Tenth Day Of Gestation Research Articles

The hyperexcitability of laterodorsal tegmentum cholinergic neurons accompanies adverse behavioral and cognitive outcomes of prenatal stress

Exposure to prenatal stress (PS) leads to the offspring's vulnerability towards the development of cognitive and behavioral disorders. Laterodorsal tegmentum (LDT) is a part of the brainstem cholinergic system that is believed to play a pivotal role in the stress-associated progression of anxiety, memory impairment, and addictive behaviors. In this study, we aimed to investigate the electrophysiological alterations of LDT cholinergic neurons and its accompanied behavioral and cognitive outcomes in the offspring of mice exposed to physical or psychological PS. Swiss Webster mice were exposed to physical or psychological stress on the tenth day of gestation. Ex vivo investigations in LDT brain slices of adolescent male offspring were performed to evaluate the effects of two stressor types on the activity of cholinergic neurons. Open field test, elevated plus maze, passive avoidance test, and conditioned place preference were conducted to assess behavioral and cognitive alterations in the offspring. The offspring of both physical and psychological PS-exposed mice exhibited increased locomotor activity, anxiety-like behavior, memory impairment, and preference to morphine. In both early- and late-firing cholinergic neurons of the LDT, stressed groups demonstrated higher firing frequency, lower adaptation ratio, decreased action potential threshold, and therefore increased excitability compared to the control group. The findings of the present study suggest that the hyperexcitability of the cholinergic neurons of LDT might be involved in the development of PS-associated anxiety-like behaviors, drug seeking, and memory impairment.

Maternal Peripartum Antibiotic Exposure Worsens Experimental Bronchopulmonary Dysplasia

Purpose: Recent reports indicate that early antibiotic exposure may worsen the severity of bronchopulmonary dysplasia (BPD) in very low birthweight infants. The mechanisms mediating worsened outcomes are unknown, but antibiotic exposure directly perturbs the intestinal microbiome stability and emerging evidence suggests the intestinal microbiota may influence systemic inflammation. Accordingly, we investigated if alteration of the intestinal microbiota altered the development of hyperoxic lung injury in neonatal mice. Methods: We created a two-exposure model that randomized pregnant C57BL/6J mice on the tenth day of gestation to maternal antibiotic treatment (penicillin 500mg/L, MAT) or control. After birth, newborn offspring were randomized to hyperoxia (FiO2 0.85 or 0.60) or FiO2 0.21 for 14 …

Induction of interleukin-6 trigger an apoptosis through IL-17 and stat3 pathway that alleviated by phycocyanin treatment

This study was to analyze the optimum dose of IL-6 induction in pregnant rats that could trigger an increase in mean arterial pressure and protein as two symptoms of preeclampsia. A total of 25 pregnant rats was divided into 5 groups, including pregnant rats the control group (without induction of IL-6), a group of pregnant rats were given an induction in IL-6 doses of 1.25 ng/day, a group of pregnant rats given doses of IL-6 induction 2.5 ng/day, a group of pregnant rats were given an induction of IL-6 doses of 5 ng/day, and groups of pregnant rats were given an induction in IL-6 doses of 10 ng/day. Induction of IL-6 was performed on the tenth day of gestation for 5 days. The expression of caspase-3, IL-17, and STAT3 were analyzed by confocal laser scanning microscopy. The expression of caspase-3, IL-17 and STAT3 was significantly higher in preeclampsia group than the control group (p<0.05). The expression of caspase-3 decreased significantly in all groups were treated by phycocyanin compared to the preeclamptic group (p<0.05), but has not been able to reach expression comparable to the control group (p<0.05). Expression of IL-17 decreased significantly in the group given the two highest doses of phycocyanin compared to the preeclampsia group (p<0.05). STAT3 expression decreased significantly in all groups were treatedby phycocyanin compared to the preeclamptic group (p<0.05), reached expression comparable to the control group in the group received phycocyanin at doses of 10 and 20 ng (p> 0.05). In conclusion, IL-6 on pregnant rats were able to increase apoptosis through IL-17 and STAT pathway. Inhibition of apoptosis due to phycoyanin treatment not only involve the formation of IL-17, this data is found in phycocyanin doses of 10 and 20 ng.

A Comparative Study of the Effects of Sodium Arsenite and Nanoparticles of Sodium Arsenite on the Apparent and Skeletal Malformations in Rat Embryos

BACKGROUND AND OBJECTIVE: Arsenic causes congenital anomalies in humans and animals, and nanoparticles of sodium arsenite are highly capable of inducing apoptosis. Since the effects of nanoparticles of sodium arsenite on fetal malformations have not been evaluated yet, this study aims to compare the effect of sodium arsenite and nanoparticles of sodium arsenite on skeletal malformations in rat embryos. METHODS: This in-vitro study was performed on four groups of pregnant rats (n= 23 rats). Mating was confirmed by observation of vaginal plug. On the tenth day of gestation, pregnant rats in different groups received intraperitoneal normal saline (n=7 rats), sodium arsenite (11 mg/kg) (n=5 rats), nanoparticles of sodium arsenite (1 mg) (n=5 rats) and nanoparticles of sodium arsenite (11 mg) (n=7 rats). All the rats were euthanized on the twentieth day of pregnancy and their embryos were removed, their weight and length were measured, and then were stained with alizarin red and alcian blue. The skeletal system abnormities of embryos such as cleft palate, malfunctions or malformations in ribs, vertebrae, spine, sternum, arms, legs, fingers and reduction of ossification were evaluated by a stereomicroscope, and then compared with the control group. FINDINGS: Sodium arsenite and nanoparticles of sodium arsenite reduced fetal weight from 5 g in the control group to 2 g (p≤0.033) and also, decreased fetal length from 38 cm to 28 cm in the control group (p≤0.023). The weight and length of fetuses were significantly reduced in nanoparticles of sodium arsenite group (11 mg), as compared to sodium arsenite group (p≤0.033). There were no skeletal malformations in the control group, while the percentage of anomalities was between 3% and 47% in the intervention group. CONCLUSION: Nanoparticles of sodium arsenite, as compared to sodium arsenite, were more effective in reducing fetal length and weight and in diminishing the rate of skeletal malformations such as cleft palate.

High oxygen environment during pregnancy rescues sickle cell anemia mice from prenatal death

Several mouse models of sickle cell disease have been developed for the study of the pathophysiology of sickle cell disease and the investigation of drug and gene therapies. In previous years, we produced a sickle cell anemia mouse model in which the endogenous mouse α- and β-globin genes were knocked out and replaced by the human α- and β s-globin transgenes. The β s-globin gene was contained in a 240 kb YAC that preserved the entire native genomic context of the β-globin locus. These mice have hemolytic anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, in the embryo, the γ-globin, like the mouse embryonic globin, declined quickly, and was replaced by β s-globin expression from 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death, we investigated the effect of placing the pregnant mothers in a high O 2 environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O 2 and kept them with the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from less than 2% to 35%. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs similar to those found in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice has facilitated physiological and therapeutic studies of sickle cell anemia.

PS13 DEVELOPMENT OF THE URINARY BLADDER IN ETU‐EXPOSED FETAL RATS IS AFFECTED BY DOWNREGULATION OF SHH AND ITS TARGET GENESCAGES

Objective The cloaca develops into the urinary bladder and distal part of the hindgut. Abnormalities of cloacal development result in a variety of anorectal and urogenital malformations. This study evaluated the formation of the urinary bladder in ETU exposed fetal rats in relation to shh and its target genes.Methodology Embryos of pregnant SD females administered 1% ETU (125 mg/kg) on the tenth day of gestation (gD10) were collected from gD12‐16 and gD21. Cloacal development was viewed with serial histology followed by qualitative and quantitative gene expression studies of shh and target genes to elucidate the expression pattern of these genes during each time point.Results ETU exposed fetuses have incomplete separation of the cloaca leading to a variety of anorectal and urinary abnormalities eg rectourethral fistula. Shh and downstream genes are expressed during urinary bladder development in fetal rats. Relative quantification showed that expression of shh, BMP4 and Hox genes are reduced in ETU‐exposed fetal rats during the process of cloacal separation and development of the urinary bladder.Conclusion This study demonstrates that ETU‐exposure in fetal rats produces abnormal cloacal development, including urinary fistulae. Shh cascade is active during the process of cloacal separation and urinary bladder development and down regulation of shh, BMP4 and Hox genes during this process may contribute to the abnormal development of urinary bladder and anorectum.

High Oxygen Environment during Pregnancy Rescues Sickle Cell Animia Mouse from Prenatal Death.

Several mouse models for sickle cell disease have been developed for the studies of the pathophysiology of sickle cell disease and the investigations of drug and gene therapies. In previous years, we have also succeeded in producing a sickle cell anemia mouse model. In this model the endogenous mouse α and β globin genes were knocked out and replaced by the human α and βs globin transgenes. The βs globin gene is contained in a 240kb YAC that preserves the entire native genomic context of the β-globin locus. These mice have anemia, reticulocytosis and irreversible sickle cells in the peripheral blood, as well as other pathological features of sickle cell disease. However, their γ globin switches to βs globin expression from around 12 days of gestation. The low level of fetal hemoglobin expression in utero led to intrauterine sickling and fetal death so that very few live-born sickle cell anemia mice could be obtained. To rescue these mice from intrauterine death we investigated the effect of placing the pregnant mothers into a high O2 environment. From the tenth day of gestation onwards, we placed the mothers into a chamber containing 50% O2 and kept the newborn pups in it for another 10 days after birth. The frequency of sickle cell anemia mice we obtained was increased from 0.2% to 32%. Moreover, 55% to 88% of the newborn sickle cell anemia mice survived in the oxygen treated group, whereas none of the sickle cell anemia mice survived in regular air breeding conditions. The survived sickle cell anemia mice develop congestion, atrophy, and infarcts in multiple organs by histological analysis. These pathological finding are very similar to those find in patients with sickle cell disease. We conclude that a high oxygen environment can be used to obtain more sickle cell anemia mice in those models that have a high perinatal mortality. The higher yield of these mice will facilitate physiological and therapeutic studies of sickle cell anemia.

Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia.

The amnionless gene, Amn, on mouse chromosome 12 encodes a type I transmembrane protein that is expressed in the extraembryonic visceral layer during gastrulation. Mice homozygous with respect to the amn mutation generated by a transgene insertion have no amnion. The embryos are severely compromised, surviving to the tenth day of gestation but seem to lack the mesodermal layers that normally produce the trunk. The Amn protein has one transmembrane domain separating a larger, N-terminal extracellular region and a smaller, C-terminal cytoplasmic region. The extracellular region harbors a cysteine-rich domain resembling those occurring in Chordin, found in Xenopus laevis embryos, and Sog, found in Drosophila melanogaster. As these cysteine-rich domains bind bone morphogenetic proteins (Bmps), it has been speculated that the cysteine-rich domain in Amn also binds Bmps. We show that homozygous mutations affecting exons 1-4 of human AMN lead to selective malabsorption of vitamin B12 (a phenotype associated with megaloblastic anemia 1, MGA1; OMIM 261100; refs. 5,6) in otherwise normal individuals, suggesting that the 5' end of AMN is dispensable for embryonic development but necessary for absorption of vitamin B12. When the 5' end of AMN is truncated by mutations, translation is initiated from alternative downstream start codons.

Endogenous electric current is associated with normal development of the vertebrate limb.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.

Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice.

The pink-eyed unstable mutation, p(un), is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.

Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice.

To explore the role of the key coagulation factor, fibrinogen, in development, hemostasis, wound repair, and disease pathogenesis, we disrupted the fibrinogen A alpha chain gene in mice. Homozygous, A alpha chain-deficient (A alpha-/-) mice are born normal in appearance, and there is no evidence of fetal loss of these animals based on the Mendelian pattern of transmission of the mutant A alpha chain allele. All of the component chains of fibrinogen (A alpha, B beta, and gamma) are immunologically undetectable in the circulation of both neonatal and adult A alpha-/- mice, and blood samples fail to either clot or support platelet aggregation in vitro. Overt bleeding events develop shortly after birth in approximately 30% of A alpha-/- mice, most frequently in the peritoneal cavity, skin, and soft tissues around joints. Remarkably, most newborns displaying signs of bleeding ultimately control the loss of blood, clear the affected tissues, and survive the neonatal period. Juveniles and young adult A alpha-/- mice are predisposed to spontaneous fatal abdominal hemorrhage, but long-term survival is variable and highly dependent on genetic background. The periodic rupture of ovarian follicles in breeding-age A alpha-/- females does not appear to significantly diminish life expectancy relative to males; however, pregnancy uniformly results in fatal uterine bleeding around the tenth day of gestation. Microscopic analysis of spontaneous lesions found in A alpha-/- mice suggests that fibrin(ogen) plays a fundamental role in the organization of cells at sites of injury.

Ultrastructural changes in the mouse placenta after administration of trichosanthin

The root tuber ofTrichosanthes kirlowiiMaxim (Family curcurbitaceae) has long been used in Chinese traditional medicine as an abortifacient and in therapy for choriocarcinoma. The active principle of the root tuber extract was recently purified and characterized. It is a basic protein of 24 KDa and is named trichosanthin. Investigations on trichosanthin action showed that it affected directly on the placenta, selectively causing necrosis of the syncytiotrophoblasts of placental villi. The present study attempts to determine the initial action of trichosanthin and follow through from the earliest sign of histopathological and ultrastructural changes of the mouse placentae after administration of trichosanthin.Mature ICR female mice were used. On the tenth day of gestation, a single dose of trichosanthin was injected intraperitoneally. The dosage used was 0.1 mg of trichosanthin in physiological saline per 25 g body weight of the mouse. At various prescribed intervals after injection, the mice were sacrificed by cervical dislocation.

The pattern of X-chromosome inactivation in the embryonic and extra-embryonic tissues of post-implantation digynic triploid LT/Sv strain mouse embryos

Spontaneously cycling LT/Sv strain female mice were mated to hemizygous Rb(X.2)2Ad males in order to facilitate the distinction of the paternal X chromosome, and the pregnant females were autopsied at about midday on the tenth day of gestation. Out of a total of 222 analysable embryos recovered, 165 (74.3%) were diploid and 57 (25.7%) were triploid. Of the triploids, 26 had an XXY and 31 an XXX sex chromosome constitution. Both embryonic and extra-embryonic tissue samples from the triploids were analysed cytogenetically by G-banding and by the Kanda technique to investigate their X-inactivation pattern. The yolk sac samples were separated enzymatically into their endodermally-derived and mesodermally-derived components, and these were similarly analysed, as were similar samples from a selection of control XmXp diploid embryos. In the case of the XmXmY digynic triploid embryos, a single darkly-staining Xm chromosome was observed in 485 (82.9%) out of 585, 304 (73.3%) out of 415, and 165 (44.7%) out of 369 metaphases from the embryonic, yolk sac mesodermally-derived and yolk sac endodermally-derived tissues, respectively. The absence of a darkly staining X-chromosome in the other metaphase spreads could either indicate that both X-chromosomes present were active, or that the Kanda technique had failed to differentially stain the inactive X-chromosome(s) present. In the case of the XmXmXp digynic triploid embryos, virtually all of the tissues analysed comprised two distinct cell lineages, namely those with two darkly-staining X-chromosomes, and those with a single darkly staining X-chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)

Asialoglycoprotein receptor expression in murine pregnancy and development.

Hepatic asialoglycoprotein receptor activity of pregnant mice increased from the tenth day of gestation, reaching 3-fold the normal adult level just prior to parturition and returning to normal within 1 week postpartum. Increases in activity involved both intracellular and cell-surface associated receptors and were associated with enhanced endocytosis of asialoorosomucoid by isolated hepatocytes. Modulation of hepatic binding protein expression during murine pregnancy represents the first situation in which this receptor's concentration has been shown to exceed normal levels in response to a physiologic change. In the fetus, receptor activity was barely detectable throughout gestation, steadily increased in the neonatal period, and reached adult levels by the fifth postpartum day.

Perinatal death and respiratory apparatus dysgenesis due to a bis (dichloroacetyl) diamine.

N, N1-bis (dichloroacetyl) diamine 1, 8-octomethylenediamine (WIN 18,446) is an experimental drug which was first investigated as a male contraceptive. It is soluble in lipid solvents but not in water. The administration of 1,200 to 1,600 mg/kg to pregnant rats on the tenth day of gestation produced multiorgan fetal malformations. Smaller doses, 400 to 800 mg/kg, especially if divided over 2 or 3 days, caused perinatal death. Thus, 60 to 100% of offspring of rats given WIN 18,446 on the tenth and 11th days of gestation died at birth or within 4 days (Taleporos et al., 78). The present study investigated such deaths. At doses of 200 mg/kg on day 10 or 50 mg/kg on days 10 and 11, 67% of offspring had defective or absent diaphragms, 48% had tracheobronchiomegaly with cystic lungs, and 67% had pleural hemorrhage. At doses of 100 mg/kg given on 1 day or 25 mg/kg each day for 2 days, 50% had tracheobronchiomegaly with cystic lungs and rudimentary acini. At lower doses (18.8 mg/kg X 2 or 12.4 mg/kg X 3), a majority of fetal lungs had rudimentary acini, thick septa, few capillaries, and wide cuffs of perivascular connective tissue. Thus, a chemical given during organogenesis produced dysgenesis of the respiratory apparatus. Varying the dose produced malformed lungs with persistently deficient acini which model such human lung faults as tracheobronchiomegaly (Mournier-Kuhn Syndrome; Mounier-Kuhn, '32), bronchiolar dysplasia (Wilson-Mikity Syndrome), and perinatal death with acinar failure resembling neonatal hyaline membrane disease.

Sister chromatid exchange frequency in mouse embryo chromosomes after in utero ethanol exposure

The frequency of sister chromatid exchange (SCE) increased in mouse embryo chromosomes following a single maternal i.p. injection of ethanol on the tenth day of gestation, indicating a possible morphogenetic action of ethanol in the developing mouse embryo.

Prenatal ethanol exposure in mice: teratogenic effects.

C57BL/6J mice were fed a liquid diet in which 17, 25, or 30% of the calories were derived from ethanol from the fifth through the tenth day of gestation. Control mice were fed lab chow or pair-fed identical diets, except that sucrose substituted isocalorically for ethanol. At term the fetuses were removed and, following fixation, examined by microdissection. The incidence of fetal resorptions and congenital malformations increased in a dose-related manner. Anomalies included skeletal, neurological, urogenital, and cardiovascular systems. These data indicate that in mice, an alcohol diet which is adequate in vitamins and protein results in increased fetal wastage and birth defects.

Early motoneuron formation in the cervical spinal cord of the mouse: an electron microscopic, serial section analysis.

AbstractCells of the ventrolateral region of the cervical spinal cord were reconstructed from serial thin sections in order to provide direct evidence concerning the early stages of motoneuron differentiation. Analysis of 321 sections from the spinal cord of a mouse embryo on the tenth day of gestation (E10) was performed. At E10 the general appearance of the ventrolateral region is that of a pseudostratified columnar epithelium. It consists primarily of radially oriented ventricular cells, while the remaining cells are either rounded mitotic cells (located at the neurocoel) or cells differentiating into either accessory root or ventral root motoneurons. The following early stages of motoneuron differentiation have been distinguished: anaxonic primitive bipolar cells (stage 1); primitive bipolar cells with an axon (stage 2); unipolar cells (stage 3); and secondary bipolar cells (stage 4). The earliest morphological change in the differentiation of both types of motoneurons appears to be the withdrawal of the apical process from the neurocoel and the migration of the Golgi apparatus, centrioles and cilium along the apical process toward the nucleus (anaxonic primitive bipolar cell). The nucleus then appears to migrate peripherally and the basal process (which never completely withdraws) to transform into an axon (primitive bipolar cell with an axon). Subsequent stages have only been identified in accessory root motoneurons; it appears that ventral root motoneurons are not as advanced in their differentiation at E10. In the unipolar stage the apical process has completely withdrawn, and the nucleus appears paler and rounder than that of earlier cells. Finally, in the secondary bipolar stage the cell is oriented dorsoventrally with an axon directed dorsally and a dendrite ventrally from the opposite pole of the cell. During differentiation of accessory root motoneurons, the Golgi apparatus, centrioles and cilium are found first in the apical process, then at the base of the axon, and finally at the base of the dendrite. The translocation of these organelles occurs secondary to the initial formation of first the axonal and then the dendritic process.

Forssman antigen and phase specific fetal antigens: an evaluation of their role in SV40 tumor immunity.

Forssman heterophile antigen was detected on hamster fetal cells which had been previously shown to be capable of eliciting transplantation resistance to syngeneic hamster SV40 tumors. The expression of Forssman antigen continued throughout fetal development and could be detected postpartum in the tissues of neonate hamsters. In contrast, fetal antigen(s) evoking immunity to SV40 tumors was also present on early fetal cells but, unlike Forssman antigen, was not expressed after the tenth day of gestation. Immunization of hamsters with guinea pig kidney cells or sheep erythrocytes which carry Forssman antigen failed to demonstrate significant protection against SV40 tumor development. Again by contrast, immunization with fetal cells was effective in evoking tumor immunity. Evaluation of serological responses to the FORSSMAN A ANTIGEN IN HAMSTERS INDICATED THAT THE HEMOLYTIC REACTIVITY PRODUCED BY IMMUNIZATION WITH GUINEA PIG KIDNEY CELLS OR SHEEP ERYTHROCYTES WAS ELICITED AGAINST ISOANTIGENS AND NOT THE Forssman antigen. A response to the Forssman determinant could only be detected in the serum from animals receiving exhaustive hyperimmunization with fetal cells or SV40 tumor cells. These data would eliminate a possible role of the Forssman heterophile antigen in the tumor protection evoked by immunization with fetal cells bearing embryonic antigens.

Incorporation of Thymidine and its Analogue, Bromodeoxyuridine, into Embryos and Maternal Tissues of the Mouse

Following the injection of pregnant mice on the tenth day of gestation with either 200 μCi/kg 3H-thymidine or 200 μCi/kg 3H-bromodeoxyuridine, the half-life of labelled nucleotides in the acid-soluble fraction was found to be of the order of 30 min in various maternal tissues and placentas, and approximately 60–80 min in the embryos. There appears to be no discrimination in turnover rate between thymidine (TdR) and bromodeoxyuridine (BrdU). The administration of a single injection containing 200 μCi/kg 3H-BrdU plus 500 mg/kg BrdU (a known teratogenic dose) resulted in increased labelled nucleotide half-lives in all of the tissues except the embryos. These increased half-lives ranged from 149 min in the maternal liver to 75 min in the kidneys. The turnover rate of labelled nucleotides in the embryos did not change significantly from those observed following a trace dose of 3H-BrdU. The time course of incorporation of label into the DNA of the various tissues was determined following the injection of the trace doses of 3H-TdR or 3H-BrdU. Significant amounts of DNA label were found in all tissues studied except maternal liver and kidneys, the bulk of the incorporation occurring within one hour of administration. The DNA specific radioactivity had begun to decline by 8 h after injection but was still demonstrable at 48 h. When the teratogenic dose of BrdU was administered along with the 3H-BrdU, the time course of incorporation of label was very similar in all tissues, with the exception of the embryo and maternal intestine, to that observed following injection of the trace dose of 3H-BrdU. The fact that radioactivity found in the DNA fractions after the injection of 3H-BrdU was due to labelled bromouracil, was established by thin-layer chromatography of DNA bases. At a time in mouse development when BrdU is capable of producing congenital defects in vivo, it is present at its presumed intracellular site of action, the DNA molecule. It is suggested that this demonstration supports the contention that, to study the role of teratogens in the interference with normal differentiation, it is essential to show their presence at their suspected site of action during morphogenesis.