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In mammals, polyamines are essential for the maintenance of cell growth. Although early studies reported the highest values of mammalian ornithine decarboxylase (ODC) activity, a key enzyme in polyamine biosynthesis, in rodent placenta, the role of this enzyme in the second half of rodent pregnancy is still controversial. In order to get new insights on polyamine metabolism during this period of pregnancy, we studied polyamine levels, ODC expression and activity and transcript profile of different polyamine-related genes in mouse placenta, fetus and yolk sac. Results indicated that ODC activity and protein levels were higher in placenta than in fetus and yolk sac, especially in the labyrinth, although no correlation between ODC activity and polyamine levels were observed. The half-life of placental ODC ( approximately 190 min) was also higher than the fetal one ( approximately 24 min). Messenger RNAs of all biosynthetic and retroconversion enzymes of polyamine metabolism were present in the three gestational compartments analyzed, as well as those of antizymes 1 and 2 and antizyme inhibitor 1. However, no expression of antizyme 3 and antizyme inhibitor 2 was detected. The catabolic enzyme diamine oxidase was expressed only in the maternal part of placenta but not in the fetal part or in the fetus. The expansion of polyamine pools in the fetus was markedly higher than in placenta, in spite of its lower biosynthetic activity. Our results suggest that the elevated polyamine biosynthetic activity of mouse placenta is required to satisfy the high demand of polyamines required by the growing fetus, during the later period of pregnancy.

Complex interrelationships exist between hyperlipidemia and the progression of renal injury. The aim of this study was to evaluate the impact of high plasma cholesterol and triglyceride levels on renal structure and the effects of atorvastatin on progression-regression of renal injury. One-hundred chickens were divided into five groups: Group A: Standard diet (SD) for 6 months; Group B: Hyperlipidemic diet (HD) for 6 months; Group C: HD for three months and SD during the next 3 months; Group D: HD for 3 months and SD during the next 3 months, when they received oral atorvastatin (3 mg/kg/d); Group E: HD for the whole 6 months, and atorvastatin (3 mg/kg/d) during the last 3 months. Increased alpha-actine immunostaining was found in glomeruli of groups B and C. An important decrease of immunostaining was observed in glomeruli of atorvastatin treated groups. Group D showed the lowest value for presence of lipids, and significant differences were found with respect to the rest of the groups. The glomeruli of group B presented the highest damage grades and those of group D showed the lowest grades and presented significant differences from the rest of the groups. The combination of atorvastatin therapy and proper diet proved to be effective in promoting renal disease regression. However, the study of several parameters indicates that neither only diet nor atorvastatin in the progression group resulted completely effective in decreasing the progression of the disease.

Objective: To study the effect of different phenolic compounds and red wine on pulmonary metastatic melanoma., Methods: Swiss mice were inoculated with 500000 melanocytes B16F10 and given oral doses of diosmin, grape seed extract (GSE) and red wine. A macroscopic count was made of the metastatic nodules on the lung surface and a microscopic study by image analysis of five sections, calculating the implantation percentage and tumoral growth and invasion indices., Results: Macroscopically, the group treated with diosmin showed the greatest reduction (52%) in the number of metastatic nodules compared with the control group, which was treated with ethanol, while GSE and red wine caused decreases of 26.07 and 28.81%, respectively. Microscopically, there was a decrease in the implantation percentage after the administration of diosmin (79.4%) and red wine (20.19%), and an increase of 2.12% after the administration of GSE, all relative to the ethanol-treated control. As regards the growth index, diosmin produced a reduction of 67.44% and red wine a reduction of 20.62%, while GSE again produced an increase (25.33%). The reductions in the invasion index were 45.23, 31.65 and 17.57% with diosmin, GSE and red wine, respectively., Conclusions: Diosmin originated the greatest reduction in pulmonary metastases, both at the macroscopic and microscopic levels.

Calcium appears to be involved in many of the cellular events which are thought to be important in atherogenesis. Calcium channel blockers have been shown to reduce arterial lipid accumulation in animals without altering serum cholesterol. Avian models of atherosclerosis offer economic and technical advantages over mammalian models. In this study, we examine the effects of nifedipine, verapamil and diltiazem at clinical and higher doses, on the extent of atherosclerosis of egg-fed chickens. In order to assess the extent of atherosclerosis quantitatively, the aortic lesions of the thoracic and abdominal aorta, aortic arch and supraaortic regions were measured by planimetry. Atherosclerotic lesions were evaluated histologically. Statistically significant reductions in the lipid deposition of the aorta were found in all the treated groups. The extent and distribution of atherosclerotic lesions were decreased in a significant way by verapamil, nifedipine and diltiazem. The higher the dosage used, the higher the regression of the atherosclerotic lesions. At clinical dosage, nifedipine showed the highest decrease of the lesions. In addition, the chicken atherosclerosis model has proved itself useful and very suitable for in vivo drug intervention studies.

Administration of the preferential mu-opioid receptor agonist, morphine, and selective K-opioid receptor agonists elicits activation of the hypothalamus-pituitary-adrenocortical axis, although the site or the molecular mechanisms for these effects have not been determined. The expression ofFos, the protein product of the c-fos protooncogene, has been widely used as an anatomical marker of monitoring neuronal activity. In the present study we evaluated 1) the effects of the mu-opioid receptor agonist, morphine, and those of the selective K-opioid receptor agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl-]benzeneacet amide methane sulfonate (U-50,488H), administration on the expression of Fos in hypothalamic nuclei; and 2) the possible modification of the activity of noradrenergic neurons known to send afferent projections to the paraventricular nucleus (PVN), the site of CRF neurons involved in initiating ACTH secretion. Using immunohistochemical staining of Fos, the present results indicate that acute treatment with either morphine or U-50,488H induces marked Fos immunoreactivity within the hypothalamus, including the medial parvicellular PVN and supraoptic and suprachiasmatic nuclei. Pretreatment with naloxone attenuated the effect of morphine, whereas nor-binaltorphimine, a selective kappa-opioid receptor antagonist, abolished the effect of U-50,488H on Fos induction. Correspondingly, morphine and U-50,488H injection increased the production of the cerebral noradrenaline metabolite 3-methoxy-4-hydroxyphenylethylene glycol as well as noradrenaline turnover in the PVN. These effects were antagonized by naloxone and nor-bin-altorphimine, respectively. All of these findings are discussed in terms of specific events that couple opioid-induced activation of the hypothalamus-pituitary-adrenocortical axis and noradrenergic activity with changes in gene expression in selective hypothalamic nuclei.

Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcbeta1,4Galbeta1,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcbeta1,4Galbeta1,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcbeta1,4Galbeta1,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcbeta1,4Galbeta1,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcbeta1,4Galbeta1,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.

In the present study, the formation and development of the acrosome during spermiogenesis in four different rodent species (rat, mouse, hamster and guinea pig) was compared by means of cytochemical and blotting techniques using a lectin from soybean (SBA). This lectin recognizes specifically the acrosome of the four species at all steps of formation. At the ultrastructural level, SBA-binding pattern was similar in the acrosome of the rat, mouse and hamster. SBA preferentially labelled the electron-lucent area of the acrosome in early-spermatids (Golgi and cap phases) and the outer region of the acrosome in mature spermatids (acrosome and maturation phase). The lectin binding pattern was more complex in the guinea pig acrosome. Three different subdomains can be established in the early acrosome of the guinea pig. The lectin bound the three subdomains but mainly a thin fold which spreads over the nucleus during the cap phase. In the acrosome phase, SBA strongly reacted with the principal segment. In contrast, no reactivity was observed in most of this segment in maturation phase spermatids. In this phase, SBA bound preferentially a thin area covering the dorsal region of the apical segment. Lectin blots of detergent-extracted testes indicated that SBA only recognizes proteins of high molecular weight (> 100 kD) in the four species studied. The results obtained in the present study suggest that the development of acrosomal subdomains is very similar in the mouse, rat and hamster but shows a more complex pattern in the guinea pig.

Hypokalemia produced different effects on steroid sex hormone concentrations in plasma and ovary in the mouse. Estradiol levels were slightly increased, whereas circulating progesterone was markedly decreased in all estrous periods. The preovulatory surge of gonadotropins and the secondary surge of follicle-stimulating hormone (FSH) at estrus were also decreased, but basal levels of both gonadotropins were unaffected. Supplementation with luteinizing hormone (LH), FSH, or gonadotropin-releasing hormone (GnRH) at proestrus rapidly normalized plasma and ovarian progesterone levels at this stage of the estrous cycle. Plasma progesterone levels at diestrus were restored only by combined treatment, at the periovulatory stage, with LH and FSH or GnRH but not by LH or FSH alone. The results demonstrate a lack of steroidogenic activity in the corpus luteum of the potassium-deficient mice and, furthermore, that FSH plays an important role in luteinization in the hypokalemic mice. We conclude that alteration of the transcellular potassium gradient may affect the regulation of the periovulatory surge of gonadotropins and progesterone secretion, probably by altering the release of GnRH from the hypothalamus. In addition, the results suggest that FSH may play a certain role as a luteotropic hormone in mice.

In a previous study, evidence was provided for the presence of a novel plasma-membrane-associated neutral-pH-optimum alpha-L-fucosidase in rat sperm. In the present study, rat sperm alpha-L-fucosidase was characterized during epididymal maturation. The pH 7 activity optimum of alpha-L-fucosidase and its subunit composition (one or two closely spaced immunoreactive protein bands of about 53+/-2 kDa) did not appear to change during transit through the epididymis. Isoelectric focusing of alpha-L-fucosidase indicated the presence of a major isoform (B) with a pI near 7 in sperm from testis, caput, corpus and the proximal half of the cauda. alpha-L-Fucosidase from sperm from the distal half of the cauda, which contained a significant enrichment of sperm and alpha-L-fucosidase activity, contained isoform B and an additional minor isoform (A) with a pI near 5.2. Isoform B and small amounts of isoform A were present in sperm from the vas deferens. The two fucosidase isoforms present in sperm from the distal cauda were separated by isoelectric focusing and comparatively characterized. They had similar pH-activity curves (with optima near pH 7) and comparable apparent KM values (0.4+/-0.04 mM) for 4-methylumbelliferyl alpha-l-fucopyranoside. Preincubation of the isoforms at different temperatures indicated that isoform A is considerably more thermostable than isoform B. Immunoprecipitation studies using polyclonal antibodies against human liver alpha-L-fucosidase indicated that approx. 90% of the enzymic activity for both isoforms was immunoprecipitable under conditions that immunoprecipitated essentially all the human liver enzyme. Neuraminidase treatment of sperm alpha-L-fucosidase from distal cauda (when compared with the appropriate heat-treated control) led to disappearance of isoform A and a concomitant increase in isoform B. The overall results suggest that isoform A is derived by sialylation of isoform B near the end of epididymal maturation.

Oligosaccharide side chains of zona pellucida (ZP) glycoproteins play a key role in the sperm-egg interaction phenomena during fertilization. In the present study, modifications of the ZP glycoproteins during the fertilization process in the mouse were studied by the lectin-gold technique and immunocytochemistry in conjunction with quantitative analysis. Binding of PNA, RCA I, DSA, LFA, MAA, AAA, and anti-ZP2 and anti-ZP3 antibodies was observed throughout the ZP of both unfertilized and fertilized eggs. However, HPA and BSAIB4 labeling was found only in the inner region of the ZP. After neuraminidase treatment (Neu), HPA showed an affinity for the entire ZP. Labeling by LFA, WGA, MAA, PNA, BSAIB4, and AAA decreased in the ZP of fertilized eggs; however, there was an increase in the binding of RCA I. HPA and Neu-HPA increased only in the inner region of the ZP. Immunoreactivity to antibodies against ZP2 and ZP3 also decreased after fertilization. The present results demonstrate that 1) terminal carbohydrate residues contained in the ZP glycoproteins are modified after fertilization and 2) inner and outer regions of the ZP contain different oligosaccharide side chains.

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.

The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte. It is involved in the sperm-egg adhesion phenomenon, induces the acrosome reaction, and participates in the late blockage to polyspermy. Thus, during the process of fertilization the cortical reaction is induced and the biochemical and biological properties of the ZP are modified. Some of these changes have been suggested to prevent the polyspermy. However, the mechanisms behind most of these changes are not well understood. Carbohydrate residues of the ZP glycoproteins have been shown to play a key role in the early step of fertilization. In the present study, the changes produced in the terminal oligosaccharide sequences of the rat ZP glycoproteins after in vivo fertilization were investigated by means of lectin-gold cytochemistry. A comparative quantitative analysis of the density of labeling in the ZP before and after fertilization was carried out by automatic counting of gold particles. The ZP of fertilized and unfertilized eggs were labeled by a battery of lectins including PNA, LFA, MAA, AAA, DSA, RCA I, and WGA. For all lectin studied in both fertilized and unfertilized eggs the labeling was preferentially located in the inner region of the ZP. After fertilization, binding of PNA, LFA, MAA, AAA, and DSA decreased in both inner and outer regions of the ZP. Labeling of RCA I-binding sites only decreased in the inner ZP, whereas reactivity to WGA was increased in the inner ZP, whereas reactivity to WGA was increased in the inner area of the ZP. Digestion of the thin-sections with neuraminidase prior to labeling with WGA resulted in a decrease of labeling for WGA binding sites. However, the labeling density of WGA binding sites was similar in both unfertilized and fertilized eggs upon treatment with neuraminidase. The present results demonstrate that the oligosaccharide chains contained in the rat ZP are modified after fertilization of the oocyte. Cortical granules of the oocytes might be involved in these modifications by two mechanisms: 1) by hydrolysis of terminal carbohydrate residues of ZP glycoproteins by specific glycosidases contained in the granules; and 2) by addition of new glycoproteins to the ZP after the exocytosis of the cortical granules (cortical reaction).

Background: The zona pellucida (ZP), an extracellular matrix which surrounds mammalian oocytes, is formed by different glycoproteins. Several studies have revealed that carbohydrate residues present in glycoproteins of ZP play a key role in the sperm-egg recognition. However, the origin and the biochemical composition of ZP remain to be completely resolved., Methods: ZP glycoproteins from rat ovarian follicles were investigated at light and electron microscopic level by the application of lectins conjugated to peroxidase, digoxigenin, and colloidal gold in combination with enzyme and chemical treatment. A quantitative analysis was also performed., Results: ZP shows reactivity to WGA, DSA, LFA, AAA, RCA I, and MAA. SBA and PNA showed a variable reactivity ranging from negative to strongly positive. A uniform pattern of binding throughout ZP was observed with DSA, Con A, AAA, MAA, and LFA. However, labeling by RCA I and SBA was higher in the outer ZP while PNA and WGA showed a higher binding in the inner ZP. Lectin reactivity was detected in cortical granules, endoplasmic reticulum, Golgi apparatus, vesicles, and multivesicular bodies of oocytes., Conclusions: ZP contained the terminal disaccharides Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, and GalNAc beta 1,3Gal and the trisaccharides Neu5Ac alpha 2, 3Gal beta 1,4GlcNAc, Neu5Ac-Gal beta 1,3GalNAc, and Neu5Ac-GalNAc beta 1,3Gal sequences. The occurrence of Fucose residues alpha 1,6 linked to the inner core region of N-linked glycoproteins of ZP was demonstrated by the use of several fucose-specific lectins. Methylation-saponification treatment in combination with lectin cytochemistry reveals that Gal, GalNAc, and polyllactosamine residues of rat ZP glycoproteins contain sulphated groups. The reactivity observed in ooplasmic vesicles was similar to that of ZP, thus suggesting that the oocyte is the site of synthesis of ZP glycoproteins.

In the present study, lectin cytochemistry in combination with enzyme and chemical treatments and ultrastructural immunocytochemistry were applied to investigate the formation of acrosomal glycoproteins in endoplasmic reticulum (ER) and Golgi apparatus (GA) of early rat spermatids. In addition, the vesicles involved in glycoprotein traffic were investigated using a monoclonal antibody against clathrin. The results obtained suggest the occurrence of high mannose and complex type N-linked oligosaccharides and mucin type O-linked oligosaccharides. In N-linked glycoproteins, Man residues are incorporated into the nascent oligosaccharide in the ER, Fuc residues of the inner core of the oligosaccharide in the cis region of GA, GlcNAc in medial cisternae of GA and Gal residues in the transmost cisternae of GA. In O-linked glycoproteins, the addition of GalNAc occurs in cis and trans cisternae of GA. Gal beta 1,3GalNAc sequence was detected in medial and trans cisternae of GA. Sialic acid was detected in both N- and O-linked oligosaccharides in medial and trans cisternae of GA but not in acrosomes. Immunoreactivity to clathrin was observed in the intermediate zone between ER and GA and in vesicles of the trans side of GA.

The glycoconjugates of the testes of four species of vertebrates including amphibians (Bufo calamita), reptiles (Mauremys caspica), birds (Columba livia) and mammals (Mesocricetus auratus) were investigated by means of lectin histochemistry. Spermatogonia of the animals studied were labelled by Con A and WGA. DBA showed a specific affinity for the spermatogonia Aal of the hamster indicating that DBA might be an useful marker for this cell type. The acrosomes of the hamster spermatids were strongly reactive to PNA, SBA and WGA. LTA and UEA-I did not show affinity for the acrosomes of any of the species studied. Lectin reactivity of spermatids and spermatozoa was similar in pigeon and hamster. Sertoli cells were reactive to Con A in all species studied. Affinity to WGA of the spermatozoa tails suggests the addition of the glycoprotein SMA4 in the testis, instead of the epididymis as has been indicated in the mouse. The present results suggest an increase in the glycosylation processes with the differentiation of the spermatogenic lineage being more marked in amphibians and reptiles in the spermatid-spermatozoa step, while in mammals the differences are greater in spermatocyte-spermatid step. The low reactivity to the lectins in the bird studied suggests a low content of glycoconjugates in the testis of this avian species.

The epithelium of the extrapulmonary airways of a Chelonia (Mauremys caspica) and a Squamata (Lacerta lepida) was investigated by means of conventional light and transmission electron microscopy, scanning electron microscopy, histochemistry and immunocytochemistry. The epithelium of Mauremys caspica is composed of basal, ciliated, endocrine and mucous cells. Serotonin-immunoreactivity was detected in the endocrine cells. Mucous cells were found to contain either sialo-mucins or both sialo- and sulphomucins. The extrapulmonary airways of Lacerta lepida were lined by an epithelium composed of basal, ciliated and secretory cells. No endocrine cells were detected. Secretory cells showed a different morphology to that observed in the mucous cells of Mauremys caspica suggesting a possible serous role for these cells. Migratory cells (plasma and mast cells, leucocytes and macrophages) and small intraepithelial nerves were also detected within the epithelium of both species. The present results show that marked morphological differences occur between the epithelia of the extrapulmonary airways of reptiles belonging to the genus Chelonia and Squamata.

The lung of the tortoise, Testudo graeca (Chelonia) was studied by means of light and electron microscopy, histochemistry and immunocytochemistry. The lung showed the typical faviform structure of the reptilian lung. Three orders of trabeculae were observed. The epithelium of primary and secondary trabeculae was composed of ciliated, mucous, basal and endocrine cells. Mucous cells contained sialo- and sulpho-mucins and were reactive to the lectins Con-A, WGA, DBA, PNA and SBA. Endocrine cells were observed as solitary cells or forming neuroepithelial bodies. By means of immunocytochemistry, endocrine cells were demonstrated to contain serotonin. In the gas-exchange area Types I and II pneumonocytes and undifferentiated cells were observed. Free macrophages were detected in the faveolar lumen. The lung interstitium contained smooth muscle cells, fibrocytes, pigment cells, myelinated and unmyelinated nerves and intrapulmonary ganglia. Nerve terminals containing clear and dense-cored vesicles were observed in the adventitia of the blood vesicles and interspersed between the smooth muscle bands. The lung of the hibernating specimens showed a marked vacuolisation of pneumonocytes. In conclusion, the lung of Testudo graeca showed a complex histological organisation. Marked differences from mammalian lung were found.