The floral nectary of Hymenaea stigonocarpa (Fabaceae, caesalpinioideae): Structural aspects during floral development

Background and Aims Considering that few studies on nectary anatomy and ultrastructure are available for chiropterophilous flowers and the importance of Hymenaea stigonocarpa in natural 'cerrado' communities, the present study sought to analyse the structure and cellular modifications that take place within its nectaries during the different stages of floral development, with special emphasis on plastid dynamics.Methods For the structural and ultrastructural studies the nectary was processed as per usual techniques and studied under light, scanning and transmission electron microscopy. Histochemical tests were employed to identify the main metabolites on nectary tissue and secretion samples.Key Results The floral nectary consists of the inner epidermis of the hypanthium and vascularized parenchyma. Some evidence indicates that the nectar release occurs via the stomata. The high populations of mitochondria, and their juxtaposition with amyloplasts, seem to be related to energy needs for starch hydrolysis. Among the alterations observed during the secretory phase, the reduction in the plastid stromatic density and starch grain size are highlighted. When the secretory stage begins, the plastid envelope disappears and a new membrane is formed, enclosing this region and giving rise to new vacuoles. After the secretory stage, cellular structures named 'extrastomatic bodies' were observed and seem to be related to the nectar resorption.Conclusions Starch hydrolysis contributes to nectar formation, in addition to the photosynthates derived directly from the phloem. In these nectaries, the secretion is an energy-requiring process. During the secretion stage, some plastids show starch grain hydrolysis and membrane rupture, and it was observed that the region previously occupied by this organelle continued to be reasonably well defined, and gave rise to new vacuoles. The extrastomatic bodies appear to be related to the resorption of uncollected nectar.

Pulvinus functional traits in relation to leaf movements: A light and transmission electron microscopy study of the vascular system

Previous studies on legume pulvini suggest that the vascular system plays an important role in the redistribution of ions and transmission of stimuli during leaf's movements. However, the number of anatomical and ultrastructural studies is limited to few species. The aim of this paper is to investigate the structure and cellular features of the pulvinus vascular system of nine legume species from Brazilian cerrado, looking for structural traits pointing to its participation in the leaf's movements. Samples were excised from the medial region of opened pulvinus of Bauhinia rufa, Copaifera langsdorffii, Senna rugosa (Caesalpinioideae), Andira humilis, Dalbergia miscolobium, Zornia dilphylla (Faboideae), Mimosa rixosa, Mimosa flexuosa and Stryphnodendron polyphyllum (Mimosoideae), and were prepared following light microscopy, transmission electron microscopy and histochemical standard techniques. The vascular system occupies a central position, comprises phloem and xylem and is delimited by a living sheath of septate fibers in all the species studied. This living cells sheath connects the cortex to the vascular tissues via numerous plasmodesmata. The absence of fibers and sclereids, the presence of phenolic idioblasts and the abundance and diversity of protein inclusions in the sieve tube members are remarkable features of the phloem. Pitted vessel elements, parenchyma cells with abundant cytoplasm and living fibriform elements characterize the xylem. The lack of lignified tissues and extensive symplastic continuity by plasmodesmata are remarkable features of the vascular system of pulvini of the all studied species. (c) 2007 Elsevier Ltd. All rights reserved.

Can sieve-element plastids in Panicum maximum (Poaceae) leaves act in the blockage of injured sieve-tube elements?

The ultrastructural features and the plastid changes caused by sample preparation were studied in sieve elements of Panicum maximum leaves. Samples of expanded leaves, taken near the ligule region, were fixed and processed by common light and transmission electron microscopy methods. In mature sieve-tube elements, the protoplast is electron-translucent and plastids are the most frequent organelles. Mitochondria and smooth endoplasmic reticulum segments are also visible and occupy a parietal position within the cell. The plastids are globular and show electron-dense proteinaceous inclusions in the stroma. The protein crystals are predominantly cuneate, but thin crystalloids and amorphous and/or filamentous proteins also occur. The presence of intact plastids plus others in different phases of plastid envelope rupture were interpreted as evidence that this rupture is a normal event in response to injury. This plastid envelope rupture is possibly activated by the release of pressure in the sieve-tube element. After plastid membrane vesiculation, the stroma and the protein crystals are dispersed within the sieve-element ground cytoplasm. The vesicles originating from the plastid envelope move to one cell pole, while protein crystalloids move to the opposite pole and agglomerate in the sieve-plate region. Our findings indicate that these protein crystalloids, which deposit in the sieve plate, may act in sieve-plate pores occlusion, preventing the release of phloem sap, similar to the role of P-protein in dicotyledons. (c) 2008 Elsevier GmbH. All rights reserved.

Two novel reports of semidry stigmatic surface in Asteraceae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intercellular pectic protuberances in Hymenaea stigonocarpa (Fabaceae, Caesalpinioideae): Occurrence and functional aspects

A study of the anatomy and ultrastructural aspects of leaf mesophyll and floral nectaries of Hymenaea stigonocarpa Mart. ex Hayne revealed the presence of intercellular pectic protuberances (IPPs) linking adjacent cells in both the leaf palisade cells and the secretory parenchyma of the floral nectary. Samples of the middle third of the leaf blade and of floral nectaries in anthesis were collected, fixed, and processed using standard procedures for light, transmission, and scanning electron microscopies. The IPPs of palisade cells of the mesophyll and the secretory parenchyma cells of the floral nectary take the form of scalae or strands, respectively. No evidence of the specific synthesis of these structures was observed, and they are apparently formed by the separation of adjacent cells due to cell expansion, when intercellular spaces develop. The IPPs observed in H. stigonocarpa increase cellular contact and probably act in apoplastic transport.

Leaf glands act as nectaries in Diplopterys pubipetala (Malpighiaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epididymis-specific pathologic disorders in rats exposed to gossypol from weaning through puberty

Previous work in our laboratory revealed that the pubertal period of reproductive development in the male rat was particularly vulnerable to gossypol exposure, with a higher frequency of round structures in the lumen of the cauda epididymidis in the treated rats. Herein, we utilized hemicastration and electron microscopy to confirm that the epididymis is a definitive target of gossypol. Although exposure to gossypol from weaning through puberty caused a significant decrease in daily sperm production, as well as in the concentration of sperm in the epididymis, serum testosterone levels and reproductive organ weights were not altered. In gossypol treated rats, sperm morphology was compromised severely, but the epithelium in testis and epididymis appeared morphologically normal. Ultrastructural examination revealed that round structures, present only in gossypol exposed males, represented: (1) principal cells exfoliated from the epididymal epithelium; (2) epididymal epithelial cell cytoplasm containing degenerating sperm; and (3) degenerating epithelial cells, consisting of vesicles and particles of different sizes, forms and densities. Taken together, the data confirm that gossypol targets the epididymis, disturbing both the structure and function of this organ, and presumably disrupts sperm maturation.

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Testicular interstitial cells, and steroidogenic detection in the protogynous fish, Synbranchus marmoratus (Teleostei, Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a freshwater protogynic diandric species. Primary males develop directly as males while secondary males arise from the sex reversal of females. Fishes from Argentine and Brazil inland waters were collected, examined and compared for this study. In order to characterize the interstitial testicular compartment, light and electron microscopy techniques and an enzyme histochemical examination for steroidogenic cells detection were used. The interstitial compartment of S. marmoratus is composed of Leydig and myoid cells, collagen fibers, blood cells, macrophages,and amyelinic nerves. At the ultrastructural level, no differences were observed in the interstitial tissue, either between specimens from the different sampling sites or between primary and secondary males. Leydig cells are present in all testes examined throughout the year. A cytoplasmatic reaction of 3beta-HSD was detected only in Leydig cells during sex reversal and in both type of males, mainly during the regressed and early maturation classes (autumn and winter). Leydig cells possess the typical fine structural characteristics associated with steroidogenesis. Furthermore, in both type of males, during sex reversal and after the spawning period, the number of granulocytes and macrophages present in the testes increased, suggesting that they could be involved in phagocytosis and resorption of damaged cells. (C) 2004 Elsevier Ltd. All rights reserved.

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups. (c) 2005 Published by Elsevier Ltd.

Ultrastructure of spermatogenic cells and spermatozoa in Hoplias malabaricus (teleostei, characiformes, erythrinidae)

The differentiation of spermatids in Hoplias malabaricus is characterized by chromatin compaction, flagellum development, nuclear rotation, nuclear fossa formation, and excess cytoplasm elimination. In the resulting spermatozoon, the head is round and the nucleus contains chromatin compacted in thick filaments, peripherically arranged, to a central electron-lucent area. The acrosome is absent. The nuclear fossa is eccentric but not pronounced. The proximal centriole penetrates it and is oblique to the flagellum. The long midpiece has several converging elongate vesicles, forming membranous hoops in the initial segment of the flagellum, but has no cytoplasmic channel. The mitochondria are elongate and branched or C-shaped and located around the initial segment of the axoneme. The lateral flagellum does not show lateral projections. The ultrastructural characteristics of H. malabaricus spermatozoa are similar to the Cypriniformes. (C) 2001 the Fisheries Society of the British Isles.

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.

Morphological aspects of cluster formation in the germarium of the sugarcane borer Diatraea saccharalis Fabricius (Lepidoptera : Pyralidae)

Diatraea saccharalis F. is one of the greatest pests of the sugar cane culture. This report aimed to characterize the germanium region of the sugarcane borer by light and transmission electron microscopy, emphasizing the morphological steps of the ovarian cluster formation. In the germanium of this insect, four zones could be morphologically identified during the cluster formation. In the most apical end of each ovariole - Zone I - the germ line stem cells undergo complete mitotic division, originating the cystoblasts. In the Zone II, each cystoblast produces a group of eight cells, the cystocytes, which are interconnected by the ring canals. Clusters containing all the cystocytes in the meiosis, characterizes the Zone III. Germ cells with ultrastructural features of apoptosis are also detected in this Zone. In the Zone IV the cystocytes differentiate, morphologically, into one oocyte and seven nurse cells. Interstitial somatic cells and pre-follicle cells exhibit, in their cytoplasm, heterogeneous vacuoles containing degenerated cellular fragments, characterized as apoptotic bodies. Our results pointed out to the morphological evidences related with important control mechanisms for new clusters/follicles production and for the cellular arrangement into the germanium, resulting from the programmed cell death. We believe that the morphological characterization of ovarian cluster formation in D. saccharalis provided valuable information for the understanding of the initial steps of oogenesis and contributed for the knowledge of the cellular mechanisms related with the oocyte production and with reproduction in insects.

Morphological investigations of the surface epithelium of ductuli efferentes of black isogenic mice (Mus musculus)

Morphological investigations of the epithelial cell types that line the ductuli efferentes (DE) of black isogenic mice confirm absorption of the luminal fluid phase by endocytosis as the main function of ductuli efferentes (DE) in this species. :Furthermore, all the histochemical and ultrastructural observations on the DE epithelial histoarchitecture indicate other cellular functions such as exocytosis and probably secretion: including an aprocrine secretory process.

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.