The use of whole rat embryo cultures to identify and characterize causes of reproductive failure.

The most important problem facing human teratology today is to identify the actual causes of this health problem. We have used cultures of whole rat embryos to address this problem using blood sera from individuals at risk as embryo culture media for this purpose. Through serum fractionations and nutrient supplementations to the serum we have studied drugs (dilantin, valproic acid), nutrient deficiencies (methionine) and an embryotoxic autoantibody to the protein laminin. In addition to identifying these factors it has been possible to address their mechanisms of action and to provide recommendations for treatment.

Effects of methionine on the cytoplasmic distribution of actin and tubulin during neural tube closure in rat embryos.

Research has previously shown that, without methionine supplements, neural tube proteins of rat embryos cultured on bovine sera were hypomethylated and neural tubes failed to close. In the present study, to identify the proteins that became methylated during neurulation, rat embryos were first cultured on methionine-deficient bovine serum for 40 hr, then incubated with puromycin for 1 hr, and, finally, incubated with [methyl-14C]methionine and puromycin for 5 hr. On the basis of molecular weights, isoelectric points, and Western immunoblots, the methyl-14C-labeled proteins were identified as actin, alpha beta-tubulin, and neurofilament L. Indirect immunofluorescence studies indicated that without the addition of methionine to the culture, localization of actin and alpha beta-tubulin in the basal cytoplasm did not occur and these neuroepithelial cells lost their columnar morphology.

The direct embryotoxicity of immunoglobulin G fractions from patients with systemic lupus erythematosus.

PROBLEM: To determine if IgG fractions from sera of individuals with systemic lupus erythematosus (SLE) were toxic to cultures of whole rat embryos. METHODS: Head-fold stage rat embryos (9.5 days of gestation) were cultured on media consisting of 50% rat serum containing IgG fractions isolated from plasmapheresis plasma of six subjects with SLE and six with other autoimmune diseases. Each fraction was tested at 11 mg/ml and those toxic were also tested at 7.5 and 4 mg/ml. RESULTS: Of the six SLE IgG fractions, four were embryotoxic (embryolethal or teratogenic) while only one of the six non-SLE fractions were embryotoxic. CONCLUSION: IgG fractions from subjects with SLE can be toxic to cultures of whole rat embryos in the absence of maternal tissues or influence. Such cultures of whole embryos may be useful to identify those antibodies that represent a risk for fetal loss as well as to understand their mechanisms of embryotoxicity.

Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.

Methionine overcomes neural tube defects in rat embryos cultured on sera from laminin-immunized monkeys.

Sera from laminin-immunized monkeys were previously found to cause neural tube defects in cultures of whole rat embryos by unknown mechanisms. In the present study, adding L-methionine to either the culture media or to the diets of the monkeys overcame the toxicity of the serum from one of these monkeys (LAM3) but not the other (LAM4). The antilaminin antibody levels and avidities for isolated murine laminin of sera from the two monkeys were comparable. However, when yolk sac homogenates were tested on ELISA, antibodies from LAM4 had greater binding than LAM3, which was further supported by immunoelectron microscopy. These differences in antibody binding were explained by the findings that antibodies from LAM4 recognized more epitopes than LAM3 and that LAM4 recognized specific epitopes not recognized by LAM3. These antibodies caused reductions in the number of microvilli on the cells and the cell sizes of the yolk sac endoderm. In addition, uptake of [14C]methionine, [14C]sucrose and [14C]valine by yolk sacs from embryos cultured on serum from LAM4 was less than that for LAM3. We suggest that the neural tube defects caused by the antilaminin antibodies were a result of reduced nutrient flow caused by the reduction in the number of microvilli on the cells of the yolk sac endoderm.

Effects of laminin monoclonal antibodies on the development of cultured rat embryos.

Whole rat embryos (9.5 days of gestation) were exposed to six different monoclonal antibodies to laminin during 48 hr of culture. Four (LAM I, LAM V, 5A2, 9D2) of the six were teratogenic or lethal and two (LAM II, 5D3) were not toxic at comparable levels. Teratogenicity and lethality were not related to antibody level, subclass or affinity for whole laminin. Indirect immunofluorescence studies using mouse embryo sections revealed that the toxic antibodies bound in a diffuse manner, while the nontoxic antibodies showed distinct labeling of tissues. These observations suggest that previous varied responses seen in cultured rat embryos exposed to laminin antibodies obtained from humans, monkeys, and rats were the result of differences in the epitope specificity of those antibodies.

Rat embryo development on human sera is related to numbers of previous spontaneous abortions and nutritional factors.

OBJECTIVES: The objectives were to determine (1) if sera from women with histories of spontaneous abortions were teratogenic to cultured embryos more often than were sera of nonaborters, (2) if the teratogenicity could be corrected by adding nutrients to the sera, and (3) if these findings were relevant to reproductive outcomes. STUDY DESIGN: Rat embryos were cultured for 48 hours on sera from 102 subjects who had experienced spontaneous abortions. Samples from 48 were retested with nutrients added and 10 took dietary supplements, were again tested with embryo cultures, and reported on their pregnancy outcomes. RESULTS: The frequencies of teratogenic sera increased with numbers of spontaneous abortions (0 to > or = 5) in a manner that did not deviate from linearity (27% to 89%) (chi 2 p > 0.957). Nutrient supplements were added to 48 samples, and 40 were corrected and 10 subjects were given dietary supplement. Sera from six showed improved embryo cultures, and these women completed their pregnancies. CONCLUSIONS: Rat embryo cultures may provide unique insights into the causes and treatment of spontaneous abortions.

Role of laminin autoantibodies on the embryo toxicity of sera from mercuric chloride treated brown Norway rats.

In previous studies, antilaminin antibodies were found to be toxic to cultured rat embryos. In order to extend these studies, Brown Norway rats were treated with mercuric chloride, which led to the production of laminin autoantibodies. Sera samples from brown Norway rats treated with mercuric chloride were found to be teratogenic as well as lethal to cultured rat embryos. This embryotoxicity was not associated with sera mercury levels, but was related to the levels of antilaminin antibodies in sera. Affinity purified laminin antibodies from these mercuric chloride treated Brown Norway rats, when added to control sera, were found to be teratogenic but not lethal. These antibodies were found to bind to the laminin sequences IKVAV (A chain) and YIGSR (B1 chain), but not RGD (A chain) or YD (B1 chain). These observations suggested the possibility that an environmental pollutant such as mercury could cause the formation of embryotoxic autoantibodies that could persist in the body as embryotoxic factors for extended periods of time.

Methionine decreases the embryotoxicity of sodium valproate in the rat: in vivo and in vitro observations.

Methionine provided in the drinking water of pregnant rats injected with sodium valproate reduced the frequency of resorptions but did not improve embryo growth. Rats drinking methionine supplemented water had approximately twice the level of serum-free methionine and consumed only one-half the volume of water of controls. Using whole rat embryo cultures, the simultaneous addition of methionine and sodium valproate to the medium provided no protection from neural tube defects, nor did the addition of methionine to a medium of serum obtained from rats previously dosed with sodium valproate. However, protection from the teratogenic effects of sodium valproate was afforded by methionine when the culture medium was sera from rats consuming methionine and was particularly striking when embryos for culture were taken from pregnant rats that had been consuming methionine. These observations along with those of others indicated the importance of dietary and culture media methionine levels in evaluating experimental and regulatory teratology studies and suggested the possibility that methionine may play an important role in human teratology where multifactorial causes have been implicated in problems such as neural tube closure defects.

Toxicity of sera from individuals with Chagas' disease to cultured rat embryos: role of antibodies to laminin.

In previous studies antilaminin antibodies in the sera of immunized monkeys and rats were found to be toxic to cultured rat embryos. In order to extend these studies to humans, head-fold stage rat embryos were cultured for 48 hours on ten different serum samples from individuals with Chagas' disease. All embryos (n = 20) cultured on these sera were found to be abnormal. Using ELISA, Western immunoblot, and indirect immunofluorescence it could be shown that antibodies in these sera reacted with laminin. That these antilaminin antibodies were, at least in part, responsible for the toxicity was indicated 1) by reduced cultured embryo toxicity for six of seven serum samples after pre-absorption with purified laminin, 2) by demonstrating a relationship between the amount of affinity-purified antilaminin IgG added to control serum for culture and the severity of embryo abnormalities seen at the end of culture, and 3) by the sera's failing to react with other basement membrane proteins, including type IV collagen, fibronectin, osteonectin, and heparan sulfate proteoglycan.

Methionine and neural tube closure in cultured rat embryos: morphological and biochemical analyses.

When headfold-stage rat embryos were cultured on cow serum, their neural tubes failed to close unless the serum was supplemented with methionine. Methionine deficiency did not appear to affect the ability of the neural epithelium to fuse as a type of fusion was observed between anterior and posterior regions of the open neural tube in methionine-deficient embryos. Although methionine deficiency reduced the cell density and mitotic indices of cranial mesenchyme and neural epithelial cells, this did not appear to be a factor in failure of the neural tube to close. For example, embryos cultured on diluted cow serum also had fewer mesenchymal cells yet could complete neural tube closure if provided with methionine. Examination of the tips of the neural folds suggested that microfilament contraction could be involved; in the absence of methionine the neural folds failed to turn in. This possibility was supported by the reductions in neurite extension of isolated neural tubes cultured without methionine and by the reductions in microfilament associated methylated amino acids contained in embryo neural tube proteins.

Acetaminophen toxicity to cultured rat embryos.

We tested the effects of acetaminophen on cultured rat embryo development. When added directly to culture media at 300 microM, a concentration approximately twice the human therapeutic blood level, acetaminophen caused abnormalities in the cultured embryos. Sera from both rats and monkeys following gavage with acetaminophen were also toxic to cultured embryos. The sera toxicities were related to acetaminophen concentrations, and the toxicity could be removed by serum dialysis. With regard to the metabolism of acetaminophen, glutathione levels in the yolk sac decreased in a concentration related fashion with addition of the drug. Also, buthionine sulfoximine, an inhibitor of glutathione synthesis, appeared to enhance acetaminophen embryo toxicity, and N-acetylcysteine, a glutathione precursor, appeared to protect embryos from acetaminophen toxicity. These results suggested that acetaminophen embryo toxicity resulted from direct exposure of embryos to acetaminophen and not a maternal metabolite.

Whole rat embryos require methionine for neural tube closure when cultured on cow serum.

Headfold-stage rat embryos, when cultured on cow serum without supplemental methionine, failed to close their neural tubes, lacked eyes and branchial arches, were abnormally shaped and were reduced in protein content compared to methionine-supplemented embryos. Methionine was essential during the first 18 h of culture, a period in which neural tube closure was initiated in supplemented cultures. All cow serum samples tested were found to require methionine addition, and the methionine was not replaced by other amino acids or vitamins, including folate. Methionine was not toxic to cultured rat embryos at concentrations up to at least 500 micrograms/ml. Analyses of serum free amino acids revealed lower levels of free methionine in cow serum compared to rat serum, and cow serum proteins contained less methionine relative to other amino acids than did rat serum proteins. Dialysis of cow serum reduced but did not eliminate the requirement for methionine. This suggested either that the free amino acids of cow serum were imbalanced or that a dialyzable component in serum interfered with the availability/utilization of methionine. Dietary supplementation of cows with rumen-protected DL-methionine increased the serum methionine level, and serum drawn from supplemented cows supported normal rat embryo development without additional methionine.

Laminin immunized monkeys develop sera toxic to cultured rat embryos and fail to reproduce.

In a previous study antilaminin antibodies in a monkey with a poor reproductive history were found to be the cause of serum toxicity to cultured rat embryos. In the present study four monkeys were immunized with murine tumor laminin and a fifth with bovine serum albumin. Subsequently, sera from only the laminin immunized monkeys became toxic to cultured rat embryos. This serum toxicity was not mediated by complement but did require the antibody to have a divalent structure. Finally, mating trials were conducted with two of the laminin immunized monkeys that previously had excellent reproductive histories. Based on progesterone levels and observation the monkeys continued to have normal menstrual cycles but failed to initiate a successful pregnancy following immunization in over 2 years of mating trials. These data demonstrated that antibodies against laminin could have prevented conception or could have interrupted pregnancy because of embryotoxicity or failure of implantation.

Autoantibodies to laminin and other basement membrane proteins in sera from monkeys with histories of reproductive failure identified by cultures of whole rat embryos.

Head-fold-stage rat embryos cultured on sera taken from monkeys with histories of reproductive failure had an abnormality frequency of 97% compared with only 7% on sera taken from monkeys with excellent reproductive histories. For a group of these poor reproducers, the toxicity of their sera was associated with the immunoglobulin G (IgG) fraction. These IgG fractions bound to Reichert's membrane and other basement membranes of the embryo. For one monkey, the IgG specifically reacted with a 41 kDa polypeptide of Reichert's membrane, while for two others binding was to laminin, type IV collagen, and several other minor polypeptides of Reichert's membrane. For serum from one monkey, the toxicity to cultured rat embryos was eliminated by absorption with laminin but not type IV collagen.

In vitro and in vivo embryo toxicity of antilaminin antibodies in the rat.

Rats mated after laminin immunization had higher frequencies of resorptions (57%) than those immunized with bovine serum albumin (20%) and had sera that were toxic to cultured rat embryos. In addition, sera from rats immunized with laminin A chains but not B chains were toxic to cultured embryos. The toxicity of sera to embryos was related to the reactivity of sera to specific laminin fragments rather than to sera IgG levels against intact laminin. In addition, resorptions in pregnant rats immunized with laminin were not related to the sera antilaminin IgG levels. However, levels of uterine antilaminin IgA, the predominant uterine isotype, increased considerably during the first 3.5 days of pregnancy, while sera antilaminin IgG remained constant. (Am J Reprod Immunol.

Methionine and iron as growth factors for rat embryos cultured in canine serum.

Development of headfold-staged rat embryos cultured in canine serum containing various supplements was compared with development in rat serum to seek suitable alternatives to rat serum in rodent embryo culture and to identify nutritional factors for cultured rodent embryos that may have relevance for normal mammalian embryonic growth and development. Supplementation of canine serum with glucose, methionine, and a lipophilic iron chelate allowed growth and development of cultured rat embryos, approximating those obtained with rat serum. These findings suggest that properly supplemented canine serum can serve as a suitable rodent embryo culture medium and that glucose, iron, and methionine may be important nutrients in mammalian embryonic development.

Heat shock and thermotolerance during early rat embryo development.

Effects of heat shock on the development of early pre-somite embryos have been studied using cultured rat embryos. The results illustrate the sensitivity of the developing head and brain to elevated temperatures prior to neural tube closure and the capacity of embryos to acquire thermotolerance. Embryos exposed briefly to an elevated temperature (43 degrees C for 7.5 min) developed severe craniofacial defects including microphthalmia, microcephaly, gross reduction of the forebrain region, and open neural tubes. In contrast, a nonteratogenic heat shock (42 degrees C for 10 min) caused embryos to acquire thermotolerance during a 15-min recovery period at 38.5 degrees C. Acquired thermotolerance was effective in protecting embryos from a subsequent more severe heat treatment which would have been teratogenic in an unprotected embryo. Recovering embryos mounted a heat shock response as evidenced by the induction of a 71 kilodalton heat shock protein. Activation of the heat shock response was not a teratogenic event in the developing embryo.