Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization.

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.

Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway.

Epidermal growth factor (EGF), circulating in the blood, is taken up by rat liver hepatocytes by means of specific and saturable receptor-mediated endocytosis. These experiments were undertaken to determine (a) the transport pathway(s) of EGF taken up by rat liver and (b) the effects of lysosomal inhibition on its transport. 125I-EGF was injected into rat portal veins, and bile samples were collected and analyzed for both total and immunoprecipitable radioactivity. In addition, the livers were examined by electron microscopic autoradiography. Some animals received injections of chloroquine before surgery, to disrupt lysosomal function. The results indicate that most of the EGF taken up by the hepatocytes is transported to lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a pathway independent of the lysosomal system, resulting in secretion of intact EGF: (a) Both degraded and immunoprecipitable EGF are secreted into bile. (b) Immunoprecipitable radioactivity peaks at 20 min after EGF injection, whereas degradation-associated radioactivity does not peak until 40 min postinjection. (c) EGF isolated from bile is specifically taken up by isolated hepatocytes in monolayer culture, indicating that it is still recognizable by the EGF receptor. (d) When the lysosomal system is inhibited with chloroquine, secretion of degraded EGF is significantly inhibited, whereas the amount of intact EGF secreted into bile is unchanged. The utilization by liver of a dual transport process for EGF represents an unusual system of intracellular ligand processing, whose physiological significance has yet to be determined.

Evidence for a vesicular transport mechanism in hepatocytes for biliary secretion of immunoglobulin A.

Quantitative electron microscopic autoradiography and diaminobenzidine cytochemistry provide evidence for an uptake and vesicular transport mechanism for iodine-125-labeled immunoglobulin A from plasma to bile by hepatocytes in vivo. The data confirm the existence of a hepatobiliary pathway for secretion of immunoglobulin A into the intestine and are consistent with a vesicular transport mechanism for biliary proteins within liver parenchymal cells.

Entry of insulin into human cultured lymphocytes: electron microscope autoradiographic analysis.

Electron microscope autoradiographs were prepared of IM-9 human cultured lymphocytes incubated with iodine-125-labeled insulin. With the use of [125I]insulin and Ilford L-4 emulsion, the technique had a resolution half-distance of approximately 0.085 micrometer. Autoradiographs revealed a time-dependent entry of insulin into the cell interior that was maximal after 30 minutes of incubation. At this time point nearly 40 percent of the [125I]insulin was in the interior of the cell at a distance 1 micrometer or greater from the plasma membrane. Grain distribution and volume density analyses revealed that the intracellular insulin was concentrated in the endoplasmic reticulum and nuclear membrane.

Quantitative analysis of spiral ganglion projections to the cat cochlear nucleus.

A quantitative examination of the tonotopic organization of primary afferent projections to the cochlear nucleus (CN) in adult cats was conducted by using focal extracellular injections of Neurobiotin (NB) into the spiral ganglion of the basal cochlea. One to three injections separated by intervals of at least 2 mm were positioned along the basal one-third of the cochlea. Each injection produced discrete projection laminae that appeared as parallel horizontal sheets of labeled axons terminals distributed sequentially dorsally to ventrally across each major CN subdivision: the anteroventral, posteroventral, and dorsal cochlear nucleus, (AVCN, PVCN, and DCN, respectively). The length (rostrocaudal dimension), width (mediolateral dimension), thickness (dorsoventral dimension), and relative placement of 18 "frequency-band" laminae were measured in 10 adult cochlear nuclei. The average AVCN projection thickness was approximately twice that of the PVCN and DCN projections. In double injection cases, the center-to-center separation between AVCN laminae was also approximately twice that in the PVCN and equal to that in the DCN. Lamina thickness did not differ significantly as a function of frequency representation. However, in both width and length, mid-frequency laminae were up to two times larger than high-frequency laminae. Thus, the results indicate that DCN projections are the most discrete (i.e., are the thinnest and have the least overlap between adjacent frequency projections), whereas the AVCN projections are the largest but are as discrete as PVCN projections. In addition, high-frequency projections are smaller and more discrete than mild-frequency projections, which are larger and have greater overlap with adjacent frequency projections.

Chronic electrical stimulation by a cochlear implant promotes survival of spiral ganglion neurons after neonatal deafness.

This investigation examined the consequences of neonatal deafness and chronic intracochlear electrical stimulation delivered by a cochlear implant during maturation. Kittens were bilaterally deafened by an ototoxic drug administered daily for 2 weeks immediately after birth. Unilateral electrical stimulation was initiated at 7-10 weeks of age and continued over periods of 22-47 weeks (4 hours/day; 5 days/week). Bipolar intracochlear electrodes delivered one of several different electrical signals designed to be temporally challenging to the central auditory system. Morphometric evaluation of spiral ganglion (SG) cell somata within Rosenthal's canal demonstrated a mean of approximately 50% of normal cell density maintained in the chronically stimulated ears, compared with approximately 30% on the control deafened side. This 20% difference in density was highly significant and was greater than differences reported in earlier studies using 30 pps stimulation delivered by either intracochlear bipolar or round window monopolar electrodes. However, the duration of stimulation was also longer in the present study, so it is unclear to what extent the nature of the temporally challenging stimulation vs. its duration contributed to the marked increase in survival. Measurements of the SG cell somata revealed a pronounced decrease in cell diameter in neonatally deafened cats studied about 1 year after deafening, and an additional decrease after long-term deafness (2.5-6.5 years). Furthermore, in the cochlear regions with the greatest stimulation-induced differences in SG cell density, direct measurements of cross-sectional soma area of the largest cells revealed that cells were significantly larger in the stimulated ears. Thus, in addition to the marked increase in the number of surviving SG cells, larger soma area contributed modestly to the pronounced increase in neural density following chronic electrical stimulation.

Spatial organization of inner hair cell synapses and cochlear spiral ganglion neurons.

The morphological organization of the central projections of the cat cochlear spiral ganglion into the cochlear nucleus was previously investigated by creating restricted lesions in the anteroventral cochlear nucleus (AVCN) to ablate selectively either the lateral or the medial aspect of isofrequency projection laminae. Such lesions resulted in highly selective retrograde degeneration of spiral ganglion cells. Ablation of the lateral part of the AVCN caused degeneration of cells within the scala tympani part of the ganglion, whereas medial ablations within the AVCN induced degeneration of the scala vestibuli aspect of the ganglion. The peripheral axons also degenerated and this fiber loss exhibited selective topographies that paralleled the cell loss within the spiral ganglion, although this phenomenon was more prominent in the proximal part of the osseous spiral lamina near the ganglion and less obvious more distally near the habenula perforata. In this investigation, inner hair cells (IHCs) from these selective lesion cases were evaluated by electron microscopy of serial sections through the basal synaptic regions. Results demonstrated differential degeneration of afferent synapses, with greater (but not completely selective) loss of pillar synapses after lateral AVCN lesions and greater loss of modiolar synapses after medial lesions. Because auditory nerve fibers of different spontaneous discharge rates (SRs) have different spatial distributions on the IHC (Liberman, Science 216:1239, 1982), our results suggest that this SR-based organization is maintained in a topographic organization across the vertical (scala tympani-to-scala vestibuli) dimension of the spiral ganglion cell cluster and carried into the ventral cochlear nuclei (VCN). Thus, in addition to the spiral frequency organization represented by the dorsal-to-ventral frequency map in the VCN, there is also an orderly organization of inputs from high- and low-SR fibers across the lateral-to-medial dimension of the VCN such that the lateral isofrequency laminae receive a proportionately greater input from high-SR fibers, whereas medial isofrequency laminae receive preferential input from low- and medium-SR fibers.

Cochlear pathology of long term neomycin induced deafness in cats.

The long term sequelae of hair cell destruction consequent from administration of the ototoxic aminoglycoside antibiotic, neomycin sulfate, were evaluated in histological and ultrastructural studies of cochlear morphology in cats. Complete hearing loss, as defined by an absence of brainstem evoked responses to click stimulation at 120 dB peak SPL, was induced by intramuscular injections of neomycin at 50 mg/kg body weight/day, and cochlear pathology was studied at 6 months and 1, 3 and 4 years following onset of profound deafness. In these long term ototoxicity cases the organ of Corti was collapsed and resorbed over the basal one-quarter to three-quarters of the cochlear spiral, depending on duration of deafness. Significant progressive reduction in the spiral ganglion cell population and sequential degenerative alterations in the remaining neurons were observed with increasing time elapsed after induced hearing loss. The sequence of pathological alterations in spiral ganglion neurons appeared to be: a) swelling, demyelination and degeneration of the peripheral dendrites; b) demyelination and shrinkage of the cell soma with preservation of the central axon; and c) demyelination of the central axon and degeneration of the cell perikaryon. In apical cochlear regions, severe degeneration of the spiral ganglion preceded the collapse of the tunnel of Corti and regional loss of pillar cells. Residual populations of spiral ganglion neurons were as low as 1-2% of the normal values in the most severely degenerated cochleae in the series. Light microscopic and ultrastructural studies revealed a selective survival advantage for the unmyelinated type II neurons over the myelinated type I neurons with these long survival periods. The prolonged time course and atrophic nature of these pathological alterations suggests that degeneration of spiral ganglion neurons progresses continuously following drug-induced insult to the cochlea. Some possible factors contributing to this long term progressive degeneration will be discussed.

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study.

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n = 3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base cochlear locations which in the adult correspond to best frequencies of approximately 20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0-P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of approximately 25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.

Chronic intracochlear electrical stimulation in neonatally deafened cats: effects of intensity and stimulating electrode location.

An earlier study conducted in this laboratory suggested that chronic intracochelear electrical stimulation at moderate current levels can at least partially delay or prevent the retrograde degeneration of primary auditory (spiral ganglion) neurons that otherwise is progressive after neonatal deafness induced by ototoxic drug administration. Increased survival of spiral ganglion neurons was observed within the basal cochlear region near the stimulating biopolar electrode pairs, while in more apical regions there was no significant difference between the stimulated and control cochleas. The mechanisms underlying this maintenance of spiral ganglion neurons induced by chronic electrical stimulation are uncertain, especially since increased neuronal survival was observed over broader sectors of the ganglion than would be expected to be directly activated by the bipolar electrodes and moderate stimulation intensity (6 dB above electrically evoked auditory brainstem response threshold) used. In this report, data are presented from a second series of neonatally deafened and chronically stimulated cats. The parameters for chronic electrical stimulation were manipulated in two simple ways. First, the intensity of the electrical stimulus was reduced from the earlier study, while the duration of chronic stimualtion periods was increased; and secondly, two different intracochlear positions of stimulating electrodes were employed in different experimental groups. Results indicate that elecrical stimulation of the cochlea at an extremely low intensity (2 dB above electrically evoked auditory brainstem response threshold) is sufficient to at least partially prevent or delay ganglion cell degeneration in the deafened cochlea. In addition, data suggest a differential distribution of the maintained or conserved ganglion cells, such that when the stimulating electrode pair was positioned near the base of the cochlea increased ganglion survival in a more basal cochlear sector, while stimulation at a more apical site resulted in increased neuronal survival extending to more apical regions.

Chronic intracochlear electrical stimulation induces selective survival of spiral ganglion neurons in neonatally deafened cats.

Ten newborn kittens were deafened by systemic administration of neomycin sulfate. Profound hearing losses were documented by ABR and FFR (500 Hz) testing. At 9-17 weeks of age, the young deafened cats were unilaterally implanted with a multichannel scala tympani electrode. Six of the animals were chronically stimulated at 6 dB above electrically evoked ABR thresholds for 1 h/day for periods of 1 month or 3 months. Stimuli were charge-balanced biphasic pulses (200 microseconds/phase, 30 pps.) The remaining 4 cats underwent identical deafening and implantation schedules but were not stimulated. Results indicate that administration of neomycin in neonatal cats induced degeneration of hair cells and spiral ganglion cell loss that was bilaterally symmetrical between the two cochleas of each individual animal, although there was variation between animals in the severity of the ototoxic drug effect. In animals receiving passive (unstimulated) implants, morphometric analysis of spiral ganglion cell density showed no significant difference in ganglion cell survival between the implanted cochleas and the contralateral control ears. In contrast, animals that were chronically stimulated for 3 months showed significantly better neuronal survival in implanted and stimulated cochleas as compared to contralateral deafened control ears. The induced conservation of spiral ganglion neurons was observed consistently within the basal cochlear region near the stimulating electrodes. In more apical regions there was no significant difference between the stimulated and control cochleas. The mechanisms underlying this selective conservation of spiral ganglion neurons induced by chronic intracochlear electrical stimulation are uncertain. Since no comparable chronic stimulation studies have been conducted in adults, it is not known whether similar conservation effects could be induced in mature animals.

Consequences of chronic extracochlear electrical stimulation in neonatally deafened cats.

This investigation examined the consequences of neonatal deafness and chronic electrical stimulation of the cochlea in the developing auditory system. Cats were bilaterally deafened by daily ototoxic drug administration for two weeks after birth. Electrical stimulation was initiated at 6-9 weeks of age and continued for up to 6 months, using monopolar round window electrodes that synchronously excited auditory neurons throughout the cochlea. Morphometric evaluation of the density of spiral ganglion cell somata within Rosenthal's canal demonstrated that chronic stimulation induced an increase of about 6% in neuronal survival. Although this difference was statistically significant, extracochlear stimulation in these cats was less effective in preventing neural degeneration than lower intensity, more restricted intracochlear stimulation that was shown in a previous study to induce an average increase of about 13% in neuronal survival. Electrophysiological recording experiments conducted in the inferior colliculus in these animals indicated that monopolar extracochlear stimulation can induce profound alterations in the spatial (frequency) selectivity of the auditory midbrain. On average, results were similar to those previously reported for bipolar intracochlear stimulation, showing about a two-fold expansion of the central representation of chronically stimulated electrodes. However, results with extracochlear stimulation showed much greater variability among individual animals. The results presented suggest that it is problematic to effect consistent 'whole' nerve stimulation using monopolar round window electrodes. Moreover, this mode of stimulation can induce profound functional alterations in the central nervous system and is substantially less effective in forestalling the degeneration of auditory neurons than intracochlear stimulation. Both these results contraindicate the implantation of such electrodes in young children for the purpose of maintaining the integrity of the auditory system for later application of a multichannel cochlear implant.

Insulin receptors on the endolymphatic sac: an autoradiographic study.

Light microscopic autoradiographs were prepared of isolated guinea pig endolymphatic sac (ES) tissue incubated with iodine (I)-125-labeled insulin (insulin I 125). These demonstrated specific binding of the insulin I 125 to receptors in the ES. Receptor-mediated endocytosis of the insulin-receptor complex is postulated to occur, and the relevance of insulin receptors in the ES is discussed, with particular emphasis on the previously observed synthesis of hyaluronan from glucose by the ES.

Hyaluronan synthesis in the adult guinea pig endolymphatic sac.

The endolymphatic sac is believed to play a major role in membranous labyrinth homeostasis by controlling the volume of endolymph, removing debris, and participating in the immune response of the inner ear. The endolymphatic sac is postulated to absorb endolymph and to synthesize and secrete high-molecular-weight and osmotically active glycosaminoglycans (GAGs). The present study examines the ability of in vitro adult guinea pig endolymphatic sac cells to synthesize complex proteins and polysaccharides. The intent is to characterize the nature of these compounds by studying carbon-14 (14C) glucose incorporation in tissue cultured endolymphatic sac specimens using autoradiographic and specific enzymatic digestion techniques. Our results suggest that sac cells can synthesize GAGs and proteins in vitro in proportionately larger amounts than surrounding connective tissue and dura. The principal GAG synthesized by the endolymphatic sac appears to be hyaluronan.

Degradative intracellular transport of antisecretory component in cultured hepatocytes. An alternate pathway for the immunoglobulin A receptor.

The liver efficiently transports dimeric immunoglobulin A (dIgA) from blood to bile in a direct, nonlysosomal pathway involving smooth-surfaced vesicles. Secretory component (SC), the plasma membrane receptor for dIgA, is released into bile still bound to its ligand by disulfide bridges. Rabbit IgG antirat SC binds specifically to plasma membrane SC, yet the biliary secretion of anti-SC is markedly lower than that of dIgA, suggesting that the IgG antibodies utilize an alternate transhepatocellular pathway. Uptake of commercially available antihuman SC conjugated to horseradish peroxidase was examined by quantitative electron microscopic immunocytochemistry using primary rat hepatocyte monolayer cultures. Coincubation with human polymeric IgA, rabbit antiserum to rat SC, free human SC, human secretory IgA, and rat bile, all significantly suppressed uptake of anti-SC-horseradish peroxidase, thus demonstrating the specificity of the labeled antibody. Coated vesicles accounted for greater than 70% of the total uptake of either the anti-SC-horseradish peroxidase preparation or colloidal gold-labeled IgG antirat SC. Both compounds could also be observed in other structures associated with the degradative pathway, i.e., multivesicular bodies and lysosomes. Moreover, the extent to which 125I-anti-SC was degraded was significantly greater than that of 125I-dIgA. These data demonstrate that dIgA and anti-SC utilize different intracellular pathways, with anti-SC undergoing lysosomal degradation.

Intracellular processing of human vs. rat immunoglobulin A in the rat liver.

It is well established that in the rat, rat dimeric IgA is transported from blood to bile across rat liver parenchymal cells via a series of minute smooth membrane-limited vesicles. This pathway is unique from that taken by a number of other ligands, which are internalized for degradation, in that there appears to be little involvement of coated vesicles, multivesicular bodies and lysosomes. The transmembrane receptor for IgA, secretory component, is not recycled but is secreted in part with the ligand into bile and must be produced continuously within the liver cell. Several recent studies have suggested that the receptor for asialoglycoproteins, as well as the structures involved in its processing, may play an important role in IgA processing. It was noted, however, that in all of these studies human polymeric IgA1 was used in the rat model. Using purified rat and human IgA preparations, we have demonstrated by light and quantitative electron microscopic autoradiography, as well as by certain biochemical procedures, that the two ligands are processed quite differently from one another in the rat. Human IgA disappears from the plasma at a slower rate and is much less efficiently transported into the bile. In addition, up to as much as 30% of the human IgA is diverted to the lysosomal pathway. This diversion of human polymeric IgA may be related to either the association of a serine-linked oligosaccharide at the hinge region of the human polymeric IgA1 or that large polymers, often found in human IgA preparations may initiate secretory component receptor aggregation, which in turn, interferes with the normal physiological processing of the IgA molecule.

The fate of polymeric and secretory immunoglobulin A after retrograde infusion into the common bile duct in rats.

In the rat, plasma IgA is rapidly endocytosed by hepatocytes and translocated to the bile via a receptor-mediated vesicular transport system which appears to remain intact even during cholestasis. During the latter phenomenon, there is an accumulation of secretory IgA (sIgA) in plasma. These data suggest that biliary IgA can be regurgitated into the plasma compartment. The present study was designed to determine the location and mechanism(s) by which this might occur. 125I-labeled human polymeric IgA (pIgA) or sIgA was infused retrograde into the rat common bile duct at a flow rate of 20 microliter per min (5 to 10 micrograms per min pIgA; 7 micrograms per min sIgA) over 1 hr. Blood and liver samples were collected 10, 30 and 60 min, and radioactivity determined. Radioactive label appeared in the blood by 10 min and increased linearly with time. By 30 min, however, the liver had reached saturation. All of the label found in the blood was intact starting material, i.e., pIgA or sIgA. Electron microscopic autoradiography analysis clearly demonstrated the presence of grains in vesicles in hepatocyte pericanalicular cytoplasm, as well as in vesicles near the sinusoidal plasma membrane. No grains were observed associated with bile duct or ductule epithelium at any time period. Further, there was no grain accumulation near the parenchymal cell intercellular spaces indicating that paracellular flow plays little or no role in large protein regurgitation. In addition, by 60 min, there were grains associated with Kupffer cells. These data provide the first evidence that hepatocytes, during times of elevated biliary pressure can readily transport macromolecules from bile to plasma via nonreceptor-mediated membrane-limited vesicles.

Hyaluronan synthesis by in vitro cultured endolymphatic sac cells.

The endolymphatic sac (ELS) has been the subject of investigation for many years and yet its overall function remains unclear. It is believed mainly to be involved in the regulation of endolymph through fluid resorption and secretion of osmotically active substances. The present study was performed using in vitro cultured, fetal ELSs of 18 to 19 day gestational mice, to assess whether the ELS cells can synthesize the osmotically active polysaccharide, hyaluronan (HA). The ELS and portions of the membranous labyrinth were dissected from whole otocyst specimens and placed in 14C glucose-enhanced tissue culture media. A light microscopic (LM), autoradiographic study was performed to assess whether 14C glucose could be incorporated by the tissue into HA. Both the ELS cells and the adjacent cartilage demonstrated radiolabel incorporation within 4 hours of incubation in tissue culture medium, with increased radiolabel density in ELS cells after 24 hours of incubation. HA-specific hyaluronidase (HAase) resulted in removal of HAase-sensitive compounds in the ELS in both 4-hour and 24-hour cultured specimens when compared to adjacent cartilage cells (p = 0.001). Approximately 43 percent of the radiolabel was incorporated into HA in ELS specimens, as compared to a 22 percent HA synthesis in the adjacent cartilage tissue, suggesting preferential synthesis by ELS cells. The dissected murine otocysts demonstrate viability in vitro as measured by their ability to incorporate 14C glucose from tissue culture medium. Under these conditions the cultured ELS demonstrates an ability to synthesize HA. A theory of ELS function is proposed.

Changes in hyaluronan synthesis by in vitro cultured endolymphatic sac cells.

The endolymphatic sac (ELS) has been implicated in the maintenance of endolymph volume and pressure in the membranous labyrinth through fluid resorption and secretion of osmotically active substances, known as glycosaminoglycans (GAGs). To assess whether the ELS cells synthesize the GAG, hyaluronan (HA), and to further elucidate the secretory function of the ELS, a series of experiments were carried out on in vitro tissue-cultured, fetal murine ELSs. In phase 1 of the investigation, the ELSs that were attached to a small portion of the posterior labyrinth, were resected from whole otocyst specimens and studied in tissue culture. This model was chosen to determine whether a change in endolymph homeostasis affects ELS activity. Radiolabeled 14C glucose incorporation was used to evaluate HA synthesis by ELS cells when cultured in vitro. Approximately 43 percent of the incorporated 14C glucose radiolabel was digested by Streptomyces hyaluronidase (an HA-specific hyaluronidase), suggesting HA synthesis by sac cells. In phase 2 of our experiments, the ELSs were not resected from the otocysts. Instead, they were left attached to intact membranous labyrinths, and whole otocysts were cultured. Studies analogous to those of phase 1, assessing the ability of the ELS cells to incorporate 14C glucose into HA, were performed on these specimens. Streptomyces hyaluronidase treatment of these ELS specimens resulted in a reduction in the removal of radiolabel. Therefore, the ELS cells of intact otocysts incorporated less 14C glucose into HA when compared to the ELS cells of the resected specimens.(ABSTRACT TRUNCATED AT 250 WORDS)