Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.     

Effects of lysosomal proteinase inhibition on the development of the rat embryo in vitro

Altered lysosomal function in the visceral yolk sac can result in abnormal development. As proteolysis is an important function of the rodent visceral yolk sac during early and mid-gestation, we characterized the lysosomal proteolytic enzyme activity of this extraembryonic membrane and determined the effects of inhibitors of protein degradation on embryonic development. Constituent activities of cysteine and aspartic acid proteinases were measured in rat visceral yolk sac on gestation day 12, and the effects of the cysteine proteinase inhibitors leupeptin, E-64 [trans-epoxysuccinyl-l-leucylamido(4-guanido)butane] and N-ethylmaleimide and the aspartic acid proteinase inhibitor pepstatin were determined in Sprague-Dawley rat embryos cultured in vitro from gestation days 10-12. It was determined that only cysteine proteinases, primarily cathepsins B and L, are active in the mid-gestation visceral yolk sac. The cysteine proteinase inhibitors leupeptin and E-64 both produced a concentration-related decrease in embryonic growth, as measured by crown-rump length, somite number, and embryonic protein content, and a concentration-related increase in incidence of abnormalities. A characteristic pattern of abnormalities was produced which involved a decrease in neural tube volume and the formation of a subectodermal blister opposite the point of attachment of the vitelline vessels. At high concentrations, anophthalmia was also observed. The decreased neural tube volume was associated with increased osmolality of the exocoelomic fluid, the major extraembryonic fluid compartment. It is possible that the osmotic change decreased neural tube volume by causing water to move to the compartment with a higher solute concentration, out of the embryo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol-induced inhibition of pinocytosis and proteolysis in rat yolk sac in vitro

Pinocytic capture of 125I-labelled polyvinylpyrrolidone and of formaldehyde-denatured 125I-labelled bovine serum albumin by 17.5-day rat visceral yolk sacs incubated in vitro was rapidly and strongly inhibited by low concentrations (0.01 and 0.05%, v/v) of ethanol. The induced inhibition of pinocytosis was readily reversible, but a marked lag was observed before ethanol-exposed tissue regained its full proteolytic capacity towards the exogenous protein. These observations suggest that the acute administration of ethanol to a pregnant rat may give rise to concentrations of ethanol in the maternal blood and/or uterine fluid that induce dysfunction of the yolk sac. In late gestation such inhibition of yolk-sac function may interfere with the transfer of passive immunity across the yolk sac. If similar dysfunction is induced earlier in gestation, in the period before the chorioallantoic placenta is functional, this could cause a transient period of inhibition of histiotrophic nutrition that may be important to the pathogenic mechanism of action of ethanol as a teratogen.     

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.     

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.     

Induced cell injury and cell death as a cause of congenital malformations in rats

Summary Enbryopathy has been produced by inhibition of histiotrophic nutrition in the rat using a number of agents which prevent this process. The experiments were carried outin vitro at various stages of development before the inception of a chorio allantoic placenta. Dose-dependent effects on the embryo were demonstrated using the acid bisazo dye Trypan Blue, which inhibits endocytosis, and an enzyme inhibitor of bacterial origin known as leupeptin which inhibits cathepsin B, H and L. Homogenates of rat kidney and placenta also produced congenital defects; the concommitant electron microscopical changes in the yolk sac suggest that these effects too are due to alterations in the availability or quality of histiotroph.     

The effects of high-sucrose diets and of maternal diabetes on the ultrastructure of the visceral yolk sac endoderm in rat embryos developing in vivo and in vitro

The ultrastructure of the visceral yolk sac endoderm of in vivo developing 9- to 13-day-old embryos from 2 diabetic rat models (streptozotocin diabetes and Cohen--genetically determined--diabetes) and from nondiabetic rats fed high sucrose diets have been studied. This was compared to yolk sacs from 9.5-day-old embryos cultured for 48 h in sera from diabetic and nondiabetic rats fed a high-sucrose diet. Light-microscopic, TEM and SEM studies showed that the pathological cellular changes in the visceral yolk sac endoderm from diabetic rats were first observed on day 9 and were most severe among 11-day-old embryos. In vitro culture of control rat embryos in serum from experimental animals induced a reduction in the number of microvilli, of vacuolar intracellular inclusions and an increase in the number of degenerated endodermal cells. SEM studies showed that in addition to disappearance of microvilli, the majority of cells were collapsed and had degenerated cell membranes. Culture of embryos from diabetic animals in control serum only slightly reversed the pathological changes in the visceral yolk sac endoderm. A good correlation exists between the rate of embryonic malformations in diabetic rats and an index of endodermal-cell damage in the visceral yolk sac.     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and morphology of the inverted yolk sac in the guinea pig (Cavia porcellus)

Although the guinea pig is an important animal model for human placentation, aspects of fetal nutrition are not fully understood, especially in regard to the yolk sac that is regarded to be essential for early development of the embryo. We investigated differentiation by means of histology, histochemistry, immunohistochemistry, and transmission electron microscopy. Data suggest that the guinea pig's yolk sac was not sufficiently developed to facilitate substantial fetal nutrition in early pregnancy. On Day 12, it was a flat, inverted, but avascular structure. This was followed by differentiation to form the typical, highly villous and vascularized condition of advanced gestation. Finally, the yolk sac degenerated toward term. We suggest that the guinea pig and other caviomorphs rely predominantly on hemotrophic nutrition via the placenta even in very early pregnancy. In contrast to the general pattern of mammals, histiotrophic nutrition via yolk sac routes seems to be most essential during mid-gestation.     

Role of the mouse visceral yolk sac in nutrition: inhibition by a somatomedin inhibitor

A low molecular weight somatomedin inhibitory serum fraction (SI), obtained from streptozotocin-induced diabetic rats, causes morphological abnormalities and growth reduction in mouse embryos grown in whole embryo culture (WEC). These abnormalities are thought to be caused, at least in part, by a failure of the visceral yolk sac (VYS) to properly degrade proteins, a process that normally provides the conceptus with amino acids and peptides for de novo protein synthesis (histiotrophic nutrition). To test this hypothesis, embryos exposed to the SI were provided with a mixture of ten essential amino acids (supplemented group) in an attempt to circumvent SI-induced VYS dysfunction. Results showed that 82.4% (14/17) of embryos in the amino acid-supplemented group exhibited improved growth and development compared to those embryos exposed to medium containing the SI alone (unsupplemented group). Supplemented embryos showed greater expansion of the brain regions, improved visceral arch development, and increased protein content compared to nonsupplemented SI-treated embryos. However, these parameters were still reduced compared to controls. VYSs from both the unsupplemented and amino acid-supplemented groups were identical with respect to alterations in morphology and increased protein content compared to VYSs from conceptuses cultured in control medium (with or without amino acid supplementation). The improvement in embryonic growth and development due to amino acid supplementation in spite of VYS abnormalities supports the hypothesis that nutritional deprivation is one aspect of SI-induced teratogenesis.     

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 (≤12.5 mM) of GA was without effect, which contrasts with reported effects in rat WEC at ≥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. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.     

Altered visceral yolk sac function produced by a low-molecular-weight somatomedin inhibitor

A fraction from diabetic rat serum containing a low-molecular-weight (800-1000) somatomedin inhibitor (SI) alters growth and development in both neurulation and early limb bud staged mouse embryos in vitro. Previous studies suggested that an accumulation of serum proteins and morphological changes of the visceral yolk sac (VYS) were produced following exposure to the SI in early limb bud staged conceptuses. The morphological changes, characterized by the presence of large endosomes in the endodermal cells, suggested that the SI altered histiotrophic nutrition, whereby proteins are pinocytosed by the endodermal VYS cells and degraded to constituent amino acids. Therefore, the effects of the SI on pinocytosis and protein degradation by the VYS were evaluated using the whole embryo culture system. Results showed that the SI reduced fluid phase pinocytosis as determined by the uptake of [U-14C]sucrose, but that accumulation of [3H]leucine-labeled hemoglobin ([3H]Hb) by the VYS was greater following exposure to the SI than in controls. In contrast, the accumulation of 3H-labeled amino acids in the embryo (produced from the degradation of [3H]Hb by the VYS) was reduced by the SI. The extent of amino acid reduction in embryonic accumulation is dependent upon the concentration of SI in the culture medium and correlates with the incidence of malformations produced by the SI, i.e., high rates of malformations occur with large reductions in embryonic 3H-labeled amino acid accumulation. The apparent paradox of high [3H]Hb accumulation in the presence of decreased pinocytosis appears to be the result of altered processing of the [3H]Hb in the endodermal cells. The altered processing decreases the "elimination" of the proteins from the VYS and results in the decrease in 3H-labeled amino acid present in the embryo proper. Therefore, the SI appears to alter two processes of VYS histiotrophic function. (1) decreased pinocytosis and (2) altered protein processing, ultimately resulting in a decreased availability of substrates for the embryo. During the early stages of embryogenesis in the human, the trophoblast cells of the placenta are responsible for the transport of nutrients from the maternal to embryonic systems. Since these cells show high phagocytic and pinocytotic activities, the SI may also disrupt these processes in the chorioallantoic placenta and contribute to diabetes-induced embryopathies.     

Maternal transferrin uptake by and transfer across the visceral yolk sac of the early postimplantation rat conceptus in vitro

Uptake and transfer of maternal transferrin by rat embryos during organogenesis in vitro was investigated using radiolabelled rat transferrin and rocket immunoelectrophoresis. Colloidal gold to which rat transferrin was adsorbed was used as an electron microscopical marker in order to follow the route taken by internalised transferrin across the visceral yolk sac. Culture of rat conceptuses from 9.5 to 11.5 days of gestation in rat or human sera resulted in the passage of rat or human transferrin from the culture medium into the extraembryonic coelom as determined by quantitative immunoelectrophoretic analysis of exo-coelomic fluid. The concentration of human transferrin which was transferred to the exo-coelomic fluid of conceptuses cultured in whole human serum at 10.5 days and 11.5 days of gestation was similar to the concentration of rat transferrin in the fluid of conceptuses cultured in rat serum which had been diluted with Hanks' saline to 50% in order to match the levels of transferrin found in human serum. Growth of rat embryos in 50% rat serum was identical to embryonic growth in 100% rat serum. Uptake of radiolabelled rat transferrin by the visceral yolk sac at 11.5 days of gestation, following culture for 60 min in radiolabelled medium, was much greater than nonspecific uptake of radiolabelled bovine serum albumin. Accumulation of radiolabelled transferrin by the embryo was reduced by the inclusion of unlabelled transferrin into the culture medium. Uptake of transferrin adsorbed 18 nm gold particles was mediated by attachment to coated pits on the apical cell surface of the extraembryonic endoderm. Transferrin-adsorbed gold colloid was internalised via coated vesicles and found in cisternal structures of the peripheral and juxtanuclear areas, as well as in smooth and coated vesicles deep within the cell. The intercellular presence of gold particles in the endodermal layer of the visceral yolk sac and their presence in the mesoderm after 60 min of incubation suggested that passage of transferrin was rapid and mediated by vesicular evagination from the extraembryonic endoderm. These findings suggest that maternal transferrin is the primary source of transferrin for the early rat embryo and its passage to the exo-coelom and embryo is mediated by specific receptors on the apical surface of the extraembryonic endoderm.     

WHOLE-EMBRYO CULTURE AND THE STUDY OF MAMMALIAN EMBRYOS DURING ORGANOGENESIS

1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive-streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation. 2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head-fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta. 3. The embryos of some other species, including the guinea-pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice. 4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue-culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat-inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac. 5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose-shunt activity. During the following days these decline while activity of the Krebs' cycle and electron-transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate. 6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2-day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac. 7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb-bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds. 8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially embryopathic agents.     

Yolk sac failure in embryopathy due to hyperglycemia: ultrastructural analysis of yolk sac differentiation associated with embryopathy in rat conceptuses under hyperglycemic conditions

Diabetes mellitus in pregnancy is associated with an increased incidence of various congenital anomalies that occur during organogenesis. Because a well functioning yolk sac is crucial to embryonic growth and development during this period, we performed an ultrastructural study of the effects of excess glucose (total glucose 750 mg/dl, osmolality 305 mOsm/kg) on pregnancy day 10 (Witschi stage 13) rat conceptuses cultured for 48 hr in heat-inactivated male rat serum with and without added d- or l-glucose. Embryos exposed to excess d-glucose demonstrated decreased conceptus size (P less than 0.001), and gross malformations in a dose-related fashion. The visceral yolk sac capillaries and vitelline vessels of conceptuses in excess d-glucose were sparse, patchy, and nonuniformly located. Ultrastructurally, the visceral yolk sac endodermal cells had reduced numbers of rough endoplasmic reticulum, ribosomes, and mitochondria. These obvious defects in yolk sac structure suggest that hyperglycemia during organogenesis has a primary deleterious effect on yolk sac function with resultant embryopathy.     

Development, morphology, and function of the yolk-sac placenta of laboratory rodents

A review of current knowledge of the unusual structure and several functions of the yolk-sac membranes of common laboratory rodents, viz., rats, mice, hamsters, guinea pigs and gerbils, enables a better assessment of the significance of this maternofetal exchange system in the experimental production of congenital anomalies. The anatomy of both visceral and parietal walls of the rodent yolk-sac placenta--specifically the anatomical relationships of each wall with maternal and with other fetal tissues--depends on the mode of origin and subsequent development of the yolk sac in these several species. Accordingly, the developmental biology of the rodent yolk sac is described. Since both fine structure and anatomical relationships also determine in large measure the functioning of the membrane as a whole in the absorption of selected materials either for intracellular digestion or for cellular translocation and transport to the developing embryo, the anatomy of the yolk sac is considered in detail. Similarly, since available evidence strongly suggests that teratogenic agents induce perturbations in the cellular mechanisms that control these several functions of the yolk-sac placental system in the production of birth defects, additionally an account is given of the cell biology of the membrane, i.e., endocytosis and targeting/trafficking of materials either for digestion within the epithelium at the maternal surface of the visceral yolk sac or for translocation across the yolk-sac membrane as a whole.     

Ultrastructure and function of the rat yolk sac: damage caused by teratogenic anti-VYS serum and recovery

It was hypothesized that heterologous anti-rat visceral yolk sac serum (AVYS) exerts its teratogenic effect by reducing the endocytosis of serum proteins by the visceral yolk sac (VYS), thus reducing the supply of amino acids to the embryo and VYS. To evaluate this hypothesis, we studied the effect of teratogenic AVYS on the endocytic function of the VYS and the ultrastructure of the VYS and parietal yolk sac (PYS). Rat conceptuses were exposed to a teratogenic dose of AVYS on the 10th day of gestation in vivo or in vitro. Control and AVYS-exposed specimens were collected 24-192 hr later and prepared for scanning and transmission electron microscopy (SEM and TEM, respectively) utilizing standard procedures. The Endocytic Index was calculated for the VYS utilizing standard procedures. Approximately 97% of the in vivo exposed and 94% of the in vitro exposed embryos were morphologically abnormal. Ultrastructural observations showed that exposure to AVYS in vivo or in vitro caused severe damage to the VYS endodermal epithelial cells with loss of cellular borders, reduction in the number and length of microvilli, and increased cellular inclusions; and some damage to PYS endodermal cells with increased blebbling and decreased cell number. Recovery was evident at 72 hr and complete by 96 hr. The Endocytic Index was significantly reduced in the VYS 24 and 48 hr after injecting AVYS into the pregnant rat but was not significantly different at 96 and 192 hr. Our results show that the AVYS antiserum damaged visceral endodermal epithelium experienced ultrastructural recovery with parallel functional recovery. These studies suggest that transient yolk sac placental ultrastructural damage and dysfunction was probably sufficient to cause irreversible damage to the developing embryo during early organogenesis. We conclude that the proximate effect of the AVYS was on the plasma membrane of the visceral endoderm and that decreased pinocytosis is a consequence of this effect.     

Nitroxide radicals protect cultured rat embryos and yolk sacs from diabetic-induced damage

BACKGROUND: Diabetic teratogenicity relates, partly, to embryonic oxidative stress and the extent of the embryonic damage can apparently be reduced by antioxidants. We investigated the effects of superoxide dismutase-mimics nitroxides, 2,2,6,6-tetramethyl piperidine-N-oxyl (TPL) as an effective antioxidant, on diabetes-induced embryopathy. METHODS: Embryos (10.5 day old) and their yolk sacs from Sabra female rats were cultured for 28 h in the absence or in the presence of nitroxides at 0.05-0.4 mM in control, diabetic subteratogenic, or diabetic teratogenic media, and monitored for growth retardation and congenital anomalies. The oxidant/antioxidant status was examined by oxygen radical absorbance capacity and lipid peroxidation assays, whereas the yolk sac function was evaluated by endocytosis assay. RESULTS: Diabetic culture medium inhibited embryonic and yolk sac growth, induced a high rate of NTDs, reduced yolk sac endocytosis and embryonic antioxidant capacity, and increased lipid peroxidation. These effects were more prominent in the embryos with NTD compared to those without NTD. TPL added to diabetic teratogenic medium improved embryonic and yolk sac growth, reduced the rate of NTDs, and improved yolk sac function. The oxidant/antioxidant status of embryos was also improved. TPL at 1 mM did not damage the embryos cultured in control medium. CONCLUSIONS: In diabetic culture medium, oxidative damage is higher in the malformed rat embryos compared to those without anomalies; the nitroxide provides protection against diabetes-induced teratogenicity in a dose-dependent manner. The yolk sac damage, apparently caused by the same mechanism, might be an additional contributor to the embryonic damage observed in diabetes.