Osmotic Swelling Stimulates Ascorbate Efflux from Cerebral Astrocytes

Abstract: Ascorbate (reduced vitamin C) is an important enzyme cofactor, neuromodulator, and antioxidant that is stored at millimolar concentrations in the cytosol of cerebral astrocytes. Because these cells swell during hyponatremia, cerebral ischemia, and trauma, we investigated the effects of osmotic stress on astrocytic transport of ascorbate. Ascorbate efflux from primary cultures of rat astrocytes was rapidly (within 1 min) increased by incubation in hypotonic medium. Efflux also increased when astrocytes, which had been adapted to a hypertonic environment, were swollen by transfer to isotonic medium. Swelling-induced ascorbate efflux was inhibited by the anion-transport inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). The pathway that mediates ascorbate efflux was found to be selective because a larger anion, 2',7'-bis(carboxyethyl)-5-(or -6)-carboxyfluorescein (BCECF), was retained in the swollen astrocytes. Na⁺-dependent ascorbate uptake into astrocytes was inhibited slightly during the first minute of hypotonic stress, indicating that the sodium ascorbate cotransporter does not mediate swelling-induced efflux. Cell concentration of authentic ascorbate was measured by HPLC with electrochemical detection. When astrocytes were incubated in ascorbate-free medium, hypotonicity decreased cell ascorbate concentration by 50% within 3 min. When astrocytes were incubated in ascorbate-supplemented hypotonic medium, intracellular ascorbate concentration was restored within 10 min because uptake remained effective. Many pathological conditions cause brain cell swelling and formation of reactive oxygen species. Ascorbate release during astrocytic swelling may contribute to cellular osmoregulation in the short-term and the scavenging of reactive oxygen species.     

Cerebral Astrocytes Transport Ascorbic Acid and Dehydroascorbic Acid Through Distinct Mechanisms Regulated by Cyclic AMP

Abstract: Cerebral ischemia and trauma lead to rapid increases in cerebral concentrations of cyclic AMP and dehydroascorbic acid (DHAA; oxidized vitamin C), depletion of intracellular ascorbic acid (AA; reduced vitamin C), and formation of reactive astrocytes. We investigated astrocytic transport of AA and DHAA and the effects of cyclic AMP on these transport systems. Primary cultures of astrocytes accumulated millimolar concentrations of intracellular AA when incubated in medium containing either AA or DHAA. AA uptake was Na⁺-dependent and inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), whereas DHAA uptake was Na⁺-independent and DIDS-insensitive. DHAA uptake was inhibited by cytochalasin B, d -glucose, and glucose analogues specific for facilitative hexose transporters. Once inside the cells, DHAA was reduced to AA. DHAA reduction greatly decreased astrocytic glutathione concentration. However, experiments with astrocytes that had been previously depleted of glutathione showed that DHAA reduction does not require physiological concentrations of glutathione. Astrocyte cultures were treated with a permeant analogue of cyclic AMP or forskolin, an activator of adenylyl cyclase, to induce cellular differentiation and thus provide in vitro models of reactive astrocytes. Cyclic AMP stimulated uptake of AA, DHAA, and 2-deoxyglucose. The effects of cyclic AMP required at least 12 h and were inhibited by cycloheximide, consistent with a requirement for de novo protein synthesis. Uptake and reduction of DHAA by astrocytes may be a recycling pathway that contributes to brain AA homeostasis. These results also indicate a role for cyclic AMP in accelerating the clearance and detoxification of DHAA in the brain.     

Differential Expression of Type A and Type B Monoamine Oxidase of Mouse Astrocytes in Primary Cultures

Abstract: Type A and type B monoamine oxidase (MAO) activities were determined in mouse brain and in primary cultures of mouse astrocytes. Thirty-one-day-old astrocyte cultures exhibited predominantly type A MAO activity. In cultures of the same age, treated with 0.25 mM dibutyryl cyclic AMP under the same culturing conditions, 30% type B MAO was expressed, although dibutyryl cyclic AMP up to 1 mM does not affect MAO activity in vitro. The specific activity of type B MAO increased significantly in older cultures, while type A MAO changed only slightly.     

Effect of Dibutyryl Cyclic AMP on the Kinetics of myo-Inositol Transport in Cultured Astrocytes

Dibutyryl cyclic AMP (dBcAMP) is known to induce maturation and differentiation in astrocytes. As myo-inositol is an important osmoregulator in astrocytes, we examined the effects of maturation and biochemical differentiation on the kinetic properties of myo-inositol transport. Treatment of astrocytes with dBcAMP significantly decreased the Vmax of myo-inositol uptake, but the effect on Km was not significant. The myo-inositol content of astrocytes was significantly decreased in cells treated for 5 days with dBcAMP as compared with untreated controls. Maximum suppression of myo-inositol uptake occurred 7 days after exposure of astrocytes to dBcAMP; this was gradually reversible when dBcAMP was removed from the medium. After exposure to hypertonic medium for 6 h, mRNA expression of the myo-inositol co-transporter was diminished by approximately 36% in astrocytes treated with dBcAMP as compared with untreated cells. It appears that myo-inositol transporters in astrocytes treated with dBcAMP are either decreased in number or inactivated during maturation and differentiation, suggesting that the stage of differentiation and biochemical maturation of astrocytes is an important factor in osmoregulation.     

Intracellular Free Calcium Dynamics in Stretch-Injured Astrocytes

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca²⁺]_i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca²⁺]_i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca²⁺]_i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca²⁺]_i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca²⁺]_i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca²⁺]_i, is responsible for some of the pathological consequences of traumatic brain injury.     

Dibutyryl cyclic AMP triggers Ca2+ influx and Ca2+-dependent electrical activity in pancreatic B cells

In the presence of 7 mM glucose, dibutyryl cyclic AMP induced electrical activity in otherwise silent mouse pancreatic B cells. This activity was blocked by cobalt or D600, two inhibitors of Ca2+ influx. Under similar conditions, dibutyryl cyclic AMP stimulated 45Ca2+ influx (5-min uptake) in islet cells; this effect was abolished by cobalt and partially inhibited by D600. The nucleotide also accelerated 86Rb+ efflux from preloaded islets, did not modify glucose utilization and markedly increased insulin release. Its effects on release were inhibited by cobalt, but not by D600. These results show that insulin release can occur without electrical activity in B cells and suggest that cyclic AMP not only mobilizes intracellular Ca, but also facilitates Ca2+ influx in insulin secreting cells.     

Biotin Transport in Primary Culture of Astrocytes: Effect of Biotin Deficiency

The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of less than 60 nM and linear at greater than 60 nM. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated diffusion. Moreover, biotin uptake in astrocytes grown in biotin-restricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10-fold) in the Vmax of the biotin uptake without any change in the apparent Km.     

Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes

Abstract: Rilmenidine, a ligand for imidazoline and α₂-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca²⁺]_i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca²⁺]_i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca²⁺-free medium, indicating that rilmenidine evokes release of Ca²⁺ from intracellular stores. However, the sustained elevation of Ca²⁺ was completely dependent on extracellular Ca²⁺, consistent with rilmenidine activating Ca²⁺ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca²⁺ influx pathway. The α₂-adrenergic antagonist rauwolscine attenuated the increase in [Ca²⁺]_i induced by clonidine (a selective α₂ agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca²⁺ release from intracellular stores and Ca²⁺ influx by a mechanism independent of α₂-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca²⁺ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

Lysophosphatidic Acid-Induced Proliferation-Related Signals in Astrocytes

Abstract: Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 n M LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca²⁺]_i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca²⁺]_i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca²⁺]_i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca²⁺]_i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, Gö 6976. The increase in [Ca²⁺]_i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca²⁺]_i, lipid peroxidation, and DNA synthesis.     

Effect of Intracellular Glutamine on the Uptake of Large Neutral Amino Acids in Astrocytes: Concentrative Na+-Independent Transport Exhibits Metastability

To examine whether the concentration gradient of glutamine (Gln) drives concentrative Na(+)-independent uptake of neutral amino acids (NAA) in mouse cerebral astrocytes, uptake was compared in "Gln-depleted" and "Gln-replete" cultures. Uptake (30 min in Na(+)-free buffer) of histidine, kynurenine, leucine, tyrosine, and a model substrate for System L transport was 70-150% greater in Gln-replete cultures. Phenylalanine uptake was not affected. All of these NAA trans-stimulated the export of Gln from astrocytes. However, the increase in NAA uptake was sustained even though the Gln content of Gln-replete cultures declined. Also, uptake of Gln itself was enhanced in Gln-replete cultures. Thus, countertransport of Gln was insufficient to explain the enhancement of NAA uptake. Enhanced uptake was restored, and could be magnified, by reloading Gln-depleted cultures either with Gln or with histidine. It is suggested that substrate-induced asymmetry and molecular hysteresis in the Na(+)-independent carrier could account for the sustained enhancement of NAA uptake. Only histidine and kynurenine were concentrated comparably to Gln (15- to 29-fold at 1 mM in Na(+)-free buffer). The other NAA were four to six times less concentrated. At least two Na(+)-dependent transport systems also supported the concentration gradient of Gln in regular buffer.     

Ascorbic Acid Uptake by a High-Affinity Sodium-Dependent Mechanism in Cultured Rat Astrocytes

Ascorbic acid (vitamin C) is synthesized in rodent liver, circulates in the blood, and is concentrated in the brain. Experiments were performed to characterize the mechanism of ascorbate uptake by rat cerebral astrocytes in primary culture. Astroglial uptake of L-[14C]ascorbate was observed to be both saturable and stereoselective. In addition, uptake was dependent on both the incubation temperature and the concentration of Na+ because it was largely inhibited by cooling to 4 degrees C, by treatment with ouabain to increase intracellular Na+, and by the substitution of K+, Li+, or N-methyl-D-glucamine for extracellular Na+. The affinity for ascorbate was relatively high in cells incubated with a physiological concentration of extracellular Na+, because the apparent Km was 32 microM in 138 mM Na+. However, the affinity for ascorbate was significantly decreased when the extracellular Na+ concentration was lowered. Treatment of astrocytes with dibutyryl cyclic AMP induced stellation and increased the maximum rate of ascorbate uptake by 53%. We conclude that astrocytes possess a stereoselective, high-affinity, and Na+-dependent uptake system for ascorbate. This system may regulate the cerebral ascorbate concentration and consequently modulate neuronal function.     

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism.     

Potassium-Induced Stimulation of Glutamate Uptake in Mouse Cerebral Astrocytes: The Role of Intracellular pH

Abstract: The Na⁺-glutamate cotransporters are believed to countertransport OH^? and K⁺. Previous evidence that the velocity of glutamate uptake can exceed the acid extrusion capacity of astrocytes raised the question of whether intracellular pH can become rate limiting for glutamate uptake. Cytoplasmic buffering capacity and acid extrusion in astrocytes are partially HCO₃^? dependent. Also, it was reported recently that raising extracellular [K⁺] alkalinizes astrocyte cytoplasm by an HCO₃^?-dependent mechanism. Here, we have compared glutamate uptake in HCO₃^?-buffered and HCO₃^?-depleted solutions at varying [K⁺]. We observed a pronounced stimulation of glutamate uptake by extracellular K⁺ (3–24 mM) that was substantially HCO₃^? dependent and affected preferentially the uptake of high concentrations (>25 µM) of glutamate. Stimulation of uptake by low extracellular [K⁺] (1.5–3 mM) was less dependent on HCO₃^?. Potassium-induced stimulation of uptake was weaker in rat astrocyte cultures than in mouse. The effects of Ba²⁺ and amiloride on glutamate uptake, as well as the HCO₃^?-dependent stimulatory effects of K⁺ and the species difference, all related consistently to effects on intracellular pH. The effects on uptake, however, were much larger than predicted by the associated changes in electrochemical gradient of OH^?. A “bimodal” scheme for glutamate transport can account qualitatively for the observed correlation between intracellular pH and velocity of glutamate uptake.     

Inhibition of Amino Acid Transport and Protein Synthesis by HgCl2 and Methylmercury in Astrocytes: Selectivity and Reversibility

Abstract: The previously reported observation that submi-cromolar concentrations of HgCl₂ inhibit glutamate uptake reversibly in astrocytes, without effect on 2-deoxyglucose uptake, suggested that elemental mercury vapor, which is oxidized to mercuric mercury in the brain, might cause neurodegenerative change through the mediation of glutamate excitotoxicity. Here, selectivity is explored further by measuring the inhibition of other amino acid transporters and protein synthesis as a function of HgCl₂ concentration. The properties of MeHgCl were compared under identical conditions, and some morphological correlates of function were examined. Inhibition of amino acid transport by HgCl₂ was selective, whereas MeHgCl was nonselective. The 50% inhibitory concentrations of HgCl₂ for uptake of α-aminoiso-butyric acid by system A, uptake of α-aminoisobutyric acid or kynurenine by a system L variant, and uptake of γ-ami-nobutyric acid were all two- to fourfold greater than that for uptake of glutamate. The submicromolar concentrations of HgCl₂ that inhibited glutamate transport also inhibited protein synthesis, but in a rapidly reversible fashion, and elicited only discrete ultrastructural changes (heterochromatin. increased numbers of lysosomal bodies, and increased complexity of cell surface). In contrast, inhibition of protein synthesis by MeHgCl was acutely (1-h) irreversible and became marked only at concentrations higher than those that elicited gross morphologic change in the form of "bleb"-like swellings. The results lend support to the proposed excitotoxic mediation of mercury vapor neurotoxicity and reveal a sharp contrast between the effects of HgCl₂ and MeHgCl on astrocytes.     

Regulation of Glycogenolysis in Transformed Astrocytes In Vitro

Cultured astrocytes, transformed by Herpesvirus, were used as a model system to study several aspects of the control of glycogenolysis. Adrenergic agonists such as norepinephrine and isoproterenol caused an immediate and dose-dependent increase in the intracellular levels of cyclic AMP. Concomitant with the initial phase of cyclic AMP increase, conversion of phosphorylase b to a and glycogenolysis were observed. The elevation of cyclic AMP, phosphorylase conversion, and glycogenolysis were simultaneously blocked by beta-adrenergic blockers, but not by alpha-adrenergic blocking agents. Repeated administration of norepinephrine caused an attenuated response in both cyclic AMP accumulation and glycogenolysis. Glycogen degradation is also partially regulated by glucose availability. In the presence of glucose, norepinephrine-induced glycogenolysis is blocked, despite elevations in cyclic AMP. The direct role of glucose is postulated, since glucose analogs mimic the effects of glucose.     

Regulation by Dibutyryl Cyclic AMP of Carnosine Synthesis in Astroglia-Rich Primary Cultures Kept in Serum-Free Medium

The synthesis of carnosine (beta-Ala-His) by astroglia-rich primary cultures was much higher if the cells were cultivated in Ham's nutrient mixture F-12 than if they were grown in Dulbecco's modified Eagle's medium. Carnosine synthesis was not affected by the presence of insulin, transferrin, phorbol myristate acetate, or dexamethasone. However, dibutyryl cyclic AMP and other agents that can, directly or indirectly, activate cyclic AMP-dependent protein kinases strongly lower the rate of carnosine synthesis. The depression of carnosine synthesis was dependent on the concentration of dibutyryl cyclic AMP. The effect was maximal (approximately 80% inhibition) in cultures preincubated with 1 mM dibutyryl cyclic AMP for 4 days. The adenylate cyclase activator forskolin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and 8-bromo-cyclic AMP caused the same depression as dibutyryl cyclic AMP, whereas neither butyrate nor dibutyryl cyclic GMP elicited any effect.     

Glutamine Transport in Mouse Cerebral Astrocytes

Abstract: We measured initial influx and exchange of [¹⁴C]glutamine in primary astrocyte cultures in the presence and absence of Na⁺. Kinetic analysis of transport in Na⁺-free solution indicated two saturable Na⁺-independent components, one of which was identifiable functionally as system L₁ transport. In the presence of Na⁺, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na⁺-dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by α-(methylamino)isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na⁺-dependent uptake, preferential inhibition of an Li⁺-intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and α-(methylamino)isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na⁺, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 µ M glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li⁺ in place of Na⁺.     

小鼠大脑皮质星形胶质细胞原代培养与鉴定

为探讨简便、高效的大脑皮质星形胶质细胞体外培养方法,本研究取新生24 h内的ICR小鼠大脑皮层,采用物理方法将其分成约1 mm^3,震荡过滤后进行培养。通过拍照的方式记录原代培养1 d、3 d、7 d、14 d、21 d、28 d、35 d和原代培养14 d后再传代培养14 d(记为P2-14 d)细胞形态;通过实时定量PCR和Western blotting比较原代培养1周、2周、3周、4周、5周和原代培养2周后再传代培养2周(即P2-2)的星形胶质细胞内胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因和蛋白水平变化。选取GFAP、S100-β和谷氨酸转运蛋白(excitatory amino acid transporter 1,EAAT1)标记星形胶质细胞,微管相关蛋白(microtubuleassociated protein 2,MAP-2)、离子钙接头蛋白-1(ionized calcium-binding adapter molecule 1,Iba-1)和髓鞘相关糖蛋白(myelin associated glycoprotein,MAG)抗体分别标记神经元、小胶质细胞和少突胶质细胞。通过免疫荧光染色鉴定细胞种类及纯度。研究结果显示细胞生长良好,原代培养4周星形胶质细胞内GFAP比2周、3周、5周和传代培养2周的细胞更加稳定。经免疫荧光鉴定,星形胶质细胞纯度在95%以上。本实验采用相对较简单经济的方法培养出高纯度且生理状态相对较稳定的原代星形胶质细胞,该细胞模型不仅可以用于星形胶质细胞生理功能研究,还可以用于中枢神经系统相关疾病的体外研究。     

Inhibition of spermiation in the syrian hamster using dibutyryl cyclic-AMP

Summary Young adult male Syrian hamsters were given intraperitoneal injections of 50 mg dibutyryl cyclic AMP twice daily for a period of three days. On the fourth day the animals were sacrificed and their testes were processed for light and electron microscopy. The results indicate that the mature spermatozoa were retained within the seminiferous epithelium after the stage in the seminiferous cycle in which spermiation normally occurs. The unreleased spermatozoa were ultrastructurally normal. Typical Sertoli-spermatid junctional specializations remained associated with the retained spermatozoa. These findings indicate that normally spermiation is initiated by the disappearance of the junctional specializations. In addition, the present results demonstrate that spermiation can be controlled.Supported in part by NIH Grant RR05654 and by NIH Grant P 30 HD 10202 (Morphology Core)The skillful technical assistance of Pam Duke is gratefully acknowledged