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.     

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.     

Synthesis of Substituted Aminomalonates by the Reaction of Chloramine with Malonate Carbanions

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.     

Teratogenic effects of avidin-induced biotin deficiency in mice

Teratogenic effects of maternal biotin deficiency were examined at different levels of severity by adding three levels of avidin (10, 20, or 40 mg/kg) in the basal diet. There was a considerable increase of fetuses with multiple congenital malformations (micrognathia, cleft palate, and micromelia) with increasing amounts of avidin. The dose-response relationship was observable in the incidence of each malformation as well. The body weight of live fetuses was also significantly reduced. However, the dams did not exhibit any typical signs of biotin deficiency, such as loss of hair, dermatitis, or nervous irritability. These results suggest that biotin is important for early embryonic development in the mouse.     

Digit development and embryonic weight in mice: analysis of sex-related time difference and mating period-related interlitter variability

The embryonic growth and digit formation in limb buds were more advanced in male embryos than in female embryos at a specific time (day 12.0) of midgestation. Furthermore, when the number of digits was compared between the sexes according to their body weight, male embryos were found to be more advanced than females in the differentiation of the digit in limb buds. This is the first demonstration of the presence of a time difference in digit development between the sexes of mouse embryos. In the short-period, morning-mating group, embryonic weights at day 12.0 were lower than those in the overnight-mating group. However, the digit development was not very much delayed in proportion to the difference in body weights, and some "catch-up" phenomena were observed in this group. Interlitter and intralitter variability in body weights of mouse embryos at day 12.0 was greater in the overnight-mating group than in the short-period-mating group. These findings suggest that, in embryonic stage-related teratological experiments in mice, a short-period-mating schedule is advised and that the incidence of developmental anomalies should be analyzed separately for male and female fetuses.     

Biotin deficiency and biotin excess: Effects on the female reproductive system

Biotin deficiency and biotin excess have both been found to affect reproduction and cause teratogenic effects. In the reproductive tract, however, the effects of biotin have not been well established yet. We investigated the effects of varying biotin content diets on the oestrus cycle, ovarian morphology, estradiol and progesterone serum levels, and the uterine mRNA abundance of their nuclear receptors, as well as on the activity of the estradiol-degrading group of enzymes cytochrome P450 (CYP) in the liver. Three-week-old female BALB/cAnN Hsd mice were fed a biotin-deficient, a biotin-control, or a biotin-supplemented diet (0, 7.2 or 400 μmol of free biotin/kg diet, respectively) over a period of nine weeks. Striking effects were observed in the biotin-deficient group: mice showed arrested estrous cycle on the day of diestrus and changes in ovary morphology. Estradiol serum concentration increased 49.2% in biotin-deficient mice compared to the control group, while the enzymatic activities of CYP1A2 and CYP2B2 increased (P < 0.05). The mRNA abundance of nuclear estrogen and progesterone receptors decreased in the biotin-deficient mice. In the biotin-supplemented group we found that, in spite of a significant (P < 0.05) decrease in the number of primary and Graafian follicles and in CYP1A2 activities, mice exhibited 105.4% higher serum estradiol concentration than the control group. No changes in the expression of the nuclear receptors were observed. No significant differences were observed in serum progesterone among the groups. Our results indicate that both the deficiency and the excess of biotin have significant effects on the female mouse reproductive system.     

Biotin deficiency and biotin excess: Effects on the female reproductive system

Biotin deficiency and biotin excess have both been found to affect reproduction and cause teratogenic effects. In the reproductive tract, however, the effects of biotin have not been well established yet. We investigated the effects of varying biotin content diets on the oestrus cycle, ovarian morphology, estradiol and progesterone serum levels, and the uterine mRNA abundance of their nuclear receptors, as well as on the activity of the estradiol-degrading group of enzymes cytochrome P450 (CYP) in the liver. Three-week-old female BALB/cAnN Hsd mice were fed a biotin-deficient, a biotin-control, or a biotin-supplemented diet (0, 7.2 or 400 μmol of free biotin/kg diet, respectively) over a period of nine weeks. Striking effects were observed in the biotin-deficient group: mice showed arrested estrous cycle on the day of diestrus and changes in ovary morphology. Estradiol serum concentration increased 49.2% in biotin-deficient mice compared to the control group, while the enzymatic activities of CYP1A2 and CYP2B2 increased (P < 0.05). The mRNA abundance of nuclear estrogen and progesterone receptors decreased in the biotin-deficient mice. In the biotin-supplemented group we found that, in spite of a significant (P < 0.05) decrease in the number of primary and Graafian follicles and in CYP1A2 activities, mice exhibited 105.4% higher serum estradiol concentration than the control group. No changes in the expression of the nuclear receptors were observed. No significant differences were observed in serum progesterone among the groups. Our results indicate that both the deficiency and the excess of biotin have significant effects on the female mouse reproductive system.     

Growth and differentiation schedule of mouse embryos obtained from delayed matings.

We previously showed that digit formation in mouse embryos from early morning mating seemed to progress faster than those from overnight mating. In this study, to confirm this phenomenon, we examine whether the embryos from normal (0 hr from ovulation to fertilization) and delayed matings (3, 6, or 9 hr from ovulation to fertilization) respond differently to some acute teratogens when they are treated at the same time point from mating. Five mg/kg of cytosine arabinoside (Ara-C) was given to pregnant mice intraperitoneally at 246, 249, or 252 hr (day of gestation (dg) 10) after mating. The patterns of Ara-C induced digit malformations in embryos from the delayed mating groups were those of more advanced stages, when compared with normal mating groups with the same time intervals from mating to Ara-C treatment. In other words, oocytes fertilized up to 9 hr after the presumed time of ovulation could grow similarly to those of normally fertilized oocytes. This catch-up phenomenon suggests that the ovulation clock should be used for the startpoint of the time scale of the growth and differentiation of embryos rather than fertilization clock.     

Dose- and stage-related sex difference in the incidence of cytosine arabinoside induced digit anomalies in the mouse fetus

Pregnant mice of the Jc1:ICR strain were injected intraperitoneally with a single dose of 2.5, 5, or 10 mg/kg of cytosine arabinoside (Ara-C) on day 9.5 or 10.5 of gestation (dg). Mice were killed on 18.4 dg, and the incidences of digit anomalies were compared between male and female fetuses. In the group treated with 10 mg of Ara-C on 9.5 dg, oligodactyly of the hindlimbs was produced more frequently in male fetuses. In the group treated with 5 mg of Ara-C on 10.5 dg, oligodactyly of the forelimbs tended to occur more frequently in male fetuses. Also, in this group, oligodactyly of the hindlimbs occurred more frequently in male fetuses, whereas polydactyly occurred more frequently in female fetuses. The observed difference in the incidences of digit anomalies between male and female fetuses may be explained either in terms of the sex-related time lag in the critical period for digit development or by a probable sex difference in embryonic susceptibility to Ara-C.     

Biotin deficiency decreases life span and fertility but increases stress resistance in Drosophila melanogaster

Biotin deficiency is associated with fetal malformations and activation of cell survival pathways in mammals. In this study we determined whether biotin status affects life span, stress resistance, and fertility in the fruit fly Drosophila melanogaster. Male and female flies of the Canton-S strain had free access to diets containing 6.0 (control), 4.8, 2.5, or 0 pmol biotin/100 mg. Biotin concentrations in diets correlated with activities of biotin-dependent propionyl-CoA carboxylase and biotin concentrations in fly homogenates, but not with biotinylation of histones (DNA-binding proteins). Propionyl-CoA carboxylase activities and biotin concentrations were lower in males than in females fed diets low in biotin. The life span of biotin-deficient males and females was up to 30% shorter compared to biotin-sufficient controls. Exposure to oxidative stress reversed the effects of biotin status on survival in male flies: survival times increased by 40% in biotin-deficient males compared to biotin-sufficient controls. Biotin status did not affect survival of females exposed to oxidative stress. Exposure of flies to cold, heat, and oxidative stress was associated with mobilization of biotin from yet unknown sources. Biotin deficiency decreased fertility of flies. When biotin-deficient males and females were mated, the hatching rate (larvae hatched per egg) decreased by about 28% compared to biotin-sufficient controls. These findings are consistent with the hypothesis that biotin affects life span, stress resistance, and fertility in fruit flies.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death

The role of collagen I for midgestation development was studied in homozygous Mov 13 embryos, which cannot synthesize alpha 1(1) mRNA as a result of insertional mutagenesis and most of which die between day 12 and 14 of gestation. No type I collagen was detected in mutant embryos, while the distribution of other collagens, laminin, and fibronectin was not affected. Mutant embryos develop normally up to day 12 of gestation, suggesting that collagen I has no essential role in the early phase of morphogenesis. The first pathological events were detected in hemopoietic cells of the liver, followed by necroses of mesenchymal cells in other parts of the embryo. The sudden death is caused by the rupture of a major blood vessel, indicating an important role for collagen I in establishing the mechanical stability of the circulatory system. Our results furthermore suggest that complex cell interactions in embryonic development such as those in early hemopoiesis may depend on the presence of collagen type I.     

Assessing the effects of low boron diets on embryonic and fetal development in rodents using in vitro and in vivo model systems

To date, boron (B) essentiality has not been conclusively shown in mammals. This article summarizes the results of a series of in vitro and in vivo experiments designed to investigate the role of B in mammalian reproduction. In the first study, rat dams were fed either a low (0.04 μg B/g) or an adequate (2.00 μg B/g) B diet for 6 wk before breeding and through pregnancy; reproductive outcome was monitored on gestation day 20. Although low dietary B significantly lowered maternal blood, liver, and bone B concentrations, it had no marked effects on fetal growth or development. The goal of the second study was to assess the effects of B on the in vitro development of rat postimplantation embryos. Day 10 embryos collected from dams fed either the low or adequate B diets for at least 12 wk were cultured in serum collected from male rats exposed to one of the two dietary B treatments. Dams fed the low B diet had a significantly reduced number of implantation sites compared to dams fed the B-adequate diet. However, embryonic growth in vitro was not affected by B treatment. The aim of study 3 was to define the limits of boric acid (BA) toxicity on mouse preimplantation development in vitro. Two-cell mouse embryos were cultured in media containing graded levels of BA (from 6 to 10,000 μM). Impaired embryonic differentiation and proliferation were observed only when embryos were exposed to high levels of BA (>2000 μM), reflecting a very low level of toxicity of BA on early mouse embryonic development. Study 4 tested the effects of low (0.04 μg B/g) and adequate (2.00 μg B/g) dietary B on the in vitro development of mouse preimplantation embryos. Two-cell embryos obtained from the dams were cultured in vitro for 72 h. Maternal exposure to the low B diet for 10, 12, and 16 wk was associated with a reduction in blastocyst formation, a reduction in blastocyst cell number, and an increased number of degenerates. Collectively, these studies support the concept that B deficiency impairs early embryonic development in rodents.     

Effects of age and biotin status on postnatal development of plasma biotinidase activity in rats

Biotinidase activity was measured in plasmas of 1-, 7-, 14-, and 21-day-old rats from control dams and dams that had been fed a biotin-depleting diet from Day 15 of gestation. Biotinidase activity increased significantly in the plasma of rats from control and depleted mothers until Postnatal Day 14, after which there was a small but significant decline at Day 21. Differences between the mean activities of the two groups of pups on each sampling day were not significant and there were no significant differences in activity levels attributable to sex. Plasma albumin concentrations increased from birth until Day 21, and plasma biotinidase activity and albumin concentration were significantly correlated (r = +/- 0.43). We suggested that these two proteins may be controlled by a common mechanism in the early postnatal period, and that biotin deficiency does not affect the development of biotinidase activity. Because biotin-depleted neonatal pups show developmental changes in biotinidase activity similar to those of human newborns, and they can be produced reliably by depleting dams from Day 15 of gestation, they may be useful models for studying the developmental abnormalities associated with human biotinidase deficiency.     

Teratogen susceptibility of XO mothers and XO embryos in mice

We examined whether the chromosomal imbalance inherent in an XO constitution in mice is more susceptible to teratogenic influence of biotin deficiency using a newly established mouse colony with pure X monosomy. We hypothesized that XO mothers or XO embryos might be more susceptible to certain teratogens. Contrary to our expectation, the incidence of external malformations induced by biotin deficiency did not differ either between XX dams and XO dams or between XX fetuses and XO fetuses.     

Griseofulvin-induced aggregation of microtubule protein.

The mechanism of the vitamin K-dependent post-translational carboxylation of the gamma-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15% of normal. However, one-stage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14CO2 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate; or via a yet to be identified intermediate, or perhaps via CO2 itself.     

Effects of biotin deficiency on pancreatic islet morphology, insulin sensitivity and glucose homeostasis

Several studies have revealed that physiological concentrations of biotin are required for the normal expression of critical carbohydrate metabolism genes and for glucose homeostasis. However, the different experimental models used in these studies make it difficult to integrate the effects of biotin deficiency on glucose metabolism. To further investigate the effects of biotin deficiency on glucose metabolism, we presently analyzed the effect of biotin deprivation on glucose homeostasis and on pancreatic islet morphology. Three-week-old male BALB/cAnN Hsd mice were fed a biotin-deficient or a biotin-control diet (0 or 7.2 μmol of free biotin/kg diet, respectively) over a period of 8 weeks. We found that biotin deprivation caused reduced concentrations of blood glucose and serum insulin concentrations, but increased plasma glucagon levels. Biotin-deficient mice also presented impaired glucose and insulin tolerance tests, indicating defects in insulin sensitivity. Altered insulin signaling was linked to a decrease in phosphorylated Akt/PKB but induced no change in insulin receptor abundance. Islet morphology studies revealed disruption of islet architecture due to biotin deficiency, and an increase in the number of α-cells in the islet core. Morphometric analyses found increased islet size, number of islets and glucagon-positive area, but a decreased insulin-positive area, in the biotin-deficient group. Glucagon secretion and gene expression increased in islets isolated from biotin-deficient mice. Our results suggest that biotin deficiency promotes hyperglycemic mechanisms such as increased glucagon concentration and decreased insulin secretion and sensitivity to compensate for reduced blood glucose concentrations. Variations in glucose homeostasis may participate in the changes observed in pancreatic islets.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

High-protein diet during gestation and lactation affects mammary gland mRNA abundance, milk composition and pre-weaning litter growth in mice

We evaluated the effect of a high-protein diet (HP) on pregnancy, lactational and rearing success in mice. At the time of mating, females were randomly assigned to isoenergetic diets with HP (40% w/w) or control protein levels (C; 20%). After parturition, half of the dams were fed the other diet throughout lactation resulting in four dietary groups: CC (C diet during gestation and lactation), CHP (C diet during gestation and HP diet during lactation), HPC (HP diet during gestation and C diet during lactation) and HPHP (HP diet during gestation and lactation). Maternal and offspring body mass was monitored. Measurements of maternal mammary gland (MG), kidney and abdominal fat pad masses, MG histology and MG mRNA abundance, as well as milk composition were taken at selected time points. HP diet decreased abdominal fat and increased kidney mass of lactating dams. Litter mass at birth was lower in HP than in C dams (14.8 v. 16.8 g). Dams fed an HP diet during lactation showed 5% less food intake (10.4 v. 10.9 g/day) and lower body and MG mass. On day 14 of lactation, the proportion of MG parenchyma was lower in dams fed an HP diet during gestation as compared to dams fed a C diet (64.8% v. 75.8%). Abundance of MG α-lactalbumin, β-casein, whey acidic protein, xanthine oxidoreductase mRNA at mid-lactation was decreased in all groups receiving an HP diet either during gestation and/or lactation. Milk lactose content was lower in dams fed an HP diet during lactation compared to dams fed a C diet (1.6% v. 2.0%). On days 14, 18 and 21 of lactation total litter mass was lower in litters of dams fed an HP diet during lactation, and the pups' relative kidney mass was greater than in litters suckled by dams receiving a C diet. These findings indicate that excess protein intake in reproducing mice has adverse effects on offspring early in their postnatal growth as a consequence of impaired lactational function.