Early Fetal Stages Research Articles

Basis of Changes in Left–Right Coordination of Rhythmic Motor Activity during Development in the Rat Spinal Cord

The basic neuronal networks generating coordinated rhythmic motor activity, such as left-right alternate limb movement during locomotion in mammals, are located in the spinal cord. In rat fetuses, the spatial pattern of the rhythmic activity between the left and right sides is synchronous at and shortly after rhythmogenesis before the pattern becomes alternate by birth. The neuronal mechanisms underlying these developmental changes in the left-right coordination were examined in isolated spinal cord preparations. Calcium imaging of commissural neurons at the early fetal stages revealed that the intracellular Ca2+ concentration of the commissural neurons was elevated by bath-application of 5-hydroxytryptamine (5-HT) in synchrony with the simultaneously recorded rhythmic activity of the ventral root, suggesting that the commissural neurons mediate the left-right coordination of the rhythmic activity from onset of the rhythmogenesis. Using a longitudinal split-bath setup, we show that the synchronicity in pattern of the rhythmic activity is the result of excitatory connections being formed via commissural neurons between the rhythm-generating networks located in the left and right spinal cord. During this period, such connections were found to be mediated by excitatory synaptic transmission via GABA(A) receptors. When the pattern of rhythmic activity became left-right alternate at later fetal stages, these connections, still via GABA(A) receptors, were mediating reciprocal inhibition between the two sides. Nearer birth, glycine receptors took over this role. Our results reveal the nature of the neuronal mechanisms forming the basis of the left-right coordination of rhythmic motor activity during prenatal development.

Critical periods for foetal mortality in gilts identified by analysing the length distribution of mummified foetuses and frequency of non-fresh stillborn piglets

The objective of this study was to investigate the timing of foetal mortality in gilts of a segregating F2 cross of Large White and Meishan pigs on the basis of the length distribution of mummified foetuses and the frequency of non-fresh stillborn piglets in order to establish whether critical periods for foetal mortality exist. All expelled conceptuses and placentae of 192 farrowing gilts with a normal health status were meticulously investigated to recover all mummified foetuses. The length of each mummified foetus was measured. The predicted number of foetuses present per gilt at the early foetal stage of gestation was calculated as the sum of numbers of mummified foetuses and non-fresh stillborn, fresh stillborn and liveborn piglets. Foetal loss was calculated as the sum of mummified foetuses and non-fresh stillborn piglets. The average foetal mortality rate per gilt was 8.7%. In total 162 mummified foetuses were found (average 0.84 per litter), ranging in length from 0.4 to 33.0 cm. This indicates a range in foetal age at death of ∼35–100 days. Although mummified foetuses of all lengths within the above mentioned range were found, relatively many had a length of less than 4 cm or of 10–21 cm. The total number of non-fresh stillborn piglets (i.e. late foetal deaths) was 58 (average 0.30 per litter). It can be concluded that foetal mortality occurred in these gilts throughout the period from day 35 to term, with relatively high incidences at the early foetal stage (days 35–40), shortly after mid-pregnancy (days 55–75) and after ∼day 100 of gestation. These three periods coincide with reported periods of change in porcine placental growth.

Hyperbaric oxygenation prevented brain injury induced by hypoxia–ischemia in a neonatal rat model

The occurrence of hypoxia–ischemia (HI) during early fetal or neonatal stages of an individual leads to the damaging of immature neurons resulting in behavioral and psychological dysfunctions, such as motor or learning disabilities, cerebral palsy, epilepsy or even death. No effective treatment is currently available and this study is the first to use hyperbaric oxygen (HBO) as a treatment for neonatal HI. Herein, we sought out to determine if HBO is able to offer neuroprotectivity against an HI insult. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O 2 at 37 °C). HBO treatment was administered by placing pups in a chamber (3 ATA for 1 h) 1 h after hypoxia exposure. Brain injury was assessed based on ipsilateral hemispheric weight divided by contralateral hemispheric weight, light microscopy, and EM. Sensorimotor functional tests were administered at 5 weeks after hypoxia exposure. After HI, the ipsilateral hemisphere was 52.65 and 57.64% ( P<0.001) of the contralateral hemisphere at 2 and 6 weeks, respectively. In HBO treated groups, the ipsilateral hemisphere was 77.77 and 84.19% ( P<0.001) at 2 and 6 weeks. There was much less atrophy and apoptosis in HBO treated animals under light or electron microscopy. Sensorimotor function was also improved by HBO at 5 weeks after hypoxia exposure (Chi-square, P<0.050). The results suggest that HBO is able to attenuate the effects of HI on the neonatal brain by reducing the progression of neuronal injury and increasing sensorimotor function.

Low dose fetal irradiation, chromosomal instability and carcinogenesis in mouse

A study was carried out to see if acute low dose fetal irradiation of mouse could induce long lasting chromosomal anomalies and adult cancers. The abdomen of pregnant Swiss mice was exposed to 0.1–1 Gy of 60Co gamma radiation on day 14 of gestation. The animals were killed 24 h later or allowed to deliver their young. Hemopoietic cell death and chromosomal aberrations were studied in the fetal liver. Chromosomal instability was traced in the 1 Gy exposed group from the fetal liver cells through their CFU-S to postnatal bone marrow. The F1 mice were observed for chromosomal aberrations in the bone marrow and for solid tumor incidence. Irradiation produced a significant dose-dependent increase in the hemopoietic cell death and aberrant metaphases in the fetal liver. The main types of aberrations were chromatid breaks and fragments. The aberrations persisted in the spleen colonies developed from these cells. Their frequency decreased in the postnatal bone marrow, however, showed a significant increase at 12 months of age. Some exposed animals had abnormally high leukocyte counts and after 1 Gy their bone marrow had eight times more polyploid cells than the control. Prenatal exposure also significantly increased the incidence of solid tumors, the ovaries showing the highest risk. It is concluded that a single exposure below 1 Gy of gamma radiation at the early fetal stage of these mice can induce persistent chromosomal instability in the hemopoietic cells, and significantly increase the incidence of solid tumors in adults.

Meckel Gruber syndrome: a first trimester diagnosis of a recurrent case

We report of a case of Meckel Gruber Syndrome (MGS) in a woman, who suffered previously from a pregnancy with the same disorder. MGS, consisting of an occipital encephalocele, bilateral cystic kidneys and postaxial polydactyly, is a rare autosomal recessive disorder, with a recurrence risk of 25%. With the present technology, a targeted ultrasound in the late embryonic or early fetal stages of pregnancy has the potential to diagnose this syndrome. Clinical screening in further pregnancies is of utmost importance and the management of such cases is presented.

Cooperation and competition between the binding of COUP-TFII and NF-Y on human epsilon- and gamma-globin gene promoters.

The nuclear receptor COUP-TFII was recently shown to bind to the promoter of the epsilon- and gamma-globin genes and was identified as the nuclear factor NF-E3. Transgenic experiments and genetic evidence from humans affected with hereditary persistence of fetal hemoglobin suggest that NF-E3 may be a repressor of adult epsilon and gamma expression. We show that, on the epsilon-promoter, recombinant COUP-TFII binds to two sites, the more downstream of which overlaps with an NF-Y binding CCAAT box. Binding occurs efficiently to either the 5' or the 3' COUP-TFII site but not to both sites simultaneously. However, adding recombinant NF-Y induces the formation of a stable COUP-TFII.NF-Y-promoter complex at concentrations of COUP-TFII that would not give significant binding in the absence of NF-Y. Mutations of the promoter indicate that COUP-TFII cooperates with NF-Y when bound to the 5' site, whereas binding at the 3' site is mutually exclusive. Likewise, in the gamma-promoter, COUP-TFII binds to a site overlapping the distal member of a duplicated CCAAT box, competing with NF-Y binding. Transfections in K562 cells show that both the mutation of the 5' COUP-TFII or of the NF-Y site on the epsilon-promoter decrease the activity of a luciferase reporter; the mutation of the 3' COUP-TFII site has little effect. These results, together with transgenic experiments suggesting a repressive activity of COUP-TFII on the epsilon-promoter and the observation that, on the 3' site, COUP-TFII and NF-Y binding is mutually exclusive, suggest that COUP-TFII may exert different effects on epsilon transcription depending on whether it binds to the 5' or to the 3' site. At the 5' site, COUP-TFII might cooperate with NF-Y, forming a stable complex, and stimulate transcription; at the 3' site, COUP-TFII might compete for binding with NF-Y and, directly or indirectly, decrease gene activity.

Development of electrophysiological and morphological diversity in autonomic neurons.

The generation of neuronal diversity requires the coordinated development of differential patterns of ion channel expression along with characteristic differences in dendritic geometry, but the relations between these phenotypic features are not well known. We have used a combination of intracellular recordings, morphological analysis of dye-filled neurons, and stereological analysis of immunohistochemically labeled sections to investigate the development of characteristic electrical and morphological properties of functionally distinct populations of sympathetic neurons that project from the celiac ganglion to the splanchnic vasculature or the gastrointestinal tract of guinea pigs. At early fetal stages, neurons were significantly more depolarized at rest compared with neurons at later stages, and they generally fired only a single action potential. By mid fetal stages, rapidly and slowly adapting neurons could be distinguished with a topographic distribution matching that found in adult ganglia. Most rapidly adapting neurons (phasic neurons) at this age had a long afterhyperpolarization (LAH) characteristic of mature vasomotor neurons and were preferentially located in the lateral poles of the ganglion, where most neurons contained neuropeptide Y. Most early and mid fetal neurons showed a weak M current, which was later expressed only by rapidly-adapting and LAH neurons. Two different A currents were present in a subset of early fetal neurons and may indicate neurons destined to develop a slowly adapting phenotype (tonic neurons). The size of neuronal cell bodies increased at a similar rate throughout development regardless of their electrical or neurochemical phenotype or their topographical location. In contrast, the rate of dendritic growth of neurons in medial regions of the ganglion was significantly higher than that of neurons in lateral regions. The apparent cell capacitance was highly correlated with the surface area of the soma but not the dendritic tree of the developing neurons. These results demonstrate that the well-defined functional populations of neurons in the celiac ganglion develop their characteristic electrophysiological and morphological properties during early fetal stages of development. This is after the neuronal populations can be recognized by their neurochemical and topographical characteristics but long before the neurons have finished growing. Our data provide strong circumstantial evidence that the development of the full phenotype of different functional classes of autonomic final motor neurons is a multi-step process likely to involve a regulated sequence of trophic interactions.

Effect of early fetal irradiation on the postnatal development of mouse.

An earlier study has shown that irradiation at the late fetal stage of Swiss albino mice disturbed postnatal growth and appearance of physiological markers. The present study was done to determine the effect of irradiation at the early fetal stage on the postnatal development of mouse. Fourteen-day pregnant Swiss albino mice were exposed to 0.1-1.5 Gy of cobalt-60 gamma-rays. F1 pups were observed for < or = 6 weeks of age. Postnatal mortality, body weight and length, head length and width, tail length, and the time of appearance of physiological markers (pinna detachment, eye opening, fur development, vaginal opening and testes descent) were noted. There was no increase in congenital anomalies. Postnatal mortality and percentage of growth-retarded pups increased significantly at doses of 0.5-1.5 Gy. A significant delay in the appearance of all the physiological markers was also noted at these doses. Body length, head length, and tail length remained significantly lower than in the controls throughout the observation period at doses of 0.3-1.5 Gy, whereas body weight and head width showed such a persistent change only at > or = 0.5 Gy. The early fetal day 14 in mouse is sensitive to radiation-induced postnatal mortality and impairment of growth and temporal development of physiological markers, but not to induction of congenital anomalies. While mortality and physiological markers are not affected at <0.5 Gy, growth retardation appears to have a lower threshold of approximately 0.3 Gy.

Stimulating Effect of Glycoprotein Hormone Free α‐Subunit and Daily Gonadotropin Releasing Hormone Treatment on Prolactin Release from 50‐Day Ovine Foetal Pituitary Explants

AbstractThe aim of our study was to determine whether free α of glycoprotein hormones (free α) plays a role in lactotroph function during early pituitary development in the sheep foetus. Detection and quantification of free α, luteinzing hormone β‐subunit (LHβ) and prolactin immunolabelling were determined by immunocytochemistry at days 32, 37, 42, 50 and 63 of gestation. Free α‐ and LHβ‐containing cells were first detected in the ovine foetal pituitary gland on day 37 of gestation, while prolactin‐containing cells were first identified on day 42. Analysis of serial sections suggested that free α immunoreactive cells were also LHβ‐positive, indicating that free α was mainly synthesized by gonadotrophs. In early foetal stages, free α occurred in the antero‐medio ventral region of the pituitary gland, whereas prolactin‐containing cells were more dorsally and more caudally localized. The free α‐, LHβ‐ and prolactin‐immunostained area increased markedly between days 50 and 63 of gestation. To evaluate a possible functional relationship between gonadotrophs and lactotrophs, the effects of free α or gonadotropin releasing hormone (GnRH) on prolactin release were assayed. Chronic treatment of pituitary explants from male and female 42‐day‐old ovine foetuses for 8 days with 10−9 or 10−7 M ovine free α did not affect prolactin release. By contrast, free α administration on pituitary explants from male and female 50‐day‐old foetuses resulted in enhanced prolactin release. At this age, a daily (2 h per day) treatment with 10−8 M GnRH had similar stimulatory effect to free α whereas a ‘first day’ treatment (24 h on the first day) reduced prolactin release throughout the culture in males and had no effect in females. These results indicate that, despite early detection of free α at day 37 in the ovine foetal pituitary, its stimulatory effect on prolactin release occurs from day 50 of gestation, corresponding to the first period of lactotroph development in vivo. A daily treatment with GnRH mimics the effect of free α on prolactin release.

Stimulating effect of glycoprotein hormone free alpha-subunit and daily gonadotropin releasing hormone treatment on prolactin release from 50-day ovine foetal pituitary explants.

The aim of our study was to determine whether free alpha of glycoprotein hormones (free alpha) plays a role in lactotroph function during early pituitary development in the sheep foetus. Detection and quantification of free alpha, luteinzing hormone beta-subunit (LHbeta) and prolactin immunolabelling were determined by immunocytochemistry at days 32, 37, 42, 50 and 63 of gestation. Free alpha- and LHbeta-containing cells were first detected in the ovine foetal pituitary gland on day 37 of gestation, while prolactin-containing cells were first identified on day 42. Analysis of serial sections suggested that free alpha immunoreactive cells were also LHbeta-positive, indicating that free alpha was mainly synthesized by gonadotrophs. In early foetal stages, free alpha occurred in the antero-medio ventral region of the pituitary gland, whereas prolactin-containing cells were more dorsally and more caudally localized. The free alpha-, LHbeta- and prolactin-immunostained area increased markedly between days 50 and 63 of gestation. To evaluate a possible functional relationship between gonadotrophs and lactotrophs, the effects of free alpha or gonadotropin releasing hormone (GnRH) on prolactin release were assayed. Chronic treatment of pituitary explants from male and female 42-day-old ovine foetuses for 8 days with 10-9 or 10-7 M ovine free alpha did not affect prolactin release. By contrast, free alpha administration on pituitary explants from male and female 50-day-old foetuses resulted in enhanced prolactin release. At this age, a daily (2 h per day) treatment with 10-8 M GnRH had similar stimulatory effect to free alpha whereas a 'first day' treatment (24 h on the first day) reduced prolactin release throughout the culture in males and had no effect in females. These results indicate that, despite early detection of free alpha at day 37 in the ovine foetal pituitary, its stimulatory effect on prolactin release occurs from day 50 of gestation, corresponding to the first period of lactotroph development in vivo. A daily treatment with GnRH mimics the effect of free alpha on prolactin release.

Effect of irradiation at the early foetal stage on adult brain function of mouse: learning and memory

Purpose : To study the long-term effect of early foetal irradiation on the learning and memory in the adult mouse. Materials and methods : The abdominal area of pregnant Swiss albino mice was exposed to a single dose of 0.25-1.5Gy γ-radiation on the 14th day of gestation and the mice were left to deliver their offspring. At 6 months of age, the learning and memory functions of the F 1 mice were tested by hole-board activity, conditioned avoidance response and radial arm maze performance. The animals were again subjected to the radial arm maze test at 12 and 18 months of age. Results : There was a significant dose-dependent decrease in the learning ability and memory retention of 6-month-old mice at doses > 0.25Gy. The significant changes persisted to 18 months of age in mice exposed to ≥0.5Gy. All changes showed a linear dose-response at doses < 1Gy. Conclusions : The gestational day 14 of Swiss albino mice is a sensitive stage in brain development to γ-ray-induced impairment of learning and memory during the adult life. Permanent deficits in these functions can be induced by a dose of ~0.5Gy at this stage, when the developmental activity of the cerebral cortex is at its peak.

Differentially expressed endoderm and mesenchyme genes along the fetal rat intestine

Developmental studies have shown that morphological and functional regionalization occurring along the mammalian intestine is defined at early fetal stages and that some aspects of this patterning are dependent on epithelial-mesenchymal cell interactions. The molecular basis of these processes are largely unknown. In this study, a differential display approach was used to identify genes differentially expressed along the longitudinal axis in the intestinal endoderm and mesenchyme moieties of 14-day-old rat fetuses at a stage prior to morpho-functional differentiation of the gut. Fifty-eight genes were identified, 36 being identical or similar to known genes and 13 corresponding to ESTs or genome sequences with unknown function. Nine cDNAs could not be assigned to any previously described nucleotide sequence. The selected genes are involved in several aspects of cell physiology, including metabolic pathways, cytoskeleton organization, signal transduction, protein biosynthesis, and regulation of gene transcription.

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis.

Parental-specific epigenetic modifications are imprinted on a subset of genes in the mammalian genome during germ cell maturation. However, the precise timing of their establishment remains to be determined. Methylation of CpG dinucleotides has been shown to be a part of the parental imprint. We have examined how the methylation pattern characteristic of the paternal allele in germ cells are established during human spermatogenesis. Two representative imprinted genes, H19 and MEST/PEG1, were studied. The experiments were performed using the bisulphite sequencing method on microdissected individual cells at different stages of male germ cell differentiation. We show that both genes are unmethylated in fetal spermatogonia, suggesting that all pre-existing methylation imprints are already erased by this stage. The MEST/PEG1 gene remains unmethylated at all subsequent post-pubertal stages of spermatogenesis, including mature spermatozoa. The methylation of H19 typical of the paternal allele first appears in a subset of adult spermatogonia and then is maintained in spermatocytes, spermatids and mature spermatozoa. Our results suggest that the methylation imprint inherited from the parents is first erased in the male germ line at an early fetal stage. The paternal-specific imprint is re-established only later, during spermatogonial differentiation in the adult testis.

Rat Embryo Cultures for In Vitro Teratology

Mammalian embryos provide a particular challenge to those who wish to study developmental processes because development takes place inside the mother's body, thus limiting the investigator's ability to directly affect and observe the embryonic stages of development. Fortunately, as this unit illustrates, methods have been developed for in vitro culture of rodent embryos during early postimplantation (gestation days 9 to 11) and early fetal (gestation days 12 to 14) stages, which are the periods during which the major systems of the embryo are established. Embryos at these stages are particularly suitable for screening studies to determine the effects of teratological agents.

Molecular and Biochemical Characterization of a Serine Proteinase Predominantly Expressed in the Medulla Oblongata and Cerebellar White Matter of Mouse Brain

A full-length cDNA clone of a serine proteinase, mouse brain serine proteinase (mBSP), was isolated from a mouse brain cDNA library. mBSP, which has been recently reported to be expressed in the hair follicles of nude mice, is most similar (88% identical) in sequence to rat myelencephalon-specific protease. The mBSP mRNA was steadily expressed in the brain of adult mice with a transient expression in the early fetal stage during development. The genomic structure of the mouse gene for mBSP was determined. The gene, which is mapped to chromosome 7B4-B5, is about 7.4 kilobases in size and contains 7 exons. Interestingly, the 5'-untranslated region of the mBSP gene was interrupted by two introns. In situ hybridization analyses revealed that mBSP is expressed in the white matter of the cerebellum, medulla oblongata, and capsula interna and capsula interna pars retrolenticularis of mouse brain. Further, mBSP was immunolocalized to the neuroglial cells in the white matter of the cerebellum. Recombinant mBSP was produced in the bacterial expression system and activated by lysyl endopeptidase digestion, and the activated enzyme was purified for characterization. The enzyme showed amidolytic activities preferentially cleaving Arg-X bonds when 4-methylcoumaryl-7-amide-containing peptide substrates were used. Typical serine proteinase inhibitors, such as diisopropyl fluorophosphates, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, strongly inhibited the enzyme activity. The recombinant mBSP effectively hydrolyzed fibronectin and gelatin, but not laminin, collagens I and IV, or elastin. These results suggest that mBSP plays an important role in association with the function of the adult mouse brain.

Gene expression of Na+/Ca2+ exchanger during development in human heart

In immature animal hearts, lower activity of sarcoplasmic reticulum and lower densities of Ca2+ channels highlight the potentially vital role of the Na+/Ca2+ exchanger (NCX) to excitation-contraction coupling. To date, studies on NCX expression have been restricted to late developmental stages. The distribution and gene expression of NCX during early ontogeny is not known, especially in humans. In the present report, we systematically characterized changes in NCX gene expression in human heart during development, with particular emphasis in early ontogeny. Human hearts during early gestation (9- to 20-week gestation), neonatal (1 to 2 days after birth) and adulthood (18-40 years old) were used. NCX mRNA levels were studied using RNase Protection Assay (RPA) and NCX protein levels were assessed by Western blot. Wet weight was also used as the tissue base. Immunolocalization studies using confocal microscopy were performed in isolated fetal cardiac myocytes. Normalization of NCX mRNA derived from ventricles against an early gestational age (10-week gestation) shows that NCX mRNA levels nominally increased from 1 to 1.13 at 19-week gestation then decreased to 0.74 (P < 0.05) at neonate and further decreased to 0.23 (P < 0.05) at adult stages. NCX protein levels increased from 1 at 9-week gestation to 3 (P < 0.05) at 20-week gestation and then decreased to 1.8 (P < 0.05) at neonate and to 1.87 (P < 0.05) at adult stages. Confocal imaging of fetal cardiac myocytes revealed intense homogeneous membrane staining and abundance of NCX protein at this stage. The data demonstrate changes in NCX transcript and NCX protein levels as well as total RNA and proteins during human heart development. Per wet weight, NCX mRNA was 4.5 times greater at early fetal than adult stages and NCX protein was 2 times greater at adult than the early fetal stage indicating considerable post-transcriptional regulation. These findings provide new insights into the understanding of temporal changes in NCX in the developing heart at the gene level. The functional significance remains to be determined.

Effect of irradiation at the early fetal stage on adult brain function in the mouse: locomotor activity

Purpose : To study long-term changes in the adult locomotor activity of mice after exposure to γradiation at the early fetal stage of development. Materials and methods : Pregnant Swiss albino mice were exposed locally on the abdominal area to a single dose of 0.25-1.5 Gy of 60 Co gamma radiation at the dose rate of 1 Gy/min. When the F1 offspring were 6 months old, their locomotor and exploratory behaviour was assessed by the open-field and dark/bright arena tests. Animals were again subjected to the dark/bright arena test at 12 and 18 months of age in order to study the persistence of the effects. Results : Irradiation produced a noticeable disturbance in the normal behaviour pattern of the mice. There was a significant dose-dependent decrease in the open-field activity of 6-month old mice. In the dark/bright arena test, the time spent and lines crossed in the dark area showed a significant decrease, while their activities in the brightly lit area increased significantly, indicating a reduced aversion to bright light. These effects were evident even at a dose of 0.3Gy and increased linearly with dose. The significant behavioural changes persisted at 12 months, but at 18 months the difference in the time spent and lines crossed in the dark and bright areas were not significantly different from sham-irradiated control values below 0.5 Gy. Conclusions : These results show that day 14 of gestation in Swiss albino mice is a time of high risk for inducing long-term changes in the adult locomotor function by γ-radiation doses below 1Gy. Using a range of radiation doses and different observation times we have demonstrated that the effect increases linearly with dose, but there appears to be a threshold of 0.3-0.5Gy for producing significant persistent changes in the adult ambulatory activity.

Neurochemical differentiation of functionally distinct populations of autonomic neurons.

The coeliac ganglion of guinea pigs displays a unique topographical arrangement of neurochemically and functionally distinct populations of sympathetic neurons. The authors used multiple-labeling immunohistochemistry to investigate the neurochemical differentiation of these neurons during embryonic and fetal development. Sympathoadrenal precursors, located on either side of the abdominal aorta, were intensely immunoreactive for tyrosine hydroxylase (TH-IR), neurofilament, and the human natural killer 1 antibody at midembryonic stages (Carnegie stages 16-19). During late embryonic stages (stages 20-23), a single bilobed ganglion had formed. At this time, neuropeptide Y immunoreactivity (NPY-IR) was widely expressed in sympathetic neurons (with moderate TH-IR) and chromaffin cells (with intense TH-IR). The onset of somatostatin (Som-IR) expression followed that of NPY-IR and was restricted to sympathetic neurons. However, at late embryonic stages, most TH-IR neurons with Som-IR also expressed NPY-IR (a combination of peptides not found in the mature coeliac ganglion). Between late embryonic stages and the end of the early fetal period, there was a significant increase in the proportion of neurons in lateral regions that had both NPY-IR and TH-IR. At the same time, there was an increase in the proportion of neurons in medial regions that had both Som-IR and TH-IR. Neurons expressing both Som-IR and TH-IR were rarely observed in lateral regions of the coeliac ganglion. Thus, a clear topography within the coeliac ganglion is established during late embryonic and early fetal stages of development and reflects that found in the mature animal by the end of the early fetal period.

Partial blockade of T-cell differentiation during ontogeny and marked alterations of the thymic microenvironment in transgenic mice with impaired glucocorticoid receptor function

Glucocorticoids (GCs) are widely known to be potent modulators of the immune system. The role of GCs in thymopoiesis as well as the integration of the thymus with the neuroendocrine system is, however, poorly understood. In the present work, we have studied, in transgenic mice with an impaired GC function, the alterations which occur in both T-cell differentiation and thymic stroma maturation, throughout ontogeny as well as in adult condition, analyzing their possible rebounding on the status of adult splenic T lymphocyte populations. These transgenic mice have been described to present a significant decrease (60–70%) of thymic and splenic GC receptor binding capacity but maintain normal their basal plasma ACTH and corticosterone levels. The animals showed a partial blockade of T-cell differentiation and decreased percentages of apoptotic cells during fetal development but not in adult life, when thymic cellularity was significantly increased although thymocyte apoptosis response was not affected. In contrast, thymic stroma was profoundly altered from early fetal stages and large epithelium-free areas appeared in adult thymus. On the other hand, our study revealed a reduction of the splenic TcRαβ population accompanied by an increase in the CD4/CD8 ratio. The analysis of different adhesion molecules as well as activation markers demonstrated that most of them (CD5, CD11a, CD11b, CD69 and MHC Class II) were normally expressed in transgenic lymphocytes, whereas CD44 and CD62L expression was altered indicating the existence of an increased proportion of primed T-cells in these animals. In view of the mutual interdependence of thymic stroma and thymocyte maturation, the partial blockade of T-cell differentiation during ontogeny and the profound alterations of the stromal cell compartment in transgenic mice with impaired GR function suggest a key role for GCs in coordinating the physiological dialogue between the developing thymocytes and their microenvironment.

Morphogenesis of the submucosal gland of cattle (Bos primigenius f. taurus)

In the 10th week of gestation the development of duodenal glands in the bovine intestinum starts by circumscript epithelial proliferation. Their secretory channels are in contact with the crypts. Already in the early fetal stage ultrastructurally there are an inconspicuous equipment with organelles in the apical cytoplasm and single globular granules, which demonstrate the differentiation of epithelial cells into duodenal glandular cells. Until the mid-point of the pregnancy the granular endoplasmic reticulum strongly develops as a condition for the impressive increase in the formation of the prosecretory granules. Finally, in the second half of the pregnancy, granules varying in structure and electronic density, but mostly showing an intensively osmiophilic and fine granular material, are the predominant cytoplasmic structures. Like in goblet cells capsulated vacuoles seem to play a role in the formation of the secretory granules. Recent, extensive studies concerning our results have been discussed.