Contemporary pollen and seed dispersal in natural populations of Bertholletia excelsa (Bonpl.).

Due to the nutritional content and commercial value of its seeds, Bertholletia excelsa is one of the most important species exploited in the Amazon region. The species is hermaphroditic, insect pollinated, and its seeds are dispersed by barochory and animals. Because the fruit set is dependent on natural pollinator activity, gene flow plays a key role in fruit production. However, to date, there have been no studies on pollen and seed flow in natural populations of B. excelsa. Herein, we used microsatellite loci and parentage analysis to investigate the spatial genetic structure (SGS), realized pollen and seed dispersal, and effective pollen dispersal for two B. excelsa populations in the Brazilian Amazon forest. Two plots were established in natural forests from which adults, juveniles, and seeds were sampled. Realized and effective pollen flow was greater than realized seed flow. The distance of realized pollen dispersal ranged from 36 to 2060 m, and the distance of realized seed dispersal ranged from 30 to 1742 m. Both pollen and seeds showed a dispersal pattern of isolation by distance, indicating a high frequency of mating among near-neighbor trees and seed dispersal near to mother trees. Both populations present SGS up to 175 m, which can be explained by isolation by distance pollen and seed dispersal patterns. Our results suggested that fragmentation of these forest populations may result in a significant decrease in gene flow, due to the isolation by distance pollen and seed dispersal patterns.

Diversity and genetic structure of mangaba remnants in states of northeastern Brazil.

In this study, we analyzed the genetic diversity and structure of remnants of mangaba populations in states of northeastern Brazil by applying 9 microsatellite markers previously developed to establish conservation strategies for germplasm and species preservation. Six to 20 individuals per population were analyzed, with a total of 94 individuals and 6 populations from the states of Ceará, Pernambuco, and Sergipe, Brazil. The intra-population positive fixation index (f) in all populations indicated inbreeding resulting from the lack of random mating. The mean genetic diversity index values GST, FST, and RST estimated for divergence among the 6 populations were 0.14 (P < 0.05), revealing moderate genetic differentiation. The smallest FST value (P ≥ 0.05) was observed between the Jacarecoara and Tapera populations (0.005) and the highest between the Barra dos Coqueiros and Jacarecoara populations (0.287). The Jacarecoara population was the most divergent among the populations analyzed. According to analysis of molecular variance results, the largest variation percentage resulted from variability within populations (83.18%). Bayesian clustering analysis showed the formation of 2 sets (K = 2). Our results are important for developing strategies for in situ conservation of the species, seed collection, and ex situ conservation. For both methods, conservation of the greatest possible genetic variability of the species is essential.

Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and pH.

The molecular basis of bilirubin toxicity to nerve cell function is still unclear. Since astrocytes are the main transporters of synaptically released glutamate and impaired glutamate uptake results in neuronal death, we investigated the effect of unconjugated bilirubin (UCB) on [(3)H]glutamate uptake in cultured rat astrocytes and the role of bilirubin ionization on toxicity. Astrocytes were incubated for 5-15 min, with UCB concentrations from 17 to 342 microM and UCB/albumin molar ratios of 0.2-3.0, at pH 7.0, 7.4, and 8.0. Exposure of astrocytes for 15 min to 85.5 microM UCB and 28.5 microM albumin resulted in a 63.1% decrease of glutamate uptake (p < 0.01). Interestingly, the effect demonstrated to be correlated with the UCB/albumin molar ratio (r = -0.986, p < 0.01) and a significant decrease was observed for a UCB/albumin molar ratio as low as 0.8. Inhibition of glutamate transport was also pH-dependent as it occurred at 7.4 (p < 0.05) and 8.0 (p < 0.01), but not at 7.0, suggesting that the monoanionic species of UCB accounted for the inhibition. These findings indicate that UCB, and more precisely the monoanionic species, impairs a crucial function of astrocytes such as glutamate transport and support a potential role of astrocyte function in the pathogenesis of UCB-related brain damage (kernicterus).

Distribution of vasoactive intestinal peptide and calcitonin gene-related peptide immunoreactive nerve fibers and binding sites in the hamster seminal vesicle during post-natal development.

The distribution of vasoactive intestinal peptide (VIP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and 125I-labeled VIP- and CGRP-binding sites was studied in the hamster seminal vesicle of 12-, 30- and 60-day-old animals. In addition, the general innervation of the seminal vesicle was examined using the general neuronal marker synaptophysin. Our results show that the densities of the overall (synaptophysin immunoreactive) and CGRP-immunoreactive innervation is constant during the post-natal development of the gland. However, a significant decrease in VIP-containing nerves is observed at the end of puberty. The autoradiographic study revealed that in 12-day-old animals, the epithelium presents VIP binding sites. However, in 30-day-old animals, VIP binding sites are observed in the epithelium of only a few clumps of acini. In 60-day-old animals, the gland is composed of acini with dilated lumina where VIP binding sites are not detected. In all groups studied the epithelium does not exhibit CGRP binding sites. The seminal vesicle muscle layer displays specific binding sites for both VIP and CGRP at all post-natal developmental times, but the density of VIP binding sites is higher in 12- than in 30- and 60-day-old animals. Our results, showing the presence of specific VIP and CGRP binding sites during the development of the hamster seminal vesicle, suggest that these neuropeptides may be involved in the growth and differentiation of the gland.

Neuropeptides in the seminal vesicles: locations, binding sites and functional implications.

The importance of neuronal factors in the normal physiology of the seminal vesicles has been traditionally underestimated when compared to the trophic role of androgens. Immunohistochemical, autoradiographical and pharmacological experiments have, however, raised the possibility that neuropeptides, such as vasoactive intestinal polypeptide (VIP), neuropeptide tyrosine (NPY) and calcitonin gene-related peptide (CGRP), are necessary for full seminal vesicle function and development. These neuropeptides may be involved in the regulation of secretion, smooth muscle tone and blood flow. Furthermore, neuropeptides may have functional interactions with androgens affecting, probably, androgen receptor-dependent gene expression in these glands. It is now timely to focus attention on the biological relevance of neuropeptides in the seminal vesicles.

Effect of castration on the VIPergic innervation and 125I-labelled vasoactive intestinal peptide (VIP) binding sites in the hamster seminal vesicle. A quantitative immunohistochemical and receptor autoradiographic study.

In the present work we have investigated the effects of medium- (15 days) and long-term (2 months) castration on vasoactive intestinal peptide (VIP)-immunoreactive nerve fibres and 125I-labelled VIP binding sites in the adult hamster seminal vesicle. The density of VIP- and synaptophysin (general neuronal marker)-containing nerve fibres was determined in immunofluorescently stained cryostat sections using a computerised image analysis system. The morphological analysis of 125I-VIP binding sites in seminal vesicle cryostat sections was performed by quantitative receptor autoradiography. Our results show that the densities of the overall (synaptophysin immunoreactive) and VIPergic innervation increase in both medium and long-term castrated animals. In absolute terms, the quantity of VIP- and synaptophysin- containing nerves is not altered in medium-term castrates, but decreases for synaptophysin in long-term castrates. Medium-term castration does not affect the density of 125I-VIP binding sites in the gland muscular coat, but a significant decrease is observed after long-term castration. In conclusion, our results indicate that whereas VIP nerves are apparently unaffected by castration, 125I-VIP binding sites in the muscular coat of hamster seminal vesicle are sensitive to androgen levels.

Calcitonin gene-related peptide in the hamster seminal vesicle and coagulating gland: an immunohistochemical, autoradiographical, and pharmacological study.

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerves and CGRP binding sites, as well as the effect of CGRP on the muscle tension, was studied in the hamster seminal vesicle and coagulating gland. The use of an immunofluorescence staining technique on cryostat sections revealed that in the hamster seminal vesicle and coagulating gland, CGRP-positive nerve fibers are found in the connective interstitium and in the muscular and mucosal layers. Using an in vitro receptor autoradiographic technique, CGRP binding sites were found associated with the muscular coat. CGRP (10 pM to 1 microM) relaxed the seminal vesicle and the coagulating gland precontracted by either noradrenaline (10-30 microM) or the alpha 1-agonist, phenylephrine (10 microM). In preparations contracted by carbachol (10 microM), CGRP relaxed the seminal vesicle but not the coagulating gland. In both preparations, CGRP (1 microM) did not affect the muscle resting tension. These results suggest that CGRP may act as an inhibitory modulator of the autonomic control of contractility in the male accessory sex glands of the hamster.

Polarized epithelial cells of the hamster seminal vesicle in a serum-free bicameral culture system: evidence of secretory and endocytic activities.

Primary cultures of epithelial cells from the hamster seminal vesicle were established in a chemically defined medium supplemented with hormones and growth factors. Epithelial cell clusters were prepared combining enzymatic dissociation and mechanical disaggregation and then seeded in bicameral systems equipped with collagen-membrane inserts. A growth curve was generated and the cells were characterized morphologically and morphometrically by light and electron microscopy. The immunocytochemical detection of cytokeratins and the measurement of transepithelial electrical resistance were also performed. The secretory activity was studied by fluorography using L-[35S]methionine as a precursor, and endocytosis was approached using horseradish peroxidase as a tracer. Our results show that epithelial cells of the seminal vesicle can be grown as a monolayer of morphological and functionally polarized cells which retain secretory and endocytic activities. These cell cultures might therefore prove useful to investigate further the regulation of secretion and endocytosis in the seminal vesicle and are a promising model to approach, in a broader scope, cell polarity and protein sorting and targeting.

Dynamics of the seminal vesicle epithelium.

In newborn rodents, seminal vesicle epithelium (SVEP) cells display a poorly developed rough endoplasmic reticulum (RER) and Golgi complex, and they show no signs of secretion. From puberty onward, secretory material starts to appear, and the RER and Golgi complex progressively develop and reorganize until the adult ultrastructure is established around 40-60 days of age. Multivesicular bodies and lysosomes follow in this development but lysosomes evolve to lipofucsin granules with aging. The duration of the secretory cycle in SVEP cells is shorter than in other exocrine cells and the secretory protein pattern depends on the animal species, androgen status, and sexual activity. SVEP cells are also involved in endocytosis, which is coupled to exocytosis, and their endocytic pathway intersects the exocytic pathway in Golgi cisterns. The structure and function of SVEP cells depends mainly on testosterone, but other hormones and factors, such as the neuropeptide VIP, also influence their activity. Castration leads to programmed death and regression of SVEP cells to an extent that depends on the animal species. In addition, castration induces changes in the secretory protein pattern and delays its intracellular transport. Endocytic kinetics is also delayed following castration. Primary cultures of SVEP cells in a bicameral system are proposed as a model to investigate the activities of SVEP cells further.

Vasoactive intestinal peptide in the hamster seminal vesicle: distribution, binding sites and possible functions.

The presence and functional role of vasoactive intestinal peptide in the hamster seminal vesicle were studied by a combination of structural and functional approaches. The use of an immunofluorescence staining technique in both cryostat sections and whole-mount preparations revealed that vasoactive intestinal peptide-immunoreactive nerve fibres were mainly localized in the lamina propria of the mucosal layer. In double-stained preparations, vasoactive intestinal peptide immunoreactivity was found to be localized in nerves also containing acetylcholinesterase activity. At the ultrastructural level, the use of an immunogold staining method showed that vasoactive intestinal peptide immunoreactivity occurred in large granular vesicles (80-150 nm in diameter) in nerve varicosities which also contained small pleomorphic agranular vesicles. In order to evaluate the anatomical distribution of vasoactive intestinal peptide binding sites in the seminal vesicle, we have utilized an in vitro receptor autoradiographic technique. Vasoactive intestinal peptide binding sites were localized in the basal region of the secretory epithelium, in the muscle layer and in the wall of blood vessels. In vitro incorporation of [3H]L-leucine into protein by tissue slices revealed that vasoactive intestinal peptide (1 microM) significantly increases the amount of released protein. Vasoactive intestinal peptide (0.1-1 microM) did not affect the resting tension of the muscle but significantly inhibited the increase in muscle tension induced by carbachol. Atropine prevented the effect of carbachol, indicating that the latter is mediated by muscarinic receptors. Our results suggest that in the hamster seminal vesicle, vasoactive intestinal peptide is involved in the modulation of muscarinic function and in the control of secretion.

Castration-resistant secretion in the hamster seminal vesicle does not depend on androgens.

The secretory activity of seminal vesicles (SV) in the castrated hamster was studied by stereological analysis and biochemical approaches following treatment with cyproterone acetate (CPA) and adrenalectomy in order to investigate whether extra-testicular androgens are responsible for castration-resistant protein secretion. Treatment of castrated animals with CPA decreased the size of secretory granules and increased the number of apical granules, though neither the absolute nor the relative volume of all the components analysed was changed. In addition, CPA-treatment increased the amount of protein exocytosed by SV in castrated animals, though total protein synthesis remained unchanged. Adrenalectomy neither suppressed secretion nor induced any further ultrastructural changes in the SV epithelium. Our results demonstrate that secretory activity of the hamster SV following castration is not controlled by extra-testicular androgens and suggest that SV secretory proteins, which are heterogeneous with regard to their sensitivity to androgen withdrawal, might be regulated differentially by androgens.

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells.

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0 degrees C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37 degrees C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0 degrees C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37 degrees C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37 degrees C, 30% of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37 degrees C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the chi-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.

Testosterone interferes with the kinetics of endocytosis in the hamster seminal vesicle.

Endocytosis was studied in the seminal vesicle secretory cells of castrated and control hamsters in order to investigate the effect of testosterone withdrawal in the endocytic activity of these cells. Horseradish peroxidase was injected into the glands lumen after removal of their contents, and tracer distribution was qualitatively studied, and the number of labeled endocytic vesicles quantitatively analyzed, following 5, 20, 40 and 60 min incubation. The following compartments are labeled both in castrate and control cells: 1), endocytic vesicles; 2), vacuoles with or without secretory material; 3), multivesicular bodies; 4), Golgi cisternae; 5), intercellular spaces; 6), sub-epithelial space. The pattern of labeling is lighter in castrate than in control cells and the labeling of Golgi cisternae, which correlates with a significant peak in the number of endocytic vesicles, is observed later in castrated animals than in controls: 40 min vs 20 min. Exocytosis, as evaluated through the fraction of secretory protein released in vitro, decreases following castration. Endocytosis performed in castrated, pilocarpine treated animals shows that the Golgi labeling, coinciding with numerous labeled endocytic vesicles, is advanced from 40 to 20 min after stimulation of exocytosis. The results show that, in the seminal vesicle secretory cells a) the endocytic pathway does not depend on testosterone; b) testosterone withdrawal decreases endocytosis and delays the kinetics of labeling and; c) endocytosis couples to exocytosis, probably so regulating the apical cell membrane area.

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study.

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.