Morphological and physiological effects of beta-hydroxybutyrate on rat embryos grown in vitro at different stages

Diabetic women are more likely to give birth to infants with congenital malformations than are nondiabetic women. Rodent embryos have been used as a model for the study of abnormal fetal development associated with maternal diabetes, and some of the metabolic factors which are altered in diabetes, such as raised glucose and ketones, have been shown to cause abnormal development of rodent embryos in vitro. The present work explores further the teratogenicity of beta-hydroxybutyrate to rat embryos. To determine the sensitivities of rat embryos at different stages of their development, rat embryos at 9.5 days of gestation have been cultured in vitro for 24 or 48 h, with or without 4 x 10(-2) M beta-hydroxybutyrate for all or part of the culture period. The embryos have been examined by scanning electron microscopy, and a detailed morphometric analysis of one tissue, the neuroepithelium, has been undertaken. The results confirm that beta-hydroxybutyrate causes abnormal development of rat embryos. The results of experiments in which embryos were exposed to beta-hydroxybutyrate for only part of a 48 h culture show that embryos exposed to beta-hydroxybutyrate for a complete 48 h culture are more severely affected than embryos exposed to beta-hydroxybutyrate for only part of the culture and that embryos are more vulnerable to beta-hydroxybutyrate during the first half of a 48 h culture (equivalent to 9.5 to 10.5 days of gestation) than during the second half of a 48 h culture (10.5 to 11.5 days of gestation). The results of experiments in which embryos were cultured with beta-hydroxybutyrate from 9.5 days of gestation for 24 h (equivalent to 9.5 to 10.5 days of gestation) showed that some effects of beta-hydroxybutyrate are already apparent after 24 hours in culture. Many of the abnormalities produced by beta-hydroxybutyrate can be classified as embryonic retardations rather than malformations--that is, embryos show features characteristic of normal, but younger, embryos. Embryos exposed to beta-hydroxybutyrate for the complete 48 h culture period consume less glucose and produce less lactate than control embryos on a per embryo basis, but not on a per microgram protein basis, suggesting that the reduced metabolism is an effect of beta-hydroxybutyrate-induced developmental delay rather than a cause of it.(ABSTRACT TRUNCATED AT 400 WORDS)     

The toxicity of niridazole in rat embryos in vitro

The antischistosomal drug niridazole (NDZ) was found to be teratogenic in a concentration-dependent manner from 10 to 50 micrograms/ml (47-233 microM) in rat embryos cultured from day 10 to day 11. A striking malformation consisting of axial asymmetry in which the right side of the embryo showed severe necrosis was the predominant malformation. The drug showed significantly greater dysmorphogenic activity at low (5%) compared to high (20%) oxygen tensions in cultures. Coincubation of embryos and NDZ with an exogenous source of metabolic enzymes and cofactors (NADPH) failed to modify teratogenicity. Inclusion of CO in the culture atmosphere significantly reduced the malformation incidence as did preincubation of the drug with S-9 and cofactors in medium with low O2 tension. Treatment of gravida with NDZ up to and including the maternal-lethal dose failed to result in observable malformations despite the use of several routes of exposure. These data lead us to hypothesize that rat embryos are capable of performing the reductive activation of NDZ in a fashion analogous to the schistosome but that reductive extraembryonic metabolism may result in teratogenic bioinactivation.     

The in vitro embryotoxicity of 5-fluorouracil in rat embryos

The fluorinated pyrimidine 5-fluorouracil (5-FU) is an effective chemotherapeutic agent that is teratogenic in a number of species. The mechanism for the embryopathic effect of the drug is unknown. We examined the effects of this compound on gestation day 10.5 rat embryos cultured for 48 hours in a rodent whole embryo culture system. Embryos were exposed for 1-4 hours to various doses of 5-FU. Embryolethality was minimal in all treatment groups. The malformation frequency increased with higher doses; within a dose, the malformation frequency increased with longer exposure to the drug. The tail and hindlimb bud were the most commonly affected structures in vitro; tail and leg defects are produced in several species by exposure to the drug in vivo. The embryopathic drug concentration in the culture media (2-8 micrograms/ml) is similar to the plasma level of 2-17 micrograms/ml, which is associated with embryopathy in vivo. Results from this study suggest that the whole embryo culture system is an appropriate model for developmental toxicity studies of 5-FU.     

Comparison of the effects of retinol and retinoic acid on postimplantation rat embryos in vitro

Rat embryos were explanted on day 8 or 9 of pregnancy and cultured for up to 48 hours in serum containing added retinol (vitamin A), retinoic acid (vitamin A acid), or absolute ethanol. They were examined morphologically and their protein content determined. Retinoic acid was more teratogenic and growth-retarding than retinol. Electron microscopy of embryos cultured for 30 minutes or one hour revealed that both forms of vitamin A brought about similar ultrastructural effects on the embryonic cells; however, the abnormally large intracellular lipid droplets observed in a previous study following exposure to retinol in vitro and retinyl palmitate in vivo were not observed in embryos exposed to retinoic acid. It is possible that the differential teratogenicity may be due to the inability of the embryonic cells to convert and store retinoic acid in a less teratogenic form.     

Niridazole metabolism by rat embryos in vitro

We report the results of studies on the reductive activation of the schistosomicidal agent, niridazole (NDZ). Intact rat embryos in vitro reduced this compound, generating a stable metabolite in the presence of 5% O2. By contrast, embryo and yolk sac homogenates or liver microsomes appeared to require anaerobiasis. Malformation incidence--specifically, axial asymmetry--showed a strong correlation with nitroreductase activity rates when the latter were modulated by oxygen tension. Data presented here suggest that when embryos are exposed to NDZ under conditions of low oxygen in vitro, redox cycling ensues with molecular oxygen serving to oxidize early reduction products. This process continues, regenerating the parent compound until oxygen is depleted locally. The basis of this localized depletion is unknown, but inability of the immature supply system to replete oxygen or demand by precociously aerobic tissues may be involved. Once local anaerobiasis is attained, further reduction could generate toxic metabolites capable of covalently binding cellular macromolecules. Localized hypoxia represents another potential mechanism of dysmorphogenesis.     

Morphological differences elicited by two weak acids, retinoic and valproic, in rat embryos grown in vitro

We compared in rat whole-embryo culture the morphological changes elicited by valproic acid (VPA) with those elicited by trans-retinoic acid (RA). Rat embryos explanted on day 9.5 of gestation were treated on day 10 with RA or VPA at concentrations producing equivalent reductions in embryonic protein. The concentrations selected for morphological assessment by scanning and transmission electron microscopy, 2.3 and 800 microM, respectively, for RA and VPA, produced approximately a 50% incidence of abnormally open anterior neuropores in initial range-finding experiments in the culture system. Protein and DNA analyses were also performed on corresponding groups of embryos at three different doses. With concurrent control groups used as reference standards, the two treatment groups were compared for differences in external and internal morphology, protein and DNA contents, and growth indices. While certain variables responded similarly in the two treatment groups, e.g., the growth variables, protein and DNA contents, each drug produced selective morphological effects. Whereas treatment with RA produced underdeveloped branchial arches, symmetrically cleft cranial defects resulting in openings in rhombencephalic and prosencephalic regions, and exteriorized neural tissue in the caudal neuropore region, VPA produced irregular clefts with wavy margins along the entire length of the neural tube, and an open caudal neuropore without eversion of the neuroepithelium, while producing no detectable effect on the branchial arches. The similar effects of these two drugs on protein and DNA contents suggest comparable degrees of overall toxicity; however, the dissimilar effects on neural tube and branchial arches, coupled with the large difference in concentration of the drug required to produce the effects, add to the evidence that their mechanisms for elicitation of abnormal development are qualitatively different.     

Effects of thyroid antagonists on rat embryos cultured in vitro

A literature review of individual pregnancies and recent surveys involving large cohorts reveal an association between congenital malformation and maternal hyperthyroidism, suggesting that some aspect of hyperthyroidism or its treatment might compromise the development of the fetus. Experiments have shown that the thyroid antagonist, ethylenethiourea (ETU), causes fetal malformations when administered to pregnant rats, but it is not known whether it is ETU or the imbalance in maternal thyroid hormone which it causes which is the teratogenic agent. Here we employ in vitro culture to determine the possible direct effects on rat embryos of two thyroid antagonists, ETU and methimazole (MMI), the latter being one which is used for treatment of thyrotoxicosis in humans. It was found that ETU can compromise the development of rat embryos in vitro, confirming that ETU has a direct effect on the rat embryo. It was also found that MMI can cause abnormal development of rat embryos in vitro, although the concentration at which MMI disturbs rat embryogenesis is higher than that which is reached in hyperthyroid patients treated with clinical doses of MMI or carbimazole.     

Responses to sucrose and glutamine by soybean embryos grown in vitro

Immature soybean (Glycine max [L.] Merr. cv. Ransom) embryos were grown in vitro in the presence of different concentrations of sucrose and glutamine to examine how availability of carbohydrate and nitrogen affects dry matter accumulation and embryo composition. Embryos were transferred to fresh medium every 4 days to maintain sucrose and glutamine concentrations of the culture medium. In all experiments, accumulation of dry matter and protein content increased when the sucrose concentration of the culture medium was increased from 1.5 to 150 mM: however, a relatively greater enhancement of dry matter than of protein accumulation resulted in a lower protein concentration at 150 than at 1.5 mM sucrose. Both content and concentration of protein were increased by the increases in glutamine supply to concentrations exceeding 68% protein at 120 mM glutamine. In combination with 150 mM sucrose, however, oil increased as glutamine supply was increased from 0.6 to 6 mM and then decreased as glutamine supply was increased from 6 lo 120 mM. Varying the concentration of sucrose available during seed development also affected embryo composition. Decreased availability of sucrose during either the early or late portion of the culture period resulted in lower accumulation of dry mailer as well as oil. Protein concentration was actually higher for embryos transferred from 150 to 1.5 nM sucrose than for those remaining in 150 mM throughout the culture period: however, the greater percentage of protein was due lo a decrease in accumulation of dry weight. In addition, embryo composition was affected by altering the availability of glutamine during culture, indicating that variation in the level of nitrogen assimilate delivered during seed development can change embryo composition. Decreasing the glutamine concentration of the medium lowered both protein and oil content. In contrast, increasing the glutamine concentration of the medium from 0.6 to 6 mM 8 days after initiation of culture increased the protein content and concentration of the embryo while oil content was not affected.     

Lack of attenuation of valproic acid-induced effects by folinic acid in rat embryos in vitro

The anticonvulsant drug valproic acid (VPA) is suspected to be a developmental toxicant in humans, inducing primarily neural tube defects. The mechanism for this effect is unknown, but it has been suggested that the drug may act via a deficiency of the vitamin folic acid. We examined this possibility by concurrent treatment of rat embryos in a whole embryo culture system with VPA and folinic acid (FA), a folic acid derivative. Groups of CD rat embryos were treated with various concentrations of VPA, various concentrations of FA, or a combination of a teratogenic dose of VPA plus various levels of FA. At the end of the 44 hour culture period, each embryo was evaluated for viability (presence of a heartbeat), yolk sac circulation, presence of any malformations, morphological score, crown-rump and head lengths, as well as DNA and protein contents. The anticonvulsant did not decrease viability but did decrease yolk sac circulation and all growth and developmental endpoints in a dose-responsive manner. There was also a dose-related increase in the incidence of open neural tubes. The addition of FA alone had no significant effect on growth and development. When various concentrations of FA were added simultaneously with a teratogenic dose of VPA, there was no decrease in the incidence of open neural tubes. Growth and developmental endpoints were altered in a somewhat random fashion but were never increased to the control level. The lack of attenuation by FA was not due to instability of the compound in the culture system, nor was there a difference in the amount of FA present in the exocoelomic fluid of VPA-treated and control embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of buffalo embryos using oocytes from in vitro grown preantral follicles

The present study examines the use of buffalo preantral follicles as a source of oocytes for in vitro embryo production. Preantral follicles were isolated from abattoir-derived buffalo ovaries and were grown for 100 days in five different culture systems: (1) minimum essential medium (MEM); (2) coconut water; (3) MEM + ovarian mesenchymal cell (OMC) co-culture; (4) MEM + granulosa cell (GC) co-culture; or (5) MEM + cumulus cell (CC) co-culture. Low growth rates for the preantral follicles were observed when follicles were cultured in MEM or coconut water medium. Moderate growth rates were seen for OMC and GC co-cultures, and high rates of growth were observed when follicles were grown in CC co-culture. The survival of preantral follicles was low in the MEM culture (<25%), but was over 75% in the other culture systems. Oocytes were not recovered from the MEM group, while an oocyte recovery rate of 80-100% was observed when the follicles were cultured with coconut water/somatic cells. Transferable embryos could be produced only with the oocytes obtained from preantral follicles grown in the OMC and CC co-culture systems. This study demonstrates, for the first time, that it is possible to produce buffalo embryos by in vitro fertilization of oocytes derived from in vitro grown preantral follicles.     

Study of aconitine toxicity in rat embryos in vitro

BACKGROUND: Aconitum is widely used in traditional medicine for its anti-inflammatory, analgesic, and cardiotonic properties. Knowledge is limited, however, on its effects on embryonic development. METHODS: Whole embryo culture was applied to explore the effects of aconitine on rat embryos during their critical period of organogenesis. All embryos isolated on gestational day 9.5 were exposed to 0, 1, 2.5, 5, and 10 microg/ml of aconitine with and without S9 mix, and scored for their growth and differentiation at the end of the 48-hr culture period. RESULTS: The embryonic growth and development were adversely affected at the concentration of 2.5 microg/ml aconitine without S9 mix, represented as reduced crown-rump length and head length, decreased number of somites, and lower morphologic score. When the concentration of aconitine was increased to 5 microg/ml, it induced severe dysmorphogenesis effects, including cardiac defect (undivided cardiac tube and inflated pericardial cavity), irregular somites, and brain malformation (e.g., narrow brain vesicles). In the presence of S9 mix, Aconitine toxicity to rat embryos was reduced to a certain extent. CONCLUSIONS: Our study showed that Aconitine had direct embryotoxic effects during the rat organogenetic period. NOAEL was about 1 microg/ml and metabolism in S9 mix could induce the attenuation of Aconitine toxicity. Until more is known about the effects of Aconitine in pregnant women, we suggest its use should be treated with caution.     

Effect of lithium carbonate on mouse and rat embryos in vitro

Lithium is effective in the treatment of manic-depressive psychosis but is suspected to be a developmental toxicant in humans. It is a developmental toxicant in mice and rats in vivo, but at human therapeutic serum levels of 0.6-1.6 meq/L, rats appear to be more sensitive to the effects of the drug than do mice. The species susceptibility to lithium-induced defects was evaluated by using a rodent whole embryo culture system employing mouse and rat embryos treated at comparable developmental stages. Mouse embryos were cultured on gestational days 8-10, and rat embryos were cultured on gestational days 10-12. Care was taken to insure that all embryos had 10 +/- 2 somite pairs at the beginning of the culture period. Embryos were cultured for 44 hours in rat serum to which lithium was added to attain final drug concentrations of 0.6, 1.2, 1.8, 2.4, or 5.0 meq/L. Control embryos were treated with distilled water, which served as the vehicle. In rats, lithium induced significant decreases in various parameters at 1.8, 2.4, and 5.0 meq/L; no malformations were observed in rats of this stage. In mice, significant decreases occurred at 2.4 and 5.0 meq/L, and embryos treated at the highest concentration had a significantly increased frequency of open neural tubes. Rat embryos were also cultured with lithium on gestational days 9-11. The lowest dose producing developmental toxicity at this stage was 0.6 meq/L. Open neural tubes were present among younger rat embryos; however, this defect occurred in all groups, including the control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ethanol and acetaldehyde on rat embryos developing in vitro

Summary Rat embryos were explanted on Days 9.5 or 10 of gestation and cultured for 48 to 30h, respectively, in rat serum containing 0, 3, 6, 9 mg/ml of Ethanol (Eth); 0, 10, 20 μg/ml of Acetaldehyde (Ach); 3 mg/ml Eth + 10 μg/ml Ach. At the end of the culture period the embryos were evaluated for vitality, and scored. Some of them were also examined histologically. Embryos exposed to Eth from Day 9.5 showed a dose-related growth retardation associated with a high frequency of malformations (open neural tube, heart defects, branchial arch hypoplasia). The exposure of 9.5-day embryos to 20 μg/ml Ach resulted in 100% embryolethality, whereas 10 μg/ml induced growth retardation and teratogenic effects. When 10-day embryos were exposed to 3 mg/ml Eth or 10 μl/ml Ach no effects were observed, but the highest levels of Eth produced a moderate growth retardation and morphologic defects. Exposure to 20 μg/ml Ach induced hypoplasia of the first arch, but did not alter the score value. The histologic examination of these embryos revealed severe lesions at the level of the neuroepithelium and of the branchial mesenchyma. Similar effects were observed in embryos exposed simultaneously to 3 mg/ml Eth and 10 μg/ml Ach. These results should make us reevaluate the role of Ach in the Eth-induced embryopathies.     

The effects of silica dust and alveolar macrophages on lung fibroblasts grown in vitro

The effect of silica (min-u-sil) on lung fibroblasts, with and without the mediation of alveolar macrophages, has been studied by measuring DNA, protein, collagen, lysosomal enzymes and secreted glycosaminoglycans. Intact macrophages were found to stimulate collagen production whether they had first been pretreated with silica or not. The dust has a direct effect on fibroblasts, an effect dependent on silica concentration and the stage of fibroblast growth. The possible relationships of the above effects to both fibrosis and emphysema are discussed.     

Nutrient requirements of rat embryos undergoing organogenesis in vitro

Rat embryos at the head-fold stage were cultured in rotating bottles for 2 days in rat serum extensively dialysed against glucose-free BSS, and to which various energy sources and nutrients were added. Optimum growth and differentiation, virtually indentical with that obtained in while serum and to that seen during the corresponding period in vivo, were obtained with the addition of glucose and certain vitamins: pantothenic acid and riboflavin had a general beneficial effect on development, i-inositol suppressed neural tube defects, and folic acid significantly improved growth of the embryos.