Canada goose posterior lobe pituicytes: seasonal ultrastructural changes in relation to axonal release of neurosecretion

Changes in ultrastructure of the posterior lobe pituicytes during six phases (Spring premigratory, Spring postmigratory, breeding, moulting, Fall premigratory and Fall postmigratory) of the annual life cycle of the migratory Canada goose, were studied. Pituicyte nuclear volume in females was significantly greater than that in males during the Spring premigratory and moulting phases. In the Fall postmigratory phase, nuclear volume in males was greater than that in females. During Spring premigratory phase, the axonal endings in males contained fewer neurosecretory granules (NSG) than those in the Fall postmigratory phase. In females, however, the number of NSG was relatively more than that in males in the Spring premigratory phase but fewer in the moulting phase. Nuclear volume is correlated with pituicyte function in releasing NSG containing arginine vasotocin (AVT) from axonal endings. The significance of AVT release is discussed in relation to initiation of reproductive and migratory behaviour, and to osmotic and metabolic regulation.     

Seasonal ultrastructural changes in the pineal gland of the migratory Canada goose

Ultrastructural changes in the pineal gland were studied in relation to migration, breeding and moult. Sizes of pinealocytes and that of their nuclei were greatest in the Spring premigratory and postmigratory, and moult phases. The ratio of nuclear size to cell size was greatest for the breeding phase and lowest for the Fall premigratory and postmigratory phases. The Golgi complex was most prominent during premigratory and breeding phases. Mitochondria which were numerous in all phases except during moult, were markedly enlarged during breeding and Fall premigratory phases. Dense-core vesicles were predominant in Spring postmigratory and moult phases. In glial cells, lipoid inclusions were abundant in Spring premigratory and postmigratory, and breeding phases. Glycogen granules were abundant in Spring postmigratory, breeding and moult phases. Large vacuoles were present in Fall premigratory phase. Lamellar membraneous whorls were often present near luminal spaces during Spring premigratory and postmigratory, and breeding phases. The endothelial cells of pineal capillaries during Spring and Fall premigratory phases, contained numerous vesicles, some of which were freely dispersed in the cytoplasm while others were fused to the cell membrane, suggestive of transendothelial transport of vesicular substances.     

Seasonal changes in serum free fatty acid level in the migratory Canada goose.

The changes in the serum level of free fatty acids (FFA) in the migratory Canada goose (Branta canadensis interior) breeding in Ft. Churchill (Manitoba, Canada) and wintering in Swan Lake (Missouri, U.S.A.), were studied during the different periods of its yearly life cycle. The lowest serum FFA level was noted during the spring premigratory phase (early March) at Swan Lake, and the highest during moult (early August) at Ft. Churchill. Serum FFA level during the spring postmigratory period (early May at Ft. Churchill) was significantly higher than that during the spring premigratory period and the breeding period (early at Ft. Churchill). No signigicant difference in FFA levels was noted between the fall premigratory (early September at Ft. Churchill) and the fall postmigratory (mid-October at Swan Lake) periods. The significance of the seasonal variations in serum FFA level is discussed in relation to the cyclic physiological events taking place in the bird.     

Distribution of arginine vasotocin in the brain of the lizard Anolis carolinensis

Summary The distribution of immunoreactive arginine vasotocin (AVT-ir) was determined in the brain of the lizard Anolis carolinensis. Cells and fibers containing AVT-ir were found in the medial septal region, lamina terminalis, lateral forebrain bundle, preoptic area, supraoptic nucleus, anterior hypothalamus, paraventricular nucleus, periventricular nucleus, arcuate nucleus, and ventromedial nucleus of the thalamus. Occasional AVT-ir cells were found in the interpeduncular nucleus. Fibers containing AVT-ir were found in the cortex, around the olfactory ventricle, in the diagonal band of Broca, amygdala area, dorsal ventricular ridge, striatum, nucleus accumbens, septum, ventromedial hypothalamus, lateral hypothalamus, medial forebrain bundle, median eminence, pars nervosa, nucleus of the solitary tract, locus coeruleus, cerebellar cortex (granular layer), dorsal part of the nucleus of the lateral lemniscus, substantia nigra, and myelencephalon. The intensity of AVT-ir staining was, in general, greater in males than in females. Comparison of AVT-ir distribution in A. carolinensis with those previously published for other reptilian species revealed species-specific differences in distribution of AVT.     

Glycoprotein secretion in the hypothalamo-neurohypophyseal system of the rat

Summary Although the secretory products of the hypothalamoneurohypophyseal system are not glycoproteins, synthesis and migration of these macromolecules occur within its secretory neurons. After being labeled with ³H-fucose in the Golgi apparatus, newly synthesized glycoproteins migrate to secretion granules, lysosomes and the plasma membrane of the secretory neurons, as demonstrated by quantitative electron-microscopic radioautography. Secretion granules bearing newly synthesized glycoproteins migrate to the pars nervosa, the labeling pattern of which was studied in rats killed from 4 h to 14 days after the isotope injection. Most of the silver grains were observed to overly the secretory axons. Labeling of pituicytes was negligible and the number of silver grains over the perivascular spaces was about 10% of the total at certain postinjection intervals. In the secretory axons, most of the silver grains were seen to overly the secretion granules. The proportion of silver grains over the different portions of the secretory axons changed with time. At the longer intervals, the percentage of silver grains increased over the nerve swellings (including Herring bodies) and decreased concomitantly in the undilated portions of the axons and in the nerve endings. This labeling pattern conforms with observations on the secretion products. Water deprivation increased the release of neurosecretion as well as glycoproteins from the pars nervosa. However, glycoproteins inside the Herring bodies were not easily releasible. There was a parallel decrease in the amount of secretion granules and ³H-fucose-labeled glycoproteins indicating that the glycoproteins are predominantly a constituent of the granule content. Some newly synthesized glycoproteins were probably also used in the renewal of the axonal membrane. The labeling of smooth vesicles in nerve endings was discussed. In conclusion, most of the glycoproteins synthesized in the perikarion of the hypothalamic secretory neurons migrate inside secretion granules along the axon to the pars nervosa where they are secreted.     

Structural analyses on the matrical organization of glycosaminoglycans in developing endocardial cushions

Extracellular glycosaminoglycans (GAG) were examined in embryonic rat valvular primordia (cushion tissue) to determine if there are specific, in situ, intermolecular associations of GAG and if the passage of migrating cushion cells alters matrical organization. Precursor incorporation studies and colloidal iron staining controlled by acidified methylation, pH, and polysaccharidase digestion indicated that both hyaluronate (HA) and chondroitin sulfate (CHS) were secreted into the premigratory matrix with the predominant GAG being HA. Premigratory matrix was revealed by scanning electron microscopy after routine fixation as a microfibrillar stroma; addition of cetylpyridinium chloride (CPCL) to the fixative resulted in the retention of an additional matrical component superimposed upon the microfibrillar stroma. TEM analysis of the CPCL-dependent matrix revealed that it was composed of intertwined 3-nm filaments, electron-dense, amorphous material, and 30-nm granules. Collagen-like microfibrils were associated primarily with the filamentous component of the CPCL-dependent matrix. Ultracytochemical results obtained with dialyzed iron binding regulated by pH and polysaccharidase and protease digestion suggests that the 3-nm filaments contain HA and the granules contain both CHS and protein. Commensurate with cushion cell formation and migration, X-ray dispersive analysis and polyanionic histochemical criteria indicated increased deposition of CHS in the postmigratory matrix (i.e., matrix transversed by cells). Ultrastructurally, the CPCL-dependent components of the postmigratory matrix became progressively restructured within the wedge of migrating cells. In contrast to premigratory matrix, fewer 3-nm filaments were evident, while 30-nm granules heavily studded the collagen-like microfibrils. Physical fixation controls confirmed the variations between pre- and postmigratory matrices. These results suggest that modification in the matrix organization of embryonic heart GAG may be correlated with the migration of cushion tissue mesenchyme.     

Expression profile of BDNF-responsive genes during cerebellar granule cell development

With the aid of microarray and PCR analysis, this investigation sought expression profiles of BDNF-regulated genes in cultured mouse cerebellar granule cells and addressed their relevance to gene regulation in developing granule cells in vivo. Many of the BDNF-upregulated and downregulated genes identified were upregulated and downregulated, respectively, during cerebellar development. This developmental change was, at least partly, prevented in the TrkB receptor-deficient cerebellum. The BDNF-upregulated genes were distributed in either postmigratory or both premigratory and postmigratory granule cells at postnatal day 8 (P8) and were still present in mature granule cells at P21. In contrast, the BDNF-downregulated genes were predominantly expressed in premigratory granule cells at P8 and disappeared at P21. Furthermore, many of the BDNF-upregulated gene products are implicated in signaling cascades of N-methyl-D-aspartate receptors and MAP kinase. The results indicate that BDNF signaling plays a pivotal role in promoting gene expression in granule cell development and maturation.     

Variation in glycogen and mucins in the equine uterus related to physiologic and pathologic conditions

Histochemical stains were applied to six equine uterine biopsies representative of the physiologic breeding season, Spring and Fall transition, and Winter anestrus periods. These were compared with uterine biopsies from six mares with intrauterine urine pooling, eight mares used to study the uterine response to indwelling catheterization, and necropsy specimens from four pregnant mares at approximately 60 or 100 d of gestation. Alcian blue staining at pH 2.5 or 1.0 was used to identify the presence of carboxylated and sulfated acid mucins or only suflated acid mucins, respectively. Periodic acid-Schiff staining was used to identify neutral mucosubstances or glycogen, with or without prior diastase digestion. The uterine glands contained glycogen, which was most abundant during the physiologic breeding season. The luminal epithelial cells during the physiologic breeding season and Spring and Fall transition contained predominately carboxylated acid mucins. Carboxylated acid mucin secretion also was stimulated by indwelling catheterization and intrauterine urine pooling. It is hypothesized that secretion of carboxylated acid mucins by the endometrial epithelium may be elicited by hormonal or irritative/inflammatory stimuli, and it may be a protective response.     

Diurnal variations in plasma arginine vasotocin (AVT) concentrations in the ovine fetus

Previously we have demonstrated the presence of AVT in the blood of fetal sheep. The source is not clear, but AVT has been identified in fetal pineal and pituitary glands. In view of the circadian secretory pattern of the pineal gland, we questioned whether fetal plasma AVT levels might vary diurnally. Plasma samples from five chronically catheterized ovine maternal ewes and fetal lambs 129-135 days' gestation were obtained at 3-19 hourly intervals for 1-2 days (mean +/- S.E.M. = 35 +/- 6 hours. Plasma AVT levels were determined by radioimmunoassay. Results were analyzed by nonlinear curve fitting procedures to relate hormone levels with time of day. Plasma AVT values for maternal ewes did not vary during the day in response to light/dark periods. The curve for mean fetal plasma AVT plotted against time showed oscillations with a period of about 25 hours (p less than 0.05). Peak fetal AVT levels were observed at 1600 hours and minimal levels at 0400 hours. These results indicate that ovine fetal AVT secretion varies diurnally. The site of AVT secretion may be the pineal gland; however, confirmation of this and identification of the physiological stimuli for secretion of fetal plasma AVT require further information.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Immunocytochemical evidence for the presence of an albumin-like substance in the rat pineal gland

Summary Two rabbits and two guinea pigs were immunized with arginine vasotocin (AVT) conjugated to bovine albumin with glutaraldehyde. Only one preparation of antiserum (anti-G 7) was of value. Anti-G 7 immunochemically defined the same rat pineal cells previously reported as presumptive AVT cells. However, absorption of anti-G 7 with bovine albumin inhibited the staining of the pineal cells demonstrating that they contained an albumin-like substance. Positive immunochemical staining of the rat pars nervosa suggested that anti-G 7 contained antibodies able to react with arginine vasopressin (AVP). Loss of a positive reaction in the posterior lobe on absorption of anti-G 7 with AVT or AVP confirmed this. However, the addition of AVT to anti-G 7 failed to inhibit the immunochemical staining of the pineal cells. This study reports the presence of an albumin-like substance in pineal cells previously described as presumptive AVT cells, and discusses possible explanations for the inability of anti-G 7 to recognize immunocytochemically the native AVT molecule. Confirmation of AVT in the pineal gland by immunocytochemistry must await the availability of more specific antisera.     

Early secretory activity in the hypothalamic nuclei and neurohypophysis in the rat; determined by selective staining

Visualization of stainable material in the neural lobe of the rat provided the most reliable index of the age at which secretory activity can first be recognized, though preceded by both hypothalamic synthesis and axonal transportation. A problem of interpretation was encountered in the neural lobes of fetal and infant animals, due to different staining responses obtained during this age period, to the two methods of staining employed; chrome alum hematoxylin-phloxin and aldehyde fuchsin after oxidation by either acidified potassium permanganate or performic acid. With aldehyde fuchsin the material of the neural lobe is stainable selectively from the eighteenth day of fetal life to adulthood. With hematoxylin phloxine the first staining response also occurred in the posterior lobe but much later, at the end of the first postnatal week. The staining situation in the pars neuralis has its counterpart in the differentiating hypothalamic nuclei; complicated by the differentiation of the supraoptic nuclei some days in advance of the paraventricular nuclei. After aldehyde fuchsin staining, evidences of neurosecretory activity were present in the perikarya of the supraoptic nuclei at birth, but mature neurons were rarely seen in the paraventricularis until at least 24 hours later. Nuclei of fetal hypothalami were not studied, but the demonstration of stainable material in the fetal neural lobes constitutes circumstantial evidence of functional competence of some neurons of either one or both types of nucleus, most likely the supraoptic.     

Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

Sex and seasonal co-variation of arginine vasotocin (AVT) and gonadotropin-releasing hormone (GnRH) neurons in the brain of the halfspotted goby

Gonadotropin-releasing hormone (GnRH) and arginine vasotocin (AVT) are critical regulators of reproductive behaviors that exhibit tremendous plasticity, but co-variation in discrete GnRH and AVT neuron populations among sex and season are only partially described in fishes. We used immunocytochemistry to examine sexual and temporal variations in neuron number and size in three GnRH and AVT cell groups in relation to reproductive activities in the halfspotted goby (Asterropteryx semipunctata). GnRH-immunoreactive (-ir) somata occur in the terminal nerve, preoptic area, and midbrain tegmentum, and AVT-ir somata within parvocellular, magnocellular, and gigantocellular regions of the preoptic area. Sex differences were found among all GnRH and AVT cell groups, but were time-period dependent. Seasonal variations also occurred in all GnRH and AVT cell groups, with coincident elevations most prominent in females during the peak- and non-spawning periods. Sex and temporal variability in neuropeptide-containing neurons are correlated with the goby's seasonally-transient reproductive physiology, social interactions, territoriality and parental care. Morphological examination of GnRH and AVT neuron subgroups within a single time period provides detailed information on their activities among sexes, whereas seasonal comparisons provide a fine temporal sequence to interpret the proximate control of reproduction and the evolution of social behavior.     

Why do fleshy-fruit plants of the mediterranean scrub intercept fall-but not spring-passage of seed-dispersing migratory birds?

Summary Production of fleshy fruits by 8 tree and shrub species, and patterns of their utilization by 6 resident and 6 transient bird species, were assessed in an Israeli Mediterranean scrub. Whereas more than half the ripe crop of plants that fruit in one relatively short burst is not removed by birds, the greater part of the crop of species that fruit throughout a long period, is removed. Of two plant species that fruit simultaneously, the one with an inconspicuous fruit is utilized by a single, year-round resident frugivorous bird, whereas the species with bright fruits is utilized by several non-resident and omnivorous bird species. A further two simultaneously fruiting species differ in fat content and the color of their conspicuous ripe fruits; the low-fat fruit being taken by a resident species and the high-fat fruit by a transient congener, just prior to the desert-crossing portion of its Fall-passage.Though more migrants pass through in Spring than in Fall, none of the fleshy-fruit plants fruit in Spring, and most fruit during the Fall. Two non-exclusive explanations for this phenomenon are (a) Fall dispersal ensures immediate germination, with least exposure to desiccation and predation; (b) Spring transients approaching their breeding territories may either be reluctant to spend much time on feeding, or mostly require proteins, amply supplied by the Spring flush of insects, typical of the Mediterranean region. Fall transients approaching the desert require mostly fats and carbohydrates, supplied by fruits. Fruits attract birds easily in the Fall, when insects are scarce.     

Age-dependent changes of ultrastructure and thyroid-stimulating hormone immunoreactivity in the pars tuberalis of the rat

The pars tuberalis of the rat adenohypophysis was investigated by immunohistochemistry and electron microscopy at different stages of the peri- and postnatal development. A characteristic pattern of changes in thyroid-stimulating hormone immunoreactivity of pars tuberalis-specific secretory cells was observed with an increase in staining intensity after birth, a marked reduction in adulthood and a subsequent increase in senium. Electron microscopy showed age-dependent changes in the number of dictyosomes per cell, in the number of large lysosomes per area of cytoplasm and in the extension of the granular endoplasmic reticulum. The number of secretory granules per area of cytoplasm was maximal perinatally; there was no correlation between granule content and immunoreactivity. Thyrotropes of the pars distalis did not show comparable immunohistochemical or ultrastructural changes.     

The anal glands of Rhapidostreptus virgator (Diplopoda: Spirostreptidae)

Summary In Rhapidostreptus virgator exocrine gland complexes are found in the anal valves of both sexes. Every gland complex consists of about 200 secretory units, each of which is comprised of four cells: two secretory cells, an intermediary cell, and a canal cell. The amount of secretion produced by these glands varies during the intermoult cycle: it is very small in freshly moulted individuals (postmoult phase), at a medial level during the following intermoult phase, and very large in the premoult phase. The secretion may be used to form the excrement clumps and above all to build the moulting chamber.     

Acetylcholine and cholinesterases (assays and light- and electron microscopical histochemistry) in different parts of the pituitary of rat,rabbit and domestic pig

Summary Acetylcholine was measured by bioassay and cholinesterases by colorimetric assay and by light and electron microscopical histochemistry in the pars nervosa, pars intermedia and pars distalis of the rat, rabbit and domestic pig pituitaries. The highest ACh concentration was found in the rat pituitary. More butyrylcholinesterases and less acetylcholinesterase was found in the rat pituitary than in the rabbit and pig pituitaries. Light microscopical histochemistry showed greater depositions of reaction products in the rat pituitary than in the other two species. This was predominantly due to butyrylcholinesterases in the pars nervosa-pars intermedia junctional region of the rat pituitary. Electron microscopical histochemistry was of limited value for quantitative estimates of distribution and localization of cholinesterases. However, the ultrastructural localization showed that most of the reaction product in the pars nervosa was associated with pituicytes with little or no reaction product in the neurosecretory terminals or in other nerve terminals. In the pars intermedia, the reaction products were primarily on the membranes of the nuclei and endoplasmic reticulum, and in some nerve terminals. No conclusions concerning the role of acetylcholine and cholinesterase in the release of the neurosecretory hormones could be made on the basis of these findings.Supported by Medical Research Council of Canada.University of Calgary Postdoctoral Fellow. Present address: Department of Pharmacology, St. Thomas's Hospital Medical School, London S.W.1., England.MRC (Canada) Postdoctoral Fellow.Associate, Medical Research Council of Canada.Research Associate of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, Argentina. Present address. Anatomisches Institut der Universität, D-6300 Gießen, Friedrichstraße 24 (Federal Republic of Germany)     

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

Summary Immunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.     

Morphometric classification of the neurosecretory granules in the rat pars nervosa

Summary The distribution of the diameters of the neurosecretory granules in the rat pars nervosa (measured from electron micrographs taken at 40 000 × ) was compared among axons by nonparametric statistical methods and the axons were classified into five groups with median granule diameters of 143, 155, 167, 180 and 193 nm. We suggested that these five axon types carried different secretory substances contained in the pars nervosa. This investigation is supported by a grant from the Population Council, New York and grant from the Ministry of Education. Authors are grateful to Japan Electron Optics Laboratory Company for their technical assistance with the electron microscopy and to Miss Kazue Yamamoto for her help in preparing the figures.