Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension.

Epidemiological studies have indicated that susceptibility of human adults to hypertension and cardiovascular disease may result from intrauterine growth restriction and low birth weight induced by maternal undernutrition. Although the 'foetal origins of adult disease' hypothesis has significant relevance to preventative healthcare, the origin and biological mechanisms of foetal programming are largely unknown. Here, we investigate the origin, embryonic phenotype and potential maternal mechanisms of programming within an established rat model. Maternal low protein diet (LPD) fed during only the preimplantation period of development (0-4.25 days after mating), before return to control diet for the remainder of gestation, induced programming of altered birthweight, postnatal growth rate, hypertension and organ/body-weight ratios in either male or female offspring at up to 12 weeks of age. Preimplantation embryos collected from dams after 0-4.25 days of maternal LPD displayed significantly reduced cell numbers, first within the inner cell mass (ICM; early blastocyst), and later within both ICM and trophectoderm lineages (mid/late blastocyst), apparently induced by a slower rate of cellular proliferation rather than by increased apoptosis. The LPD regimen significantly reduced insulin and essential amino acid levels, and increased glucose levels within maternal serum by day 4 of development. Our data indicate that long-term programming of postnatal growth and physiology can be induced irreversibly during the preimplantation period of development by maternal protein undernutrition. Further, we propose that the mildly hyperglycaemic and amino acid-depleted maternal environment generated by undernutrition may act as an early mechanism of programming and initiate conditions of 'metabolic stress', restricting early embryonic proliferation and the generation of appropriately sized stem-cell lineages.

Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform.

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. We have shown previously that individual components of the tight junction assemble in a temporal sequence, with membrane assembly of the cytoplasmic plaque protein ZO-1 occurring 12 hours before that of cingulin. Subsequently, two alternatively spliced isoforms of ZO-1 (alpha+ and alpha-), differing in the presence or absence of an 80 residue alpha domain were reported. Here, the temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1alpha- mRNA was present in oocytes and all preimplantation stages, whilst ZO-1alpha+ transcripts were first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms were detected in trophectoderm and ICM cells. Immunoprecipitation of 35S-labelled embryos also showed synthesis of ZO-1alpha- throughout cleavage, whereas synthesis of ZO-1alpha+ was only apparent from the blastocyst stage. In addition, 33P-labelling showed both isoforms to be phosphorylated at the early blastocyst stage. The pattern and timing of membrane assembly of the two isoforms was also distinct. ZO-1alpha- was initially seen as punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesced laterally along the membrane until they completely surrounded each cell with a zonular belt by the late morula stage. ZO-1alpha+ however, was first seen as perinuclear foci in late morulae before assembling at the tight junction. Membrane assembly of ZO-1alpha+ first occurred during the 32-cell stage and was zonular just prior to the early blastocyst stage. Immunostaining indicative of both isoforms was restricted to the trophectoderm lineage. Membrane assembly of ZO-1alpha+ and blastocoele formation were sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. In addition, the tight junction transmembrane protein occludin co-localised with ZO-1alpha+ at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. Moreover, the late expression of ZO-1alpha+ and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo.

Stimulation of development in vitro by platelet-activating factor receptor ligands released by mouse preimplantation embryos.

Previous reports have demonstrated that culture of mouse preimplantation embryos at high density stimulates their rate of development. The molecular basis of this phenomenon was investigated. Culture of embryos from the four-cell stage at high density in normal medium, or at low density either in embryo-conditioned medium or medium containing platelet-activating factor (PAF), significantly advanced the timing of compaction, initiation of cavitation and/or completion of zona hatching, and also increased the number of cells in blastocysts. In contrast, Lyso-PAF, an inactive metabolite of PAF, and Enantio-PAF, an enantiomer of PAF, did not have a stimulatory effect at low embryo density, but did not inhibit the stimulation of development at high embryo density. The stimulatory effect of culture at high density was inhibited in the presence of either CV-3988 or SDZ 64-412, two structurally distinct competitive PAF-receptor antagonists, while the development rate at low density was not affected. We conclude that an embryo-derived factor related to PAF is secreted by blastomeres during in vitro culture and acts in a receptor-mediated manner to stimulate the rate of development.

Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts: effects on intracellular calcium.

1. The aim of these experiments was to determine the ability of the muscle-type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium-sensitive fluorescent dye Indo-1 was used. 2. Application of the nicotine agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 +/- 22 nM (mean +/- s.e. mean; n = 40 cells) rising to 600 +/- 81 nM on addition of SDC (10 microM; n = 15 cells), whereas no increase in [Ca2+]i was seen in non-transfected control QT6 fibroblasts (before: 128 +/- 9 nM, n = 40; after; 113 +/- 13 nM, n = 15). 3. The increase in [Ca2+]i caused by application of SDC was dose-dependent, with an EC50 value of 12.7 +/- 5.9 microM (n = 14). 4. The responses to SDC in QF18 cells were blocked by prior application of alpha-bungarotoxin (200 nM), by the addition of Ca2+ (100 microM), by removal of Na+ ions from the extracellular solution, or by the voltage-sensitive calcium channel blockers nifedipine and omega-conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5. We conclude that activation of the nicotinic AChRs leads to a Na(+)-dependent depolarization and hence activation of endogenous voltage-sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.

Evidence that Fc gamma receptors in rabbit yolk sac endoderm do not depend upon an acid pH to effect IgG binding and transcytosis in vitro.

An in vitro culture system has been devised creating apical and basal compartments separated by rabbit visceral yolk sac (VYS) with an intact epithelium. Selective transcytosis and binding of heterologous IgG applied to the apical yolk sac endoderm (YSE) was demonstrated in vitro using double label immunofluorescence. Thus, whilst both human and bovine IgG could be detected in endosomes in YSE, only human IgG could be detected in the basement membrane and vascular mesenchyme. This mirrors what is found in vivo. The Fc fragment of human Ig was transcytosed but not the Fab fragment, indicating that Fc receptors were expressed in the cultured YSE. When VYS was previously chilled to 4 degrees C to prevent endocytosis and treated with rabbit serum albumin to prevent non-specific binding, human IgG, but not bovine IgG, became specifically bound to YSE apical plasma membrane; comparison of binding at pH 6.0, 7.3 (the average pH of rabbit uterine fluid) and 8.0 revealed no obvious difference. Pre-exposure of VYS for up to 5 min in monensin, followed by culture in monensin and immunoglobulin-containing medium, did not prevent the selective transcystosis of human IgG, suggesting that an acidic compartment may not be needed for transcytosis. An acid pH dependent Fc gamma receptor equivalent to that on suckling rat gut jejunal enterocyte plasma membranes could not be isolated from rabbit YSE following exposure of solubilized membrane to affinity matrix bound IgG at pH 6.0 and elution at pH 8.0. These results contradict a recent suggestion that Fc receptors on all IgG transcytosing epithelia require an acid pH to effect IgG binding and selective transcytosis.

Monoclonal antibodies directed against surface and intracellular antigens of mouse granulated metrial gland cells.

Rat monoclonal antibodies with reactivity directed against mouse GMG cells have been produced. One of the antibodies (GMG-1) reacts with a surface antigen of GMG cells and cross-reacts with T lymphocytes. Another (GMG-2) reacts with an intracellular antigen in GMG cells and with asialo-GM1 positive cells in the spleen. Three antibodies (GMG-3, -4, -5) bind to intracellular antigens in GMG cells. The cross-reactivity of these antibodies is discussed with reference to the lineage relationship of GMG cells to NK cells and T cells and the recent suggestion that NK cells and T cells have a common progenitor cell. It is proposed that GMG cells share this common progenitor cell but are otherwise independent of the NK or T cell lineages.

Extramedullary haemopoiesis in fetal and adult human spleen: a quantitative immunohistological study.

Haemopoietic cells were assessed in spleens from normal adults, adults with splenic extramedullary haemopoiesis due to chronic myeloproliferative disorders and fetuses of 17-21 weeks' gestation. A variety of antigens expressed by developing granulocytes and erythrocytes were demonstrated immunohistochemically. The relative proportions of early and late precursor cells of these two lineages were quantified. There was no significant haemopoiesis in normal adult spleen, while there was abundant (predominantly granulocytic) haemopoiesis in patients with chronic myeloproliferative disorders. Fetal spleens contained numerous late erythroid precursors but few early erythroid or granulocytic cells. The relative numbers of early and late haemopoietic cells in adult chronic myeloproliferative disorders and fetal spleens showed statistically significant differences. Our findings indicate that haemopoiesis in the spleens of adult patients with these disorders differs fundamentally from that occurring in fetal life. They support the view that the human spleen does not have a significant role in fetal haemopoiesis, but that it filters circulating nucleated erythroid precursors and is permissive of their terminal differentiation only. Our results also favour the view that adult splenic haemopoiesis originates by displacement of precursor cells from the bone marrow rather than by activation of stem cells which have lain dormant in the spleen since fetal life.

IgG transport across the gut of the suckling opossum (Monodelphis domestica).

We investigated IgG transport across the gut of suckling opossums to see whether it is likely to be Fc gamma R-mediated. Enterocytes isolated from the proximal and distal regions of the small intestine of suckling aged 12-52 days, and reacted with indicator SRBC at pH 6.0 or 7.2, bound opossum IgG in rosette assays. Considerable overall variation was observed in the numbers of enterocytes forming rosettes. No binding was seen with rabbit IgG at these ages, or with opossum and rabbit IgG when enterocytes were obtained from opossums aged 55-73 days. Opossum anti-SRBC antibody (IgG) fed to sucklings at 52 days and earlier (but not later) could subsequently be detected in the serum. However, rabbit anti-SRBC antibody (IgG) could not be detected in the blood serum when fed to sucklings of any age. Fluorescent tracing of FITC-labelled opossum and rabbit IgG fed to suckling opossums, and of endogenous opossum IgG, pointed to transport of the homologous IgG occurring across gut enterocytes of the proximal region. These results suggest that IgG is recognised and transcytosed by specific Fc gamma Rs present on opossum enterocytes prior to weaning.

Apical expression of an antigen common to rabbit yolk sac endoderm and kidney proximal tubule epithelium.

The tissue distribution, molecular weight, and biochemical nature of an antigen detected by a mouse monoclonal antibody designated 283D3 and raised against rabbit visceral yolk sac endodermal cells, has been investigated. The antigen is located on the luminal side of apical tubules and large sub-apical vesicles in rabbit yolk sac endoderm and proximal kidney tubule epithelial cells. It is expressed in a similar polarised fashion in epithelial cells lining the epididymis. Western blotting showed the antigen to comprise proteins of molecular weight 330-380 kDa. The antigen has been affinity purified from yolk sac and kidney and is predominantly protein in nature with a small percentage of N-linked carbohydrate. In terms of tissue distribution and molecular weight it has close similarity to Heymann nephritis antigen but differs in not being confined to coated pits. Its function is not known, but the association with endocytic elements implies a possible role in non-specific protein absorption.

The route of maternal IgM transport to the rabbit fetus.

The route of IgM transport to the rabbit fetus was investigated by comparing its localization with that of IgG in the yolk sac splanchnopleure and uterine tissues using direct immunofluorescence and immunodiffusion analysis. IgM was first detected in fetal serum at 20 days of gestation but was present in uterine fluid at 18 days, the earliest stage tissues and fluids were examined. IgM was co-localized with IgG in the yolk sac endoderm basement membrane and in the vascular mesenchyme of the yolk sac splanchnopleure providing evidence of its transport to fetal blood; it was also present in vesicles in the yolk sac endoderm. IgM could not be detected in uterine fluid of nonpregnant uterine horns of rabbits unilaterally pregnant. Human IgM injected into the maternal circulation was readily transported to the uterine fluid and across the yolk sac splanchnopleure to fetal blood indicating that IgM secreting plasma cells, found to be present in the uterine stroma, contributed little towards IgM in the uterine fluid. Degenerating paraplacental decidual tissue, a feature of rabbit pregnancy, is suggested to be a major route for maternal immunoglobulin transport to the uterine fluid.

Receptors for IgG2b on cells of the mouse metrial gland.

Single cells prepared from metrial glands of mice killed at days 10, 13 and 17 of pregnancy were assayed for the expression of Fc gamma receptors in a standard rosetting assay using sheep red blood cells sensitised with a mouse monoclonal IgG2b antibody. Rosettes, indicating Fc gamma receptors, were found on both granulated metrial gland (GMG) cells and non-GMG cells, comprising mainly stromal cells, from each stage of pregnancy. Some animals were given an intravenous injection of horseradish peroxidase 2 h before they were killed in order to identify endocytic cells. No GMG cells were found to have endocytosed the horseradish peroxidase. Non-GMG cells which showed endocytic activity all expressed Fc gamma receptors but these receptors were also found on some of the non-GMG cells which had not exhibited endocytosis. The finding of Fc gamma receptors on GMG cells provides further evidence that these cells may be related to NK cells.

Fc receptor-mediated transcytosis of IgG-coated liposomes across epithelial barriers.

Drug carriers such as liposomes are not readily transported across cellular barriers that constitute epithelia. However, certain epithelia (rabbit yolk sac endoderm and enterocytes of suckling rat gut proximal small intestine) are well known to transcytose maternal IgG by Fc receptor-mediated endocytic events. We have shown that coating liposomes with appropriate IgG enhances their transport across these epithelia, as measured both by radioactivity indicative of liposomal membrane or entrapped 125I-PVP and [3H]inulin, and by the hypoglycemic effect of entrapped insulin. It is suggested that these transported liposomes follow a pathway of transcytosis in clathrin-coated vesicles, thus escaping lysosomal degradation.

Effect of dexamethasone on Fc gamma receptor expression in foetal and neonatal rat gut.

When injected into 12-day-old suckling rats, dexamethasone caused a precocious disappearance of Fc gamma receptors from enterocytes of the proximal small intestine. However, dexamethasone appeared to be necessary for the maintenance or production of such receptors in foetal rat gut cultured in vitro.

Ontogeny of the Fc gamma receptor in rat small intestine.

The time course of Fc gamma receptor expression on isolated enterocytes of the small intestine of rat fetuses and sucklings has been studied. This was achieved principally using indicator sheep red blood cells (SRBC) sensitized with rabbit IgG in an erythrocyte-antibody rosette assay which detects receptors located mainly on the lateral and basal plasma membrane, and in a more limited way using binding of rabbit IgG to metabolically inhibited gut as detected by immunofluorescence and which detects receptors located on the apical brush border. From the time they were first detectable in the rosette assay (20-day-old fetuses) to the time they disappeared (22-day-old sucklings) such receptors were found always to be acid pH dependent and restricted to enterocytes from the proximal region. Acid pH, Fc-dependent binding of rabbit IgG to metabolically inhibited gut was first detectable at 21 days gestation and there were indications that receptors differentiate on enterocytes in a proximal to distal direction. This was also indicated by electron microscope studies using rabbit PAP injected into the duodenum of 21-day-old fetuses. Such studies also provided evidence for the receptor-mediated translocation of IgG across the duodenum of the fetal rat in a manner similar to that described for older sucklings.

Distribution of Fc gamma receptors on isolated gut enterocytes of the suckling rat.

An erythrocyte-antibody rosette assay has been used to study the presence and distribution of Fc gamma receptors on enterocytes isolated from 12-day-old suckling rat gut by means of a buffer medium containing EGTA. Such receptors were found to be restricted to enterocytes in the proximal region (duodenum and jejunum) of the small intestine and to be acid-pH dependent. For the majority of enterocytes indicator red cells bound in high density to the abluminal plasmalemma but not to the apical microvillous brush border. Since immunofluorescence studies revealed strong binding of added IgG to the microvillous region, a likely explanation is that there is a paucity of Fc gamma receptors from the tips of microvilli (at least under the conditions of the rosette assay) and that receptors more deeply situated as inaccessible to indicator red cells. Binding of indicator red cells was readily inhibited by rabbit, human, guinea pig and rat IgG but less so by bovine IgG, and of the two sub-classes, bovine IgG2 inhibited much more readily than bovine IgG1. Cortisone acetate injection virtually abolished Fc gamma receptor expression on isolated enterocytes within three days. These findings correlate both with selective transport of IgG of different species in vivo and the known effect of cortisone acetate to terminate such transport.

Secondary lens formation from the cornea following implantation of larval tissues between the inner and outer corneas of Xenopus laevis tadpoles.

Secondary lens formation from the cornea of larval Xenopus laevis has been used as a measure of the lens-inducing capacities of various larval Xenopus tissues. The experimental design employed involved implantation of selected body tissues between the inner and outer cornea of stage-50 tadpole eyes, in such a way that the integrity of the inner cornea and eye cup was not disrupted. Implantation of retina, pituitary, limb blastema or limb bud resulted in secondary lens formation from the outer cornea. Such lenses were similar in appearance to stage-5 lens regenerates described by Freeman (1963). No secondary lenses were observed in eyes receiving either heart or hind brain implants or in eyes which underwent corneal separation but which received no implant. It is concluded that the retina is the natural source of a stimulatory factor which initiates and maintains corneal transformation to lens during lens regeneration following lensectomy. Influences emanating from pituitary, limb blastema and limb bud, but apparently not from heart or hind brain, are able to act on cornea in a way similar to the retinal factor. Furthermore, our findings support the contention that in the normal eye, the inner cornea is a barrier to the passage of retinal factor and so maintains the single lens structure of the eye. When this barrier is by-passed by lens-inducing tissue, as in the present experimental design, lens formation from the cornea is able to take place. Electronmicroscopical studies have shown that the inner cornea, in the stage-50 tadpole eye, consists of a dense meshwork of collagen fibrils and a basal layer of cohesive elongated mesenchymal cells well suited for this barrier function.

Direct evidence for pH-dependent Fc receptors on proximal enterocytes of suckling rat gut.

By means of an erythrocyte-antibody rosette technique, Fc receptors, functional at pH 6.0 but not at 7.2, were shown to be present on enterocytes isolated from duodenum and jejunum (but absent from ileum) of 12-20-day-old suckling rats.

Lens regeneration from cornea of larval Xenopus laevis in the presence of the lens.

In Xenopus laevis tadpoles, wounding of the outer cornea failed to initiate lens regeneration. If both the outer and inner corneas were wounded or if the lens was dislocated, lens regeneration was initiated but failed to continue beyond stage III. However, lensectomy followed by re-implantation of the lens resulted in the regeneration of a fully differentiated lens in several cases, despite the presence of the re-implanted lens. Although some of the regenerates in these eyes were also arrested at stage III, those which attained full lens differentiation, i.e. stage V, developed normally and synthesized crystalline from the onset of stage IV as indicated by a positive immunofluorescence reaction. Histological examination of the dislocated and re-implanted lenses showed the majority of them to be normal in appearance. Cornea transplanted to the posterior chamber of the eye also regenerated a lens in the presence of the re-implanted lens. All these regenerates underwent lens fibre differentiation to give stage-V regenerates. These findings show that lens regeneration from the cornea can occur in the presence of lens. Results are discussed on the basis that contrary to earlier suggestions, an inhibitory lens factor does not exist in vivo, but rather that a factor for the initiation and maintenance of regeneration emanates from the eye cup and upon wounding of the inner cornea is able to reach the inner cell layer of the outer cornea and initiate lens regeneration.