Immunological factors responsible for pathogenetic cell degeneration in pregnancy

The placenta has an important role as an immunological barrier during pregnancy. When the placental barrier is disrupted, materno-embryonic transfusion takes place. Several clinical reports relate congenital malformations or abortion to intrauterine bleeding or transplacental transfusion. In an earlier experiment, pathogenetic cell degeneration was induced using an in vitro whole rat embryo culture. Transplacental transfusion was simulated by intracardiac injection of an allogeneic rat-antirat serum directed against the blood group antigens. The present study examines the morphological and immunological effects on the development of rat embryos 9 to 10 days old (stages 8-10 somites) of the separate administration of primary allogeneic antisera, obtained 10-17 days after immunization, and secondary allogeneic antisera, obtained after booster immunization on day 45-52. Rat-antirat alloantibodies were directed against the blood group antigens. Transplacental transfusion was simulated by the embryonic intracardiac microinjection of approximately 0.5 microliters serum enriched with either primary or secondary obtained allogeneic antibodies. After 48 hours' incubation, the embryos were examined microscopically, and it appeared that the secondary antisera, which had hemolytic activity, was more potent (P less than 0.005) in the induction of pathogenetic cell degeneration. It is well known that IgG antibodies display hemolytic activity. This finding was confirmed by direct immunofluorescence performed on rat embryos 2, 4, and 6 hours after injection, where incubation with rabbit-antirat anti-IgG antibodies gave a strong reaction. The hypothesis discussed is whether or not pathogenetic cell degeneration subsequent to transplacental transfusion of maternal antibodies can be initiated by similar immunological events.     

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.     

Phenytoin embryotoxicity: role of enzymatic bioactivation in a murine embryo culture model

A murine embryo culture model was developed to study the potential contribution of enzymatic bioactivation to the teratogenicity of phenytoin. To assess the relative embryonic and maternal contributions to bioactivation, embryos were cultured respectively alone or in the presence of an exogenous source of cytochromes P-450 (P-450), which are thought to bioactivate phenytoin to a teratogenic reactive intermediate. Embryological development from gestational day 9 to day 10 was assessed, and bioactivation was quantified by the irreversible binding of radiolabeled phenytoin to embryonic protein. Embryos cultured with phenytoin and an exogenous P-450 bioactivating system showed a significant decrease in the incidence of turning and closure of the anterior neuropore, yolk sac diameter, and protein content as well as growth retardation. In the absence of an exogenous P-450 system, phenytoin did not decrease the incidence of turning or anterior neuropore closure but did cause growth retardation and a lesser but significant reduction in yolk sac diameter and embryonic protein content. An exogenous P-450 system enhanced the bioactivation of phenytoin, although significant activity also was detectable in embryos cultured without an exogenous bioactivating system. These results suggest that the embryo itself can enzymatically bioactivate embryotoxically significant amounts of phenytoin, and that bioactivation and embryotoxicity can be further enhanced, qualitatively and quantitatively, by an exogenous P-450 system, implicating a possible maternal contribution to phenytoin teratogenicity.     

Roles of retinoic acid receptors in early embryonic morphogenesis and hindbrain patterning

Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) alpha and RAR gamma (A alpha/A gamma mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that we previously observed during the fetal period. We show that embryonic day (E)9.5 A alpha/A gamma embryos display severe malformations, similar to those already described in retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds; and in the closure of the primitive gut. The hindbrain of E8.5 A alpha/A gamma embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RAR alpha and RAR beta (A alpha/A beta mutants), in which we have previously shown that the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an A alpha/A beta-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an A alpha/A gamma-like phenotype. Altogether, our data suggest that RAR alpha and/or RAR gamma transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RAR beta is subsequently involved in setting up the caudal boundary of this territory.     

Ethylenethiourea-induced hydrocephalus in vivo and in vitro with a note on the use of a constant gaseous atmosphere for rat embryo cultures

Embryos were studied either after direct exposure to ethylenethiourea (ETU) during incubation of embryo cultures or after maternal ETU dosing and subsequent embryonic development in utero with a view to assess the similarity of these two systems to produce hydrocephalus. Ten-day-old rat embryos were incubated with nutrient media containing 0-2.0 mM of ETU in a constant gasseous environment following a newly modified method. The cultured embryos showed hydrocephalus in the form of dilated rhombencephalon and other anomalies at the 1.5 and 2.0 mM of ETU after 26 hours of incubation. No anomalies were seen in the control group. In in vivo studies, dilated rhombencephalon or hydrocephalus was not observed when dams, orally dosed with ETU on gestation day 10, were either killed daily for three postdosing days to examine embryos or killed at term to evaluate fetuses. This discrepancy in dilatation that was incidental to the rhombencephalon in the two systems pointed out that the fourth ventricle of the cranial neural tube responded by dilatation in vitro but remained unaffected in vivo following ETU exposure. ETU dosing of dams on the 12th day of pregnancy, when embryos are known to be sensitive to ETU-induced hydrocephalus, followed by serial gross examination of embryos, suggested that edema occurred in a generalized form but only after the appearance of both hydrocephalus (dilatation primarily in mesencephalon) and, the previously reported, neuroblastic necrosis.     

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

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

Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis

Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.     

Time-lapse photographic study of neural tube closure defects caused by xylocaine in the chick

Sequential changes in the morphology of early chick embryos were, for the first time, photographically recorded. Embryos were explanted at stage 8 (four-somite) or 9- (six-somite) of development using New's technique and grown in nutrient medium (thin albumen) with or without a teratologic dose (200 micrograms/ml) of xylocaine. They were photographed using a Nikon Diaphot inverted microscope equipped with both phase-contrast optics and photomicrographic accessories maintained in an incubator. It was found, among other things, that a characteristic neural tube closure defect often seen in the midbrain and anterior portion of the hindbrain of xylocaine (200 micrograms/ml)-treated chick embryos was a consequence of failure of the neural tube to withstand the tension generated by the rapidly expanding cephalic region, which occurred, regardless of the stage at explanation, when corresponding control embryos had advanced to stage 10+ (11-somite) of development.     

Bovine embryo morphology and evaluation

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.     

Drosophila goosecoid participates in neural development but not in body axis formation.

In vertebrate embryos, the homeobox gene goosecoid (gsc) is expressed in the gastrula organizer region and in later arising embryonic tissues including the foregut anlage. Ectopic expression and loss-of-function studies have demonstrated that Xenopus gsc elicits a dorsalizing activity that contributes to body axis formation. Here we report that the gsc gene is conserved in invertebrates. In Drosophila, D-gsc is expressed most strongly in the foregut anlage, which gives rise to the foregut proper and the stomatogastric nervous system (SNS). D-gsc expression overlaps with one of the three SNS precursor groups invaginating from the foregut anlage. Embryos mutant for D-gsc gastrulate normally but show disrupted invagination in the SNS primordium and lack one specific SNS ganglion. In addition, D-gsc mutant embryos show a less well defined defect in foregut arrangement. Our results indicate that this invertebrate homolog of gsc is not required for gastrulation but plays a role in neurogenesis in post-gastrula Drosophila embryos.     

Acceleration of early chick embryo morphogenesis by insulin is associated with altered expression of embryonic genes

In the present study, we show that insulin accelerates early morphogenesis in gastrulating chick embryo explants cultured in vitro, whereas antiserum to insulin adversely affects this process. Comparison between length of body axis of control and treated embryos clearly brings out the significant acceleration of development by excess insulin (0.175 to 17.5 nM). In embryos treated with 87.5 and 175 nM insulin, a high occurrence of abnormalities is observed. Treatment of embryos with antiserum to porcine insulin results in a high percentage of abnormalities, particularly in the forming neural tube. In situ hybridization of whole embryos using digoxigenin-labeled riboprobes showed that insulin modifies the expression of crucial developmental genes within 2 hours. While Brachyury, a pan-mesodermal marker gene, ERNI, the earliest known marker for neural induction in chick, and noggin, important in neural tube patterning, are upregulated, expression of goosecoid, necessary for gastrulation movements, does not appear to be significantly altered. During the same time, insulin does not exert any mitogenic effect on chick embryonic cells as assessed by nuclear counts. These findings demonstrate that insulin plays an important role in the early morphogenesis of the chick embryo. The function of insulin appears to be mediated by specific genes which orchestrate pattern formation during early development.     

Effects of L-asparaginase on rat embryonic development and yolk sac membrane in vitro

A comparative analysis of the teratogenic effects of L-asparaginase on 10.5- and 11.5-day rat embryos after 24 and 48 hours of exposure in vitro, respectively, were performed. Several medium concentrations of L-asparaginase (0.05, 0.25, and 1.5 IU/ml) were tested in both embryo series. Resulting embryos were submitted to morphological studies in a search for a specific route of pathogenesis. Morphological alterations of the visceral yolk sac were also studied to investigate its contribution to L-asparaginase teratogenicity in rats. Main embryonic malformations (open truncal neural tube, open encephalic vesicles, anophthalmia, lack of inversion, abnormal frontolateral protrusions, great vascular dilations at the cephalic level) and developmental retardation were already generated after the first 24 hours of culture (embryos of 10.5 days) and presented a dose-response relationship. Vascular dilations and neurulation disturbances seemed to be related to an early mesenchyme deficiency. Reduced number of mesenchymal cells was more evident in embryos of 10.5 days than those of 11.5 days, suggesting the existence of a later compensatory mechanism of cellular proliferation in the older embryo. Visceral yolk-sac endodermal cells at both embryonic stages were greatly deformed and enlarged by an increase of the high electron-dense vacuolar system. Therefore, both a blockage of the processes of lysosomal digestion and derived trophic deficiencies probably existed. A double teratogenic mechanism for L-asparaginase is postulated: a direct action mainly in younger embryos (before invagination of the embryo into the yolk sac) and a yolk sac-mediated one.     

Hatching ofCoregonus albula andC. lavaretus embryos at different stages of development

Synopsis Hatching in Coregoninae embryos is not closely connected with a determined stage of embryonic development. Electrical stimulation (AC) induced precocious hatching, and incubation at low temperature caused delay of hatching compared to controls. Embryos hatched precociously had shorter bodies and larger yolksacs than those whose hatching was normal or delayed. This was accompanied by respective changes in duration of the eleutheroembryonic phase (DEP) — inCoregonus lavaretus DEP decreased from 7 days in embryos hatched precociously to 0 days in embryos from delayed hatching.     

Embryotoxic effects of sodium arsenite and sodium arsenate on mouse embryos in culture

Embryotoxic effects of two inorganic arsenic compounds, sodium arsenite (Asi) and sodium arsenate (Asa), on the development of mouse embryos during early organogenesis were studied using the whole embryo culture technique. Embryos with three to five somites exposed to 1-40 microM Asi or to 10-400 microM Asa were cultured for 48 hours and their development was compared with that of control embryos. Asi proved to be teratogenic between 3 and 4 microM and embryolethal at higher concentrations; Asa had similar activity but at concentrations ten times higher than for Asi. Both compounds produced a growth retardation and a similar pattern of defects. Growth retardation was indicated by a statistically significant reduction in crown-rump length, head length, and yolk sac diameter. Abnormal embryos were characterized by hypoplasia of the prosencephalon with open neural tube, hydropericardium, somite abnormalities, and failure of development of limb buds and sensory placodes. These results confirm that both Asa and Asi are embryotoxic compounds and that the Asi activity occurs at concentrations ten times lower than for Asa. Our results suggest that in humans both of these compounds may be involved in part of "unaccountable" early abortions and malformations claimed to be due to the toxicity of heavy metals.     

An in vivo/in vitro evaluation of teratogenic action

Several compounds were administered to pregnant Wistar-derived rats either 24 or four hours prior to the recovery of day 10 embryos for in vitro culture in Waymouth's medium and fetal calf serum. The compounds tested were 2-amino-1,3,4-thiadiazole (thiadiazole), cadmium sulfate, 1,2-dibromo-2,2-dichloroethyl dimethyl phosphate (dibrom), 2-(sec-Butyl)-4,6-dinitrophenol (dinoseb), led nitrate, polybrominated biphenyls (PBB), sodium arsenate, and trypan blue. After 24 hours in culture, two thirds of the embryos were recovered for examination. The remaining one third were continued in culture until 42 hours. Recovered embryos were examined for rotation of the embryonic axis, heart rate, establishment of the visceral yolk sac circulation, somite number, growth of the limb buds, closure of the neural tube, and development of the allantois and amnion. All tested compounds inhibited the rate of development in vitro.     

Pomegranate peel extracts effects to reduce mono sodium glutamate toxic effects on chicken embryos: Morphological studies

BackgroundMonosodium glutamate (MSG) is a flavoring agent added to various foods. This experimental study investigated MSG effects on chicken embryos morphology and the possible ameliorative effects of pomegranate peel extracts (PPE) at different incubation periods.MethodsSeven hundred and twenty fertilized chicken eggs were used and divided into six groups: control, PPE, MSG, PPE + MSG, preventive (PPE–MSG) and therapeutic (MSG–PPE) groups. Fertile chicken eggs were injected with MSG (0.1 ml) and/or PPE (0.3 ml) twice before incubation at days 0, 1. Embryos were extracted at days 7, 10, 12, 14 and 16. Effects of MSG and/ or PPE on embryo development during different incubation periods were studied.ResultsMSG injected into embryos led to congenital anomalies that appeared mainly in MSG and MSG + PPE groups. These anomalies included growth retardation, absent eye, abdominal swelling and hernia. Mortality rate was the highest in MSG, then in MSG + PPE and MSG–PPE groups. PPE treatment reduced MSG toxic effects and these results were better in MSG–PPE and PPE–MSG groups than MSG + PPE group.ConclusionsMSG injection affected chicken embryonic development causing growth retardation and decline in total body length, break length, and total body weight in all the treated groups. These harmful actions can be ameliorated with PPE treatment depending on embryo age.     

维甲酸致小鼠胚胎畸形发生过程中维甲酸受体α/β及β-catenin和caspase-3基因的表达变化

流行病学研究显示,在胚胎发育过程中摄入过多维甲酸可致各种发育缺陷,其中神经管畸形最为常见. 因此有必要探明维甲酸致各种发育缺陷的发生机制,以便为各种生长缺陷的预防和治疗提供实验依据. 用 RT-PCR 及蛋白质印迹技术,探测了过量维甲酸对昆明小鼠胚胎神经管中维甲酸受体α/β及β-catenin 和 caspase-3 基因表达的调整. 结果显示,在神经管闭合期过量维甲酸显著降低了维甲酸受体α/β及β-catenin 和 caspase-3 的基因表达,神经管闭合后,维甲酸受体β、β-catenin 及 caspase-3 的基因表达又出现了一个明显的回升过程. 提示,过量维甲酸改变了昆明小鼠胚胎神经管中维甲酸受体α/β及β-catenin 和 caspase-3 基因的正常时间表达模式,这种异常的基因表达模式可能参与了维甲酸致昆明小鼠胚胎畸形的发生机制.     

The Role of Reactive Oxygen Species in Diabetes-Induced Anomalies in Embryos of Cohen Diabetic Rats

The role of the antioxidant defense mechanism in diabetesinduced anomalies was studied in the Cohen diabetes-sensitive (CDs) and -resistant (CDr) rats, a genetic model of nutritionally induced type 2 diabetes mellitus. Embryos, 12.5-day-old, of CDs and CDr rats fed regular diet (RD) or a diabetogenic high-sucrose diet (HSD) were monitored for growth retardation and congenital anomalies. Activity of superoxide dismutase (SOD) and catalaselike enzymes and levels of ascorbic acid (AA), uric acid (UA), and dehydroascorbic acid (DHAA) were measured in embryonic homogenates. When fed RD, CDs rats had a decreased rate of pregnancy, and an increased embryonic resorption. CDs embryos were smaller than CDr embryos; 46% were maldeveloped and 7% exhibited neural tube defects (NTDs). When fed HSD, rate of pregnancy was reduced, resorption rate was greatly increased (56%; P < .001), 47.6% of the embryos were retrieved without heart beats, and 27% exhibited NTD. In contrast, all the CDr embryos were normal when fed RD or HSD. Activity of SOD and catalase was not different in embryos of CDs and CDr rats fedRD. When fed HSD, levels of AA were significantly reduced, the ratio DHAA/AA was significantly increased, and SOD activity was not sufficiently increased when compared to embryos of CDr. The reduced fertility of the CDs rats, the growth retardation, and NTD seem to be genetically determined. Maternal hyperglycemia seems to result in environmentally induced embryonic oxidative stress, resulting in further embryonic damage.