The Formation of Otoliths in the Frog Rana esculenta. Scanning Electron Microscopic and X-Ray Diffraction Studies

Two crystal forms of calcium carbonate were observed: calcite (utricle) and aragonite (saccule, lagena, endolymphatic sac). The first step in otolith formation is the appearance of organic structures in the macula. The subsequent step is characterized by fast growing primitive crystals with a prismatic habitus that successively transform into adult or mature crystals. With the metamorphosis, the aragonite crystals of the endolymphatic organ show clear signs of erosion that can be related to a process of CaCO₃ mobilization from such deposits.     

Scanning electron microscopic and X-ray diffraction studies of otoconia in the lizard Podarcis s. sicula

Summary The otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.     

Scanning Electron Microscopy and X-ray Diffraction Studies of the Macular Crystals in the Chondrostean Fish Erpetoichthys calabaricus (Smith)

Morphologic and crystallographic studies of the otoliths of the reed-fish Erpetoichthys calabaricus showed (1) aragonite statoliths with a serrated surface, and (2) two populations of statoconia: one of numerous discoid biconvex crystals of vaterite, the other of pseudohexagonal crystals of aragonite. We suggest that the presence of two calcium carbonate polymorphs in the statoacoustic organs of this archaic fish may have an evolutionary, as well as a systematic and functional significance.     

Light- and electron-microscopic study of the endolymphatic sac of the tree frog,Hyla arborea japonica

Summary The endolymphatic sac of the tree frog and its crystals were observed by light- and electron microscopy. Scanning electron microscopy revealed that the crystals have a faceted body and two pointed ends. Light- and transmission electron microscopy revealed that the endolymphatic sac is composed of many small chambers. In their lumina, numerous ghosts of crystals that resulted from decalcification were observed. The ghosts were demarcated by a linear dense material or embedded in a flocculent substance. The epithelium of the endolymphatic sac is simple squamous or cuboidal and peculiar cytoplasmic granules are found in most cells. The granules are surrounded by a limiting membrane and have varying electron density. Some granules contain a core and/or tubular structures. Vacuoles containing large ghosts are also found in the epithelial cells. These ghosts were quite similar to those in the lumen and sometimes coexist with cell debris. The fine structure of the endolymphatic sac and its crystals is discussed.     

Developmental expression of otoconin-22 in the bullfrog endolymphatic sac and inner ear.

In amphibians, calcium carbonate crystals are present in the endolymphatic sac and the inner ear. The formation of these crystals is considered to be facilitated by a protein called otoconin-22. We examined the spatial and temporal expression of otoconin-22 during the development of the bullfrog (Rana catesbeiana) using RT-PCR, in situ hybridization (ISH), and immunofluorescence techniques. By RT-PCR, otoconin-22 mRNA was first detected in embryos at Shumway stage 20, and this expression pattern continues in late stages. The first otoconin-22 mRNA-positive reaction was detected in stage 22 embryos in the placode of the endolymphatic sac. Otoconin-22 protein was observed in the epithelial cells of the endolymphatic sac at stage 24. On the other hand, a whole-mount ISH technique showed the first expression of otoconin-22 mRNA in the inner ear, in addition to the endolymphatic sac, at the mid-phase of Shumway stage 25. We discuss the role of otoconin-22 in the formation of calcium carbonate crystals in the endolymphatic sac and inner ear.     

The ultrastructure of the statocysts in the pediveliger larvae of Pecten maximus (L.) (Bivalvia)

The pediveliger of Pecten maximus (L.) has a pair of statocysts situated at the base of the foot on either side of a bilobed pedal ganglion. The statocysts consist of a spherical sac connected to the mantle cavity by a cylindrical ciliated canal. Within the sac there are statoconia which are variable both in shape and structure. The cells of the sac are joined by septate desmosomes. There is a non-ciliated cell in each sac containing a variety of granules some of which resemble certain of the statoconia. The remainder of the sac is composed of hair cells, which bear a circular array of radiating cilia. The basal bodies and horizontal striated roots of these cilia are directed radially. The hair cells give rise to thin processes which probably join together to form the static nerve. This nerve runs from the static canal to the pleural ganglion.     

Fusion of Otoconia: a Stage in the Development of the Otolith in the Evolution of Fishes

Abstract The rigid, polycrystalline otolith of teleosts is a side-branch of the general evolution of endolymphatic otoconia that extends from the sharks and rays to the higher vertebrates including man. The transition from the otoconial mass found in the endolymphatic sac of sharks and rays to the rigid polycrystalline otolith of teleosts probably occurred by progressive fusion of otoconia from a loose aggregate to a semi-rigid mass. Traces of the primitive fused otoconia type of otolith still occur in the otherwise polycrystalline otoliths of some teleosts, and a few species of fish retain otoliths that are probably similar to the primitive fused otoconia type of otolith. The morphology of the fusion of otoconia varies according to the polymorph of calcium carbonate that is involved, as well as the particular crystal habit of the polymorph. Analyses of the size distributions of the polymorph-specific morphologies and crystal structure of otoconia suggest that three physical chemical processes, Ostwald ripening, Keith-Padden spherulitic growth and carbonate cementation are significant in the chemistry of fusion of otoconia in the evolution of the aragonite teleost otolith. Predictions of otoconia growth rate from the theory of Ostwald ripening can be compared with predictions from the Keith-Padden theory of spherulitic growth.     

Effect of locally applied drugs on the pH of luminal fluid in the endolymphatic sac of guinea pig

The aim of the present work was to assess the effect of various drugs applied locally on the pH of the luminal fluid (pH(lum)) in guinea pig endolymphatic sac. pH(lum) and transepithelial potential, when measured in vivo by means of double-barrelled pH-sensitive microelectrodes, were 7.06 +/- 0.08 and +6.1 +/- 0.34 mV (mean +/- SE; n = 84), respectively, which is consistent with a net acid secretion in the luminal fluid of the endolymphatic sac. Bafilomycin and acetazolamide increased and decreased, respectively, pH(lum). Amiloride, ethylisopropylamiloride, ouabain, and Schering 28080 had no effect on pH(lum). Results obtained with inhibitors of anionic transport systems were inconclusive; e.g., DIDS reduced pH(lum), whereas neither SITS nor triflocin had any effect. We conclude that bafilomycin-sensitive H(+)-ATPase activity accounts for the transepithelial acid gradient measured in the endolymphatic sac and that intracellular and membrane-bound carbonic anhydrase probably participates in regulating endolymphatic sac pH(lum). The relationship between acid pH, endolymph volume, and Ménière's disease remains to be further investigated.     

Studies of Biominerals Relevant to the Search for Life on Mars

The evidence of the water erosion on Mars is particularly interesting since present climatic conditions are such that liquid water cannot exist at the surface. But, if water was present on the planet in the past, there may have been life, too. Since the discovery of carbonates on Mars also may have very important implications on the possibility that life developed there, we are studying minerals that can have biotic or abiotic origin: calcite (CaCO₃) and aragonite, a metastable state of calcite. We have analysed biomineral aragonite, in the form of recent sea shells, as well as crystals of mineral aragonite. Infrared spectroscopy in the 2–25 μm wavelength range reveals that, after thermal processing, the biotic samples have a different spectral behaviour from the abiotic ones. As a result, it is possible to distinguish abiotic mineral aragonite from aragonite of recent biological origin. Obviously, if life existed in the past on the Red Planet, we could expect to find “ancient” biotic carbonates, which should therefore be investigated, in order to search for a way of discriminating them from abiotic minerals. For this reason, at the beginning we have considered samples of crushed fossil shells of aragonite composition. Afterwards, in order to take into account that fossilization processes almost always produce a transformation of metastable form (aragonite) into more stable form (calcite), we also studied samples of mineral calcite and different types of fossils completely transformed into calcite. All these biotic fossil samples show the same spectral behaviour as the fresh biotic material after thermal annealing at 485°C. Instead, the calcite behaves like abiotic aragonite. Furthermore, it is known that seashells and other biominerals are formed through an intimate association of inorganic materials with organic macromolecules. The macromolecules control the nucleation, structure, morphology, crystal orientation and spatial confinement of the inorganic phase: this differentiates biominerals from minerals. Analysing the aragonite or calcite fossils with a Scanning Electron Microscope, we found that the fossilization process did not modify the structure of the biominerals which maintain their microscopic characteristics. Looking at the morphology of fossil biominerals, it is evident that the crystals are arranged in complex architectures compared with the compact structure of the mineral crystals. In conclusion, the properties and structure of the biominerals are different from those of the minerals. The rapid increase of the crystalline structure developed under biotic conditions makes these minerals less resistant to thermal treatments, compared with samples of abiotic origin. This result holds both for recent shells as well as all fossil samples. The spectroscopic behaviour of all analysed calcium carbonates of biotic origin is different from that of the abiotic one. Therefore, the infrared spectroscopy is a valid technique to discern the origin of the samples and a powerful tool for analysing in-situ and “sample-return” Mars missions specimens. Also Optical and Scanning Electron Microscopy can be useful to support this type of studies. ^*Presented at: National Workshop on Astrobiology: Search for Life in the Solar System, Capri, Italy, 26 to 28 October, 2005     

小黄鱼内耳淋巴液液晶与耳石结晶关系的初步研究

作者以扫描电镜观察了小黄鱼耳尘的“透镜样”结构;Ｘ衍射分析表明小黄鱼耳石由ＣａＣＯ３霰石构成;以偏光显著镜发现内耳淋巴液中有液晶态存在。最后,以液晶理论对耳尘、耳石的形成进行了讨论。     

The endolymphatic duct and sac of the rat: a histological,ultrastructural, and immunocytochemical investigation

A study of the ultrastructure, vascularization, and innervation of the endolymphatic duct and sac of the rat has been performed by means of light- and electron-microscopic and immunocytochemical methods. Two different types of epithelial cells have been identified: the ribosome-rich cell and the mitochondria-rich cell. These two cell types make up the epithelium of the complete endolymphatic duct and sac, although differences in their quantitative distribution exist. The morphology of the ribosome-rich cells varies between the different parts of the endolymphatic duct and sac; the morphology of the mitochondria-rich cells remains constant. According to the epithelial composition, vascularization, and structural organization of the lamina propria, both duct and sac are subdivided into three different parts. A graphic reconstruction of the vascular network supplying the endolymphatic duct and sac shows that the vascular pattern varies among the different parts. In addition, the capillaries of the duct are of the continuous type, whereas those supplying the sac are of the fenestrated type. Nerve fibers do not occur within the epithelium of the endolymphatic duct and sac. A few nerve fibers regularly occur in the subepithelial compartment close to the blood vessels; these fibers have been demonstrated in whole-mount preparations by the application of the neuronal marker protein gene product 9.5. Single beaded fibers immunoreactive to substance P and calcitonin-gene related peptide are observed within the same compartment. Dopamine--hydroxylase-immunoreactive axons are restricted to the walls of arterioles. Morphological differences between the different portions of the endolymphatic duct and sac are discussed with regard to possible roles in fluid absorption and immunocompetence.Abbreviations CGRP Calcitonin gene-related peptide - DSP distal sac portion - DH dopamine--hydroxylase - ED endolymphatic duct - ES endolymphatic sac - EDP enlarged duct portion - IR immunoreactive - ISP intermediate sac portion - LIS lateral intercellular space - NDP narrow duct portion - PMA posterior meningeal artery - PVA posterior vestibular artery - PGP 9.5 protein gene product 9.5 - PSP proximal sac portion - SP substance P - TDP transitional duct portion - VVA vem of vestibular aqueduct     

The endolymphatic sac: its roles in the inner ear

The endolymphatic sac is a non-sensory organ of the inner ear. It is connected to the endolymphatic compartment that is filled with endolymph, a potassium-rich fluid that bathes the apical side of inner ear sensory cells. The main functions ascribed to the endolymphatic sac are the regulation of the volume and pressure of endolymph, the immune response of the inner ear, and the elimination of endolymphatic waste products by phagocytosis. Functional alteration of these functions, leading to deficient endolymph homeostasis and/or altered inner ear immune response, may participate to the pathophysiology of Ménière's disease, an inner ear pathology that causes episodes of vertigo, sensorineural hearing loss and tinnitus, and is characterized by an increase in volume of the cochleo-vestibular endolymph (endolymphatic hydrops).     

Ultrastructure of the endolymphatic sac in the larva of the Japanese red-bellied newt Cynops pyrrhogaster

The ultrastructure of the endolymphatic sac (ES) of the late stage larva of the Japanese red-bellied newt, Cynops pyrrhogaster (stage 57), was examined by light and transmission electron microscopy. The two endolymphatic sacs are located at the dorsal-medial side of the otic vesicle on the dorsal-lateral side of the midbrain in the cranial cavity. The wall of the sac is composed of a layer of cubical epithelial cells with loose, interposed intercellular spaces. The sac contains a large luminal cavity, in which endolymph and numerous otoconia are present. The epithelial cells of different portions of the sac have a similar structure. These cells contain an abundance of cytoplasmic organelles, including ribosomes, Golgi complexes, and numerous vesicles. Two types of vesicles are found in the epithelial cells: the “floccular” vesicle and the “granular” vesicle. The floccular vesicles are located in the supra- and lateral-nuclear cytoplasm and contain flocccular material. The granular vesicles have a fine granular substance and are usually situated apposed to the apical cell membrane. The granular vesicles are suggested to be secreted into the lumen, while the floccular vesicles are thought to be absorbed from the lumen and conveyed to the intercellular spaces by the epithelial cells. The apical surfaces of the epithelial cells bear numerous microvilli. Apparently floating cells, which bear long microvilli on the free surfaces, are observed in the lumen of the ES. Based on the fine structure, the function of the endolymphatic sac of the newt Cynops pyrrhogaster is discussed.     

Carbonic anhydrase is required for statoconia homeostasis in organ cultures of statocysts from Aplysia californica

A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (L15) medium supplemented with salts and Aplysia haemolymph for four days at 17°C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 × 10^–4 M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.     

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere''s disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain''s protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.Open in a separate windowClick here to view.^{(41M, flv)}     

Development of gravity-sensing organs in altered gravity conditions: opposite conclusions from an amphibian and a molluscan preparation

Researchers examined early otolith development in microgravity using fertilized eggs of the Japanese newt, Cynops pyrrhogaster in space flight. Ground experiments examined statocyst, embryonic statolith volume, and statoconia in the post-metamorphic marine mollusk Aplysia californica reared at 1-g and 2-5.7-g. Results indicate that exposure to hypergravity decreased the otolith mass to compensate for increased weight in Aplysia. In the Cynops, there was no compensatory difference in otolith mass, though otoconia production in the endolymphatic system was enhanced.     

Skeletal development inAcropora cervicornis

Scanning electron microscopy, field studies using dyes which become incorporated into the skeleton of living corals as time markers, and petrographic and mineralogic techniques were used to describe the diel pattern of calcium carbonate accretion in the extending axial corallite ofAcropora cervicornis. The axial corallite extends by the formation of randomly oriented fusiform crystals at the distal tip of the branch. Morphological and mineralogical characteristics suggest that these might be calcite crystals. They form a framework upon which needle-like aragonite crystals (initially small tufts) begin to grow. As the needles elongate, groups of them form well defined bundles, fasciculi, which compose the primary skeletal elements. There is a diel pattern in the deposition of the skeleton. At night (1800–0600 hours) the distal spines are pointed and composed primarily of fusiform crystals. During the day (0600–1800 hours) mineral accretion occurs on all surfaces of the skeleton, apparently by epitaxial growth on the aragonite needles of the fasciculi.     

Photosynthesis versus Exopolymer Degradation in the Formation of Microbialites on the Atoll of Kiritimati,Republic of Kiribati,Central Pacific

Aragonitic microbialites, characterized by a reticulate fabric, were discovered beneath lacustrine microbial mats on the atoll of Kiritimati, Republic of Kiribati, Central Pacific. The microbial mats, with cyanobacteria as major primary producers, grow in evaporated seawater modified by calcium carbonate and gypsum precipitation and calcium influx via surface and/or groundwaters. Despite the high aragonite supersaturation and a high photosynthetic activity, only minor aragonite precipitates are observed in the top parts of the microbial mats. Instead, major aragonite precipitation takes place in lower mat parts at the transition to the anoxic zone. The prokaryotic community shows a high number of phylotypes closely related to halotolerant taxa and/or taxa with preference to oligotrophic habitats. Soil- and plant- inhabiting bacteria underline a potential surface or subsurface influx from terrestrial areas, while chitinase-producing representatives coincide with the occurrence of insect remains in the mats. Strikingly, many of the clones have their closest relatives in microorganisms either involved in methane production or consumption of methane or methyl compounds. Methanogens, represented by the methylotrophic genus Methanohalophilus, appear to be one of the dominant organisms in anaerobic mat parts. All this points to a significant role of methane and methyl components in the carbon cycle of the mats. Nonetheless, thin sections and physicochemical gradients through the mats, as well as the ¹²C-depleted carbon isotope signatures of carbonates indicate that spherulitic components of the microbialites initiate in the photosynthesis-dominated orange mat top layer, and further grow in the green and purple layer below. Therefore, these spherulites are considered as product of an extraordinary high photosynthesis effect simultaneous to a high inhibition by pristine exopolymers. Then, successive heterotrophic bacterial activity leads to a condensation of the exopolymer framework, and finally to the formation of crevice-like zones of partly degraded exopolymers. Here initiation of horizontal aragonite layers and vertical aragonite sheets of the microbialite occurs, which are considered as a product of high photosynthesis at decreasing degree of inhibition. Finally, at low supersaturation and almost lack of inhibition, syntaxial growth of aragonite crystals at lamellae surfaces leads to thin fibrous aragonite veneers. While sulfate reduction, methylotrophy, methanogenesis and ammonification play an important role in element cycling of the mat, there is currently no evidence for a crucial role of them in CaCO₃ precipitation. Instead, photosynthesis and exopolymer degradation sufficiently explain the observed pattern and fabric of microbialite formation.