Rhombomere-specific patterns of apoptosis in the tree shrew Tupaia belangeri

Whether rhombomere-specific patterns of apoptosis exist in the developing hindbrain of vertebrates is under debate. We have investigated the sequence of apoptotic events in three-dimensionally reconstructed hindbrains of Tupaia belangeri (8- to 19-somite embryos). Apoptotic cells were identified by structural criteria and by applying an in situ tailing technique to visualize DNA fragmentation. Seven rhombomeres originated from three pro-rhombomeres. Among pre-migratory neural crest cells in the dorsal thirds of the neural folds, the earliest apoptotic concentrations appeared in the developing third rhombomere (r3). Dorsal apoptotic maxima then persisted in r3, extended from r3 to r2, and also arose in r5. Transverse apoptotic bands increased the total amount of apoptotic cells in odd-numbered rhombomeres first in r3 and, with a delay, also in r5. This sequence of apoptotic events was paralleled by an approximate rostrocaudal sequence of neural crest cell delamination from the even-numbered rhombomeres. Thus, large-scale apoptosis in r3 and r5 helped to establish crest-free zones that segregated streams of migrating neural crest cells adjacent to r2, r4, and r6. The sequence of apoptotic events observed in the dorsal thirds of rhombomeres matches that reported for the chick embryo. Other shared features are apoptotic peaks in the position of a circumscribed ventricular protrusion of fusing parts of the neural folds in r1 and r2, and Y-shaped apoptotic patterns composed of apoptotic maxima in the dorsal and lateral thirds of r1, r2, and r3. These rhombomere-specific patterns of apoptosis may therefore represent a conserved character, at least in amniotes.This work was supported by the Deutsche Forschungsgemeinschaft (KN 525/1–1, KN 525/1–2, BR 1185/4–1, and former Sonderforschungsbereich 89: Cardiology)     

The size of a melanin-based plumage ornament correlates with glucocorticoid receptor concentrations in the skin of that ornament

Glucocorticoid hormones such as corticosterone (CORT) play crucial roles in many physiological processes. CORT''s actions are primarily mediated via binding to two receptors (glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs)) in different target tissues. CORT receptors can be independently regulated from circulating hormone titres, from tissue to tissue and even within different regions of the same tissue type. Increasing evidence has shown relationships between circulating CORT and melanin-based pigmentation in skin and feathers, yet to our knowledge, there have been no studies of CORT receptors in the skin of melanized ornaments. Male house sparrows (Passer domesticus) have a black, melanized bib, and evidence suggests that bib size is an important intraspecific signal. We examined the relationship between bib area and tissue sensitivity to CORT by quantifying GR and MR in bib skin and in adjacent paler-feathered belly skin (as a control tissue) at different life-history stages using radioligand binding assays. Males with larger bibs relative to their life-history stage had less GR in bib skin, but not belly skin, than males with smaller bibs. These results suggest a connection between the size of a melanin-based ornament and the underlying tissue''s responsiveness to CORT.     

Intracellular glucocorticoid receptors in spleen,but not skin,vary seasonally in wild house sparrows (Passer domesticus)

Over the short-term and at physiological doses, acute increases in corticosterone (CORT) titres can enhance immune function. There are predictable seasonal patterns in both circulating CORT and immune function across many animal species, but whether CORT receptor density in immune tissues varies seasonally is currently unknown. Using radioligand binding assays, we examined changes in concentrations of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) in spleen and skin in wild-caught house sparrows in Massachusetts during six different life-history stages: moult, early winter, late winter, pre-egg-laying, breeding and late breeding. Splenic GR and MR binding were highest during the pre-laying period. This may help animals respond to immune threats through increased lymphocyte proliferation and/or an increase in delayed-type hypersensitivity reactions, both of which CORT can stimulate and in which spleen is involved. A decrease in splenic GR and MR during the late breeding period coincides with low baseline and stress-induced CORT, suggesting immune function in spleen may be relatively CORT-independent during this period. We saw no seasonal patterns in GR or MR in skin, suggesting skin''s response to CORT is modulated primarily via changes in circulating CORT titres and/or via local production of CORT in response to wounding and other noxious stimuli.     

Thermogenic characteristics and evaporative water loss in the tree shrew (Tupaia belangeri)

Thermogenic characteristics and evaporative water loss were measured at different temperatures in Tupaia belangeri. The thermal neutral zone (TNZ) of T. belangeri was 30–35 °C. Mean body temperature was 39.76±0.27 °C and mean body mass was 100.86±9.09 g. Basal metabolic rate (BMR) was 1.38±0.03 ml O₂/g h. Average minimum thermal conductance (C_m) was 0.13±0.01 ml O₂/g h °C. Evaporative water loss in T. belangeri increased when the temperature rose; the maximal evaporative water loss was 3.88±0.41 mg H₂O/g h at 37.5 °C. The results may reflect features of small mammals in the sub-tropical plateau region: T. belangeri had high basal metabolic rate and high total thermal conductance, compared with the predicted values based on their body mass whilst their body temperatures are relatively high; T. belangeri has high levels of evaporative water loss and poor water-retention capacity. Evaporative water loss plays an important role in temperature regulation.     

Structure and postnatal development of photoreceptors and their synapses in the retina of the tree shrew (Tupaia belangen)

Summary The all cone retina of the tree shrew (Tupaia belangeri) was examined in the adult and early postnatal stages by light and electron microscopy. Rods are not as rare as previously thought, but make up about 4% of the photoreceptors. They are relatively short and narrow cells, which stain (toluidine blue) more intensively and lie more proximal than cones. Among the cones three morphological varieties could be distinguished. Most cones stain lightly but have a light or a dark giant mitochondrion in their inner segment; a third type stains darker but occurs only rarely. All cones possess extensive radial processes (lateral fins) around the basal part of their inner segments. Such fins are well known from reptiles and birds, but have only once been described in a mammal (gray squirrel).The maturation of the retina in Tupaia belangeri proceeds centrifugally, i.e., from the vitreal to the scleral side, as in most mammals. A few synapses are already present at birth in the outer and inner plexiform layers, but seem to be more advanced in the latter. Such early synapses are small and have only few synaptic vesicles; they appear almost mature by day 14. The light-sensitive outer segments develop last. The first disks are seen by day 10, but regular membrane stacks are only present by day 18. Thus, it seems that the retina is functional when the young first open their eyes, which occurs around day 18.     

Cone photoreceptor shedding in the tree shrew (Tupaia belangerii)

Summary Tree shrews were sacrificed at various times during a 12 h light-12 h dark cycle and the retinal pigment epithelium (RPE) was examined for phagosomes. Analysis of photoreceptor densities showed that the tree-shrew retina consists of approximately 96% cone photoreceptors. Therefore, phagosomes in the RPE were assumed to be mostly those of cones. A peak in the number of RPE phagosomes was found about one hour after the onset of light. The number of phagosomes/mm RPE during the light cycle varied from 17.02 at the peak to 2.49 ten hours after light onset. During the dark cycle, values ranged from 0.10 to 0.61 phagosomes/mm RPE. Size profiles of phagosomes showed that large phagosomes peak in number 1/2 h after light onset, while smaller sizes peak at about 1 h after light onset. This may indicate that maximal shedding and phagocytotic activity occurs sometime before the peak in the total number of phagosomes is reached. Statistical corrections for phagosome size, section thickness and phagosomal degradation time were applied to the data in order to assess outer segment renewal time for tree shrew cones.     

Fiber type distribution in the shoulder muscles of the tree shrew, the cotton-top tamarin, and the squirrel monkey related to shoulder movements and forelimb loading

Muscle fiber type composition of intrinsic shoulder muscles was examined in tree shrews, cotton-top tamarins, and squirrel monkeys with respect to their shoulder kinematics and forelimb loading during locomotion. Enzyme- and immunohistochemical techniques were applied to differentiate muscle fiber types on serial cross-sections of the shoulder. In the majority of the shoulder muscles, the proportions of fatigue resistant slow-twitch fibers (SO) and fatigable fast-twitch fibers (FG) were inversely related to each other, whereas the percentage of intermediate FOG-fibers varied independently. A segregation of fatigue resistant SO-fibers into deep muscle regions is indicative of differential activation of histochemically distinct muscle regions in which deep regions stabilize the joint against gravitational loading. In all three species, this antigravity function was demonstrated for both the supraspinatus and the cranial subscapularis muscle, which prevent passive joint flexion during the support phase of the limb. The infraspinatus muscle showed a high content of SO-fibers in the primate species but not in the tree shrew, which demonstrates the "new" role of the infraspinatus muscle in joint stabilization related to the higher degree of humeral protraction in primates. In the tree shrew and the cotton-top tamarin, a greater proportion of the body weight is carried on the forelimb, but the squirrel monkey exhibits a weight shift to the hind limbs. The lower amount of forelimb loading is reflected by an overall lower proportion of fatigue resistant muscle fibers in the shoulder muscles of the squirrel monkey. Several muscles such as the deltoid no longer function as joint stabilizers and allow the humerus to move beyond the scapular plane. These differences among species demonstrate the high plasticity of the internal muscle architecture and physiology which is suggested to be the underlying reason for different muscle activity patterns in homologous muscles. Implications for the evolution of new locomotor modes in primates are discussed.     

Postnatal differentiation of the gametogenic and endocrine functions of the testis in the tree-shrew (Tupaia belangeri)

Summary Testicular development was studied in Tupaia belangeri (tree-shrew) from birth to sexual maturity. At birth the seminiferous cords contained peripheral supporting cells and centrally located gonocytes. Large foetal Leydig cells were prominent in the interstitium. The mitotic index of the gonocytes was low at birth and rose to peak levels at Day 20, following the regression of the foetal generation of Leydig cells, and during the nadir in circulating testosterone concentrations. Mitotic activity returned to low levels at Day 30 in association with the reappearance of differentiated Leydig cells and the first signs of increased androgenesis. The negative temporal relationship between mitogenesis and androgenic function suggests that the proliferation of the gonocytes does not require, and may be inhibited by, high titres of androgens. Post-mitotic development of the gonocytes occurred during a period of rising testosterone levels, and the first appearance of spermatogonia coincided with peak testosterone levels. This indicates that androgens may be specifically involved in the initiation of spermatogenesis. Spermatogenesis progressed to completion during a phase of declining testosterone levels. The precise temporal correlations established during post-natal development suggest that the tree-shrew is a suitable animal model for studies on the endocrine control of the initiation of spermatogenesis in primates.     

Relationship of glucagon-somatostatin and gastrin-somatostatin cells in the stomach of the monkey

The stomach of the monkey Tupaia belangeri was investigated by serial sections utilizing the indirect immunoperoxidase reaction to demonstrate the distribution of glucagon, gastrin and somatostatin immunoreactive cells. A striking topographical distribution was found. Glucagon and somatostatin immunoreactive cells were located in the upper parts, whereas gastrin and somatostatin immunoreactive cells were situated in the lower parts of the stomach. The remaining regions of the stomach did not contain cells immunoreactive to the antisera applied. Similarly, the ultrastructural study revealed the same distribution of endocrine cell types identified as A-cells, D-cells, and G-cells. Thus, there may be a glucagon-somatostatin area in the upper part and a gastrin-somatostatin endocrine surface in the lower part of the stomach. This spatial relationship of the endocrine cells suggests a functional cell interaction between glucagon and somatostatin cells in the cranial stomach and between gastrin and somatostatin in the caudal parts of the stomach.     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Ultrastructural and immunohistochemical demonstration of pancreatic polypeptide-containing F-cells in the stomach and pancreas of Tupaia belangeri

The gastro-entero-pancreatic (GEP) system in Tupaia belangeri contains a specific cell which reacts with antisera to pancreatic polypeptide (PP). The cells are scattered between exocrine pancreatic cells and also found in the pancreatic islets. Furthermore, they are also located in the fundic glands and to a very small extent in the corpus mucosa and in the glands of the upper duodenum. The cell reacting with antisera against PP is identified as the F-cell which has a specific ultrastructure especially with respect to its secretion. The present identification of the F-cell as the PP-cell in pancreas and stomach is discussed with respect to its possible functional implications.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Maternal psychosocial stress during pregnancy alters the epigenetic signature of the glucocorticoid receptor gene promoter in their offspring: a meta-analysis

Prenatal stress has been widely associated with a number of short- and long-term pathological outcomes. Epigenetic mechanisms are thought to partially mediate these environmental insults into the fetal physiology. One of the main targets of developmental programming is the hypothalamic-pituitary-adrenal (HPA) axis as it is the main regulator of the stress response. Accordingly, an increasing number of researchers have recently focused on the putative association between DNA methylation at the glucocorticoid receptor gene (NR3C1) and prenatal stress, among other types of psychosocial stress. The current study aims to systematically review and meta-analyze the existing evidence linking several forms of prenatal stress with DNA methylation at the region 1_F of the NR3C1 gene. The inclusion of relevant articles allowed combining empirical evidence from 977 individuals by meta-analytic techniques, whose methylation assessments showed overlap across 5 consecutive CpG sites (GRCh37/hg19 chr5:142,783,607-142,783,639). From this information, methylation levels at CpG site 36 displayed a significant correlation to prenatal stress (r = 0.14, 95% CI: 0.05–0.23, P = 0.002). This result supports the proposed association between a specific CpG site located at the NR3C1 promoter and prenatal stress. Several confounders, such as gender, methylation at other glucocorticoid-related genes, and adjustment for pharmacological treatments during pregnancy, should be taken into account in further studies.     

Studies on the juxtaglomerular apparatus VI. Sympathetic innervation,catecholamines and the renin-angiotensin-system in rats and tree-shrews (Tupaia belangeri)

Summary It has previously been reported that the primitive primate Tupaia belangeri develops a renal failure when exposed to psychosocial stress. In order to learn if this high susceptibility to stress of the Tupaia kidney can be correlated with morphological and functional parameters of the Juxtaglomerular apparatus (JGA) and the renin-angiotensin system, comparative experiments were performed on Tupaia and rat.Our results reveal an outstandingly high potency of the JGA and the renin-angiotensin system in Tupaia as evident from the following findings: The Tupaia JGA contains a great number of epithelioid cells abounding in renin granules (electron microscopy). The renin content of the Tupaia kidney is considerably higher than in the rat (radio-immunoassay). The sympathetic innervation of the kidney and especially of the JGA is abundant in Tupaia (fluorescence and electron microscopy). Catecholamine contents of the kidney and other organs are significantly higher in Tupaia than in rats (spectrophotofluorometry).Our results support the previously developed concept of a potent intrarenal neuroendocrine interaction at the JGA level favouring, under certain conditions of social stress, the development of acute renal failure in Tupaia belangeri.The studies were supported by the German Research Foundation within the SFB90 Cardiovasculäres System     

The morphological basis of fluid balance in the interstitium of the juxtaglomerular apparatus

Summary The morphological basis of fluid balance in the interstitium of the juxtaglomerular apparatus (JGA) was reevaluated in rats, mice and Tupaia. Three ultrastructural features in the region of the vascular pole of the renal corpuscle are described that may be important for the fluid balance in this region: (1) podocyte foot processes in the parietal layer of Bowman's capsule, (2) endothelial fenestrations in the wall of the incoming afferent arteriole, both facing Goormaghtigh and epithelioid cells, and (3) the mesangial type lining of the glomerular stalk. With respect to the relevant pressure gradients, this morphology may provide the basis of bulk-fluid flow directed to the interstitium of the JGA including the Goormaghtigh cell field. Thus, the fluid balance in the lacis area and, consequently, the tubulo-glomerular feedback mechanism, probably does not solely depend upon the reabsorptive transport of the macula densa. Similar considerations may be valid for the humoral control of renin secretion from juxtaglomerular epithelioid cells.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid