Skeletal Development Retardation Research Articles

Fam83h mutation causes mandible underdevelopment via CK1α-mediated Wnt/β-catenin signaling in male C57/BL6J mice.

Truncation mutations in FAM83H are the major cause of autosomal dominant hypocalcified amelogenesis imperfecta. Some studies also indicated that FAM83H could be involved in osteogenic differentiation; however, the function of FAM83H in bone formation was rarely explored. This study aimed to explore the effect of Fam83h mutation on skeletal development. We generated Fam83h c.1186C>T (p.Q396*) knockin C57/BL6J mice by CRISPR/Cas9 technology and found that the Fam83hQ396⁎/Q396⁎ male mice presented skeletal development retardation that was inconspicuous at birth but progressively worsened as they grew up. Alcian and Alizarin Red staining of the whole-mount skeleton showed Fam83hQ396⁎/Q396⁎ mice presented obvious skeletal development retardation. Moreover, Micro-computed tomography (Micro-CT) analysis and H&E staining showed that the mandible of Fam83hQ396⁎/Q396⁎ mice exhibited decreased bone trabecula and slight bone rarefaction compared with wild-type mice. Calcium and phosphorus content of serum and bone, and serum ALP activity analysis showed that the serum ALP activity and value of bone calcium were decreased in Fam83hQ396⁎/Q396⁎ mice. The reduced expression of mineralization markers of RUNX2, OSX, OCN, and COL1, the reduced ALP activity and the weakened ARS staining exhibited in osteoblasts isolated from 3-day-old Fam83hQ396⁎/Q396⁎ mice. The increased protein expression of casein kinase 1α (CK1α) in the cytoplasm and the decreased expression of β-CATENIN in the nucleus indicated the inhibiting Wnt/β-catenin signaling in osteoblasts from Fam83hQ396⁎/Q396⁎ mice. Furthermore, agonists of Wnt/β-catenin signaling and Ck1α siRNA partially reversed the mineralization inhibition and the decreased expression of key signaling molecules in osteoblasts of Fam83hQ396⁎/Q396⁎ mice. In conclusion, Fam83h mutation caused the increase of cytoplasmic CK1α (as one of the components of the degradation complex), which in turn promoted degradation of β-CATENIN in the cytoplasm and reduced β-CATENIN translocation into the nucleus, subsequently inhibited Wnt/β-catenin signaling in osteoblast differentiation, and thus resulted in the mandible underdevelopment in Fam83hQ396⁎/Q396⁎ male mice.

Altered gut microbiome accompanying with placenta barrier dysfunction programs pregnant complications in mice caused by graphene oxide

The wide use of graphene oxide (GO) has raised increasing concerns about the potential risks to environmental and human health. Recent studies have shown the vital role of gut microbiome in various pathological status or even exogenous exposure, but more detailed understanding about the effects of possible gut microbiome alterations under GO exposure on reproductive toxicology evaluations in pregnant mammals remained elusive. Here we found that orally administrated GO daily during gestational day (GD) 7–16 caused dose-dependent pregnant complications of mice on the endpoint (GD19), including decreased weight of dam and live fetus, high rate of resorbed embryos and dead fetus, and skeletal development retardation. Meanwhile in placenta tissues of pregnant mice exposed to GO at dose over 10 mg/kg, the expression levels of tight junctions (Claudin1 and Occludin) and vascular endothelial growth factor (VEGFA) decreased approximately by 30%–80%, meaning impaired placenta barrier. According to the data of fecal 16s RNA sequencing in 40 mg/kg dose group and the control group, gut microbiome showed dramatically decreased α- and β-diversity, and upregulated Firmicutes/Bacteroidetes ratio owing to GO exposure. What's more, significantly differentiated abundance of Euryarchaeota is expected to be a special biomarker for failed pregnancy caused by GO. Notably, the result of Spearman correlation analysis suggested that there was a strong link (correlation coefficient>0.6) between perturbed gut microbiome with both abnormally expressed factors of placenta barrier and adverse pregnant outcomes. In summary, the damages of GO exposure to placenta barrier and pregnancy were dose-dependent. And GO exposure was responsible for gut microbiome dysbiosis in mice with pregnant complications. These findings could provide referable evidence to evaluate reproductive risk of GO to mammals.

Modulatory Mechanism of Polyphenols and Nrf2 Signaling Pathway in LPS Challenged Pregnancy Disorders.

Early embryonic loss and adverse birth outcomes are the major reproductive disorders that affect both human and animals. The LPS induces inflammation by interacting with robust cellular mechanism which was considered as a plethora of numerous reproductive disorders such as fetal resorption, preterm birth, teratogenicity, intrauterine growth restriction, abortion, neural tube defects, fetal demise, and skeletal development retardation. LPS-triggered overproduction of free radicals leads to oxidative stress which mediates inflammation via stimulation of NF-κB and PPARγ transcription factors. Flavonoids, which exist in copious amounts in nature, possess a wide array of functions; their supplementation during pregnancy activates Nrf2 signaling pathway which encounters pregnancy disorders. It was further presumed that the development of strong antioxidant uterine environment during gestation can alleviate diseases which appear at adult stages. The purpose of this review is to focus on modulatory properties of flavonoids on oxidative stress-mediated pregnancy insult and abnormal outcomes and role of Nrf2 activation in pregnancy disorders. These findings would be helpful for providing new insights in ameliorating oxidative stress-induced pregnancy disorders.

Zinc Supplementation during Pregnancy Protects against Lipopolysaccharide-Induced Fetal Growth Restriction and Demise through Its Anti-inflammatory Effect

LPS is associated with adverse developmental outcomes, including preterm delivery, fetal death, teratogenicity, and intrauterine growth restriction (IUGR). Previous reports showed that zinc protected against LPS-induced teratogenicity. In the current study, we investigated the effects of zinc supplementation during pregnancy on LPS-induced preterm delivery, fetal death and IUGR. All pregnant mice except controls were i.p. injected with LPS (75 μg/kg) daily from gestational day (GD) 15 to GD17. Some pregnant mice were administered zinc sulfate through drinking water (75 mg elemental Zn per liter) throughout the pregnancy. As expected, an i.p. injection with LPS daily from GD15 to GD17 resulted in 36.4% (4/11) of dams delivered before GD18. In dams that completed the pregnancy, 63.2% of fetuses were dead. Moreover, LPS significantly reduced fetal weight and crown-rump length. Of interest, zinc supplementation during pregnancy protected mice from LPS-induced preterm delivery and fetal death. In addition, zinc supplementation significantly alleviated LPS-induced IUGR and skeletal development retardation. Further experiments showed that zinc supplementation significantly attenuated LPS-induced expression of placental inflammatory cytokines and cyclooxygenase-2. Zinc supplementation also significantly attenuated LPS-induced activation of NF-κB and MAPK signaling in mononuclear sinusoidal trophoblast giant cells of the labyrinth zone. It inhibited LPS-induced placental AKT phosphorylation as well. In conclusion, zinc supplementation during pregnancy protects against LPS-induced fetal growth restriction and demise through its anti-inflammatory effect.

Effect of experimental diabetes on craniofacial growth in rats

Objectives The goal of this study was to assess the effect of type 1 diabetes mellitus (T1DM) on the craniofacial growth and skeletal maturation using the STZ-DM rat model. Design Experimental T1DM was induced in 3-week-old male Wistar rats by a single dose of 60 mg/kg body weight of streptozotocin (STZ). Lateral and dorsoventral X-rays of the head were taken at the age of 7 weeks. The X-rays were scanned, digitised and selected linear distances were measured and analysed statistically. Results In STZ-DM statistical analysis of results revealed a reduction in growth of most of the linear measurements in the neurocranium and mandible by X-ray analysis, and all measurements were significantly lower in viscerocranium. Conclusions Uncontrolled T1DM reduces craniofacial growth, resulting in retardation of skeletal development.

Reactive oxygen species are involved in lipopolysaccharide-induced intrauterine growth restriction and skeletal development retardation in mice

Maternal infection is a cause of adverse developmental outcomes including embryonic resorption, intrauterine fetal death, and preterm labor. Lipopolysaccharide-induced developmental toxicity at early gestational stages has been well characterized. The purpose of the present study was to investigate the effects of maternal lipopolysaccharide exposure at late gestational stages on intrauterine fetal growth and skeletal development and to assess the potential role of reactive oxygen species in lipopolysaccharide-induced intrauterine fetal growth restriction and skeletal development retardation. The timed pregnant CD-1 mice were intraperitoneally injected with lipopolysaccharide (25 to 75 microg/kg per day) on gestational day 15 to 17. To investigate the role of reactive oxygen species on lipopolysaccharide-induced intrauterine fetal growth restriction and skeletal development retardation, the pregnant mice were injected with alpha-phenyl-N-t-butylnitrone (100 mg/kg, intraperitoneally) at 30 minutes before lipopolysaccharide (75 microg/kg per day, intraperitoneally), followed by an additional dose of alpha-phenyl-N-t-butylnitrone (50 mg/kg, intraperitoneally) at 3 hours after lipopolysaccharide. The number of live fetuses, dead fetuses, and resorption sites was counted on gestational day 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Maternal lipopolysaccharide exposure significantly increased fetal mortality, reduced fetal weight and crown-rump and tail lengths of live fetuses, and retarded skeletal ossification in caudal vertebrae, anterior and posterior phalanges, and supraoccipital bone in a dose-dependent manner. Alpha-phenyl-N-t-butylnitrone, a free radical spin-trapping agent, almost completely blocked lipopolysaccharide-induced fetal death (63.2% in lipopolysaccharide group versus 6.5% in alpha-phenyl-N-t-butylnitrone + lipopolysaccharide group, P < .01). In addition, alpha-phenyl-N-t-butylnitrone significantly reversed lipopolysaccharide-induced intrauterine growth restriction and skeletal development retardation. However, aminoguanidine, a selective inhibitor of inducible nitric oxide synthase, had little effect. Furthermore, lipopolysaccharide-induced intrauterine fetal death, intrauterine fetal growth restriction, and skeletal development retardation were associated with lipid peroxidation and glutathione depletion in maternal liver, placenta, and fetal liver. Alpha-phenyl-N-t-butylnitrone significantly attenuated lipopolysaccharide-induced lipid peroxidation and glutathione depletion in maternal liver, placenta, and fetal liver. Maternal lipopolysaccharide exposure at late gestational stages results in intrauterine fetal growth restriction and skeletal development retardation in mice. Reactive oxygen species might be, at least in part, involved in lipopolysaccharide-induced intrauterine fetal growth restriction and skeletal development retardation.

Tumor necrosis factor alpha partially contributes to lipopolysaccharide-induced intra-uterine fetal growth restriction and skeletal development retardation in mice

Maternal infection is a cause of adverse developmental outcomes. Lipopolysaccharide (LPS)-induced embryonic resorption, intra-uterine fetal death (IUFD) and preterm labor have been well characterized. In the present study, we investigated the effects of maternal LPS exposure on intra-uterine fetal growth and skeletal development. All pregnant mice except controls received an intraperitoneal injection of LPS (75 μg/kg) on gestational days (GD) 15–17. The number of live fetuses, dead fetuses and resorption sites was counted on GD 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. As expected, perinatal LPS exposure resulted in 63.2% fetal death. LPS significantly lowered fetal weight, reduced crown-rump and tail lengths, and retarded skeletal ossification in caudal vertebrae, anterior and posterior phalanges, and supraoccipital bone. Additional experiment showed that a single dose of LPS (75 μg/kg, i.p.) on GD 15 increased the expression of TNF-α mRNA in maternal liver and placenta and TNF-α concentration in maternal serum and amniotic fluid. Furthermore, pentoxifylline, an inhibitor of TNF-α synthesis, significantly inhibited TNF-α production, reduced fetal mortality, and reversed LPS-induced fetal intra-uterine growth restriction and skeletal development retardation. Taken together, these results suggest that TNF-α is, at least in part, involved in LPS-induced intra-uterine fetal death, intra-uterine growth restriction and skeletal development retardation.

Ascorbic acid protects against lipopolysaccharide-induced intra-uterine fetal death and intra-uterine growth retardation in mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR) and preterm labor. Reactive oxygen species (ROS) mediate LPS-induced developmental toxicity. Ascorbic acid is an antioxidant. In the present study, we investigated the effect of ascorbic acid on LPS-induced IUFD and IUGR in mice. All ICR pregnant mice except controls received an intraperitoneal (75 μg/kg, i.p.) injection of LPS daily on gd 15–17. The experiment was carried out in three different modes. In mode A, the pregnant mice were pretreated with a single dose (500 mg/kg, i.p.) of ascorbic acid before LPS. In mode B, the pregnant mice were administered with a single dose (500 mg/kg, i.p.) of ascorbic acid at 3 h after LPS. In mode C, the pregnant mice were administered with 500 mg/kg (i.p.) of ascorbic acid at 30 min before LPS, followed by additional dose (500 mg/kg, i.p.) of ascorbic acid at 3 h after LPS. The number of live fetuses, dead fetuses and resorption sites was counted on gd 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were examined and skeletal development was evaluated. Results showed that maternally administered LPS significantly increased fetal mortality, decreased fetal weight and crown-rump and tail lengths of live fetuses, and retarded skeletal ossification in caudal vertebrae, anterior and posterior phalanges, and supraoccipital bone. LPS-induced IUFD and IUGR were associated with lipid peroxidation and GSH depletion in maternal liver, placenta and fetal liver. Pre-treatment with ascorbic acid significantly attenuated LPS-induced lipid peroxidation, decreased fetal mortality, and reversed LPS-induced fetal growth and skeletal development retardation. By contrast to pre-treatment, post-treatment with ascorbic acid had less effect on LPS-induced IUFD, although post-treatment significantly attenuated LPS-induced lipid peroxidation and reversed LPS-induced fetal growth and skeletal development retardation. Furthermore, post-treatment with ascorbic acid reduced the protective effects of pre-treatment on LPS-induced IUFD. All these results suggest that pre-treatment with ascorbic acid protected against LPS-induced fetal death and reversed LPS-induced growth and skeletal development retardation via counteracting LPS-induced oxidative stress, whereas post-treatment had less effect on LPS-induced IUFD.

Effect of N-Acetylcysteine on Lipopolysaccharide-Induced Intra-uterine Fetal Death and Intra-uterine Growth Retardation in Mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including embryonic resorption, intra-uterine fetal death (IUFD), intra-uterine growth retardation (IUGR), and preterm delivery. Reactive oxygen species (ROS) have been associated with LPS-induced developmental toxicity. N-acetylcysteine (NAC) is a glutathione (GSH) precursor and direct antioxidant. The present study investigated the effects of NAC on LPS-induced IUFD and IUGR. All pregnant mice except controls were injected with LPS (75 microg/kg, ip) on gestational day (GD) 15-17. NAC was administered in two different modes. In mode A, the pregnant mice were pretreated with two doses of NAC (either 50 plus 25 mg/kg or 200 plus 100 mg/kg) before LPS, one (either 50 or 200 mg/kg) at 12 h before LPS and the other (either 25 or 100 mg/kg) at 15 min before LPS. In mode B, the pregnant mice were administered with two doses of NAC (either 50 plus 25 mg/kg or 200 plus 100 mg/kg) in 24 h, one (either 50 or 200 mg/kg) injected immediately after LPS and the other (either 25 or 100 mg/kg) injected 3 h after LPS. The number of live fetuses, dead fetuses and resorption sites was counted on GD 18. Live fetuses in each litter were weighed. Crown-rump and tail lengths were measured and skeletal development was evaluated. Results showed that pretreatment with NAC significantly alleviated LPS-induced fetal mortality and reversed LPS-induced growth and skeletal development retardation. Correspondingly, pretreatment with NAC significantly attenuated LPS-induced elevation in TNF-alpha concentration in maternal serum and amniotic fluid and lipid peroxidation in maternal and fetal livers. By contrast to pretreatment, posttreatment with NAC had no effect on LPS-induced TNF-alpha production and lipid peroxidation. When administered after LPS, NAC did not protect against LPS-induced IUFD and IUGR and in fact aggravated LPS-induced preterm labor. All these results indicate that NAC had a dual effect on LPS-induced IUFD and IUGR. Pretreatment with NAC improves fetal survival and reverses LPS-induced fetal growth and skeletal development retardation, whereas posttreatment with NAC aggravates LPS-induced preterm labor.

Skeletal abnormalities and ultrastructural changes of cartilage in transgenic mice expressing a collagen II gene (COL2A1) with a Cys for Arg-α1-519 substitution

Objective To examine the mechanism by which the Arg→Cys 519 mutation causes the clinical phenotype employing transgenic mice that express the mutated human COL2A1. Methods A DNA construct under the control of a COL2A1 specific promoter was prepared from genomic DNA isolated from fibroblasts from the proband with primary generalized osteoarthritis (OA) associated with a mild chondrodysplasia. Transgenic mice were obtained by injection of the constructs into pro-nuclei of fertilized eggs from the FVB/N inbred mouse strain. Transgenic mice harboring two alleles of the mutated human COL2A1 were examined for morphological abnormalities and for alterations of their skeletal development. Ultrastructural examination was performed to identify changes in the organization and density of collagen II fibrils in articular cartilage of the transgenic mice. Results Transgenic mice harboring two alleles of the mutated human collagen gene were smaller than their normal littermates, had a cleft palate, and disorganized growth plate. Electron microscopy of articular cartilage showed a decreased density of collagen II fibrils and revealed chondrocytes with dilated Golgi cysternae. Conclusions Expression of a COL2A1 with an Arg→Cys 519 substitution in transgenic mice causes retardation of skeletal development and ultrastructural alterations in articular cartilage with a profound reduction of the density of the collagen II fibrils in the tissue. These alterations may be responsible for the phenotype of precocious generalized OA and chondrodysplasia displayed by patients harboring this COL2A1 mutation. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

Retardation of skeletal development and cervical abnormalities in transgenic mice expressing a dominant-negative retinoic acid receptor in chondrogenic cells.

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen alpha1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

Interactive effects of alcohol and diabetes during pregnancy on the rat fetus

To determine the effect of maternal diabetes and alcohol intake, separately and in combination, on fetal growth and development, pregnant rats were divided into four groups: diabetic (D), diabetic plus alcohol (DA), control (C), and control plus alcohol (CA). Diabetes was induced by administration of streptozotocin before mating and alcohol was administered by gavage (2 g/kg body weight/day) on days 6-11 of gestation. Both diabetic groups (D and DA) had significantly lower weight gain during pregnancy compared to the controls (C and CA), despite the fact that the former consumed more food and water. Alcohol treatment resulted in reduced water and food intake and lower weight gain in the diabetic rats (DA), but not in the non-diabetic rats (CA), compared to their respective controls (D and C). On day 21 of gestation fetal body weights were significantly less and placental weights were significantly greater in the diabetic groups (D and DA) compared with the non-diabetic groups (C and CA). Differences in fetal and placental weights between rats exposed and not exposed to alcohol (C vs. CA and D vs. DA) were not significant. The number of fetuses with external malformations was significantly greater in the litters of alcohol exposed diabetic (DA) than non-alcohol exposed (D) animals. No external or skeletal malformations were observed in fetuses of non-diabetic rats regardless of whether or not they received alcohol (C or CA). The skeletal development of fetuses of diabetic rats, judged by the number and size of ossification centers on day 21 of gestation, was retarded when compared with fetuses of non-diabetic rats. Alcohol further retarded skeletal development of fetuses of diabetic animals (DA vs. D), but not of fetuses of non-diabetic rats (CA vs. C). It is concluded that maternal alcohol administration potentiates the effects of maternal diabetes on the incidence of fetal malformations and the retardation of skeletal development.

Timed interruption of insulin therapy in diabetic BB/E rat pregnancy: effect on maternal metabolism and fetal outcome.

Experimental and clinical studies have suggested that periods of poor metabolic control in early diabetic pregnancy have an adverse effect on the developing embryo, but the precise nature and mechanism of this damaging influence have not been defined. In this study the effect of withdrawing treatment with insulin for 2 days at various times during early gestation on maternal metabolism and fetal outcome has been investigated in the spontaneously diabetic BB/E rat. Non-diabetic BB/E rats and diabetic BB/E rats treated continuously with insulin throughout pregnancy served as controls. Continuously treated diabetic rats had a higher rate of fetal resorption and bigger placentae and their offspring had fewer ossification centres, lower extractable pancreatic insulin content, larger hearts, and smaller kidneys and lungs than the offspring of non-diabetic rats. Interruption of treatment with insulin further aggravated the adverse effect of diabetes on the outcome of pregnancy by resulting in a further increase in the rate of fetal resorption, a rise in the neonatal death rate, a reduction in fetal body weight, and retardation of skeletal development. These effects were more apparent when interruption of treatment with insulin occurred during the period of organogenesis, i.e. during gestational days 8 and 9, and 10 and 11. Two severe malformations were seen, both in litters originating from mothers whose treatment with insulin was interrupted during and immediately before fetal organogenesis. We conclude that a period of disturbed maternal metabolism during fetal organogenesis is capable of affecting the survival, growth, and organ development of the fetus and that the spontaneously diabetic insulin-dependent BB rat appears to be a good model for studies of the effect of diabetes and its treatment on the outcome of pregnancy.

Diabetes in Pregnancy: Skeletal Malformations in the Offspring of Diabetic Rats After Intermittent Withdrawal of Insulin in Early Gestation

Precise timing of the teratogenic period in diabetic pregnancy is of clinical importance since correction of the glucose intolerance during this period may protect the offspring from malformations. An experimental approach to elucidate this problem with regard to skeletal development was made in groups of pregnant streptozotocin-diabetic rats (MDI), which were treated with daily insulin injections except for a 2-day period in the first half of pregnancy. The degree of metabolic derangement was estimated by measurements of serum glucose concentrations. During the insulin-free period, the rats showed severe hyperglycemia (greater than 20 mM) while during ongoing insulin treatment, only brief periods of hyper- or hypoglycemia were observed. Insulin treatment was withdrawn successively between gestational days 3 and 12. Control groups consisted of normal pregnant rats (N) or pregnant rats with manifest diabetes (MD) without insulin treatment. The serum glucose levels of the N animals were below 6 mM while those of the MD animals were above 25 mM throughout pregnancy. Skeletal malformations in the viable offspring were recorded on gestational day 20 after Alizarin staining of calcified ossification centers, which also allowed an estimate of skeletal development as a whole. Untreated diabetes in the MD rats induced a high rate of fetal resorptions, a decrease in fetal weight and viability, as well as retardation of skeletal development. Intermittent insulin treatment in the MDI rats ameliorated, but did not abolish, these changes. In the MD group 9 of 48 viable fetuses showed severe malformations of either the lower jaw (micrognathia) or of the lumbosacral region (caudal dysgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)

Teratogenic effects of proline analogue L-azetidine-2-carboxylic acid in hamster fetuses.

The teratogenic effects of L-azetidine-2-carboxylic acid (LACA) were evaluated in hamsters. Single or multiple intraperitoneal injections of LACA were given on gestational days 7-12. Fetal weights were significantly reduced in several LACA-treated groups as compared to controls. The frequency of external anomalies was significantly higher than controls in only two LACA-treated groups: 7.2% in 600 mg/kg on day 12 and 9.6% in 200 mg/kg/day on days 10-12. Subcutaneous hemorrhage and cleft palate were the most frequently occurring externally visible defects. Skeletal anomalies such as retarded ossification, synchronous vertebral ossification and shortening of bones were found in a high percentage of LACA-treated fetuses. The major teratological effect of LACA appeared to be the retardation of skeletal development.