Early Fetal Stages Research Articles

137 MACROSCOPIC AND MICROSCOPIC CHARACTERISTICS OF THE FEMALE LLAMA (LAMA GLAMA) FETAL, INFANTILE, AND ADULT GENITAL TRACTS

The higher incidence of the left uterine horn gestations that occur in llamas (Lama glama) might be due to anatomical differences between the uterine horns. A study of the macro- and micro-anatomy of the female llama genital tract was conducted to determine possible differences between the left and the right uterine horns. Female genital tracts were obtained at a local slaughterhouse and immediately kept in a fixing solution. A total of 74 genital tracts were inspected grossly and/or by translumination after fixation (54 fetuses, 2 infantiles, 1 and 30 days old, and 18 adults, 3 to 8 years old) to determine gross anatomy. Fetal genital tracts were arranged, according to the Crown-Ramp lengths (C-R), into 6 groups: Group 1, 25.0-29.5 cm (n = 3); Group 2, 30.0-39.5 cm (n = 18); Group 3, 40.0-49.5 cm (n = 18); Group 4, 52.0-59.6 cm (n = 9); Group 5, 62.7-63.5 cm (n = 2); and Group 6, 70.6-78.9 cm (n = 4). Length of the uterine septum and the outer length (flexible ruler) and diameter (caliper) of the uterine horns, body of the uterus, and cervix were taken. Uterine luminal diameter was taken at 3 previously selected places. Length and diameter of the left and right uterine horns were compared by differences of means for paired observations and used to prepare regression curves of length and diameter growth in relation to C-R length. Seventeen of the 54 fetal genital tracts (two from Group 5 and three from each of the other groups), both infantile tracts, and 11 of the 18 adult tracts were used to determine microscopic characteristics. Histological preparations of tissue from 3 previously selected areas, (3 sections/area) of the left and right uterine horns, and from 1 area of the uterine septum and cervix, were prepared. Thickness samples of the uterine wall, endometrium, and myometrium plus perimetrium of the left and right uterine horns were measured under the microscope using a micrometer and were compared by differences of means for paired observations in relation to C-R length. Results indicated that, on an anatomical basis, the outer lengths and luminal diameters of the left uterine horn, from early fetal stages to adulthood, were greater than the right uterine horn (P ≤ 0.05). The special anatomical disposition of the uterine septum appeared to restrict the diameter of the lumen of the right horn. No microscopic differences were detected between horns. Additionally, uterine glandular development was found in fetal genital tracts at an early age (from C-R length, 34.5 cm and onward) and no differences were found either between left and right uterine horns or between fetal and adult genital tracts. In summary, both uterine horns, on a microscopic basis, were not different and appeared to be competent to carry out physiological events such as gestation. On a gross anatomical basis, the left uterine horn presented a greater luminal diameter than the right uterine horn. The greater space that the left uterine horn offers and the peculiar disposition of the uterine septum appear to contribute to the higher incidence of left uterine horn gestations.

From spatial-data to 3D models of the developing human brain

Visualisation and interpretation of gene expression data have been crucial to advances in our understanding of mechanisms underlying early brain development. As most developmental processes involve complex changes in size, shape and structure, spatial-data can most readily provide information at multiple levels (cell type, cell location in relation to tissue organisation or body axes, etc.), that can be related to these complex changes. Although three-dimensional (3D) spatial-data are ideal, the restricted availability of suitable tissues makes it difficult to generate these for genes expressed at early human fetal stages. Mapping gene expression data to representative 3D models facilitates combinatorial analysis of multiple expression patterns but does not overcome the problems of sparsely sampled data in time and space. Here we describe software that allows 3D domains to be reconstructed by interpolating between sparse 2D gene expression patterns that have been mapped to 3D representative models of corresponding human developmental stages. A set of procedures are proposed to infer expression domains in these gaps. The procedures, which are connected in a serial way, include components clustering, components tracking, shape matching and points interpolation. Each procedure consists of a graphical user interface and a set of algorithms. Results on exemplar gene data are provided.

The role of glycosphingolipid metabolism in the developing brain

Glycosphingolipids (GSLs) are amphipathic lipids ubiquitously expressed in all vertebrate cells and body fluids, but they are especially abundant in the nervous system. The synthesis of GSLs generally is initiated in the endoplasmic reticulum and completed in the Golgi apparatus, followed by transportation to the plasma membrane surface as an integral component. The amount and expression patterns of GSLs change drastically in brains during the embryonic to postnatal stages. Recent studies have revealed that GSLs are highly localized in cell surface microdomains and function as important components that mediate signal transduction and cell adhesion. Also in developing brains, GSLs are suggested to play important roles in nervous system formation. Disturbance of GSL expression and metabolism affects brain function, resulting in a variety of diseases, particularly lysosomal storage diseases. In this review, we describe some aspects of the roles of GSLs, especially of gangliosides, in brain development.

Glutamine synthetase is essential for proliferation of fetal skin fibroblasts

Background. Glutamine synthetase (GS) is ubiquitously expressed in the human and plays a major role for many metabolic pathways. However, little is known about its role during the fetal period. Methods. Cultured skin fibroblasts derived from an aborted fetus deficient in GS activity due to a R324C exchange as well as fetal and mature controls were used to determine the level of GS-expression, apoptosis, and proliferation in presence or absence of exogenous glutamine. Results. Glutamine synthetase can be found at early gestational stages. Loss of GS activity either inherited or induced through l-methionine sulfoximine leads to an upregulation of the GS protein but not of the GS mRNA and results in a significant drop in the proliferation rate but has no effect on apoptosis. Exogenous glutamine does not influence the rate of apoptosis but increases proliferation rates of the fetal but not the mature fibroblasts. Conclusion. GS can be found during early human fetal stages when it displays a significant effect on cell proliferation.

Regional- and temporal-dependent changes in the differentiation of Olig2 progenitors in the forebrain, and the impact on astrocyte development in the dorsal pallium

Olig2 is a basic helix–loop–helix transcription factor essential for oligodendrocyte and motoneuron development in the spinal cord. Olig2-positive (Olig2+) cells in the ventricular zone of the ventral telencephalon have been shown to differentiate into GABAergic and cholinergic neurons. However, the fate of Olig2 lineage cells in the postnatal forebrain has not been fully described and Olig2 may regulate the development of both astrocytes and oligodendrocytes. Here, we examined the fate of embryonic Olig2+ progenitors using a tamoxifen-inducible Cre/loxP system. Using long-term lineage tracing, Olig2+ cells in the early fetal stage primarily differentiated into GABAergic neurons in the adult telencephalon, while those in later stages gave rise to macroglial cells, both astrocytes and oligodendrocytes. Olig2+ progenitors in the diencephalon developed into oligodendrocytes, as observed in the spinal cord, and a fraction developed into glutamatergic neurons. Olig2 lineage oligodendrocytes tended to form clusters, probably due to local proliferation at the site of terminal differentiation. In spite of the abundance of Olig2 lineage GABAergic neurons in the normal neocortex, GABAergic neurons seemed to develop at normal density in the Olig2 deficient mouse. Thus, Olig2 is dispensable for GABAergic neuron specification. In contrast, at the late fetal stage in the Olig2 deficient mouse, astrocyte development was retarded in the dorsal neocortex, but not in the basal forebrain. Olig2 functions, therefore, in gliogenesis in the dorsal pallium. Short-term lineage tracing experiments revealed that the majority of late Olig2+ cells were not direct descendants of early Olig2+ progenitors in the fetal forebrain. These observations indicate that embryonic Olig2+ progenitor cells change their differentiative properties during development, and also that Olig2 plays a role in astrocyte development in a region-specific manner.

Gene placement and competition control T cell receptor γ variable region gene rearrangement

The production of distinct sets of T cell receptor (TCR) γδ+ T cells occurs in an ordered fashion in thymic development. The Vγ3 and Vγ4 genes, located downstream in the TCRγ Cγ1 gene cluster, are expressed by the earliest waves of developing TCRγδ+ T cells in the fetal thymus, destined for intraepithelial locations. Upstream Vγ2 and Vγ5 genes are expressed in later waves in the adult and constitute most TCRγδ+ T cells in secondary lymphoid tissue. This developmental pattern is caused in part by a preference for rearrangements of the downstream Vγ3 and Vγ4 genes in the early fetal stage, which switches to a preference for rearrangements of the upstream Vγ2 and Vγ5 gene rearrangements in the adult. Our gene targeting studies show that the downstream Vγ genes rearrange preferentially in the early fetal thymus because of their downstream location, independent of promoter or recombination signal sequences and unrelated to the extent of germline transcription. Remarkably, gene deletion studies show that the downstream Vγ genes competitively inhibit upstream Vγ rearrangements at the fetal stage. These data provide a mechanism for specialization of the fetal thymus for the production of T cells expressing specific Vγ genes.

Anatomical considerations in TNM staging and therapeutical procedures for low rectal cancer

Separation of the mesoderm-derived muscular structures and the endoderm-derived structures of the hindgut and reclassification of their involvement based on their embryological origin may be of clinical importance in providing anatomical support for a more standardized perineal resection during abdominoperineal resection. The aim of this study was to utilize magnetic resonance images and histological studies of fetal and neonatal specimens to redefine the T3/T4 distinction by reassessment of the intersphincteric plane and the pelvic diaphragm as they pertain to cancer infiltration and as part of the embryological development of the pelvic floor muscles and their connective tissue compartments. Pelvic floor anatomy was studied in seven newborn children and 120 embryos and fetuses. Anatomical data were completed by magnetic resonance imaging in 82 patients with T3 and T4 rectal cancers (64 T3, 18 T4; 35 women and 47 men) undergoing neoadjuvant chemoradiation for locally advanced (T3 or T4) rectal cancers. Clear demarcation between mesodermal and endodermal structures of the pelvic floor, which is equally evident in plastinated sections and magnetic resonance images, is already visible in early fetal stages. There is a constitutive overlap between the endoderm- and the ectoderm-derived components of the pelvic floor. Our data suggest that the current classification of rectal cancer staging is confusing, where the routinely used TNM classification system unnecessarily differentiates between embryologically identical muscular structures. Tumor spread along the musculature of the hindgut beyond the dentate line could possibly explain the occasional involvement of lymph nodes outside the conventional mesorectum.

Developmental Changes in Testicular Sensitivity to Estrogens throughout Fetal and Neonatal Life

There is now compelling evidence that inappropriate exposure to estrogen during fetal or neonatal life could affect adult reproductive functions because the testis is sensitive to estrogens during specific periods of its development. Therefore, we investigated the effects of exogenous estrogens on gametogenesis and steroidogenesis during fetal and neonatal testicular development in the rat. We used in vitro systems, organ cultures, and dispersed testicular cell cultures, which allow the development of fetal and neonatal germ cells (gonocytes) and Leydig cells. Exogenous estrogens inhibited testosterone production in dispersed testicular cell cultures throughout fetal life, but this inhibition was observed only in the early fetal stages in organ culture. By using an aromatase inhibitor (letrozole, Novartis Pharma AG), we showed that the inhibitory effect of exogenous estrogens on testosterone production is masked in the whole testis at later stages (20.5 days postconception) due essentially to local production of estrogens. In both systems, additions of high concentrations (10(-6) M) of 17beta-estradiol or diethylstilbestrol decreased the number of gonocytes during the first fetal proliferative period but not during the neonatal period. Letrozole was without effect, suggesting that the aging-related loss of responsiveness of gonocytes is not due to any aromatase activity in the gonocytes.

Edaravone inhibits lipid peroxidation in neonatal hypoxic-ischemic rats: An in vivo microdialysis study

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. Free radical-mediated lipid peroxidation is the main cause of neurotoxicity including neonatal brain damage. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel anti-oxidant agent and the drug of choice in the treatment of acute ischemic brain disorders in adult patient. The purpose of this study is to determine the direct effects of edaravone in inhibiting the lipid peroxidation production in the neonatal rat brains during hypoxic-ischemic insult by electron paramagnetic resonance (EPR) spectoroscopy and in vivo brain microdialysis. Seven-day-old Wistar rats were subjected to left common carotid artery ligation and a probe was inserted in the rat hippocampus. Edaravone (5, 50, or 100μM) or saline was perfused with a spin trap agent (α-(4-pyridyl-N-oxide)-N-tert-butylnitrone; POBN) before, during and after hypoxia (1h of 8% O2 exposure) and then analyzed by EPR. Edaravone (100μM) did not show any EPR evidence of POBN adduct formation during and after hypoxic-ischemic insult. However, the EPR signal increased, but not significantly during the hypoxic period in the hypoxic and edaravone 50μM-treated groups compared to control. Edaravone at 5μM significantly increased the EPR signals compared to control. This study shows that edaravone directly and dose-dependently inhibited the formation of lipid free radicals produced during hypoxic-ischemic insult in the neonatal rat brain. These results suggest that edaravone is able to attenuate neuronal damage in the rat neonatal brain by inhibiting the formation of lipid radicals.

Phthalate ester toxicity in Leydig cells: Developmental timing and dosage considerations

Humans have significant exposures to phthalates, as these chemical plasticizers are ubiquitously present in flexible plastics. Recent epidemiological evidence indicates that boys born to women exposed to phthalates during pregnancy have an increased incidence of congenital genital malformations and spermatogenic dysfunction, signs of a condition referred to as testicular dysgenesis syndrome (TDS). TDS is thought to develop as a result of environmental factors that cause a testicular disturbance at an early fetal stage with a resultant spectrum of clinical testicular dysfunction, ranging from impaired spermatogenesis and genital malformations to increased risk for development of testicular cancer. Proposed environmental factors in the etiology of TDS include endocrine disrupting compounds such as the phthalates. Leydig cells have been classified as one of the main targets for phthalate ester toxicity in the body based on studies in rodents. In support of this hypothesis, two Leydig cell products – insulin-like growth factor 3 (INSL3) and testosterone (T) – are both suppressed after phthalate exposures. Both fetal and adult generations of Leydig cells are affected by phthalate esters, although their sensitivities may differ. In rodent models, when pregnant dams are exposed to phthalate esters, fetal Leydig cells form enlarged clusters that are retained in the testis even after birth, in contrast to untreated controls. Despite the retention of fetal Leydig cells, however, their numbers and average cell volume of total in exposed males are reduced, as are INSL3 production and steroidogenic competence. These alterations are directly associated with clinical features of TDS, including cryptorchidism and impaired spermatogenesis.

Head morphology in perinatal dolphins: A window into phylogeny and ontogeny

In this paper on the ontogenesis and evolutionary biology of odontocete cetaceans (toothed whales), we investigate the head morphology of three perinatal pantropical spotted dolphins (Stenella attenuata) with the following methods: computer-assisted tomography, magnetic resonance imaging, conventional X-ray imaging, cryo-sectioning as well as gross dissection. Comparison of these anatomical methods reveals that for a complete structural analysis, a combination of modern imaging techniques and conventional morphological methods is needed. In addition to the perinatal dolphins, we include series of microslides of fetal odontocetes (S. attenuata, common dolphin Delphinus delphis, narwhal Monodon monoceros). In contrast to other mammals, newborn cetaceans represent an extremely precocial state of development correlated to the fact that they have to swim and surface immediately after birth. Accordingly, the morphology of the perinatal dolphin head is very similar to that of the adult. Comparison with early fetal stages of dolphins shows that the ontogenetic change from the general mammalian bauplan to cetacean organization was characterized by profound morphological transformations of the relevant organ systems and roughly seems to parallel the phylogenetic transition from terrestrial ancestors to modern odontocetes.

Development and pH sensitivity of the respiratory rhythm of fetal mice in vitro

In newborn and adult mammals, chemosensory drive exerted by CO2 and H+ provides an essential tonic input: without it the rhythm of respiration is abolished. It is not known, however, whether this chemosensory drive and the respiratory rhythm appear simultaneously during development. In isolated brainstem–spinal cord preparations from fetal mice, we determined at what stage of fetal life the respiratory rhythm appeared in third to fifth cervical ventral roots (phrenic motoneurons) and whether this fetal rhythm was sensitive to chemosensory inputs. A respiratory-like rhythm consisting of short duration bursts of discharges recurring at 2–16 min−1 was detected in two of nine embryonic day 13 fetuses; it was abolished by transection of the spinal cord between the first to second cervical segments and was phase-related to rhythmic activity from medullary units of the ventral respiratory group. At embryonic day 13, it coexisted with a slow rhythm (0.1–2.0 min−1) of long duration bursts of action potentials which was generated by the spinal cord. At later fetal stages, the respiratory-like rhythm became more robust and of higher frequency, while the spinal cord rhythm became less obvious. At all fetal stages, acidification of the superfusion medium from pH 7.5–7.2 or 7.4–7.3 or 7.4 to 7.2 increased the frequency of both the respiratory-like and the spinal cord rhythms. In addition, acidification reduced the amplitude of the integrated burst activity of the spinal cord rhythm of embryonic day 13–embryonic day 16 fetuses and the respiratory-like rhythm of embryonic day 17 and older fetuses. Our results indicate that the rhythms transmitted by phrenic motoneurons during fetal development are chemosensitive from early fetal stages. Through its effects on induction and patterning of the rhythm, chemosensory drive may play a role in activity-dependent formation of respiratory neural networks.

Collagen fibril morphology and organization: Implications for force transmission in ligament and tendon

Connective tissue mechanical behavior is primarily determined by the composition and organization of collagen. In ligaments and tendons, type I collagen is the principal structural element of the extracellular matrix, which acts to transmit force between bones or bone and muscle, respectively. Therefore, characterization of collagen fibril morphology and organization in fetal and skeletally mature animals is essential to understanding how tissues develop and obtain their mechanical attributes. In this study, tendons and ligaments from fetal rat, bovine, and feline, and mature rat were examined with scanning electron microscopy. At early fetal developmental stages, collagen fibrils show fibril overlap and interweaving, apparent fibril ends, and numerous bifurcating/fusing fibrils. Late in fetal development, collagen fibril ends are still present and fibril bundles (fibers) are clearly visible. Examination of collagen fibrils from skeletally mature tissues, reveals highly organized regions but still include fibril interweaving, and regions that are more randomly organized. Fibril bifurcations/fusions are still present in mature tissues but are less numerous than in fetal tissue. To address the continuity of fibrils in mature tissues, fibrils were examined in individual micrographs and consecutive overlaid micrographs. Extensive microscopic analysis of mature tendons and ligaments detected no fibril ends. These data strongly suggest that fibrils in mature ligament and tendon are either continuous or functionally continuous. Based upon this information and published data, we conclude that force within these tissues is directly transferred through collagen fibrils and not through an interfibrillar coupling, such as a proteoglycan bridge.

Morphometric study of the human fetal heart I. Arterial segment

This study measured the dimensions of the great arteries of normal human fetal hearts at an early fetal stage (between 13-20 weeks post-fertilization) in 103 fetuses obtained by necropsy. Different segments of the aorta and the pulmonary artery were dissected and their external diameters measured under stereoscopic magnification. All segments showed linear growth during this period of fetal development. Ranges in median values of external diameters associated with the pulmonary artery were: 2.1-4.2 mm for the valve ring; 2.2-4.2 mm for the main pulmonary trunk; 1.2-2.5 mm for the right pulmonary artery; 0.9-2.18 mm for the left pulmonary artery; and 4.0-8.0 mm for the length of the main pulmonary trunk. Similarly, in the aorta, median diameters were: 2.1-4.2 mm for the ascending aorta; 1.92-3.8 mm for the aortic arch; 1.45-3.0 mm for the aortic isthmus; and 1.75 -3.35 mm for the descending aorta. The diameter of the ductus arteriosus ranged between 1.2-2.45 mm. The growth rates of the pulmonary artery and the aorta were similar. The diameter of the ascending aorta was found to be greater than that of the descending aorta and the right pulmonary artery was wider than the left pulmonary artery. In addition, the magnitude of growth in the various aortic segments was different and the ratios obtained between the aortic isthmus and the ascending and descending aortae, ranged between 0.66-0.93. This study provides important morphometric reference information concerning the dimensions and growth of the great arteries of the fetal heart and has clinical application in pediatric cardiac surgery and echocardiography.

Development of spontaneous mouth/tongue movement and related neural activity, and their repression in fetal mice lacking glutamate decarboxylase 67

Spontaneous body movement starts at early fetal stage, at embryonic day (E) 12-15 in mice. In the present study, the movement of the head region was studied in E13-14 mice by in utero ultrasound imaging, together with the in vitro recording of underlying neural activities in the hypoglossal nerve and the ventral root of the upper cervical cord of an isolated brainstem-spinal cord preparation. The role of gamma-aminobutyric acid (GABA) in the generation of fetal movement was assessed using mice lacking GABA-synthesizing glutamate decarboxylase 67 (GAD67). At E14, mouth opening and tongue withdrawal were observed independently at frequency of 14/h each. This movement was rarely observed in the GAD67-deficient mouse. The intraventricular administration of picrotoxin or 3-mercaptopropionic acid abolished mouth opening in the wild-type mice. In a brainstem-spinal cord preparation, three types of neural discharge were recorded: mouth/tongue-moving burst, respiratory burst and irregular activity on the basis of their waveform, regularity in occurrence and concomitant muscle activity. In the GAD67-deficient mice, the occurrence of mouth/tongue-moving burst and irregular activity was inhibited to about 15 and 40% of those in the wild-type mice, respectively. Respiratory burst was slightly inhibited but the difference was not significant. Picrotoxin greatly reduced the frequency of mouth/tongue-moving burst. These results indicate that GABA is involved in rhythm generation in movement of the head region and support the hypothesis that cleft palate in the GAD67-deficient mouse is due to the impairment of mouth or tongue movement that assists palate formation.

321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs

226 GREATER DYSREGULATION OF GENE EXPRESSION IN PRE-IMPLANTATION CLONED OVINE COMPARED WITH CLONED BOVINE CONCEPTUSES

In our experience, the cloning of sheep by somatic cell nuclear transfer has been less successful than with cattle (5 v. 10% live births from embryo transfer). Here, we compare data collected over many years on the pre-implantation development of nuclear transfer (NT) ovine and bovine embryos with contemporary in vitro produced (IVP) embryos at the elongation (bovine Day 16–18 v. ovine Day 14) and allantois formation (bovine Day 26/27 v. ovine Day 21) stages. Sheep NT conceptuses were generated from three fibroblast types (skin, kidney, and lung) derived from a Day 100 female fetus. Bovine NT embryos were generated from a granulosa cell line derived from a mature dairy cow. The expression of a subset of genes (IGF2, H19, IGF2R, IGFBP-2, PEG1/Mest and trophoblast-expressed genes such as IFN-τ, TKDP-1, COX-2, and placental lactogen) was examined in both species at the two stages of development, using either Northern or slot-blot analysis. Pairwise comparisons between groups were carried out using Student's t-test. The proportion of ovine NT embryos that developed into blastocysts from the three cell types combined was 11% (117/1052) compared with 79% (86/109) for bovine NT from the one cell line. After transfer to synchronized recipients, 50–60% developed to the elongated conceptus stage for both species. Day 14 ovine NT (n = 19) and IVP (n = 26) conceptus lengths were similar whereas Day 16 bovine NT (n = 9) lengths were slightly shorter than those of IVP (n = 3) conceptuses (P > 0.05). However, only 30% (16/52) of ovine NT embryos developed to Day 21 compared with 64% (9/14) for Day 26/27 bovine NT. Bovine NT and IVP embryo and allantoic lengths were not significantly different; however, both lengths were shorter in ovine NT than in IVP controls (P < 0.05 and < 0.005, respectively). In each species, there was considerable individual variation in expression levels of most genes at each stage, probably due to variation in developmental stage of individuals even from the same day of gestation. The difference may be due to the timing of onset or termination of gene expression. In Day 16–18 bovine NT conceptuses, only the mean level of PEG1/Mest expression was significantly different (P < 0.05) from that of IVP conceptuses. In contrast, mean expression levels for TKDP-1 (P < 0.0001) and COX-2 (P < 0.05) were higher in ovine Day 14 NT, and PEG1/Mest was higher (P < 0.05) in Day 21 NT when compared with contemporary IVP controls. H19 and IGF2 expression was coordinately regulated in bovine NT and in ovine and bovine IVP conceptuses (IGF2 and H19 mRNA abundance was directly correlated). This coordinate regulation was disrupted in certain Day 21 ovine NT conceptuses, where high H19 levels occurred in the presence of low levels of IGF2, but not the converse. Thus, greater dysregulation in gene expression and loss of coordinate regulation of two key imprinted genes may explain the lower survival rate of ovine NT embryos to the early fetal stage when compared with bovine NT embryos. The effect of donor cell type on the species difference cannot be discounted. This work was supported by FRST C10X0303 programme.

Insulin‐Like Growth Factor Promotes Engraftment, Differentiation, and Functional Improvement after Transfer of Embryonic Stem Cells for Myocardial Restoration

Insulin-like growth factor-1 (IGF-1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF-1 might enhance cellular engraftment and host organ-specific differentiation after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF-1 to the injected cell population. Green fluorescent protein (GFP)-labeled sv129 ESCs (2.5 x 10(5)) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF-1 (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF-1 (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF-1 group (p = .025). There was a trend toward higher fractional shortening in the IGF-treated group. Histological analysis demonstrated that IGF-1 promoted expression of alpha-sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF-1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

A requirement for Notch1 distinguishes 2 phases of definitive hematopoiesis during development

AbstractNotch1 is known to play a critical role in regulating fates in numerous cell types, including those of the hematopoietic lineage. Multiple defects exhibited by Notch1-deficient embryos confound the determination of Notch1 function in early hematopoietic development in vivo. To overcome this limitation, we examined the developmental potential of Notch1–/– embryonic stem (ES) cells by in vitro differentiation and by in vivo chimera analysis. Notch1 was found to affect primitive erythropoiesis differentially during ES cell differentiation and in vivo, and this result reflected an important difference in the regulation of Notch1 expression during ES cell differentiation relative to the developing mouse embryo. Notch1 was dispensable for the onset of definitive hematopoiesis both in vitro and in vivo in that Notch1–/– definitive progenitors could be detected in differentiating ES cells as well as in the yolk sac and early fetal liver of chimeric mice. Despite the fact that Notch1–/– cells can give rise to multiple types of definitive progenitors in early development, Notch1–/– cells failed to contribute to long-term definitive hematopoiesis past the early fetal liver stage in the context of a wild-type environment in chimeric mice. Thus, Notch1 is required, in a cell-autonomous manner, for the establishment of long-term, definitive hematopoietic stem cells (HSCs).

Cellular expression and specific intragranular localization of chromogranin A, chromogranin B, and synaptophysin during ontogeny of pancreatic islet cells: an ultrastructural study.

To get more insight into the differentiation patterns of pancreatic islet neuroendocrine cells and granules during ontogeny, the expression and localization of chromogranin A (CgA), chromogranin B (CgB), synaptophysin, and insulin were ultrastructurally studied with the immunogold technique in porcine and human pancreatic islet neuroendocrine cells. In porcine pancreas at early fetal stage, CgA was visualized throughout the immature granules in all neuroendocrine cells. Later, CgB also was expressed with the same pattern in most granules in all types of cells. In neonatal islets, CgA was localized in the periphery of immature alpha- and beta-cell granules and throughout the matrix of delta-cell granules; CgB was distributed throughout the matrix of these granules. In adult islets, alpha-cell granules stored CgA in the halo and CgB in both the core and the halo, beta-cell granules stored both CgA and CgB in their cores, and in delta-cell granules, both substances were mixed throughout the matrix. In all ontogenetic stages, synaptophysin was demonstrated in all cell types and granules. Insulin was expressed in early fetal cells of both pigs and humans, and colocalization with CgA, CgB, and synaptophysin was demonstrated. The early expression of CgA and synaptophysin may reflect a role at early fetal stages, and the individual localization of CgA and CgB upon differentiation indicates individual functions.