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PubMed1990

Quantitative microfocal radiographic assessment of progression in osteoarthritis of the hand.

We studied 32 patients with osteoarthritis who had 5x macroradiographs taken of their wrists and hands at 6-month intervals over an 18-month period. The higher magnification and resolution of microfocal radiography permitted the quantitative detection of progressive changes in 4 different features: subchondral sclerosis, the number and size of osteophytes, juxtaarticular radiolucencies, and joint space narrowing. Compared with normal control subjects, subchondral cortical thickness was greater in all patients at entry and showed a variable degree of change over the study period. Osteophytes and juxtaarticular radiolucencies were present in all patients at study entry; by the end of the study, osteophytes had increased in number and area, and juxtaarticular radiolucencies had increased in area, but not in number. At entry, 44% of the patients had joint space narrowing significantly greater than that in the control subjects; by 18 months, this proportion increased to 65%. No correlation was found between subchondral sclerosis, osteophytes, juxtaarticular radiolucencies, and joint space narrowing. We conclude that in osteoarthritis of the hand, the bony changes have progressed significantly before the occurrence of radiographically evident joint space narrowing indicative of cartilage thinning.

Aged↗

PubMed1995

Development of basal forebrain projections to visual cortex: DiI studies in rat.

We performed experiments using retrograde and anterograde labeling with DiI to examine the development of basal forebrain (BFB) projections to the visual cortex in postnatal rats. DiI placed in occipital cortex led to retrograde labeling of BFB neurons as early as postnatal day 0 (P0); labeled cells were found mainly in the diagonal band complex but also in the medial septum, globus pallidus, and substantia innominata. The retrogradely labeled BFB cells displayed remarkably well-developed dendritic arbors, even in younger animals, and showed increases in soma size, dendritic arbors, and dendritic spines over the first 2 postnatal weeks. DiI placements in the diagonal band led to anterogradely labeled axons in cortex. At early ages (P0-P1), labeled axons were largely confined to white matter. With increasing age, greater numbers of labeled axons were seen in the white matter and in deep cortical layers, and labeled axons extended into superficial layers. The leading edge of labeled fibers reached layer V of visual cortex by P2 and layer IV by P4 and were found throughout the cortical layers by P6. Numbers and densities of labeled axons in visual cortex were greater in older animals, at least through P14. The time of ingrowth of labeled BFB axons into visual cortex indicates that these afferents grow into particular cortical layers after those layers have differentiated from the cortical plate. These data indicate that basal forebrain projections arrive in occipital cortex after cortical lamination is well underway and after the entry of primary thalamocortical projections.

Animals↗

PubMed1990

Behavioural deficits and serotonin depletion in adult rats after transient infant nasal viral infection.

Dysfunction of subcortical serotoninergic neurons has been implicated in some behaviour disturbances. The serotoninergic neurons in the dorsal and median raphe project widely in the brain. They innervate the olfactory bulbs and can be targets for exogenous agents attacking the olfactory epithelium and bulbs. We report here an injury to the serotoninergic neurons after intranasal infection in 12-day-old rats with a temperature-sensitive mutant of vesicular stomatitis virus. The brain infection was focal and transient. Viral antigens could no longer be detected 13-15 days after infection. In spite of this the animals, as adults, had a severe serotonin depletion in the cerebral cortex and hippocampus, and showed abnormal locomotor and explorative behaviour as well as learning deficits. The neocortex was histologically intact and parameters related to other neurotransmitters such as dopamine, noradrenaline, GABA and acetylcholine showed no marked changes. A relatively selective damage to serotoninergic nuclei as a result of virus neuroinvasion through a natural portal of entry, may constitute a new pathogenetic mechanism for cortical dysfunction and behavioural deficits.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2004

Rhizobium infection: lessons from the versatile nodulation behaviour of water-tolerant legumes.

Water-tolerant legumes provide bacteria with special ways of invading roots to establish N(2)-fixing symbiosis upon flooding. On well-aerated roots, root hair curling (RHC) invasion is used, whereas, under hydroponic conditions, rhizobia enter the cortex through cracks at lateral root bases (LRBs). Here, we compare the physiological and anatomical traits of these invasions. During waterlogging, accumulating ethylene inhibits the epidermal stages of RHC invasion. LRB invasion circumvents this step by direct colonization of the cortical tissue. By avoiding the epidermis for bacterial entry under hydroponic conditions, the stringent nodulation (Nod) factor perception systems that are active within the epidermis are not needed. Consequently, LRB invasion might be useful for analysing the requirement for Nod factor perception and other signal transduction systems downstream of the epidermis.

Fabaceae↗

PubMedDate not supplied

An in vitro comparison of hollow ground and trocar points on threaded positive-profile external skeletal fixation pins in canine cadaveric bone.

OBJECTIVE: To compare the microstructural damage created in bone by pins with lathe-cut and rolled-on threads, and to determine the peak tip temperature and damage created by positive-profile external fixator pins with either hollow ground (HG) or trocar (T) tips during insertion. STUDY DESIGN: An acute, in vitro biomechanical evaluation. SAMPLE POPULATION: Twenty-seven canine tibiae. METHODS: Lathe-cut thread design with T point (LT-T), rolled-on thread design with T point (RT-T), and rolled-on thread design with HG point (RT-HG) pins were evaluated. Twenty pins of each type were inserted under constant drilling pressure into 12 canine tibiae (12 diaphyseal and 8 metaphyseal sites per pin type). Peak pin tip temperature, drilling energy, end-insertional pin torque, and pullout force were measured for each pin. For the histologic study, five pins of each type were inserted into cortical and cancellous sites in 15 additional tibiae. Entry and exit damage, and thread quality were assessed from 100 micron histologic sections by using computer-interfaced videomicroscopy. RESULTS: T-tipped pins reached higher tip temperature in both diaphyseal and metaphyseal bone compared with HG-tipped pins. RT-T pins had higher pullout strength (diaphyseal) and end-insertional torque compared with other combinations. No differences in drilling energy or insertional bone damage was found between the three pin types (P < .05). CONCLUSIONS: T-tipped pins mechanically outperformed HG-tipped pins. Pin tip and thread design did not significantly influence the degree of insertional bone damage. CLINICAL RELEVANCE: T-tipped pins may provide the best compromise between thermal damage and interface friction for maximizing performance of threaded external fixator pins.

Animals↗

PubMed2002

The fungal sheath of ectomycorrhizal pine roots: an apoplastic barrier for the entry of calcium, magnesium, and potassium into the root cortex?

The apoplastic permeability of the fungal sheath of two different ectomycorrhizal associations of Pinus sylvestris L. was analysed by laser microprobe mass analysis (LAMMA) and energy-dispersive X-ray spectroscopy (EDXS) after stable isotope labelling with 25Mg, 41K and 44Ca. Entry of 25Mg and 44Ca into the outer cortical apoplast of non-mycorrhizal roots was detected after 4 min of labelling. After a longer exposure time the endodermis with its Casparian band acted as an efficient apoplastic diffusion barrier for the radial movement of 25Mg and 44Ca into the stele. A fraction of approximately one-third of the apoplastic cations of the root cortex could not be exchanged against the external label even after longer exposure times. The ectomycorrhizal sheath of the two fungal species used, Pisolithus tinctorius (Pers.) Coker & Couch and Suillus bovinus (L. ex Fr.) Kuntze, does not completely inhibit the apoplastic movement of ions into the mycorrhizal root cortex, but retarded the penetration of isotopes into the cortical apoplast. In roots inoculated with S. bovinus, a clear labelling of the cortical apoplast could first be detected after 24 h of exposure to the stable isotope solution. At this time the labelling of the cortical apoplast in these mycorrhizal roots was higher than those of non-mycorrhizal roots and, with EDXS, changes in the element composition of the apoplast were detected. The results indicated that possibly hydrophobins localized in the fungal cell wall might be involved in the increased hydrophobicity of mycorrhizal roots and the lower permeability of the ectomycorrhizal sheath.

Calcium↗

PubMed1989

Mediated transport of long-chain fatty acids by rat renal basolateral membranes.

Transport of long-chain fatty acids (LCFA) was studied in isolated rat renal cortical membrane vesicles to determine the mechanism of entry of these metabolic substrates into renal cells. Total uptake of palmitate into basolateral (BLMV) and brush-border membrane vesicles (BBMV) was determined with the use of a rapid filtration technique. In the presence of an inwardly directed H+ gradient (pHo = 6.0, pHi = 7.4) palmitate (0.26 microM) uptake went through a maximum ("overshoot") in BLMV but not in BBMV. The amount of palmitate nonspecifically bound to vesicles was subtracted from total uptake to determine intravesicular content. Peak values for intravesicular palmitate content were 26 +/- 9 pmol/mg protein, 2.5-fold higher than 60-min values of 10 +/- 2 pmol/mg (P less than 0.05, n = 6). The overshoot could not be accounted for by changes in voltage. An inwardly directed Na+ gradient was without effect on palmitate uptake in BLMV and BBMV. pH gradient-stimulated palmitate transport (15 s) in BLMV was subject to cis-inhibition by 4,4'-diisothiocyanatostilbene-,,2-disulfonic acid. Probenecid and p-aminohippurate efflux from BLMV resulted in counter-flow accumulation of palmitate. It is concluded that at least a portion of LCFA transport across the basolateral membrane is mediated and may occur via anion exchange.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMedDate not supplied

Melatonin provides neuroprotection in the late-gestation fetal sheep brain in response to umbilical cord occlusion.

Oxygen free radicals, including the highly toxic hydroxyl radical (*OH), initiate lipid peroxidation and DNA/RNA fragmentation and damage cells. The pineal hormone melatonin is an antioxidant and powerful scavenger of *OH. We hypothesized that maternally administered melatonin could reduce *OH formation, lipid peroxidation, and DNA/RNA damage in the fetal brain in response to asphyxia. In 15 fetal sheep, extracellular *OH was measured by microdialysis in white and gray matter of the parasagittal cortex. In 10 fetuses, asphyxia was induced by umbilical cord occlusion for 10 min using an inflatable cuff - the ewes of these fetuses received either intravenous melatonin (1 mg bolus, then 1 mg/h for 2 h; n = 5) or vehicle (1% ethanol in saline; n = 5), and results were compared to fetuses with sham cord occlusion and vehicle-infused ewes (n = 5). Hypoxemia, acidemia, hypertension and bradycardia produced by cord occlusion was similar in the melatonin- and vehicle-treated groups. In the vehicle-treated group, cord occlusion resulted in a significant increase in *OH in gray matter at 8-9.5 h after occlusion (p < 0.05); in contrast, there was no *OH change in the melatonin-treated group. After cord occlusion, lipid peroxidation (4-hydroxynonenal immunoreactivity) found throughout the brain of vehicle-infused ewes was significantly less in the melatonin-infused group. Melatonin had no significant effect on the distribution of DNA/RNA fragmentation, as shown by 8-hydroxydeoxyguanosine immunoreactivity. Thus, brief asphyxia results in significant and delayed entry of *OH into the extracellular space of cortical gray matter in the fetal sheep brain, and melatonin given to the mother at the time of the insult abrogates this increase. Melatonin, in reducing O2 free radical production, may be an effective neuroprotective treatment for the fetus.

Animals↗

PubMed2003

Angiotensin I conversion to angiotensin II stimulates cortical collecting duct sodium transport.

Angiotensin (Ang) II directly stimulates epithelial sodium channel activity in the rabbit cortical collecting duct. Because Ang I and converting enzyme analogues might be present in the distal nephron, this raises the possibility of intraluminal generation of Ang II. Conversion of Ang I to Ang II was monitored by Ang II-dependent changes in intracellular sodium concentration as a reflection of sodium transport across the apical membrane. This involved imaging-based fluorescence microscopy with sodium-binding benzofuran isophthalate in isolated, perfused, cortical collecting-duct segments from rabbit kidney. Principal and intercalated cells were differentiated by rhodamine-conjugated peanut lectin. Control principal cell intracellular sodium concentration, during perfusion with 25 mmol/L NaCl and zero sodium in the bath plus monensin (10(-5) mol/L) averaged 5.8+/-0.14 mmol/L (n=156). The increase in intracellular sodium concentration, when luminal NaCl was increased from 25 to 150 mmol/L, was elevated by 3.5-fold in the presence of intraluminal Ang I (10(-6) mol/L). Also, the effects of Ang I on sodium transport were not significantly different from the effects of Ang II (10(-9) mol/L). Ang I was used in micromolar concentrations to ensure that there was sufficient substrate available for conversion to Ang II. Inhibition of the angiotensin-converting enzyme with captopril reduced the stimulatory effect of Ang I. These results suggest that intraluminal conversion of Ang I to Ang II can occur in the cortical collecting duct, resulting in enhanced apical sodium entry.

Angiotensin I↗

PubMed1982

Homocystine uptake in isolated rat renal cortical tubules.

Isolated rat renal cortical tubules were used to study the nature of homocystine entry into the tubule cell and its transport interactions with cystine and the dibasic amino acids. The uptake of homocystine with time was progressive, reaching a steady state after 60 min. of incubation. Analysis of the intracellular pool after 5 and 30 min. of incubation revealed that virtually all of the transported homocystine had been converted to other metabolites of the transsulfuration pathway. The major metabolite was cystathionine with a somewhat lesser, but still significant amount as S-adenosylhomocysteine. A kinetic analysis showed that two systems for cellular entry of homocysteine existed with a Km1 of 0.17 mM and a Km2 of 7.65 mM. Arginine and lysine inhibited homocystine uptake via the low Km, high affinity system, but appeared not to inhibit the high Km, low affinity system. Cystine inhibited the low Km, high affinity system, but had an indeterminate effect on the high Km, low affinity system. Homocystine inhibited the uptake of cystine, lysine and arginine by isolated rat renal cortical tubules. The inhibition of homocystine on cystine uptake appeared to occur on both the high and low Km system for tubule cell entry of cystine. The data suggest that the low Km system for homocystine transport is shared with cystine and the dibasic amino acids. These data extend the knowledge of homocystine metabolism and provide a rational basis for new approaches to the treatment of homocystinuria.

Amino Acids↗

PubMed1997

Repeated DOI and SR 46349B treatments do not affect elevated plus-maze anxiety despite opposite effects on cortical 5-HT2A receptors.

We report the consequences of a 4-day treatment (b.i.d) with the 5-HT2A,2B,2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 1.5 mg/kg) or the selective 5-HT2A receptor antagonist trans-4-[(3Z)3-(2-dimethylaminoethyl)oxyimino-3-(2-fluorophe nyl)propen-1-yl]phenol hemifumarate (SR 46349B, 7.5 mg/kg) on (i) anxiety-related behaviour in an elevated plus-maze, and (ii) specific [3H]ketanserin binding at central 5-HT2A receptors, in Roman rats. Neither DOI nor SR 46349B pretreatment affected the behaviour in the open arms of the elevated plus-maze; however, DOI pretreatment promoted discrete changes in the closed arm entries. The Bmax value of [3H]ketanserin binding at cortical 5-HT2A receptors was decreased by repeated DOI pretreatment. Conversely, Bmax, but also KD, values were increased by SR 46349B pretreatment. Thus, changes at central 5-HT2A receptors may occur without there being changes in anxiety-related behaviour in the elevated plus-maze.

Amphetamines↗

PubMed1982

Lithium entry into neural cells via sodium channels: a morphometric approach.

Rat cerebral cortical explants prepared from 18-day-old embryos were grown for 18 days in vitro. Cultures were exposed to Na+, Li+ and choline+ media, respectively, in the presence or absence of tetrodotoxin and/or veratridine, and processed for electron-microscopy. Veratridine (50 microM) induced an increase in summated and mean areas of neuronal profiles in Na+- and Li+-media but not in the choline+-medium: the summated perimeters did not change. The mean value of the neuronal form factor was significantly elevated following exposure to veratridine in a Na+- or Li+-dependent manner, indicating that the shape of the sectioned neuronal elements shifted towards an (ideal) circle. Qualitative assessment revealed an increased electron-lucency of the cytoplasm of neuronal profiles in Na+- and Li+-media containing veratridine. The veratridine-induced neuronal changes were inhibited by simultaneous addition of tetrodotoxin (1 microM) to the media. In the case of the glial cells, the values of the summated area and form factor did not change in the various experimental groups. The area of the extracellular space per unit area of sections significantly decreased in the Na+- and Li+-media following veratridine exposure; this did not occur in the choline+-medium. The results indicate a considerable swelling of the neuronal elements, reflecting cation, Cl- and water uptake following prolonged sodium channel activation in the presence of Na+ or Li+ ions. The quantitative ultrastructural data strongly suggests an entry of Li+ ions into cultured rat cerebral cells via sodium channels.

Animals↗

PubMed2006

Neuronal cell cycle re-entry mediates Alzheimer disease-type changes.

Evidence showing the ectopic re-expression of cell cycle-related proteins in specific vulnerable neuronal populations in Alzheimer disease led us to formulate the hypothesis that neurodegeneration, like cancer, is a disease of inappropriate cell cycle control. To test this notion, we used adenoviral-mediated expression of c-myc and ras oncogenes to drive postmitotic primary cortical neurons into the cell cycle. Cell cycle re-entry in neurons was associated with increased DNA content, as determined using BrdU and DAPI, and the re-expression of cyclin B1, a marker for the G2/M phase of the cell cycle. Importantly, we also found that cell cycle re-entry in primary neurons leads to tau phosphorylation and conformational changes similar to that seen in Alzheimer disease. This study establishes that the cell cycle can be instigated in normally quiescent neuronal cells and results in a phenotype that shares features of degenerative neurons in Alzheimer disease. As such, our neuronal cell model may be extremely valuable for the development of novel therapeutic strategies.

Alzheimer Disease↗

PubMed1986

[Significance of sensory evoked potentials in the diagnosis of polyneuropathy].

In 172 patients suffering from neuropathies of different aetiologies (diabetic, uraemic, inflammatory, hereditary, alcoholic, cryptogenic) the SEP findings (cortical median and sural nerve SEP, cervical median nerve SEP, Erb's point potential) were compared with the results of conventional sensory and motor electroneurography (ENG) and with clinical signs. SEP's yielded a high percentage of abnormalities. Thus in 5 of the 6 groups the sural nerve SEP presented an unequivocal latency prolongation in 55 to 75% of the patients, in HMSN-I-patients even in 100%. Also well over 50% of the median nerve evoked potentials were outside the normal range. In many cases the delay of the SEP's simply reflected the impairment of conduction within the peripheral nerve fibres as documented by ENG; here the ENG was naturally even more sensitive in detecting slight distal conduction disturbance, which did not shift the SEP latency outside the normal range. However, in a certain percentage that varied in the different aetiological groups, the SEP's demonstrated an impairment of conduction within the proximal segments of the sensory system not accessible to conventional ENG technique. Thus, in 15 to 25% of the patients with diabetic, uraemic, inflammatory and cryptogenic neuropathies, pathological SEP findings were combined with normal results of the ENG examination. In no case this "proximal" conduction disturbance affected the "central conduction" between the cervical spinal cord and the cortex. A more detailed differentiation was often impossible: A prolonged conduction time between brachial plexus and cervical cord could not be subdivided further due to the lack of the SEP component representing the "spinal entry of the afferent volley". SEP's--especially the cortical SEP's--can be reliably recorded even if a peripheral sensory nerve action potential is lacking; in these cases the extent of the conduction disturbance is documented only by the--practically always demonstrable--delay of the SEP. Nearly without exception, pronounced latency prolongations were seen only in cortical SEP's because in these cases the subcortical components could no longer be identified. Two types of considerably delayed cortical SEP's could be distinguished: Potentials of abnormal shape, where the complete extinction of the initial complex had to be assumed: the latency prolongation cannot be equated with the actual conduction delay. Completely normal-shaped potentials whose latency times evidently reflected the real delay.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗

PubMed2005

A store-operated Ca2+ influx activated in response to the depletion of thapsigargin-sensitive Ca2+ stores is developmentally regulated in embryonic cortical neurons from mice.

Store-operated channels (SOCs) are recruited in response to the release of Ca2+ from intracellular stores. They allow a voltage-independent entry of Ca2+ into the cytoplasm also termed capacitative Ca2+ entry (CCE). In neurons, the functional significance of this Ca2+ route remains elusive. Several reports indicate that SOCs could be developmentally regulated. We verified the presence of a CCE in freshly dissociated cortical cells from E13, E14, E16, E18 fetuses and from 1-day-old mice. Intracellular Ca2+ stores were depleted by means of the SERCA pump inhibitor thapsigargin. At E13, the release of Ca2+ from thapsigargin-sensitive compartments gave rise to an entry of Ca2+ in a minority of cells. This Ca2+ route, insensitive to voltage-gated Ca2+ channel antagonists like Cd2+ and Ni2+, was blocked by the SOC inhibitor SKF-96365. After E13 and on E13 cells kept in culture, there is a marked increase in the percentage of cells with functional SOCs. The lanthanide La3+ fully inhibited the CCE from neonatal mice whereas it weakly blocked the thapsigargin-dependent Ca2+ entry at E13. This suggests that the subunit composition of the cortical SOCs is developmentally regulated with La3+-insensitive channels being expressed in the embryonic cortex whereas La3+-sensitive SOCs are found at birth. Our data argue for the presence of SOCs in embryonic cortical neurons. Their expression and pharmacological properties are developmentally regulated.

Animals↗

PubMed2005

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion.

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.

Actins↗

PubMed2006

Histological and scanning electron microscopy analyses of bone/implant interface using the novel Bonelike synthetic bone graft.

Synthetic bone grafts provide an alternative to autografts and allografts. Bonelike is a patented synthetic bone graft that mimics the mineral composition of bone. The aim of the present work was to assess the biological behavior of Bonelike graft in humans, before using the material in orthopedic applications of bone regeneration, for example, in opening wedge high tibial osteotomies for medial knee osteoarthritis. Bonelike granules were implanted in cortical bone of 11 patients undergoing osteotomies, and new bone formation, osteoconductive properties, and resorption characteristics of the granules were analyzed. The granules ranged from 500 to 1000 microm and were implanted in the lateral aspect of the tibia. The patients' mean age was 59 years (range 48 to 70 years); there were eight women and three men, all suffering from medial compartment osteoarthritis of the knee. At surgery, a 1 x 1 x 1-cm cortical defect was created 3 cm distal to the entry point of the screws, in line with the long axis of tibia. The implanted Bonelike graft sample was extracted for analysis during removal of the metallic prosthesis after implantation times of 6, 9, and 12 months. Radiological follow-up, scanning electron microscopy, histological analysis, and histomorphometric measurements were conducted on the retrieved samples to assess bone regeneration in the defect area. Osteoconductive capacity was demonstrated by extensive mature bone formation around the implanted granules and high levels of percentage bone-to-graft contact (from 67-84%). Bonelike acted as an excellent bioactive scaffold, allowing the migration, proliferation, and differentiation of bone cells on its surface, and therefore regeneration of the defects was achieved in a rapid, controlled manner. Our results suggest that Bonelike graft is an excellent candidate for orthopedic applications where rapid new bone formation is a fundamental requirement.

Aged↗

PubMed1991

Autoradiography of progesterone and model compound entry and distribution in Xenopus laevis oocytes.

Xenopus laevis oocytes resume meiosis in response to progesterone. The initial interaction involves surface binding to numerous low-affinity receptor proteins. The mechanism of entry and functions of intracellular steroid are unknown. Because the latter are important for understanding progesterone-induced maturation, a dry-mount autoradiographic technique for analyzing entry and intracellular distribution of radiolabeled steroids was developed and tested. The distinguishing feature of this cryo-technique is sample preparation directly in incubation media using uncross-linked polyacrylamide for inert support. The external ligand functions as an internal standard, so quantitation is by simple ratio (bound/free). The entry kinetics and subcellular binding patterns in large oocytes were studied using this method at nM levels of radiolabeled steroids and model compounds. Progesterone, estradiol, corticosterone, and 1,25-dihydroxycholecalciferol all showed rapid entry (P approximately 10(-6) cm/sec). Entry rates were not saturable with unlabeled steroid. Intracellular patterns of these steroids were highly specific and negatively associated with yolk protein and lipid. Intracellular binding in animal hemisphere ooplasm was 10x that of the yolk-rich vegetative ooplasm. In contrast, dexamethasone, ponasterone-A, and ecdysone displayed entry rates 20-60x slower than progesterone with little compartmentalization. Glycerol, glucose, and leucine entered over 10x slower than progesterone. Cholesterol and Ca++ had entry rates below detection. Evidence for mediated entry of progesterone included the rapid saturation of a cortical compartment equivalent in magnitude to reported receptor numbers. The kinetics and specificity of cortical uptake were consistent with low-affinity, high capacity protein binding. Intracellular binding was seen to correlate with rhodamine 123 patterns, suggesting involvement of mitochondrial or other microtubule-associated structures in steroid responses. Mitochondrial binding is consistent with the limited steroid metabolism seen in oocytes. Since several maturation events are consistent with respiratory uncoupling, reported by others for steroids and isolated organelles, and since mitochondria contain nearly all of the oocyte DNA, a role for these organelles in steroid-induced oocyte maturation was proposed.

Animals↗