Carbamazepine toxic effects in chick cardiomyocyte micromass culture and embryonic stem cell derived cardiomyocyte systems--possible protective role of antioxidants.

The use of carbamazepine (CBZ) during pregnancy increases cardiovascular anomalies. In this study CBZ developmental cardiotoxic effects were evaluated using chick cardiomyocyte micromass (MM) culture and mouse embryonic stem cells derived cardiomyocyte (ESDC) systems. In MM culture, CBZ only inhibited the cardiomyocyte contractile activity, while in ESDC it completely ceased the contractile activity at 200 µM with decreased cell viability and protein content. The antioxidant superoxide dismutase (SOD) supplement in MM and ascorbic acid (AA) in ESDC showed protective effects on CBZ toxicity, but elevated levels of reactive oxygen species (ROS) production were recorded with CBZ treatment only in ESDC. CBZ has also affected cardiac connexin 43 expression in both in vitro systems. Our results indicated CBZ induced ROS stress as mechanism of developmental cardiotoxicity at early stage of cardiogenesis in ESDC system compared to MM system's differentiated cells. These toxic effects can be negated by using antioxidant agent.

Lithium carbonate teratogenic effects in chick cardiomyocyte micromass system and mouse embryonic stem cell derived cardiomyocyte--possible protective role of myo-inositol.

The drug lithium carbonate (Li2CO3) use during pregnancy increases the possibility of cardiovascular anomalies. The earlier studies confirm its phosphatidylinositol cycle (PI) inhibition and Wnt pathways mimicking properties, which might contribute to its teratogenic effects. In this study the toxic effects of Li2CO3 in chick embryonic cardiomyocyte micromass system (MM) and embryonic stem cell derived cardiomyocyte (ESDC) were evaluated, with possible protective role of myo-inositol. In MM system the Li2CO3 did not alter the toxicity estimation endpoints, whereas in ESDC system the cardiomyocytes contractile activity stopped at 1500 µM and above with significant increase in total cellular protein contents. In ESDC system when myo-inositol was added along with Li2CO3 to continue PI cycle, the contractile activity was recovered with decreased protein content. The lithium toxic effects depend on the role of PI cycle at particular stage of cardiogenesis, while relation between myo-inositol and reduced cellular protein contents remains unknown.

Primary cell and micromass culture in assessing developmental toxicity.

Under the European Commission's New Chemical Policy both currently used and new chemicals should be tested for their toxicities in several areas, one of which was reproductive/developmental toxicity. Thousands of chemicals will need testing which will require a large number of laboratory animals. In vitro systems (as pre-screens or as validated alternatives) appear to be useful tools to reduce the number of whole animals used or refine procedures and hence decrease the cost for the chemical industry. Validated in vitro systems exist for developmental toxicity/embryotoxicity testing. Indeed, three assays have recently been validated: the whole embryo culture (WEC), the rat limb bud micromass (MM), and the embryonic stem cell test (EST). In this article, the use of primary embryonic cell culture, and in particular micromass culture, including a relatively novel chick heart micromass (MM) culture system has been described and compared to the validated D3 mouse embryonic stem cell (ESC) test.

The effect of vascular endothelial growth factor on in vitro embryonic heart development in rats.

In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24-h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown-rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 +/- 0.87, 46.6 +/- 1.90 and 52.1 +/- 0.97, somite numbers were 26.5 +/- 0.47, 20.1 +/- 0.63 and 24.4 +/- 0.46, crown-rump lengths were 3 +/- 0.07, 2.4 +/- 0.06 and 2.7 +/- 0.06 mm, and embryo protein contents were 160.5 +/- 7.41, 98.2 +/- 4.81 and 141.1 +/- 10.96 mug per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed.

The relative embryotoxicity of 1,3-dichloro-2-propanol on primary chick embryonic cells.

1,3-Dichloro-2-propanol (1,3-DCP) is a chlorinated compound used in the fabrication of industrial products such as hard resins, celluloid or paints. It has also been detected in instant soups and soy sauce. 1,3-DCP has been associated with major necrosis of the liver in humans [Chem.-Bio. Interact. 80 (1991) 73]. In humans and laboratory animals, 1,3-DCP is metabolised to dichloroacetone (1,3-DCA) by cytochromes P450 2E1 and 1A2 [J. University Occup. Environ. Health 14 (1992) 13]. 1,3-DCA is a hepatotoxin. We suggest that 1,3-DCA could be embryotoxic at doses that do not cause adverse maternal hepatic damage. To investigate the embryotoxic effects of 1,3-DCA, we have adapted a micromass culture method from Atterwill and colleagues [1992. A tiered system for in vitro neurotoxicity testing. In: Zbinden, G. (Ed.), The Brain in Bits and Pieces. Verlag M.T.C., Vollikon, pp. 89-91], using chick midbrain cells and from Wiger et al. [Pharmacol. Toxicol. 62 (1988) 32] using chick mesenchymal cells. The basis of the micromass system is that embryotoxins in vitro are likely to affect development and differentiation of disaggregated neuronal and limb bud micromass cultures. The endpoints chosen for the midbrain assay are resazurin reduction (viability), total protein content (cell number), morphological quantification of neuronal cultures (neuronal projection number) and of limb bud cultures (cartilage nodule number). Preliminary results using chick whole embryo cultures indicated that 1,3-DCA had an inhibitory effect on whole chick embryo development. We also found that embryonic derived cells were sensitive to 1,3-DCA but not 1,3-DCP at concentrations above 1 microM, suggesting a potential teratogenic effect of 1,3-DCA. The exposure to 1,3-DCP is not limited to industrial settings, and hence a better knowledge of its effects and tissue specific actions on embryonic-derived cells would be beneficial.

Labelling of rat endothelial cells with antibodies to vWF, RECA-1, PECAM-1, ICAM-1, OX-43 and ZO-1.

Labelling with endothelium specific monoclonal antibodies, von Willebrand Factor (vWF), rat endothelial cell antigen-1 (RECA-1), platelet-endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), OX-43 and zonula occludentes-1 (ZO-1), was investigated in cryostat sections of vessels from rats of different ages using a confocal microscope. The results showed that labelling of the vWF was positive in endothelial cells from adult, fetal and different ages of embryonic rat. Labelling with RECA-1 was weakly positive in adult rat aorta and lung endothelial cells but not in embryonic yolk sac endothelial cells. Labelling using PECAM-1, ICAM-1 and OX-43 was negative in both adult and embryonic endothelial cells. ZO-1 showed positive but very weak reactivity in embryonic yolk sac endothelial cells. The expression of vWF on vessels from adult and 19.5-day fetal tissues was strongly positive. However, the expression of vWF in embryonic endothelial cells was dependent on the gestational age. While the 11.5-day yolk sac vessels stained weakly, staining gradually increased in 13.5-, 15.5- and 17.5-day-old yolk sac vessels. The results suggest that vWF is a reliable endothelial cell marker in rat vascular endothelial cells, including both fetal and embryonic stages.

Growth promoting effects of human placental lactogen during early organogenesis: a link to insulin-like growth factors.

Many maternally derived factors may be involved in the regulation of embryonic growth but the control mechanisms involved are poorly understood. Human placental lactogen (hPL) has been implicated in playing a role in the control of embryonic growth. Several investigators suggested that there may be a possible link between the effects of this hormone and insulin-like growth factors (IGFs). In order to determine the growth promoting potential of hPL and involvement of IGFs in the mechanism of action of the hormone, 9.5 d rat embryos were cultured in vitro for 48 h in depleted serum in the presence and absence of hPL with additional IGF antisera. The growth supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. Addition of hPL (3.2-25.6 ng/ml) to depleted serum significantly improved embryonic growth and development, suggesting that the developing embryo may utilise hPL. The presence of antisera against hPL, IGF-I and -II abolished the hPL-induced increase in the development in all parameters suggesting that there may be a possible link between the IGFs and the effects of hPL on rat embryonic development and this hormone may achieve its growth promoting effects via IGFs.

The growth promoting effects of bFGF, PD-ECGF and VEGF on cultured postimplantation rat embryos deprived of serum fractions.

Serum components in which embryos are cultured in vitro are very important for normal embryonic development. In this study, rat serum was fractionated using Macrosep filters to study the effect of a single growth factor. The fractionated serum, both that containing only material greater than 30 kDa molecular weight (> 30 kDa) and that from which material between 30 kDa and 50 kDa had been removed (< 30 kDa+ > 50 kDa), caused significant embryonic growth retardation. Addition of different concentrations of basic fibroblast growth factor (bFGF, 18 kDa), vascular endothelial growth factor (VEGF, 45 kDa) and platelet-derived endothelial growth factor (PD-ECGF, 45 kDa), to fractionated serum (bFGF to > 30 kDa serum and VEGF or PD-ECGF to < 30 kDa+ > 50 kDa serum) partially restored embryonic growth and development according to a morphological scoring system and protein assay. This restoration was clear by all criteria, as well as in yolk sac vascularisation and heart development. The growth promoting effects of all 3 factors were significant but did not reach the level seen in embryos grown in whole rat serum. The effect of these growth factors was also investigated on anembryonic yolk sac development using a concentration for which maximum whole embryonic growth was seen (128 ng/ml bFGF, 1.6 ng/ml VEGF and 4 ng/ml PD-ECGF), and significant anembryonic yolk sac development was found. These findings suggest that the angiogenic factors may have a growth promoting effect on total embryonic development and vascularisation.

The use of whole rat embryo culture as a technique for investigating potential serum toxicity in recurrent miscarriage patients.

Previously, the rat embryo model has been used as an experimental technique in investigations of the aetiology of idiopathic recurrent miscarriage. The aim of the present study was to validate it as a tool in the investigation of the aetiology of this condition. Subjects (n = 36) with a history of recurrent miscarriage were recruited from two dedicated recurrent miscarriage clinics and compared with control women with at least one previous pregnancy resulting in a live birth (n = 23). Serum from each woman was used as culture medium in the rat embryo model. Cultured embryos were scored for growth and differentiation. No statistical difference was found in any parameter between the two groups. Furthermore, patients from the recurrent miscarriage group whose serum demonstrated a trend towards lower scores, subsequently conceived and underwent uncomplicated pregnancies.

Growth-promoting effects of different fractions of extra-embryonic coelomic fluid on embryonic development.

In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. These factors may be supplied locally to the embryo, by the decidua, the placenta, or the yolk sac. In this study the growth-promoting potential of extra-embryonic coelomic fluid (EECF) and its fractions was investigated. The embryonic requirement of growth-promoting molecules may be studied by reducing the growth-supporting capacity of serum. Thus, ultrafiltration of rat serum was carried out for 8 h using Millipore filters with a molecular weight exclusion of 30 kDa. Rat embryos at 9.5 days of age were cultured for 8 days for anembryonic yolk sacs, and then EECF was collected and divided into three different molecular weight fractions by ultrafiltration. Rat embryos were cultured for 48 h in whole rat serum and the serum retenate (which has low growth-supporting capacity) in the presence and absence of EECF, its fractions, or in EECF only. Embryos grown in retenate showed severe growth retardation, and the addition of EECF significantly improved embryonic growth. The fraction which contained the molecules with molecular weight between 10 and 30 kDa had significantly more effect on embryonic development than the other fractions. This fraction of EECF was analysed by gel electrophoresis. Three of the four protein bands observed in this fraction were identified by amino-terminal sequencing as alpha-fetoprotein precursor (22 kDa), apolipoprotein A1 precursor (24 kDa) and fetal haemoglobin Y2 chain (14 kDa), none of which are likely to be responsible for the growth-promoting activity. To further investigate growth-promoting proteins, EECF was Western-blotted to nitrocellulose membranes and probed with antisera against rat prolactin, epidermal growth factor, insulin-like growth factors I and II and human placental lactogen. No immunoreactive bands were detected in the EECF, suggesting that either these proteins are not present or are present at levels too low to be detected. Although the growth-promoting effect of the EECF was demonstrated in this study, the molecules responsible remain uncharacterized.

Protection by free oxygen radical scavenging enzymes against salicylate-induced embryonic malformations in vitro.

Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals. In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated. Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr. ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml. Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 micromol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud. When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations. Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL. There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione. The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid. An enhanced production of these metabolites in embryonic tissues may be directly related to the increased risk of congenital malformations.

Teratogenicity of edoferon kappa A, a molecule derived from salicylate, in cultured rat embryos: differences from salicylate and interaction with free oxygen radical scavenging enzymes.

The effect of edoferon kappa A (E-KA), a non-specific immunomodulatory and anti-neoplastic chemical substance derived from the methyl form of salicylate (acetyl salicylic acid; ASA), on mammalian embryos was studied and compared to the effects of ASA. Rat embryos were cultured in vitro from 9.5 days of gestation for 48 h. E-KA (0.1-12.8 mg/ml) and ASA (0.1-0.6 mg/ml) were added to the whole rat serum. To investigate the interaction of these molecules with antioxidant agents, the lowest effective concentrations of E-KA (0.6 mg/ml) and ASA (0.3 mg/ml) for all parameters were added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 mumol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. E-KA and ASA decreased growth and development in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology (haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud). There were no statistically significant differences between the control and embryos grown in the presence of 0.1-0.4 mg/ml E-KA, although the effects of ASA started at a concentration of 0.2 mg/ml. Embryos showed significant growth retardation in all scoring criteria and severe malformations when 0.5-3.2 mg/ml E-KA and 0.3-0.6 mg/ml ASA were added. When SOD was added, there was a significant decrease in the incidence of malformations and growth and developmental parameters were increased but this decrease never reached the control level. We concluded that E-KA has direct toxic effects on the developing embryo but at much higher concentrations than ASA, and the teratogenic effects of these molecules might be related to free oxygen radicals.

The in vitro effects of low molecular weight serum fractions on embryonic Wistar rat (Rattus norvegicus) development.

The in vitro embryo culture technique has been used in many research areas as well as for teratologic and toxicologic tests. Usually, homologous serum is used as a culture medium in these techniques. In this study, the effects of low molecular weight serum fractions on embryonic rat development were tested. Using 30 and 50 kDa Macrosep centrifugal concentrators, the homologous serum was centrifuged for 8 h to separate the low molecular weight serum fractions. The embryos were cultured in the sera which included > 30 kDa, > 50 kDa and < 30 + > 50 kDa serum fractions. Whole rat serum (WRS) was also used for control. After a 48-h culture period, embryonic growth and development were assessed using a morphologic scoring system and the protein content of embryos and yolk sacs. The results showed that the embryonic growth and development during organogenesis significantly decreased in > 30 kDa and > 50 kDa serum fractions when compared to WRS. Addition of a < 30 kDa serum fraction to the > 50 kDa serum fraction improved the embryonic growth, but not to the level seen in embryos grown in WRS. While morphological scores for the embryos grown in WRS, > 30 kDa, > 50 kDa and < 30 + > 50 kDa serum fractions were 57.5 +/- 0.83, 44.53 +/- 1.06, 37.81 +/- 1.9 and 45.14 +/- 1.56, respectively, somite numbers were 26.4 +/- 0.28, 20.47 +/- 0.46, 19.15 +/- 0.58 and 21.78 +/- 0.5, yolk sac diameters were 3.35 +/- 0.06, 2.89 +/- 0.05, 2.61 +/- 0.03 and 2.71 +/- 0.04 mm, crown-rump lengths were 3.03 +/- 0.06, 2.72 +/- 0.04, 2.36 +/- 0.04 and 2.52 +/- 0.04 mm, embryo protein contents were 160.93 +/- 6.88, 119.07 +/- 5.15, 67.23 +/- 3.87 and 98.72 +/- 4.87 micrograms and yolk sac protein contents were 114.87 +/- 5.18, 86.33 +/- 1.92, 62.38 +/- 2.7 and 75.88 +/- 2.87 micrograms, respectively. These results suggest that low molecular weight serum fractions could be very important for normal embryonic development.

Angiotensin II is a growth factor in the peri-implantation rat embryo.

Angiotensin II (ANG II) is increasingly recognised as a growth factor, both in its own right and through interactions with other growth factors. There is a high density of ANG II receptors in the rat fetus, especially the AT2 receptor, the function of which is still uncertain. We have now studied the effects of ANG II on growth and development in the rat embryo in vitro between d 9.5 and 11.5, and characterised the receptor subtype mediating these effects. Embryos were cultured in whole rat serum, a high molecular weight retenate after ultrafiltration of whole rat serum, retenate with angiotensin II and retenate with ANG II and AT1 or AT2 receptor blockers. Growth and development were scored using conventional methods. Culture in retenate was associated with a marked reduction in growth and development by comparison with whole rat serum. This was partly, and significantly (P < 0.001), reversed by angiotensin II. The optimum concentration of angiotensin II was found to be angiotensin II 10(-11) M, within the physiological range. Angiotensin II had highly significant effects on both somatic (P < 0.001) and yolk sac/allantoic (P < 0.005) development. The latter effects suggest a role for angiotensin II in placentation. The effects of angiotensin II were blocked by PD123319, an AT2 blocker, but not by GR117289, an AT1 blocker. Interestingly, culture in retenate with GR117289 without added angiotensin II was also associated with some increase in growth (P < 0.05). Angiotensin II in low concentrations was measurable in the retenate, presumably arising from the action of endogenous renin on angiotensinogen. We therefore postulate that this effect of GR117289 was due to the action of endogenous angiotensin II on 'uncovered' AT2 receptors. This study has thus demonstrated a direct growth promoting effect of angiotensin II during organogenesis in the whole rat embryo in vitro. This effect is mediated through the AT2 receptors.

The mechanism of growth-promoting effects of prolactin in embryogenesis--links to growth factors.

The polypeptide hormone prolactin (PRL) has been implicated in the regulation of embryonic growth and development, but the control mechanisms involved in the effects of the hormone are poorly understood. Several investigators suggested that there may be a possible link between the effects of PRL and insulin-like growth factors (IGFs). Recent studies have also shown that ligand-induced activation of PRL receptors leads to tyrosine phosphorylation of multiple intracellular proteins, and tyrosine kinase activation takes place in mediating the mitogenic action of PRL. In order to determine whether IGFs are involved in mediating the growth-promoting effect of PRL, rat embryos were culture in vitro for 48 h in whole rat serum and serum depleted of low molecular weight molecules (30 kD retenate) supplemented with rat PRL in the presence and absence of antisera against rat PRL, IGF I and IGF II. To investigate the effects of inhibiting the signal transduction of the PRL receptors, the embryos were preincubated for 2 h in retenate in the presence of tyrosine kinase inhibitors, tyrphostin 47 and genistein, then rat PRL was added to the culture medium. Embryos cultured in retenate showed severe growth retardation, and the addition of rat PRL caused significant increase in growth and development of the embryos suggesting that embryos may be able to utilize maternally derived PRL during organogenesis. The presence of antiserum against rat PRL abolished the PRL-induced increase in development and antibodies against IGF I and II had a similar effect, suggesting that IGFs may be involved in the effect of the hormone. The 2-hour preincubation with genistein and tyrphostin also abolished the PRL-induced increase in development. These results indicate that functional PRL receptors are present in rat embryos at this stage which may play an important role in the control of growth and development and this may be linked to growth factors and their receptors.

Species-specificity of growth-promoting effects of prolactin during rat embryogenesis.

In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. The molecules involved in growth processes may be either hormones or growth factors, or molecules that interact with such factors. The pregnancy related hormone, prolactin (PRL, MW 23 kDa) has been implicated in the control of embryonic growth. The growth-promoting potential of PRL and its species-specificity was investigated by culturing 9.5 d rat embryos in vitro for 48 h in depleted serum in the presence and absence of PRL from 3 different species. The growth-supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. This method provided a 'semidefined' medium (retenate) in which embryonic growth and development was significantly reduced, demonstrating that the low molecular weight fraction of serum may contain some growth-promoting factors. Addition of PRL (0.4-25.6 ng/ml) from different species (human, sheep and rat) to retenate significantly improved embryonic growth and development, suggesting that the developing embryo may utilise PRL. Amongst PRLs, rat PRL was found to be active at much lower concentrations than either of the other molecules, and human PRL had more effect in low concentrations than sheep PRL suggesting a species-specificity for this hormone. It may be that the PRL receptors of the rat embryos have greater affinity for the rat hormone as different responses for hormones from different species have been shown. These findings suggest that embryos may be able to utilise maternally derived PRL during organogenesis.

The role of exogenous growth-promoting factors and their receptors in embryogenesis.

During organogenesis, the cells of the embryo may require growth factors that promote a cascade of intracellular events. An absolute requirement for exogenous insulin by presomite 9.5-d rat embryos grown in culture has been demonstrated. The uptake and processing of insulin and insulin-like growth factor-I showed different uptake and localization patterns. When epidermal growth factor (EGF) or "long EGF" is added to media depleted of low molecular weight material, a dose-dependent improvement in growth is observed. Furthermore, the specific EGF receptor signal transduction inhibitor Tyrphostin 47 can inhibit embryonic growth when it is administered in culture. When Tyrphostin 47 was microinjected into embryos on Day 11 and their growth and differentiation evaluated on Day 12 of gestation, a dose-dependent decrease in developmental score was observed. Thus, exogenous growth factors may be essential to normal rat development and these may be synthesized locally in the decidua or placental tissues. Perturbations to ligand-receptor interactions may be a mechanism for dysmorphogenesis.

Uptake of microparticles by rat visceral yolk sac.

The visceral yolk sac (VYS) is responsible for a major part of the amino acid nutrition of the early post-implantation rat embryo and possibly also at the fetal stage of gestation. The mechanism involves endocytic uptake of proteins by the tissue's epithelial cells followed by intralysosomal digestion to amino acids. The amino acid so generated are used for protein synthesis in both the embryo and the VYS. Previous reports had indicated that the endocytic capacity of the VYS might be limited to exclude larger macromolecules. This study demonstrates that Percoll, which comprises 30-nm silica particles coated with polyvinylpyrrolidone (PVP), is as effectively captured by the 17.5-day rat VYS cultured in vitro as PVP itself. Uptake of 125I-labelled Percoll was progressive with time over 5 h and was inhibited by a low incubation temperature, 2,4-dinitrophenol (50 micrograms/ml), EGTA (5 mM), colchicine (10 micrograms/ml) or cytochalasin B (10 micrograms/ml). After uptake of 125I-labelled Percoll, VYSs released only 20 per cent of their radioactivity when re-incubated in fresh medium for 3 h. These data, and electron micrographs showing Percoll in intracellular vacuoles, are all consistent with uptake by endocytosis. Percoll's rate of uptake by the VYS indicates that, like 125I-labelled PVP, it enters the cell chiefly by fluid-phase pinocytosis. It is concluded that endocytosis by the VYS will efficiently capture even the largest globular proteins, and that previous indications of a relatively low size exclusion reflected the loosely coiled configuration of the synthetic polymers used in the earlier studies.

The role of maternally derived epidermal growth factor and the epidermal growth factor receptor during organogenesis in the rat embryo.

Epidermal growth factor (EGF) has been implicated in the control of embryonic development, but although the receptor is expressed from an early stage, there is little evidence of embryonic expression of EGF. In order to investigate the role of maternally derived EGF during organogenesis, rat embryos were explanted at d 9.5 and cultured in serum depleted of low molecular weight molecules (retenate) which was then supplemented with EGF. Serum depleted of low molecular weight molecules by prolonged filtration loses its capacity to support normal embryonic development, possibly due to the loss of growth promoting factors. The addition of EGF to retenate significantly improved embryonic development with a maximal effect at 8 ng/ml. The addition of an analogue of EGF, long EGF, to retenate also caused a significant increase in development, although at higher concentrations a decrease in its effect was observed, possibly due to down regulation of the EGF receptor. Therefore, embryos may be able to utilise maternally derived EGF during organogenesis. To test the effects of inhibiting the EGF receptor during organogenesis, d 9.5 embryos were cultured in the presence of tyrphostin 47, a specific EGF receptor inhibitor. Tyrphostin 47 caused a significant dose-dependent decrease in the development of embryos which was also observed when tyrphostin 47 was injected into the vitelline circulation at d 11.5 to bypass the effects of the yolk sac. These findings suggest that the EGF receptor is essential for normal organogenesis and may play a role in the control of proliferation and differentiation. Although EGF is not expressed in the rat embryo at this stage, maternally derived EGF may be the ligand for the embryonic EGF receptor.

The role of exogenous growth-promoting factors and their receptors in organogenesis.

The mechanisms involved in the regulation of early embryonic development are poorly understood. Certain growth promoting molecules are known to be produced within the embryo itself. It is clear, however, that at the early stages of embryonic development, many additional growth promoting factors have to be provided by the maternal system. Since the levels of factors such as epidermal growth factor and insulin in the maternal circulation are not linked with gestational age of the offspring, it is likely that regulation of receptors in the embryonic tissues may provide the key to the regulation of development. The expression of any receptor may depend on its synthetic rate, turnover or its distribution between the cell surface and intracellular pools. The study of the role of exogenous growth promoting molecules and receptor distribution and regulation for such growth factors, in particular insulin, insulin-like growth factor-I and epidermal growth factor, in embryos has been addressed using whole embryo culture, supported by anembryonic yolk sac culture and intravitelline injection of rat embryos.