Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice

Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.     

Viability and development of 'tube-locked' mouse embryos

'Tube-locked' morulae and blastocysts were recovered from the ampulla of the oviduct of centchroman-treated mice between Days 4 and 12 post coitum and transferred to the uteri of pseudopregnant female mice. Pregnancy and implantation rates were lower and the post-implantation resorption rate was higher in the treated than in the control group. There was little difference in the pregnancy or implantation rates between embryos recovered on Days 4 or 12 post coitum, but the resorption rate increased with increasing duration of embryos in the oviducts and was 100% for the Day-12 embryos. The resorption rate was similar even when these embryos were transferred to the sterile uterine horn of unilaterally pregnant mice. Centchroman did not produce any deleterious effect on embryos which survived until Day 19 of pregnancy in foster mothers. The average fetal weight was also comparable to those of control fetuses.     

Neuromuscular development following tetrodotoxin-induced inactivity in mouse embryos

Developmental aspects of the neuromuscular system in mouse embryos chronically paralyzed in utero with tetrodotoxin (TTX) between embryonic days 14 and 18 were studied using biochemical and histological methods. The number of lumbar spinal motoneurons (MNs) was higher in inactive embryos than in controls suggesting a decreased motoneuron cell death. In association with the increase in MN number, choline acetyltransferase activity was significantly increased in both spinal cord and peripheral synaptic sites. Paralyzed muscles exhibited a decreased number of mature myofibers and the nuclei were centrally located. Creatine kinase activity was greatly decreased and total acetylcholine receptor and receptor cluster numbers per myofiber were significantly increased in paralyzed muscles. A similar pattern of changes occurs in the neuromuscular system of the mutant mouse muscular dysgenesis (mdg). However, in contrast to the mdg mutant, tetrodotoxin-treated muscles were similar to controls in their innervation pattern, in the ultrastructural aspects of the excitation–contraction coupling system (i.e., dyads and triads) and in the extent of dihydropyridine binding. Thus, neuromuscular inactivity is not sufficient to impair the pattern of muscle innervation or the appearance of either the triadic junctions or dihydropyridine receptors. These results indicate that alterations of dihydropyridine binding sites and triads in muscular dysgenesis cannot be accounted for by inactivity but rather must reflect a more primary defect involving the structural gene(s) regulating the development of one or more aspects of muscle differentiation.     

Abnormal development of cardiovascular systems in rat embryos treated with bisdiamine

Administration of N,N'-bis(dichloroacetyl)-1,8-octamethylenediamine, bisdiamine, in pregnant Donryu rats on day 10 of gestation induces a high incidence of cardiovascular anomalies in fetuses. Bisdiamine administration induced aplasia of the sixth aortic arch artery, with both the right and left primitive pulmonary arteries being directly linked to the truncus, and resulting in four types of malformation of pulmonary arteries (PAs). When two primitive PAs shared a single root, the consequence was either pulmonary trunk hypoplasia, as is seen in tetralogy of Fallot, or type I persistent truncus arteriosus (PTA) as classified by Collet and Edwards. When root portions of two PAs did not fuse, either type II or type III PTA resulted. In controls, the right dorsal aorta (DA) between the right seventh intersegmental artery (IA) and the site where both DAs fuse degenerated and the left aortic arch (AA) and the right subclavian artery (SA) were formed. Bisdiamine administration induced two additional types of vascular anomalies. In one of these, the right DA between the right 4AA and the right 7IA degenerated and a left AA accompanied by an aberrant right SA resulted. In the other type, the left DA between the left 4AA and the left 7IA degenerated and a right AA accompanied by an aberrant left SA resulted. These results indicate that administration of bisdiamine induces malformation in the great blood vessels by disturbing persistency and degeneration of aortic arch arteries and DAs.     

Cardiac hypertrophy in copper-deficient rats is owing to increased mitochondria

Dietary copper depletion results in cardiac hypertrophy and ultrastructural alterations. The objective of this study was to determine the components that contribute to cardiac enlargement. Two groups (n = 4) of male, weaning, Sprague-Dawley rats were fed ad libitum with copper-adequate or copper-deficient diets for five weeks. Cross sectional transmission electron micrographs from both groups were evaluated using image analysis to quantify absolute area occupied by myocyte, mitochondria, myofibril, and other intracellular material. Copper-deficient rats had larger myocytes, increased area of mitochondria, and increased ratio of mitochondria :myofibril as well as mitochondria:myocyte. Copper deficiency did not change the absolute area occupied by myofibrils. These data suggested that increase in the absolute mitochondria area is the major contributory factor to the cardiac hypertrophy in copper deficiency. Under the conditions used, myofibril has minimal role toward contributing to the hypertrophic state. The pathology reported resembles human forms of genetic mitochondrial cardiomyopathies. The copper-deficient rat may be a useful model to investigate the underlying biochemical or molecular responses when peptides of enzymes are deleted.     

The development of XO gynogenetic mouse embryos

Diploid gynogenetic embryos, which have two sets of maternal and no paternal chromosomes, die at or soon after implantation. Since normal female embryos preferentially inactivate the paternally derived X chromosome in certain extraembryonic membranes, the inviability of diploid gynogenetic embryos might be due to difficulties in achieving an equivalent inactivation of one of their two maternally derived X chromosomes. In order to investigate this possibility, we constructed XO gynogenetic embryos by nuclear transplantation at the 1-cell stage. These XO gynogenones showed the same mortality around the time of implantation as did their XX gynogenetic counterparts. This shows that the lack of a paternally derived autosome set is sufficient to cause gynogenetic inviability at this stage. Autosomal imprinting and its possible relation to X-chromosome imprinting is discussed.     

Abnormal corneal and eyelid development in the repeated epilation mouse

Development of the eyelids and cornea in repeated epilation (Er/Er) mice is characterized by fusion of the tarsal conjunctiva to the epithelia of the bulbar conjunctiva and cornea. We investigated the ultrastructural features of this malformation and tested for abnormal expression of filaggrin by immunofluorescence. Heterozygous (Er/+) breeding stock were mated and 13,14,15, and 19-day-old embryos were studied by light and electron microscopy. Fusion occurred in all Er/Er specimens and was associated with abnormal migration of surface ectodermal cells onto the cornea. Immunofluorescence studies with antimouse filaggrin antibody on day 13 and day 15 revealed the presence of filaggrin precursors in the fused epithelia of mutants, but not in normal corneal or conjunctival epithelia. The results suggest defective regulation of the synthesis of cellular proteins and altered cell surface properties in the Er/Er ocular epithelia.     

Early development and X-chromosome inactivation in mouse parthenogenetic embryos.

Early development and X-chromosome inactivation were studied in ethanol-induced mouse parthenogenones. About 24% of oocytes transferred to 0.5-day pseudopregnant recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6- to 8-day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X-inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.     

Purines inhibit the development of mouse embryos in vitro

The first cleavage of embryos derived from random-bred, inbred, and hybrid-inbred female mice was not arrested by purines at concentrations as high as 30 microM. Development after the first or second cleavage was arrested by hypoxanthine, adenosine or inosine, but not guanosine. In agreement with previous results, the purine-induced block was reversed when arrested embryos were transferred to purine-free media after 24 h in culture. The cleavage arrest was not due to elevations of cAMP as a result of inhibition of phosphodiesterase activity since similar concentrations of phosphodiesterase inhibitors or dibutyryl cAMP did not block development. Treatment with inhibitors of enzymes that convert IMP to AMP or to GMP did not reverse the hypoxanthine-induced block, thus demonstrating that mitotic arrest is mediated by a mechanism different from the hypoxanthine arrest of meiosis. Thymidine incorporation studies showed that the block did not prevent the onset of DNA synthesis. The results reveal a profound sensitivity to purine inhibition of a cell process that occurs during the first 30 h of mouse embryo development and is necessary for progession through the G2 or M phases of the second or third cleavage.     

Effects of irradiation on facial development in mouse embryos

The purpose of this study was to observe the effects of irradiation on the craniofacial development of NMRI mouse embryos. Two populations of pregnant mice were irradiated with a single dose of 2 Gray on day 8 of gestation for the first population (Po. 1) and on day 9 of gestation for the second population (Po. 2). On gestational days 9 to 17, embryos were submitted to histological and scanning electron microscope examinations. The two populations of embryos presented a high percentage of centro-facial hypoplasia (74.7% for Po. 1 and 75% for Po. 2) which was more pronounced in the latter one. Ocular anomalies were present in 16% of the first population. Cases of anencephaly, cleft palate and anomalies of the central nervous system were found in both populations.     

Teratogenicity of 3-hydroxynorvaline in chicken and mouse embryos

Summary. 3-Hydroxynorvaline (HNV; 2-amino-3-hydroxypentanoic acid), a microbial L-threonine analogue, is toxic to mammalian cells and displays antiviral properties. In view of this, we investigated the toxicity and/or potential teratogenicity of HNV in developing chicken and mouse embryos. HNV was administered to chicken embryos (in ovo; dose 75–300 μmole/egg; 48 h post-incubation) and pregnant Hanover NMRI mice (per os; total dose 900–1800 mg/kg body mass; gestation days 7–9). Control animals received sterile saline solutions. Harvested embryos (chicken embryos, 10 days post-incubation; mouse embryos; gestation day 18) were fixed in glutaraldehyde and stereomicroscopically inspected for signs of dysmorphogenesis. Body mass, body and toe length and mortality of chicken embryos, and the body mass and mortality of mouse embryos were recorded. HNV exposure significantly increased the incidence of embryotoxic (growth retardation, toxic mortality) and congenital defects in both chicken and mouse embryos. All the observed effects were dose-dependent. In conclusion, HNV is an embryotoxic and teratogenic compound, which caused significant developmental delay and congenital defects in developing chicken and mouse embryos.     

Cell and tissue-autonomous development of the circadian clock in mouse embryos

The emergence of the circadian rhythm is a dramatic and physiologically essential event for mammals to adapt to daily environmental cycles. It has been demonstrated that circadian rhythms develop during the embryonic stage even when the maternal central pacemaker suprachiasmatic nucleus has been disrupted. However, the mechanisms controlling development of the circadian clock are not yet fully understood. Here, we show that the circadian molecular oscillation in primary dispersed embryonic cells and explanted salivary glands obtained from mPER2^Luc mice embryos developed cell- or tissue-autonomously even in tissue culture conditions. Moreover, the circadian clock in the primary mPER2^Luc fibroblasts could be reprogrammed by the expression of the reprogramming factors. These findings suggest that mammalian circadian clock development may interact with cellular differentiation mechanisms.