Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract

Lipoprotein(a) [Lp(a)] biogenesis was examined in primary cultures of hepatocytes isolated from mice transgenic for both human apolipoprotein(a) [apo(a)] and human apoB. Steady-state and pulse-chase labeling experiments demonstrated that newly synthesized human apo(a) had a prolonged residence time (approximately 60 min) in the endoplasmic reticulum (ER) before maturation and secretion. Apo(a) was inefficiently secreted by the hepatocytes and a large portion of the protein was retained and degraded intracellularly. Apo(a) exhibited a prolonged and complex folding pathway in the ER, which included incorporation of apo(a) into high molecular weight, disulfide-linked aggregates. These folding characteristics could account for long ER residence time and inefficient secretion of apo(a). Mature apo(a) bound via its kringle domains to the hepatocyte cell surface before appearing in the culture medium. Apo(a) could be released from the cell surface by apoB-containing lipoproteins. These studies are consistent with a model in which the efficiency of post-translational processing of apo(a) strongly influences human plasma Lp(a) levels, and suggest that cell surface assembly may be one pathway of human Lp(a) production in vivo. Transgenic mouse hepatocytes thus provide a valuable model system with which to study factors regulating human Lp(a) biogenesis.     

Immunohistochemical localization of androgen receptor in mouse testicular germ cells during fetal and postnatal development

BACKGROUND: Determination of the cellular distribution of the androgen receptor (AR) in testicular cells is necessary for understanding the mode of AR action in the testis. We here investigated immunohistochemically the localization of AR by use of anti-human AR polyclonal antibody NH27, with special reference to the AR in germ cells in the developing mouse testis. METHODS: ICR mouse testes taken from day 14 post coitum (p.c.) to day 56 post partum (p.p) were used for AR immunohistochemistry by the routine immunoperoxidase method at the light microscopic level and the pre-embedding method at the electron microscopic level. RESULTS: On day 14 p.c., AR immunoreactivity was present in nuclei of prospermatogonia but not in those of Sertoli cells or interstitial cells. On day 14 p.p., the AR was detected in the nuclei of spermatogonia, Sertoli cells, and myoid cells. AR immunoreactivity in nuclei of Leydig cells appeared on day 21 p.p. In the mature mouse testis, the AR was present in the nuclei of spermatogonia, Sertoli cells, myoid cells, and Leydig cells. CONCLUSIONS: AR was present both in germ cells and in somatic cells during fetal and postnatal development of the mouse testis. In the fetal testis, AR was localized exclusively in prospermatogonia and spermatogonia, suggesting that androgen may act directly on germ cells during prespermatogenesis and the early stage of spermatogenesis. Based on the fact that AR is expressed in Sertoli cells, myoid cells, and Leydig cells around the onset of spermatogenesis, the regulation of AR expression in the germ cells seems to be different from that in the somatic cells. Furthermore, our present data suggest the ultrastructural localization in nuclei of mouse testicular cells is similar to that of some other steroid receptors, both in germ cells and somatic cells.     

Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.     

LacZ and OMP are co-expressed during ontogeny and regeneration in olfactory receptor neurons of OMP promoter-lacZ transgenic mice

The ontogeny and cellular specificity of expression of beta-galactosidase activity and olfactory marker protein (OMP) are compared in olfactory tissue of the H-OMP-lacZ-3 line of transgenic mice. In this line the expression of lacZ is driven by a 0.3 kb fragment of the rat OMP promoter. During fetal development, lacZ expression is detectable in olfactory receptor neurons (ORNs) shortly after the initial appearance of endogenous OMP. The beta-galactosidase marker was observed only in mature olfactory receptor neurons where it co-localized with endogenous OMP. It was absent from immature neurons that express the growth associated phosphoprotein B50/GAP43. Lesion of the peripheral olfactory pathway by intranasal irrigation with Triton X-100 eliminated expression of both OMP and lacZ in the olfactory neuroepithelium. Subsequent regeneration of the full complement of olfactory receptor neurons was associated with co-expression of both OMP and beta-galactosidase activity. Neither OMP nor beta-galactosidase activity was induced in any other cell type of the regenerating olfactory mucosa. Thus, as little as 0.3 kb of the OMP promoter has the ability to target lacZ expression to olfactory receptor neurons in a temporally and spatially defined manner. We discuss the potential utility of this transgenic line for future studies of the olfactory system.     

Electrofusion of zona-free mouse embryonic cells in electrolytes and their development in vitro

The influence of increasing the physical electrofusion parameters, direct current (DC) pulse strength, pulse duration, pulse number, alternating current (AC) voltage and alignment time, in electrolytes on the rates of fusion, degeneration and development of zona-free mouse 2-cell embryos were examined. Furthermore, the effects of physiological saline and mannitol as fusion media and various mouse strains were also evaluated. Dulbecco's phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum was used as the main fusion solution. A significant increase in the rate of fusion (P < 0.05) was obtained by increasing pulse strength from 30 to 300 V/mm. The embryos fused at the pulse strengths of 30 to 70 V/mm had significantly higher development rates to blastocysts compared with those fused at 100 to 300 V/mm (P < 0.05). There were no significant differences in the rates of fusion, degeneration and development to blastocysts when the pulse duration was increased from 30 to 90 microseconds. Although fusion rates were increased (P < 0.05) by increasing the pulse number up to 4, a significant decrease (P < 0.05) in development to blastocysts was observed when the pulse number was 5. Application of AC voltage prior to the DC pulse tended to increase the fusion rate (89.2-93.8%), compared with fusion with the DC pulse only (75.0%). Prolongation of alignment time from 5 to 15 sec had no effect on the fusion rate. Under the optimum conditions (2 pulses of DC of 70 V/mm, 70 microseconds pulse duration and AC of 5 V/mm for 5 sec), no significant difference was obtained in the fusion and development rates in different mouse strains, nor were fusion and development rates significantly different among PBS, physiological saline and mannitol solutions (P > 0.05).     

Immunohistochemical localization of transforming growth factor-alpha, epidermal growth factor (EGF), and EGF receptor in the human fetal ovary

Peptide growth factors are thought to be involved in adult ovarian regulatory functions. However, little is known about the role of growth factors in human fetal ovarian development. This study is an attempt to identify and localize transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), and EGF receptor (EGF-R) in human fetal ovaries. Ovaries were obtained from first and second trimester elective abortuses. Immunohistochemistry was performed on paraffin sections of these specimens after fixation. We examined the sections microscopically using the specific antibodies against TGF alpha, EGF, and EGF-R. Phosphate-buffered saline and preimmune IgG were used as negative controls. First and second trimester ovaries stained positively for all three proteins. Staining was significantly more intense in the oocytes than in the stroma. Negative controls did not stain. These results combined with our previous demonstration of messenger ribonucleic acid for these growth factors suggest roles for TGF alpha, EGF, and EGF-R in human fetal ovarian development. The strong staining in the oocytes suggests a possible autocrine or paracrine role of these growth factors in human oocyte growth in utero.     

Effect of chronic duodenal ulceration and its treatment with lanzoprazole or sucralfate on gastroduodenal mucosal protein turnover and TGF-alpha, bFGF, and EGF receptor expression in humans

In order to investigate whether chronic duodenal ulcer disease is a consequence of disturbed mucosal turnover and growth factor expression, we studied 16 patients with duodenal ulcers before, during, and after endoscopic healing with lansoprazole or sucralfate. Before treatment, gastric fundal and antral mucosal protein turnover rates were higher in patients than controls, without parallel increases in growth factors. Both forms of therapy produced similar changes, with overall increases in duodenal mucosal turnover and transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGF-r) levels. Measurements after healing showed persistent elevations of mucosal turnover in the antrum and duodenum and depressions of basic fibroblast growth factor (bFGF) in gastric fundal and duodenal mucosa. We conclude that mucosal turnover is abnormally high in patients with chronic duodenal ulcer disease and is not easily explained by growth factor changes. The failure of lansoprazole and sucralfate to normalize rates, despite endoscopic healing, may explain the high ulcer relapse rates in non-HP-eradicated patients.     

Comparative investigations on water-soluble crystallins of the embryonic, fetal, and postnatal human lens during development and ageing

To compare the crystallin composition of embryonic and fetal human lenses with those of postnatal and adult lenses, we investigated the crystallins of lenses of various ages (from the 5th gestational week to 55 years) by gel chromatography, isoelectric focusing, immunodiffusion, and immunoelectrophoresis. Age-related changes were calculated as relative percentages of the different classes and subclasses of crystallins. During prenatal lens development the percentages of both high- and low-molecular-weight alpha-crystallins as well as gamma-crystallins gradually increased, whereas the percentage of beta-crystallins decreased. A considerable change in crystallin composition was found immediately after birth: the relative percentage of beta-crystallins increased, whereas that of gamma-crystallins decreased. Gel-filtration analysis of crystallins from juvenile and adult lenses showed a high-molecular-weight peak, which was not found in extracts from fetal and new-born lenses.     

Noninvasive, in utero imaging of mouse embryonic heart development with 40-MHz echocardiography

BACKGROUND: The increasing number of transgenic and targeted mutant mice with embryonic cardiac defects has resulted in the need for noninvasive techniques to examine cardiac structure and function in early mouse embryos. We report the first use of a novel 40-MHz ultrasound imaging system in the study of mouse cardiac development in utero. METHODS AND RESULTS: Transabdominal scans of mouse embryos staged between 8.5 and 13.5 days of gestation (E8.5 to E13.5) were obtained in anesthetized mice. Atrial and ventricular contractions could be discerned from E9.5, and changes in cardiac morphology were observed from E9.5 to E13.5. Hyperechoic streaming patterns delineated flow through the umbilical, vitelline, and other major blood vessels. Diastolic and systolic ventricular areas were determined by planimetry of the epicardial borders, and fractional area change was measured as an index of contractile function. Significant increases in ventricular size were documented at each stage between E10.5 and E13.5, and the ability to perform serial imaging studies over 3 days of embryonic development is described. Finally, the detection of vascular cell adhesion molecule 1 (VCAM-1) homozygous null mutant embryos demonstrates the first example of noninvasive, in utero analysis of cardiac structure and function in a targeted mouse mutant. CONCLUSIONS: We used 40-MHz echocardiography to identify key elements of the early mouse embryonic cardiovascular system and for noninvasive dimensional analysis of developing cardiac ventricles. The ability to perform serial measurements and to detect mutant embryos with cardiac defects highlights the usefulness of the technique for investigating normal and abnormal cardiovascular development.     

Changes in the content of progesterone receptor isoforms and estrogen receptor alpha in the chick brain during embryonic development

Progesterone and estradiol participate in the regulation of several reproductive functions through interaction with intracellular progesterone receptors (PR) and estrogen receptors (ER), respectively. In this work, we determined PR and ER-alpha isoforms content in the brain of chicks of both sexes on days 8 and 13 of embryonic development as well as on the day of hatching by Western blot analysis. PR isoforms protein content increased during embryonic development in both female and male chick brain. The highest PR isoforms content was observed on the day of hatching in both sexes. Interestingly, PR-A content was higher in the brain of chick males than in that of females on day 8 of embryonic development. PR-A/PR-B ratio was higher in the brain of males than in that of females at all ages. We found two ER-alpha isoforms of 66 and 52 kDa; the content of both isoforms was higher in the brain of females than in that of males on days 8 and 13 of embryonic development. An opposite pattern of ER-alpha isoforms content was observed. In males, ER-alpha content increased during embryonic development whereas in the females it decreased during this process. These results indicate that the content of PR and ER-alpha isoforms is related to the degree of brain development in chicks, and suggest that PR and ER-alpha isoforms should exhibit sexual dimorphism in the brain of chicks during embryonic development.     

Expression of nerve growth factor and its high-affinity receptor Trk-A in the rat pancreas during embryonic and fetal life

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes. To clarify the mechanism of the IL-1-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-1-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it. Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3. The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release. An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-1-stimulated bFGF release even if increased production of bFGF is related to the release. IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order. NG-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.     

Changes in myocardial A1 adenosine receptor and message levels during fetal development and postnatal maturation

Dysembryoplastic neuroepithelial tumor (DNT) is a recently described rare brain neoplasm with characteristic clinical and morphological features and favorable prognosis. We report here two cases of DNT. The first concerned a 12 years old girl who presented complex seizures preceded by acoustic aura (melodies). Computed tomography revealed a hypodense tumor measuring 2 x 2.5 cm in diameter, located paracortically in the left temporal lobe. The second tumor was removed from a 21-year-old man with partial complex seizures. Nine years earlier patient underwent neurosurgery with partial removal of the tumor The tumor's histopathologic diagnosis is unfortunately lacking. Computed and magnetic resonance imaging showed a mass occupying the cortex and paracortical areas of the anterior pole of the temporal lobe. Histologically, both tumors consisted of small, S-100 protein immunopositive oligodendrocyte-like cells (OLCs) arranged between synaptophysin- and, to a lesser degree, NFP-immunopositive axons (glioneuronal element). In the second case, an area of pilocytic astrocytoma-like appearance was also found, these cells were immunopositive for GFAP. The present study provides clinical, radiological and histological data, which may be helpful in differential diagnosis of this newly recognised brain tumor.     

Several levels of EGF receptor signaling during photoreceptor specification in wild-type, Ellipse, and null mutant Drosophila

Dominant Ellipse mutant alleles of the Drosophila EGF receptor homologue (DER) dramatically suppress ommatidium development in the eye and induce ectopic vein development in the wing. Their phenotype suggests a possible role for DER in specifying the founder R8 photoreceptor cells for each ommatidium. Here we analyze the basis of Ellipse mutations and use them to probe the role of DER in eye development. We show that Elp mutations result from a single amino acid substitution in the kinase domain which activates tyrosine kinase activity and MAP kinase activation in tissue culture cells. Transformant studies confirmed that the mutation is hypermorphic in vivo, but the DER function was elevated less than by ectopic expression of the ligand spitz. Ectopic spi promoted photoreceptor differentiation, even in the absence of R8 cells. Pathways downstream of DER activation were assessed to explore the basis of these distinct outcomes. Elp mutations caused overexpression of the Notch target gene E(spl) mdelta and required function of Notch to suppress ommatidium formation. The Elp phenotype also depended on the secreted protein argos and was reverted in Elp aos double mutants. Complete loss of DER function in clones of null mutant cells led to delay in R8 specification and subsequently to loss of mutant cells. The DER null phenotype was distinct from that of either spitz or vein mutants, suggesting that a combination of these or other ligands was required for aspects of DER function. In normal development DER protein was expressed in most retinal cells, but at distinct levels. We used an antibody specific for diphospho-ERK as well as expression of the DER target gene argos to assess the pattern of DER activity, finding highest activity in the intermediate groups of cells in the morphogenetic furrow. However, studies of mutant genotypes suggested that this activity may not be required for normal ommatidium development. Since we saw distinct phenotypic effects of four different levels of DER activity associated with wild-type, null mutant, Elp mutant, or fully activated DER function, we propose that multiple thresholds separate several aspects of DER function. These include activation of N signaling to repress R8 specification, turning on argos expression, and recruiting photoreceptors R1-R7. It is possible that during normal eye development these thresholds are attained by different cells, contributing to the pattern of retinal differentiation.     

Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat

We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis.     

Changing patterns of gap junctional intercellular communication and connexin distribution in mouse epidermis and hair follicles during embryonic development

This paper is concerned with whether transport accident risk tends to peak at particular times, in relation to both time of day and time on task, and with the underlying causes of such peaks. Macro-analyses confirmed the presence of a clear circadian (ca 24 hour) rhythm in road accident risk with a major peak at ca 03:00 but suggested that this rhythm could not be entirely accounted for in terms of drivers falling asleep at the wheel. Sleep propensity clearly shows a pronounced circadian rhythm and performance efficiency in wakeful subjects shows a similar trend implying that the 03:00 road accident peak may simply reflect lowered performance capabilities. However, there are 'residual' peaks in accidents at certain times of day that are difficult to account for in terms of circadian rhythmicity. It is suggested that these may reflect a time on task effect which shows a pronounced, but transient, 2-4 hour peak in risk. Only when individuals had been on duty for 12 hours or more did the risk exceed that found during the 2-4 hour peak. While an explanation for this transient peak is offered, the underlying reason for it is, as yet, uncertain and clearly warrants investigation in view of its practical implications. It is concluded that there are 'black times' when accidents are far more likely and that there is a strong need to investigate possible countermeasures.     

The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm

The Drosophila EGF receptor (DER) is activated by secreted Spitz to induce different cell fates in the ventral ectoderm. Processing of the precursor transmembrane Spitz to generate the secreted form was shown to be the limiting event, but the cells in which processing takes place and the mechanism that may generate a gradient of secreted Spitz in the ectoderm were not known. The ectodermal defects in single minded (sim) mutant embryos, in which the midline fails to develop, suggested that the midline cells contribute to patterning of the ventral ectoderm. This work shows that the midline provides the site for Spitz expression and processing. The Rhomboid and Star proteins are also expressed and required in the midline. The ectodermal defects of spitz, rho or Star mutant embryos could be rescued by inducing the expression of the respective normal genes only in the midline cells. Rho and Star thus function non-autonomously, and may be required for the production or processing of the Spitz precursor. Secreted Spitz is the only sim-dependent contribution of the midline to patterning the ectoderm, since the ventral defects observed in sim mutant embryos can be overcome by expression of secreted Spitz in the ectoderm. While ectopic expression of secreted Spitz in the ectoderm or mesoderm gave rise to ventralization of the embryo, increased expression of secreted Spitz in the midline did not lead to alterations in ectoderm patterning. A mechanism for adjustment to variable levels of secreted Spitz emanating from the midline may be provided by Argos, which forms an inhibitory feedback loop for DER activation. The production of secreted Spitz in the midline, may provide a stable source for graded DER activation in the ventral ectoderm.     

Partial cloning of the hormone-binding domain of the cortisol receptor in tilapia, Oreochromis mossambicus, and changes in the mRNA level during embryonic development

The success of managing the infectious complications of acute leukemia has permitted oncologists to develop new approaches to induction and high-dose therapy. The single most important risk factor for infection is the duration of absolute neutropenia. Historically, most attention was directed towards gram negative aerobes, especially Pseudomonas aeruginosa, but in recent years gram positive bacteria, generally considered to be less virulent, have become the most frequent isolates in most centers. A recent disturbing trend is the isolation of vancomycin-resistant enterococci. A recent controversy has been whether to use empirical vancomycin; the Centers for Disease Control has issued a formal recommendation discouraging empirical vancomycin in the febrile neutropenic patient. Empirical monotherapy has replaced combination therapy in many institutions except where there has been an increase in resistant isolates. In patients who remain profoundly neutropenic, fungal infections represent a serious source of secondary infection, especially species of Candida and Aspergillus. Recently lipid-based formulations of amphotericin B have offered reduced nephrotoxicity. Less toxic antifungals, the azoles, which include fluconazole and itraconazole, offer an attractive alternative to amphotericin B. New patterns of invasive mycoses have emerged, as for example hepatosplenic candidiasis, presenting new problems in diagnosis and therapy. The successful management of virus infections with herpes simplex, cytomegalovirus, varicella zoster, and Epstein Barr virus is based on early recognition and careful attention to prevention.     

Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A deficiency in the rat are prevented by feeding pharmacological levels of all-trans-retinoic acid

Vitamin A is required for reproduction and normal embryonic development. We have determined that all-trans-retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 micrograms of atRA per g of diet (230 micrograms per rat per day) or 250 micrograms of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 micrograms of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 micrograms of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 micrograms/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 micrograms of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 micrograms of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 micrograms of atRA per g of diet fed to VAD dams (approximately 4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.