tight junctions.

Freeze-fracture replicas of visceral yolk sac from rats, mice and hamsters in late stages of gestation were studied by electron microscopy. Special attention was directed toward determining the types of junctional specializations that exist between the columnar endoderm cells of this placental membrane. In all three species, well-developed, zonular tight (occluding) junctions were found on the contiguous lateral surfaces of the endoderm cells. The tight junctional network, located in an immediate subluminal position, was from 0.2--0.5 micrometers in depth and consisted at any point of 2--5, interconnecting, approximately 9 eta wide, strands (P-face) or shallow furrows (E-face). Patch-like aggregations of irregular intramembrane particles, characteristic of desmosomes (maculae adherentes), also were observed at scattered sites below the tight junctions. However, no evidence of gap (communicating) junctions was encountered. The endoderm cells of the rodent visceral yolk sac have been shown to play a central role in the selective transport of macromolecular substances from the maternal to the fetal system. Tight junctions may be vital to this endodermal cell function by preventing random paracellular fluxes of macromolecules.

Whole-body autoradiography of 63NI in mice throughout gestation

Whole-body autoradiography was used to study nickel uptake and retention in mice throughout gestation. After an intraperitoneal injection of 50 μCi 63NiCl 2 into a 16-day-pregnant mouse, nickel appeared rapidly in connective tissues. Prominent sites of radioactivity 72 h after injection included the visceral yolk sac, lung, gastrointestinal tract and kidney. Nickel crossed the placental barriers throughout gestation, i.e. the visceral yolk sac during early gestation, the visceral yolk sac and chorioallantoic placenta during late gestation. A marked uptak of nickel was seen already in the 5- and 6-day embryo. Fetal accumulation of nickel took place up to 16 days gestation. Whereas nickel was distributed throughout the early embryo, distribution became more differentiated with increasing gestation, imitating that in the mother. Penetration of nickel into the mouse conceptus makes possible a similar ability in the human conceptus.]

90Sr placentas, embryos and foetuses of mice, evaluated by whole-body autoradiography.

90Sr is a hazardous fission product from nuclear reactions. Its transplacental passage is of particular interest because of the high sensitivity of embryos/foetuses to ionizing radiation. We have used whole-body autoradiography to study the foetal uptake and retention of 90Sr and the embryonic/foetal transfer of this isotope as a function of gestational age. Fifteen minutes after an intraperitoneal injection of 50 muci 90Sr-nitrate into a 14-day-pregnant mouse, radioactivity was present in the placenta, but not in the foetus. During a 72-hour observation period, 90Sr gradually accumulated in the hard foetal tissues. In the soft foetal tissues, accumulation occurred up to 12--24 hours. After 72 hours, no 90Sr could be detected in the soft tissues. 90Sr crossed the visceral yolk sac placenta during early gestation, the visceral yolk sac and the chorioallantoic placentas during late gestation. Transfer increased considerably at the end of the latter period. There was a marked uptake of 90Sr in all structures of early embryos, whereas the isotope concentrated in the preskeletal and skeletal parts of the foetuses. Intake of 90Sr during early gestation may therefore be more injurious to the offspring than intake during late gestation.

The effect of heterologous antisera on embryonic development. XIII. Lack of effect of antisera to alpha fetoprotein.

This investigation was carried out to determine whether heterologous antisera to alpha fetoprotein (AFP) are embryotoxic to developing rat embryos. Homogeneous rat AFP was isolated and antisera directed against this glycoprotein were produced in rabbits, horse and goat. The effect of the antisera on embryonic development was examined by injecting the antisera intraperitoneally into pregnant rats on the ninth, eleventh and thirteenth days of gestation. The results demonstrated that there was no evidence of increased incidence of fetal abnormalities in 472 surviving fetuses of 42 injected rats. There was no evidence of increase embryonic death or retardation of intrauterine growth following administration of the antisera on the ninth, eleventh and thirteenth days of gestation. The localization of the injected antisera was examined by the indirect immunofluorescent method. The results showed that the heterologous AFP antibodies localized specifically in the visceral yolk sac placenta. No antibody localization was observed in the embryo proper or the chorioallantoic placenta. It is speculated that the localization of AFP antibodies in the visceral yolk sac does not interfere with the embryotrophic function of the visceral yolk sac placenta.

Localization of rabbit gamma globulins in the mouse visceral yolk sac placenta.

The yolk sac placenta has been implicated previously in transmission of passive immunity to the fetus. This work uses an immunohistochemical technique devised by Sternberger et al. ('69) to study this problem. Rabbit serum containing gamma globulins was injected into the uterine lumen of the White Swiss mouse during the last third of pregnancy. Two or four hours later yolk sacs were removed and fixed in 2% paraformaldehyde or freeze-dried and fixed with paraformaldehyde vapors. Finely chopped tissue was treated with (1) sheep antiserum to rabbit serum gamma globulin, (2) an antigen-antibody complex consisting of horseradish peroxidase and rabbit anti-horseradish peroxidase and (3) hydrogen peroxide and 3,3'-diaminobenzidine. Reaction product was heavily concentrated in visceral yolk sac endodermal cells, frequently deposited in endothelial cells of vitelline vessels, and rarely in the serosal basement membrane and mesothelial cells which border the exocoelomic cavity. This supports evidence of other workers that the yolk sac membrane rapidly absorbs substances with which it comes in contact, transport into vitelline vasculature is a route of transfer from mother to fetus, and possible transfer into the exocoelomic cavity and thence to the amniotic cavity may occur in mice.

Ruthenium labeling of micropinocytotic activity in the rat visceral yolk-sac placenta

Visceral yolk-sac placentas of rats were autopsied from 12-, 17-, and 22-day pregnancies. The membrane is known to transport proteins from maternal to fetal systems only during the last of these 3 stages. Protein absorption previously has been shown to be initiated by micropinocytosis at the apices of the epithelial cells which cover the membrane. The pits and vesicles which are formed by this process are fuzzy-coated, or alveolate. In the present study, the yolk-sac membrane from each selected stage was treated with ruthenium red which binds at the site of micropinocytotic vesicle formation and which catalyzes osmium reduction during subsequent processing of the tissue. By this method, the micropinocytotic process was observed in detail. Morphologically, the alveolate vesicles come into close relationship with a subapical system of microvesicles and canaliculi, a system which, in turn, communicates with large storage vacuoles. By the ruthenium method, relative micropinocytotic activities at different gestational ages also were determined. The 12-day membrane appeared micropinocytotically very active. At the 17-day stage, on the other hand, the membrane was relatively quiescent. At 22 days, the placental membrane exhibited a degree of activity about like that at 12 days. Consequently, at the gestational ages which were examined, no correlation was observed between the rate of micropinocytotic uptake of protein and known protein transport rates.