Cumulus expansion during in vitro maturation of bovine oocytes: relationship with intracellular glutathione level and its role on subsequent embryo development.

Glutamine (GLN) is a metabolic precursor for hexosamine synthesis and its inclusion in culture medium has been reported to improve cumulus expansion. Glutamine and cysteine share the same transport system. Excess external GLN may act as a competitive inhibitor for the uptake of cysteine and stimulate loss of cellular cysteine, interfering this with GSH synthesis. Experiments were designed to evaluate the effect of 1-3 mM GLN during in vitro maturation (IVM) on bovine-cumulus expansion, intracellular GSH levels in both oocytes and cumulus cells, and subsequent embryo development up to blastocyst stage. Also, GSH content was measured in 6- to 8-cell embryos and a possible relationship between cumulus expansion and GSH synthesis was studied. Intact cumulus cell-oocyte complexes were incubated for 24 hr and cumulus expansion was measured by a computerized image-digitizing system either before or after IVM. IVM/IVF bovine oocytes were cultured up to 6- to 8-cell stage embryos for assessment of GSH content or for 8 days up to blastocyst stage for embryo development. The measurement of total GSH content was performed by an enzymatic method in oocytes, cumulus cells and 6- to 8-cell embryos. The maximal expansion was achieved by addition of 2 mM GLN without affecting GSH levels, in both oocytes and cumulus cells. At 3 mM, the degree of cumulus expansion was lower and the GSH levels decreased. The addition of 2 mM GLN improves cleavage and blastocyst rates, whereas no differences were found between O, 1, and 3 mM GLN. Moreover, the GSH content in 6- to 8-cell embryos was similar at any GLN concentrations. In order to study the relationship between GSH and cumulus expansion: 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of hexosamine synthesis, or buthionine sulfoximide (BSO), an inhibitor of GSH synthesis, either alone or with GLN was added to IVM medium. GSH level was not affected by the presence of DON. However, the degree of cumulus expansion was reduced in the presence of BSO. In conclusion, bovine oocytes matured in the presence of 2 mM GLN improve their capacity for subsequent embryo development. Nevertheless, GSH level was altered when GLN was added to IVM medium at a high concentration with a reduction in the degree of cumulus expansion. This study provides evidence that optimal cumulus expansion in vitro is partially dependent on hexosamine production and intracellular GSH content.

A lineal equation for the classification of progressive and hyperactive spermatozoa.

Capacitation of spermatozoa, a complex sequence of events that render them able to fertilize the egg, is generally associated with a switch from lineal, progressive movement to a vigorous, non-progressive pattern characterized by starlike tracks, a process known as hyperactivation. Development of a method for the analysis of progressive and hyperactive tracks is thus important for the assessment of capacitation in biochemical, physiological and clinical studies. In this study, we have applied a two-step heuristic model to deduce a lineal equation that discriminates hyperactive from progressive spermatozoa. The kinetic parameters (curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH), straightness (STR), wobble (WOB), mean 'dance' (DAN) and velocity of the average path (VAP)) of ram spermatozoa were evaluated with a computerized motility analyzer, and classified one by one as progressive or hyperactive by the appearance of their tracks. In a first step, a discriminating plane was defined by minimizing the number of misclassified spermatozoa ('conflicting points'); then, the plane was adjusted by an iterative process to minimize the distance from conflicting points to it. The resulting plane showed a discriminating capacity of over 95% for both classes, higher than that achieved by setting a threshold value for the parameters taken separately or in group. When included in a standard semen analysis, application of the equation allowed a rapid assessment of the percentage of hyperactive spermatozoa. The method described, developed in ram spermatozoa, can be applied to different species for a variety of purposes.

Treatment of human spermatozoa with seminal plasma inhibits protein tyrosine phosphorylation.

It has long been known that seminal plasma contains factors that influence the fertilizing capacity of spermatozoa in many different ways. However, little is understood of the biochemical cascades triggered when spermatozoa and seminal plasma interact. In this study, we examined how incubation with seminal plasma affected protein tyrosine phosphorylation in human spermatozoa. Increased protein tyrosine phosphorylation is a hallmark of sperm capacitation in several mammalian species, including human. Seminal plasma blocks protein tyrosine phosphorylation when added to washed, non-capacitated spermatozoa. Removal of seminal plasma and incubation in capacitating medium led to partial recovery of the tyrosine phosphorylation cascade. Addition of seminal plasma to a suspension of spermatozoa previously incubated for 5 h under capacitating conditions decreased the level of tyrosine phosphorylation on all proteins in a dose-dependent manner. In this case, the phosphotyrosine signal did not increase upon removal of seminal plasma followed by overnight incubation in fresh capacitating media, indicating that removal of seminal plasma was necessary but not sufficient for protein tyrosine phosphorylation to occur. These results indicate that human seminal plasma contains factors that influence the tyrosine phosphorylation status of human spermatozoa.

Actin localization in ram spermatozoa: effect of freezing/thawing, capacitation and calcium ionophore-induced acrosomal exocytosis.

We have analyzed, by immunofluorescence, the localization of actin in ram spermatozoa, its colocalization with the actin-binding protein, gelsolin, and the effect of freeze/thawing, in vitro capacitation, and induced acrosomal exocytosis on its distribution. The monoclonal anti-actin and anti-gelsolin antibodies used recognized single bands at 43,000 and 90,000 kDa, respectively. In all spermatozoa, intense actin staining was observed in the whole length of the flagellum and, depending on the protocol used, in the neck and postacrosomal region of the head. Comparison of three staining methods, together with the use of NBD-phallacidin, allowed us to characterize ram sperm actin as a monomeric, intracellular, membrane-associated protein. Gelsolin was also present in ram spermatozoa and precisely colocalized with actin. Processes involving alterations in membrane structure such as freezing/thawing, in vitro capacitation, and calcium ionophore-induced acrosomal exocytosis provoked changes in the exposure of actin to the antibody. This strongly suggests a physical association of this protein to the plasma membrane, most likely by its intracellular side. The possible role of actin in sperm function is discussed.

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Ram spermatozoa were treated with fluorescein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIC) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC. Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved samples.

Glutathione synthesis during in vitro maturation of bovine oocytes: role of cumulus cells.

Glutathione (GSH) synthesis during in vitro maturation (IVM) has been shown to play an important role in embryo development. The present study was carried out to evaluate the role of cumulus cells in GSH synthesis during IVM of bovine oocytes in the presence of cystine, cysteine, the cysteine analogue N-acetylcysteine, and cysteamine. For this purpose, cumulus-oocyte complexes (COCs), denuded oocytes (DOs), and DOs in coculture with a cumulus cell monolayer were used. An increase in GSH level stimulated by cystine was observed only in the presence of cumulus cells, either with COCs or in DOs matured on a coculture monolayer. Addition of cysteine and cysteamine to IVM medium increased GSH levels in COCs and DOs. N-Acetylcysteine increased GSH levels only in DOs. Moreover, cumulus cells contributed to the stimulatory effect exerted by cysteine and cysteamine on GSH synthesis in COCs. These results indicate that cumulus cells during IVM play an important role in oocyte GSH synthesis, allowing the oocytes to use cystine and contributing to the stimulatory effect exerted by cysteine and cysteamine. In addition, these results demonstrate that IVM medium supplemented with cysteine or cysteamine increased GSH content in oocytes without cumulus mass (DO) and in the absence of a cumulus cell monolayer. This may be useful to increase the efficacy of IVM of those oocytes having few cumulus cell layers, in a system without coculture.

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability.

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.

In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay.

We have described the different patterns of chlortetracycline (CTC) binding to ram spermatozoa, immediately after ejaculation and upon in vitro capacitation and calcium ionophore-induced acrosomal exocytosis. Four different forms of CTC distribution were found. Form I showed an even distribution of fluorescence over the entire head, with a brighter band in the equatorial region. In Form II, uniform fluorescence was observed without equatorial band. Form III consisted of fluorescence in the anterior portion of the head. Form IV showed no fluorescence over the head. In all cases, fluorescence in the middle piece of the flagellum was observed as well. Immediately after ejaculation, Form I was the most abundant one (78%) in fresh semen with Forms II and III being relatively scarce (less than 15%). Form IV was virtually absent or appeared only occasionally. Incubation under in vitro capacitating conditions led to a significant decrease in Form I and to a significant increase in Forms II and III. Form II was mainly associated to intact acrosomes, while most spermatozoa in Form III showed intermediate forms of acrosomal status. Incubation of spermatozoa with the calcium ionophore A23187 resulted in 55% of spermatozoa showing Form IV, suggesting that it represents the acrosome-reacted stage. Form I was abruptly decreased at 30 min of incubation and was neglectible after 60 min. In contrast, Forms II and III increased at 30 min but decreased later on, suggesting that both forms represent intermediate stages before the acrosomal exocytosis. Analysis of acrosomal status in spermatozoa from individual CTC forms revealed that all spermatozoa that remained in Form II after incubation had intact acrosomes. Intermediate stages were predominant in Form III-spermatozoa, while most Form IV-spermatozoa underwent full acrosomal exocytosis. These results show that CTC binding can be used to monitor changes in ram spermatozoa during capacitation and acrosome-reaction.

Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro.

The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and O (control), 25, 50, or 100 muM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.01) for oocytes matured in medium containing 100 muM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P < 0.05) in oocytes matured with 100 muM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P > 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH.

Use of computerized motility analyzer for the evaluation of frozen-thawed ram spermatozoa.

A computerized motility analyzer (CellTrak/STM) was calibrated for its use with ram semen. Adjustment of the several setup variables allowed an accurate measurement of kinetic parameters such as percentage of motile cells, straight velocity, curvilinear velocity, linearity and amplitude of lateral displacement of the head. All kinetic parameters, except the lateral displacement of the head, showed significant changes after freezing and thawing. The curvilinear velocity exhibited the least significative post-thaw decrease. The alterations in kinetic parameters provoked by freezing and thawing could account for the low success obtained with frozen semen by cervical insemination, as it is accepted that during the initial steps of fertilization high motility and linearity are required.

Fluorescent staining as a method of assessing membrane damage and post-thaw survival of ram spermatozoa.

We studied the relationship between motility and membrane damage, as assessed by fluorescent staining, in fresh and in frozen-thawed ram spermatozoa. Semen from Merino rams was incubated with 6-carboxyfluorescein diacetate and propidium iodide. In both fresh and frozen-thawed samples, the percentage of intact spermatozoa was lower than the motility rate, thus indicating the presence of damaged but motile spermatozoa. Freezing and thawing resulted in a marked loss of membrane integrity, whereas motility decreased to a lesser extent. There was a positive relationship (r=0.64; P<0.001) between membrane integrity immediately after thawing and motility after 8 h of incubation at 37 degrees C. These results demonstrate the usefulness of the fluorescent staining method for the prediction of ram sperm quality and post-thaw survival.

Regulation of glucocorticoid receptors in human mononuclear cells: effects of glucocorticoid treatment, Cushing's disease and ketoconazole.

Glucocorticoid receptors (GcR) were determined by a whole cell assay in human mononulear leukocytes (hMNL) from control subjects, patients receiving glucocorticoid therapy for systemic diseases and Cushing's disease patients with or without ketoconazole therapy. Prolonged corticosteroid treatment resulted in down-regulation of GcR, while the mean level of GcR in Cushing's disease was normal. In this group, however, receptor levels and morning plasma cortisol values showed a negative correlation, indicating a subtle down-regulatory effect. Furthermore, GcR were unaltered after these patients received ketoconazole, in spite of a marked reduction in morning plasma cortisol and urinary free cortisol. We also observed that ketoconazole was a weak competitor of GcR in intact cells, although it significantly inhibited [3H] dexamethasone binding in cytosolic preparations from rat tissues. The results suggested that GcR in hMNL are down-regulated by synthetic steroids given in vivo, but they showed very mild down-regulation in hypercortisolemic patients suffering from Cushing's disease. Finally, we did not observed either up-regulation or antagonism of GcR by ketoconazole treatment, at the time that cortisol levels of patients with Cushing's disease were reduced. This indicates that the beneficial effects of ketoconazole in Cushing's disease are due to adrenal cortisol suppression and not to interaction with GcR of target cells, and that the process of GcR regulation in hMNL is a complex phenomenon awaiting further elucidation.

Glucocorticoid type II receptors of the spinal cord show lower affinity than hippocampal type II receptors: binding parameters obtained with different experimental protocols.

We have used three experimental protocols to determine binding parameters for type I and type II glucocorticoid receptors in the spinal cord and hippocampus (HIPPO) from adrenalectomized rats. In protocol A, 0.5-20 nM [3H]dexamethasone (DEX) was incubated plus or minus a 1000-fold excess of unlabeled DEX, assuming binding to a two-site model. In protocol B, [3H]DEX competed with a single concentration of RU 28362 (500 nM), whereas in protocol C, we used a concentration of RU 28362 which varied in parallel to that of [3H]DEX, such as 500 x. Results of protocols A and C were qualitatively similar, in that: (1) Bmax for type I receptors favored the HIPPO, while the content of type II sites was comparable in the two tissues; (2) Kd was consistently lower for type I than for type II sites in both tissues; and (3) type II receptors from the spinal cord showed lower affinity than their homologous sites from HIPPO. This last result was also obtained when using protocol B. In contrast, protocol B yielded binding data indicating that type II sites were of similar or higher affinity than type I sites. Computer simulation of the binding protocols demonstrated that protocols A and C were the most theoretically reliable for estimating the Kd and Bmax of type I sites, and the predicted error was smaller for protocol C, in comparison with protocol B. We suggest that the noted differences in the Kd of type II receptors between the spinal cord and HIPPO could account for a difference in sensitivity of the two systems in the physiological adrenal hormone range.

Glucocorticoid receptors and enzyme induction in the spinal cord of rats: effects of acute transection.

The spinal cord is a glucocorticoid-responsive tissue, as demonstrated by hormonal effects on enzyme induction and by the presence of type II and type I glucocorticoid receptors in cytoplasmic extracts of this CNS region. Using microdissection techniques, we have found in the present investigation that glucocorticoid type II receptors are the most abundant class detected in gray (ventral and dorsal horns) and white (lateral funiculus) matter and that the distribution of type II sites among these regions was quantitatively similar. Type I sites were also quantified, with a slight prevalence in gray matter as opposed to white matter. Furthermore, stimulation of an inducible enzyme, ornithine decarboxylase (ODC), was found in ventral horn and lateral funiculus but not in dorsal horn after administration of dexamethasone (DEX), a type II receptor ligand. We also found that surgical transection of the spinal cord, while markedly increasing ODC activity per se, did not prevent the stimulatory effect of DEX administration on ODC activity measured in the lumbar enlargement of the spinal cord located below the surgical lesion. Taken together, the results suggest a direct effect of glucocorticoids on ODC activity in the spinal cord of rats, probably mediated by glucocorticoid receptors (type II) found in target cells of the ventral horn and lateral funiculus. The results also indicate that glucocorticoid receptors of the dorsal horn were not involved in ODC induction, and a function for these receptors awaits the results of further experimentation.

Adrenocorticoid action in the spinal cord: some unique molecular properties of glucocorticoid receptors.

1. Glucocorticoid hormones affect several functions of the spinal cord, such as synaptic transmission, biogenic amine content, lipid metabolism, and the activity of some enzymes (ornithine decarboxylase, glycerolphosphate dehydrogenase), indicating that this tissue is a target of adrenal hormones. 2. Corticosterone, the main glucocorticoid of the rat, is detected at all regional levels of the spinal cord, and cold stress increases this steroid, predominantly in the cervical regions. 3. Intracellular glucocorticoid receptors have been found in the spinal cord, with higher concentrations in the cervical and lumbar enlargements. Prima facie, these receptors presented biochemical, stereospecifical, and physicochemical properties similar to those of receptors found in other regions of the nervous system. The prevalent form in the spinal cord is the type II receptor, although type I is also present in small amounts. 4. The type II glucocorticoid receptor of the spinal cord shows an affinity lower (Kd 3.5 nM) than that of the hippocampal type II site (Kd 0.7 nM) when incubated with [3H]dexamethasone. This condition may impair the nuclear translocation of the spinal cord receptor. 5. Another peculiar property of spinal cord type II site is a greater affinity for DNA-cellulose binding than the hippocampal receptor during heat-induced transformation. Also, the spinal cord receptor shows resistance to the action of RNAse A, an enzyme which increases DNA-cellulose binding of the hippocampal receptor, indicating that both receptors may be structurally different. 6. Therefore, it is possible that a different subclass of type II, or "classical glucocorticoid receptor," is present in the spinal cord. This possibility makes the cord a useful system for studying diversity of glucocorticoid receptors of the nervous system, especially the relationship between receptor structure and function.

Heterogeneity and properties of transformation of corticosteroid receptors in spinal cord and hippocampus.

The central nervous system contains two classes of corticoid receptors, named types I and II following terminology accepted for the kidney. Phenotypically, type I sites are differentiated into a corticosterone (CORT)-preferring species (Ia) and a mineralocorticoid receptor (Ib). These populations were tentatively compared in the spinal cord and hippocampus. Using [3H]dexamethasone (DEX) and selective blockage of sites, we have observed that type II receptors were comparable in both tissues, while Ia was almost exclusive of the hippocampus. Saturation analysis using [3H]DEX demonstrated that type Ia was a low affinity receptor (Kd approximately equal to 2-5 nM) while type II was a higher affinity site (KdII less than KdI). Using [3H]CORT, or [3H]aldosterone (ALDO), as ligand, preferential labeling of type I sites was achieved, always showing higher concentrations in the hippocampus. Therefore, [3H]DEX seems a ligand of choice to visualize types Ia and II receptors. Another difference noted between the spinal cord and hippocampus, pertained to the sensitivity towards the enzyme RNAse A, which increases heat-induced transformation of the bound receptor, according to the results of DNA cellulose affinity chromatography. In these experiments, type I sites of both spinal cord and hippocampus, plus type II of hippocampus, showed sensitivity toward the enzyme, whereas type II of the spinal cord was refractory to RNAse A enhancement of transformation. These results indicate that the dynamics of transformation is different among receptors showing similar affinity and competition, suggesting further heterogeneity due to receptors themselves, or to tissue factors regulating their biochemical properties.

A correlative study between glucocorticoid receptor levels in human mononuclear leukocytes and biochemical data in Cushing's disease.

UNLABELLED: We determined glucocorticoid receptors in human mononuclear leukocytes in 9 patients with Cushing's disease, in order to correlate them with laboratory data. Receptors were measured by a whole-cell assay method, after incubation with [3H]-dexamethasone in the presence or absence of excess unlabelled hormone. In Cushing's disease, there were 4425 +/- 364 sites/cell (N = 9), similar to in the controls: 4473 +/- 476 (N = 10); average Kd was 2.42 +/- 0.52 nmol/l (N = 3) similar to in the controls: 2.0 +/- 0.20 nmol/l (N = 3). In Cushing's patients we found significant negative correlations between basal glucocorticoid receptors and: 1) morning blood cortisol (r = -0.67, P less than 0.05), and 2) 17-ketogenic steroids after 2 mg of dexamethasone (r = -0.85, P less than 0.01). No correlations were observed with afternoon blood cortisol, free urinary cortisol, basal and post-8-mg dexamethasone 17-ketogenic steroids, TRH-TSH area, urinary calcium, plasma glucose, or systolic blood pressure. CONCLUSIONS: In Cushing's disease, a subtle receptor down-regulation may exist, as suggested by the inverse relationship between glucocorticoid receptors and morning blood cortisol. Secondly, the relationship between basal receptors and 17-ketogenic steroids after 2 mg of dexamethasone suggests that glucocorticoid receptors in human mononuclear leukocytes could reflect the sensitivity of the nervous system-pituitary-adrenal axis to dexamethasone inhibition.

A comparison of the glucocorticoid receptor system in the spinal cord and hippocampus.

Studied under in vivo conditions, uptake of [3H]corticosterone (CORT) by purified cell nuclei of the hippocampus was much higher than in the spinal cord, although the latter may contain in cytosol up to 50% of glucocorticoid receptors found in cytosol of hippocampus. Experiments were undertaken to explain these differences. First, the in vivo affinity of receptors for exogenous CORT was comparable in both tissues. Second, an inhibitor of translocation, although present, was not preferentially concentrated in the spinal cord as compared to the hippocampus. However, the sensitivity towards RNAase A, an enzyme that increased binding to DNA-cellulose (taken as a measure of increased affinity for nuclear components), was preserved in the hippocampus but absent in the cord. We discuss the possibility that refractoriness to RNAase A may play a role in the reduced nuclear uptake of [3H]CORT shown by the spinal cord in vivo, but also consider possible that heterogeneity of receptor types binding CORT in the spinal cord and hippocampus may account for the differences observed in both tissues.

Glucocorticoid regulation of glycerol phosphate dehydrogenase and ornithine decarboxylase activities in the spinal cord of the rat.

We examined the effects of glucocorticoids on induction of glycerol phosphate dehydrogenase (GPDH) and ornithine decarboxylase (ODC) in the spinal cord of rats. After a single subcutaneous dose of 5 mg/kg of dexamethasone (DEX) phosphate, GPDH activity was maximally increased at 20 h with the effect still persisting for 46 h, in contrast to ODC activity, which was already stimulated at 4 h. The enzyme induction was accompanied by a reduction in number of cytosolic glucocorticoid receptors already at 1 h after DEX treatment, with replenishment at 22 h. A dose-response curve for DEX demonstrated that the minimal effective dose (0.2 mg/kg) for enzyme induction also reduced the number of cytosolic receptors because of occupation/depletion. The effects were specific for natural and synthetic glucocorticoids, as GPDH and ODC activities were not stimulated by aldosterone, testosterone, estradiol, or progesterone. ODC was induced in the cervical region of the spinal cord as well as in the horse tail plus filum terminale, whereas GPDH responded in the former but not the latter region. Previous work has demonstrated that glucocorticoid receptors are slightly more concentrated in the cervical spinal cord. It is suggested that glucocorticoid induction of these two predominantly glial enzymes occurs by a steroid receptor-mediated event, as postulated in other regions of the nervous system. In view of the short latency required for induction of ODC, we also examined the effect of inhibitors of transcription and translation. Whereas cycloheximide reduced the stimulatory effect of DEX, a paradoxical stimulation was obtained when DEX and dactinomycin (actinomycin D) were given concomitantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Functions and dysfunctions of receptors for adrenal corticoids in the central nervous system.

Glucocorticoids (GC) have several known effects on the function of the nervous system, and GC receptors have been identified in regions responding to hormonal action. In the spinal cord, GC receptors have been characterized in vitro, which share several biochemical properties in common with receptors in better studied areas such as the hippocampus. Moreover, enzymes which are induced by GC in the hippocampus, such as glycerolphosphate dehydrogenase and ornithine decarboxylase, are also under specific GC control in the spinal cord. Yet GC receptors in the latter tissue divert from those in hippocampus during some in vivo as well as in vitro studies. In vivo, uptake of [3H]corticosterone by purified cell nuclei was 5-8-fold higher in the hippocampus as compared to the cord. In vitro, a higher percentage of GC receptors previously transformed by heating, showed affinity towards DNA-cellulose in the spinal cord than in the hippocampus. The enzyme RNAse A effectively increased receptor binding to DNA-cellulose in hippocampus, whereas the cord was insensitive to its action. These results suggest that there is a "receptor dysfunction" in the spinal cord, the significance of which is poorly understood in terms of the accepted model of steroid hormone action.