Teratogenic effects of maternal biotin deficiency on mouse embryos examined at midgestation

Pregnant mice were fed a basal diet that not only did not contain biotin, but also contained the spray-dried egg white including avidin that caused the biotin deficiency. The effects of maternal biotin deficiency on craniofacial and limb development in embryos were examined at two stages of midgestation. On day 12.6 of gestation, male and female embryos weighted less and digit development was retarded in the biotin-deficient group. On day 15.6 of gestation (dg), the embryos also weighted less and external malformations, such as micrognathia (94.8%), micromelia (41.4%), and exencephaly (11.4%), were observed. The inhibition of palatal and digit formation by biotin deficiency at midgestation is responsible for later formation of cleft palate and micromelia. On dg 12.6 the liver biotin level of biotin-deficient dams was reduced to 20% of control values. Interestingly, the biotin content of the whole embryonic body was about ninefold greater than liver biotin levels in their dams.     

Marginal biotin deficiency is teratogenic

Recent studies of biotin status during pregnancy provide evidence that a marginal degree of biotin develops in a substantial proportion of women during normal pregnancy. Several lines of evidence suggest that, although the degree of biotin deficiency is not severe enough to produce the classic cutaneous and behavioral manifestations of biotin deficiency, the deficiency is severe enough to produce metabolic derangements in women and that characteristic fetal malformations occur at a high rate in some mammals. Moreover, our analysis of data from a published multivitamin supplementation study provide significant albeit indirect evidence that the marginal degree of biotin deficiency that occurs spontaneously in normal human gestation is teratogenic. Investigation of potential mechanisms provides evidence that biotin transport by the human placenta is weak. Further, proliferating cells accumulate biotin at a rate five times faster than quiescent cells; this observation suggests that there is an increased biotin requirement associated with cell proliferation. Perhaps this requirement arises from the need to synthesize additional biotin-dependent holocarboxylases or provide additional biotin as a substrate for biotinylation of cellular histones. Reduced activity of the biotin-dependent enzymes acetyl-CoA carboxylase and propionyl-CoA carboxylase can cause alterations of lipid metabolism and might theoretically lead to alterations of polyunsaturated fatty acid and prostaglandin metabolism that derange normal skeletal development.     

Sex difference in digit and palate formation of mouse embryos at midgestation

We investigated whether there is a difference in timing between the sexes of mouse embryos with regard to digit and palatal formation at midgestation by using a simple method of sex chromatin analysis for rodent embryos. At day 14.4 of gestation, although the mean body weight of male embryos was greater than that of female embryos, digit and palatal formation of female embryos was found to be more advanced than in males when compared according to their body weight. This was contrary to our previous finding of digit formation at the earlier stage (day 12.0 of gestation): namely, that digit development was more advanced in male embryos than in female embryos even when two sexes were compared according to their body weight. Thus, the development of female embryos catches up with that of male embryos at the later stages of midgestation. The period for digit development must be longer in male embryos than in female embryos. If mothers are exposed to some teratogens, a sex difference in incidence of digital defects might be produced.     

Marginal biotin deficiency is teratogenic in mice and perhaps humans: a review of biotin deficiency during human pregnancy and effects of biotin deficiency on gene expression and enzyme activities in mouse dam and fetus

Recent studies of biotin status during pregnancy provide evidence that a marginal degree of biotin deficiency develops in a substantial proportion of women during normal pregnancy. Several lines of evidence suggest that although the degree of biotin deficiency is not severe enough to produce the classic cutaneous and behavioral manifestations of biotin deficiency, the deficiency is severe enough to produce metabolic derangements in women and may be teratogenic. In studies of mice, a similar degree of biotin deficiency induces characteristic fetal malformations at a high rate. Fetal hepatic biotin content and PCC activity decrease indicating that the fetuses also become biotin deficient. Fetal hepatic acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and beta-methylcrotonyl-CoA carboxylase abundances determined by Western blotting decreased more than the dam holocarboxylase abundances (10% of sufficient vs. 50% of sufficient); however, hepatic mRNA for the carboxylases and for HCS did not change significantly in either dams or fetuses. These observations suggest that maternal biotin deficiency results in a lack of adequate biotin to biotinylate apocarboxylases in the fetus despite the normal expression of genes coding for the apocarboxylases and holocarboxylase synthetase.     

Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7-30 microM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) micrograms Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 microM Zn medium. Embryos from the -Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from -Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 microM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed -Zn diets for a 3- or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.     

Strain difference of mouse in susceptibility to Japanese encephalitis virus infection

Eight strains of mice were examined for their susceptibilities to intraperitoneal infection with AS-6 strain of Japanese encephalitis virus (JEV). 1) C3H/He mice suffered from a high mortality as well as infection rate. 2) C57BL/6, RR, NC and KK mice showed approximately the same infection rates as C3H/He, while these strains showed significantly lower mortalities than C3H/He. 3) AA, BALB/c and ddY mice showed no death and had the lowest infection rates among the eight strains. There was no difference in the virus recovery from six visceral organs (except the brain) between C3H/He, C57BL/6 and AA. Despite the equal degree of preceding viremia, the incidence of encephalitis was much lower in C57BL/6 than in C3H/He. The same strain difference as the above was also observed in C3H/He and C57BL/6 by intravenous inoculation with JEV. However, there was no difference in mortality between C3H/He and C57BL/6 mice when intracerebrally inoculated with JEV. The incubation period and survival time in the intracerebral inoculation were shorter than in the intraperitoneal and intravenous inoculations. The three types of strains were characterized: the first (C3H/He) was highly susceptible to both visceral phase infection (VI) and nervous phase infection (NI): the second (C57BL/6) was susceptible to VI but resistant to NI, and the third (AA) was probably resistant to VI and highly resistant to NI.     

Sex difference in mouse embryonic development at neurulation

Sixty-seven mouse embryos from 10 litters collected on the morning of Day 9 of gestation, when neurulation is beginning, were classified according to the precise stage of development reached, and sex-chromatin analysis was performed on the yolk sac. Within litters, the least developed embryos were more likely to be female than male, while the most advanced embryos were predominantly male. Taking all embryos, the mean somite number was greater in males than females.     

Propylthiouracil is teratogenic in murine embryos

Background

Hyperthyroidism during pregnancy is treated with the antithyroid drugs (ATD) propylthiouracil (PTU) and methimazole (MMI). PTU currently is recommended as the drug of choice during early pregnancy. Yet, despite widespread ATD use in pregnancy, formal studies of ATD teratogenic effects have not been performed.

Methods

We examined the teratogenic effects of PTU and MMI during embryogenesis in mice. To span different periods of embryogenesis, dams were treated with compounds or vehicle daily from embryonic day (E) 7.5 to 9.5 or from E3.5 to E7.5. Embryos were examined for gross malformations at E10.5 or E18.5 followed by histological and micro-CT analysis. Influences of PTU on gene expression levels were examined by RNA microarray analysis.

Results

When dams were treated from E7.5 to E9.5 with PTU, neural tube and cardiac abnormalities were observed at E10.5. Cranial neural tube defects were significantly more common among the PTU-exposed embryos than those exposed to MMI or vehicle. Blood in the pericardial sac, which is a feature indicative of abnormal cardiac function and/or abnormal vasculature, was observed more frequently in PTU-treated than MMI-treated or vehicle-treated embryos. Following PTU treatment, a total of 134 differentially expressed genes were identified. Disrupted genetic pathways were those associated with cytoskeleton remodeling and keratin filaments. At E 18.5, no gross malformations were evident in either ATD group, but the number of viable PTU embryos per dam at E18.5 was significantly lower from those at E10.5, indicating loss of malformed embryos. These data show that PTU exposure during embryogenesis is associated with delayed neural tube closure and cardiac abnormalities. In contrast, we did not observe structural or cardiac defects associated with MMI exposure except at the higher dose. We find that PTU exposure during embryogenesis is associated with fetal loss. These observations suggest that PTU has teratogenic potential.     

Hypothermia: teratogenic and protective effects on the development of mouse embryos in vitro

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.     

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.