Eighth Day Of Gestation Research Articles

Teratogenic effects of retinoic acid on neurulation in mice embryos

Retinoic acids (RA) are natural chemicals that exert a hormone-like activity and a variety of biological effects on early development of mouse. In this study, the probable teratogenic effects of RA on CNS have been investigated in pregnant mice ( n = 20) divided into four groups: (1) untreated controls, (2) controls which received a single dose of DMSO, (3) a group that received 40 mg/kg, and (4) a group that received 60 mg/kg of all-trans RA in DMSO, respectively on the eighth day of gestation. Embryos whose dams had received 40 and 60 mg/kg doses of RA, showed malformations and decreased size. At 40 mg/kg dosage level, 50% of the embryos had closed neural tubes while at 60 mg/kg dosage level the neural tube failed to close. The neuroblast mantle layers were disorganized in the 40 mg/kg and even more in the 60 mg/kg exposed group compared to the controls. In mitosis, the density of chromatin was increased in the 60 mg/kg dose group. Compared to controls the 40 and 60 mg/kg dose groups of RA treated dams decreases in the luminal longitudinal and internal measures were observed. Also the thickness of ventricular, mantle and marginal layers was smaller. Wide intercellular spaces due to the degenerated cells at high doses of RA as well as an accumulation of intercellular fluid were observed. Therefore, the wedge shape of neuroepithelium was abolished, preventing the elevation of the neural wall.

Pre- and postnatal expression of glycoconjugates (3-fucosyl-N-acetyllactosamine and HNK-1 epitopes) in the mouse inner ear

The distribution of two glycoconjugates 3-fucosyl-N-acetyllactosamine (CD15) and HNK-1 epitope (CD57) in the inner ear of the NMRI mouse was analysed from the eighth day of gestation to the 16th day after birth. CD15 epitope distribution is developmentally regulated. The up- and down-regulation of expression, the change in the number of cells which are positive, the ingrowth of CD15-positive cells and their distribution, intracellular and/or cell-surface-associated expression, all assume a characteristic appearance at each developmental stage. Distribution of CD57 documented the nerve outgrowth and formation of the innervation of the vestibular apparatus and cochlear duct. Correlation between CD15 and CD57 expression patterns revealed differences in the interaction of the ingrowing fibres and epithelial tissue between the vestibular organ and the cochlea and differences in the development of the cristae and maculae.

Relationship between Decidual Leukocyte Infiltration and Spontaneous Abortion in a Murine Model of Early Fetal Resorption

The relationship between the early cellular response to embryo implantation and subsequent embryo survival was explored. Immunohistochemistry using the anti-CDI 1b antibody (Mac-I) was used to localize and quantify maternal inflammatory cells present at the fetoplacental interface. CDI 1b is expressed mostly on macrophages, but is also present on natural killer (NK) cells, neutrophils, and B cells. The occurrence of CDI 1b-positive cells at the fetoplacental interface was quantified in CBA/J females mated by DBA/2 males (20-30% embryo loss) and CBA/J females mated by BALB/c males (5-10% embryo loss) in order to investigate the relationship between infiltration by these types of cells and subsequent embryo loss. CDI 1b-positive cells were found to infiltrate decidua of each embryo starting at Day 6 of gestation. Their numbers sharply increased on Days 7 and 8, to a plateau on Days 8 to 10, well before any damage to the embryo is macroscopically visible on Days 10 to 12 of gestation. The resorption-prone mating of CBA/J female by DBA/2 male showed a significantly elevated number of CDI 1b-positive cells in 26% of the embryos on the eighth day of gestation compared to CBA/J female by BALB/c male matings which were taken as the reference mating. Moreover, experimental conditions modulating fetal survival in CBA/J mothers such as poly (I:C) treatment of DBA/2-mated females (lower survival) or mating with BALB/c males (higher survival than with the mating with DBA/2 males), were found to be associated with high or low numbers of CDI 1b-positive cells at the fetoplacental interface. Furthermore, injection of anti-CDI 1b into pregnant mice at Day 6 of gestation significantly reduced the subsequent incidence of resorption in the resorption prone CBA/J x DBA/ 2 mating. These results suggest that CDI 1b-positive cells are associated with the etiology of spontaneous abortion in this system.

Effects of Hyperbaric Oxygen on Rat Embryos

The aim of the study was to investigate the toxic effects of hyperbaric oxygen (HBO) on rat embryos. Two groups of Fischer pregnant rats were exposed to oxygen at pressures of 324 and 426 kPa for 90 min per day for 5 consecutive days (8-12 days of gestation). The third group was exposed to normobaric oxygen for 12 h on the eighth day of gestation. Two control groups were used; the first was comprised of intact and the second of sham-treated animals. The HBO treatment did not significantly affect maternal weight gain or reduce litter size, nor did it induce any embryonic abnormalities. However, the fetal body weight was reduced and the placental weight increased in the groups exposed to HBO at pressures of 324 and 426 kPa. When female fetuses which had been exposed to oxygen at 324 or 426 kPa were compared to the intact control group, a reduction in wet weights of 9.2 (p < 0.05) and 12.1% (p < 0.01), respectively, was noted. Male fetuses exposed to oxygen at 324 and 426 kPa displayed a reduced body weight of 11.7 (p < 0.01) and 16.6% (p < 0.01), respectively. Placental weight was increased by 18.9 (p < 0.01) and 23.6% (p < 0.01) in the groups exposed to oxygen at 324 and 426 kPa, respectively. These data suggest that HBO, either at 324 or 426 kPa, is not potent at inducing malformations and that the largest embryotoxic effects are upon fetal body weight and placental weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute ethanol intoxication during pregnancy: Postnatal effects on the behavioral response to serotonin agents

Pregnant wistar rats were treated on the eighth day of gestation (GD 8) with two IP injections, spaced by 4 h, of either ethanol (2.9 g/kg in 24% v/v saline solution, EG) or saline (SG). Other pregnant females did not received any type of IP injections (absolute control group, ACG). Offspring were tested at 45 or 90 days of age. At 45 days of age, EG showed an increased behavioral response (forepaw treading and hindlimb abduction) to the 5-HT 1 agonist, 5-methoxy- N, N-dymethyltryptamine. In addition, an enhanced “wet-dog” shakes behavioral response to 5-HT 2 agonist, 5-hydroxy- l-tryptophan, was also observed in EG as compared to ACG and SG. On the contrary, at 90 days, EG exhibited a diminished behavioral reactivity to 5-HT 1 and 5-HT 2 agonists as compared to SG. These results demonstrated that acute administration of ethanol on GD 8 induced long-lasting changes in the functioning of central serotonergic systems.

Cocaine-induced disturbances of corticogenesis in the developing murine brain

Exposure of fetuses to cocaine can induce behavioral disturbances and structural modifications of the CNS. Cocaine was injected into pregnant mice from the eighth day of gestation until term and the brains of their offspring were examined at the light and EM levels. Cocaine severely disturbs neocortical architecture, disrupting horizontal and vertical lamination, and inducing abnormal array of the axonal-dendritic bundles. Cocaine also alters several steps of gliogenesis. The severity of the malformations is variable, but evident in all exposed animals.

Response to an ethanol challenge dose on sleep time and blood alcohol level in Wistar rats prenatally exposed to ethanol during gestational day 8

Pregnant albino rats were treated during the eighth day of gestation (GD 8), with two IP injections, spaced by 4 hours, of either ethanol (2.9 g/kg in 24% v/v saline solution) or saline. Maternal blood alcohol levels reached a peak of 457 mg/dl 60 min after the second dose. At the age of 45 days, an equal number of male and female offspring were injected with 3.5 g/kg ethanol and sleep time and blood ethanol levels were determined upon awakening. Ethanol metabolic rate was studies in other individuals injected with the same dose of ethanol and the slope of the linear descending portion of the curves was calculated. Animals that received ethanol in utero exhibited shorter sleep time and higher blood ethanol levels at the moment of awakening than controls. The rate of ethanol metabolism was similar in both groups. These results show that an acute intoxication with ethanol during GD 8 induced long-term changes in the CNS of offspring which caused reduced sensitivity to ethanol hypnotic effects.

Post-implantation development and cytogenetic analysis of diandric heterozygous diploid mouse embryos.

Diandric heterozygous diploid mouse embryos were produced by standard micromanipulatory techniques using eggs from female mice with a normal chromosome constitution and fertilised by homozygous Rb(1.3)1Bnr males containing a pair of large metacentric marker chromosomes in their karyotype. The constructed diandric eggs were transferred to the oviducts of pseudopregnant recipients and subsequently autopsied midday on the eighth day of gestation. From a total of 85 eggs transferred to females that subsequently became pregnant, 30 implanted. Eighteen implantation sites were found to contain resorptions, and 12 egg cylinder stage embryos were recovered. These were cytogenetically examined. In two cases, no mitoses were observed, and in a third embryo of normal size, only a single paternally-derived marker chromosome was present in its mitoses, indicating that this embryo had a normal chromosome constitution. This presumably resulted from a technical error during the micromanipulatory procedure. The remaining nine morphologically small but normal embryos were diploid, and each had two paternally-derived marker chromosomes, thus establishing their ploidy and confirming their diandric origin. G-banding analysis revealed that all of these embryos had an XY sex chromosome constitution. Since the expected XX:XY:YY ratio of 1:2:1 was not observed, it is clear that the XX class embryos were lost at some stage during the pre- or early post-implantation period, though whether they are represented by the resorption sites is not yet established. The YY class would not be expected to be recovered in any case, as these embryos are believed to be lost during early cleavage. The cytogenetic findings reported here are therefore similar to the results of the chromosomal analyses of the human complete hydatidiform moles of dispermic origin, all of which apparently have an XY karyotype. It is unclear why, both in the human and in the mouse, the XX diandric heterozygous diploid group should develop poorly compared to similar embryos with an XY karyotype.

Concentration and chemical status of arsenic in the blood of pregnant hamsters during critical embryogenesis: 2. Acute exposure

We have determined the concentration and chemical composition of arsenic in the blood of pregnant hamsters between 0.2 and 6 hr after an intraperitoneal injection of a teratogenic dose of radiolabeled sodium arsenate on the morning of the eighth day of gestation. Arsenic was present in plasma and red cells 0.20 hr postinjection. The plasma arsenic concentration reached a maximum of 220 mumole/kg blood near 0.5 hr postinjection. Plasma arsenic existed entirely as low-molecular-weight species. Both arsenite and dimethylarsinate (DMA) were present in plasma 0.20 hr postinjection, indicating that arsenate reduction and methylation of arsenic are rapidly initiated. However, the arsenite contribution remained small while the DMA contribution increased with time. Red cell arsenic included macromolecular arsenic (AsP) as well as three low-molecular-weight forms. The contribution of DMA remained small, but arsenite and AsP contributions increased with time. These findings identify the maternal blood concentration and chemical status of arsenic following the administration of a teratogenic dose of arsenate during the period of organogenesis. They could prove useful for predicting the likelihood of a teratogenic outcome in other mammalian species.

Sirenomelia: analysis in the cadmium- and lead-treated golden hamster.

Sirenomelia, a fusion of the lower extremities, is believed to result from a median, bilateral symmetric defect of the caudal portion of the embryo at a very early stage in development. Anomalies of the gastrointestinal and urogenital systems are commonly associated with this malformation. Sirenomelia is not an embryo-lethal condition but typically is incompatible with postnatal life when combined with the associated malformations. In this study, intravenous treatment of hamsters with a combination of cadmium and lead on the morning of the eighth day of gestation resulted in 1.4, 22.2, and 35.6% of the viable fetuses displaying sirenomelia in litters recovered on days 15, 12, and 10 of gestation, respectively. With the exception of several fetuses with exencephaly, most structures cranial to the level of the umbilicus were normal. Caudal abdominal and pelvic structures were severely affected, with agenesis or dysgenesis of the kidneys, bladder, umbilical arteries, and external genitalia frequently noted. The administration of a combination of cadmium and lead has proven to be an effective and consistent means of inducing sirenomelia in the golden hamster.

Acute alcohol exposure during gestational day 8 in the rat: Effects upon physical and behavioral parameters

Pregnant rats on the eighth day of gestation (GD 8) received 2 intraperitoneal injections (0.015 ml/g body weight) spaced by an interval of 4 hours, of either 0, 6, 12, 18, or 24% (v/v) alcohol solution. Brains of pups sacrificed 24 hours after delivery revealed a dose-related impairment in hemispheric and cerebellar length, width and weight. Statistically significant differences were also observed in body weights of culled pups during Post-partum Day 1. An open field test performed when offspring were 40–45 days old demonstrated a dose-dependent increase in ambulation and rearing scores for both males and females and in defecation scores for females only. Although behavioral studies revealed no effects on acquisition or retention of an active avoidance response, there was a dose-dependent impairment in transfer to passive avoidance conditioning. The present results demonstrate that in the rat, an alcohol insult during GD 8 is sufficient to induce morphological and behavioral alterations in the offspring.

Teratogenic effect of AY 9944 in rats: importance of the day of administration and maternal plasma cholesterol level.

An inhibitor of cholesterol synthesis, AY 9944 (trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride) is teratogenic. A single dose of AY 9944 (50 mg/kg or 75 mg/kg) given to Wistar pregnant rats on the second, fourth, sixth, seventh, or eighth day of gestation induced malformations such as holoprosencephaly. They were often limited to isolated pituitary agenesis. The highest percentage of holoprosencephalic fetuses was found when AY 9944 was given on the fourth day of gestation. Whatever the dose and the day of administration, the lower the maternal plasma cholesterol level, the more frequent were holoprosencephalic fetuses. Therefore, it is suggested that the decrease in maternal plasma cholesterol level is at least one of the factors provoking holoprosencephaly.

Dose-response relationships of retinol in production of the Arnold-Chiari malformation

A single oral dose of 5000, 10000, 15000, 20000 or 30000 I.U. vitamin A alcohol (retinol) on the eighth day of gestation in the pregnant hamster resulted in the production of the Arnold-Chiari malformation types I and II in 0, 6.6, 57, 56 and 85%, respectively, of all fetuses examined. The pattern and severity of the malformations of the chondrocranium and viscerocranium changed as the dose of retinol was increased. The smallest dose of retinol used in the present study which induced gross terata in hamsters was approximately 11 times greater than the highest oral daily dose of retinol used in clinical cancer chemoprevention trials.

Ultrasound biologic effects: a suggestion of strain specificity.

Pregnant hybrid LAF1/J mice were exposed to continuous-wave ultrasonic energy at a frequency of 1 MHz at a spatial peak intensity of either 2.5 W/cm2 or O W/cm2 (sham) for 20 seconds on the eighth day of gestation. Fetal weight (day 18 of gestation) and postpartum pup weight (21, 29 and 42 days post conception) were determined. No significant differences in weight were observed. This result differs from findings in others studies using outbred mouse strains. It is suggested that hybrid mice exposed to ultrasonic energy in utero may be more resistant to alterations in fetal and pup weight progression as compared with outbred strains.

Normal and abnormal midfacial development in the cadmium-treated hamster.

Clefts of the midface ranging in severity from a notched lip to complete facial disorganization were observed in the offspring of golden hamsters injected with cadmium chloride on the morning of the eighth day of gestation. Other malformations frequently observed were microphthalmia, anophthalmia, and diencephalic encephaloceles. Histologic examination of embryos on the 10th, 11th, 12th, and 13th days of gestation revealed a marked deficiency of mesenchyme in the frontonasal process, which led to a foreshortened and deformed nasal septum. These data confirm that the teratogenicity of cadmium is highly specific for the region of the anterior neural segment on the eighth day of gestation. The deficiency of mesenchyme in the frontonasal process may be the result of disruption in neural crest cell development in this region.

Corticosteroid-induced cleft lip in mice: a teratologic, topographic, and histologic investigation.

Unlike cleft palate, relatively few teratogens have been found to induce cleft lip in mice. The present study was designed to assess the teratologic, topographic (SEM), and histologic effects on lip morphogenesis following the administration of triamcinolone hexacetonide on the eighth day of gestation. The frequency of cleft lip in treated A/J mice was found to be more than three times greater than the spontaneous frequency in untreated controls. Comparable studies with other murine strains suggest no association between the cleft lip response and either a maternal effect or the H-2 complex. Affected A/J embryos showed a severe reduction in the size of the lateral nasal processes; affected embryos also demonstrated localized cell type-specific alterations, particularly in the epithelia and at the interface between epithelium and mesenchyme.

Controlled release of tetracycline--III: A physiological pharmacokinetic model of the pregnant rat.

A controlled tetracycline delivery device, consisting of a membrane enclosed trilaminate disc fabricated from a series of 2-hydroxyethylmethacrylate/methylmethacrylate copolymers, demonstrated the ability to deliver tetracycline at zero-order rates in vitro and in vivo in rats and was applied to study the pharmacokinetics of tetracycline in the pregnant rat. The trilaminate discs containing tetracycline were implanted in pregnant Sprague-Dawley rats on the eighth day of gestation. The animals were sacrificed on days 19, 20, and 21 of gestation in order to measure the distribution of the controlled release tetracycline in the maternal, fetal, and placental tissues. Constant plasma tetracycline levels were attained in both the maternal and fetal circulations after 4 to 5 days postimplantation of the trilaminate discs. Placental transfer of tetracycline appeared rapid and no partitioning of the drug was observed between the maternal and fetal soft tissue (liver, kidney, G.I. tract, muscle, placenta) as measured over the last 3 to 4 days of the animal's gestational period. Highest tetracycline concentrations were determined in the fetal bone samples. In addition, some accumulation of the drug occurred in the amniotic fluid. A flow-limited pharmacokinetic model was constructed to simulate the distribution of tetracycline, delivered at a constant rate from the trilaminate device, in the pregnant rats. Predictions of fetal growth and maternal and fetal tissue tetracycline concentrations were in good agreement with the experimental measurements. The ability of these copolymer systems to deliver tetracycline at zero-order rates over extended periods offers numerous potential therapeutic and investigational applications, especially where such drug delivery characteristics are beneficial to the elucidation of physiological rate mechanisms, as in the pregnant animal.

Syndromes of cardiovascular malformations induced by copper citrate in hamsters.

Pregnant golden hamsters were injected intraperitoneally on the morning of the eighth day of gestation with a solution of copper citrate (2.7 mg/kg) or deionized water. Embryos were removed from the mothers by laparotomy on the 12th and 13th days of gestation, serially sectioned through the thorax, and examined for cardiac malformations. Twenty-one of the 37 edematous embryos from copper-treated mothers were affected with a total of 58 major cardiac anomalies. Sixteen of these embryos were affected with double outlet right ventricle in association with a membranous ventricular septal defect, two embryos contained hearts with overriding aortas, and three embryos were affected with isolated ventricular septal defects. Eleven of the 16 embryos with double outlet right ventricle were also affected with pulmonary trunk hypoplasia. The concept that these cardiac anomalies arise from a common pathogenetic basis is supported by the present model.

Induction of monozygotic twinning in the mouse.

VINCRISTINE SULPHATE (Oncovin, Lilly) is a drug commonly used in the chemotherapy of acute leukaemias in children, lymphomas and certain other malignant conditions. We describe here experiments investigating the teratogenicity of this drug during the early stages of embryogenesis in the mouse. We report that a low incidence of gross anomalies was observed in fetuses on days 10 and 12 of gestation when pregnant females were treated with a single dose of this agent on the sixth, seventh or eighth day of gestation. However, contrary to expectation, a high proportion of litters from females treated on the seventh and eighth day of gestation contained at least one set of monozygotic twins. In this respect the present study is unique in that an experimental method of inducing a low but significant incidence of identical twinning in a mammal is reported. Furthermore, this work provides direct evidence regarding the comparatively late stage of embryonic development when identical twin formation may be induced in the mouse, as the most advanced stage at which twinning could be induced with vincristine was at the early headfold stage. However, the highest incidence of monozygotic twinning was obtained when pregnant females were treated on the morning of the seventh day. of gestation, when embryos would be expected to be at the advanced egg-cylinder stage. It was in this latter group that a single conjoined twin of the janiceps type (cephalothoracopagus) was obtained. The possible aetiology of monozygotic and conjoined twin formation is briefly discussed here in the light of these experimental findings.

Light and electron microscopic observations on the morphogenesis of the chorioallantoic placenta of the golden hamster (Cricetus auratus). Days seven through nine of gestation.

AbstractIn the mature placental labyrinth of common myomorph rodents (rat, mouse and hamster) fetal capillaries are separated from maternal blood spaces by trabeculae composed of three distinct cytoplasmic layers. To resolve the controversy regarding the embryonic origin(s) of these layers a correlated light and electron microscopic study was made of the developing placental tissues of the golden hamster during the eighth and ninth days of gestation, the critical period of labyrinth morphogenesis in this species. It was determined that the syncytial inner layer (layer III) of the trabeculae, the layer bordering the fetal capillaries, is derived from the “true” chorionic ectoderm which fuses with the undersurface of the Träger (ectoplacental cone) early on the eighth day of gestation. Both the syncytial middle layer (layer II) and the cellular outer layer (layerI) of the trabeculae, on the other hand, were found to differentiate from the trophoblastic cells of the Träger. Allantoic mesenchyme, which approximates the fused chorionic‐Träger tissues late on day eight and is the source of the fetal capillaries and mesenchyme of the labyrinth, was noted to exert a critical inductive effect on the morphogenesis of syncytial layers II and III. On the basis of fine structure, the developing labyrinth appears not to be a major site for maternal‐fetal transfer of macromolecules nor to be involved to a significant degree in the production of proteins or steroids for secretion.