Transcriptomic analysis of polyamine-related genes and polyamine levels in placenta, yolk sac and fetus during the second half of mouse pregnancy.

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.

The effect of the flavonoid diosmin, grape seed extract and red wine on the pulmonary metastatic B16F10 melanoma.

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.

Glycosylation in Golgi apparatus of early spermatids of rat. A high resolution lectin cytochemical study.

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.

Transforming growth factor beta1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation.

Transforming growth factor-beta1 (TGFbeta1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by alpha-melanocyte stimulating hormone (alphaMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFbeta1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFbeta1 (10(-10) M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFbeta1 and alphaMSH on melanosome number, volume density and maturation degree. alphaMSH (10(-7) M, 48 h) increased 7-fold melanosome volume density, whereas TGFbeta1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or alphaMSH-treated cells. Moreover, TGFbeta1 blocked the alphaMSH-elicited increase in the number of melanosomes. Control and alphaMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFbeta1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with alphaMSH and TGFbeta1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFbeta1 and with its hypopigmenting effect.

Hypokalemia decreases testosterone production in male mice by altering luteinizing hormone secretion.

Potassium deficiency produced by feeding mice a low potassium diet caused a marked decrease in plasma and testicular testosterone concentrations and a concomitant fall in the weight of seminal vesicles and in renal ornithine decarboxylase activity. All of these parameters were rapidly restored when potassium supply was normalized. Immunocytochemical analysis of gonadotropes and plasma LH values suggested that the pulsatile liberation of LH by the pituitary was impaired in the potassium-deficient male mice. Because the synthesis of testosterone in the potassium-deficient mice was stimulated by exogenous LH, hCG, or GnRH, one can conclude that alteration of the transcellular potassium gradient could affect the regulation of the hypothalamo-hypophyseal-testicular axis by affecting the pulsatile release of GnRH. Our results showing that the stimulation of LH secretion after castration was similar in control and potassium-deficient male mice suggest that a testicular factor(s) different from testosterone could be implicated in the abnormal regulation of LH secretion in potassium-deficient mice. We conclude that plasma potassium concentration is an important factor in the regulation of gonadotropin secretion and testicular functions.

Activation of c-fos expression in hypothalamic nuclei by mu- and kappa-receptor agonists: correlation with catecholaminergic activity in the hypothalamic paraventricular nucleus.

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.

Ultrastructural localization of glycoconjugates in human bronchial glands: the subcellular organization of N- and O-linked oligosaccharide chains.

We investigated the glycoconjugates of the human bronchial glands at light and electron microscopic level by means of lectin histochemistry in combination with neuraminidase digestion and beta-elimination reaction. Both direct and indirect techniques using lectin-peroxidase, lectin-gold, and glycoprotein-gold complexes were applied. The binding pattern of the six lectins (ConA, HPA, DSA, WGA, LEA, and PNA) used in the present study suggests that mucous and serous cells of human bronchial glands contain both N- and O-glycosylated proteins in the secretory granules. Asparagine-linked oligosaccharides containing Gal(beta-1,4) GlcNAc and Man residues were abundant in serous cells. The demonstration of both the terminal Neu 5Ac (alpha-2,3, or 6) Gal (beta-1,4) GlcNAc sequence in the N-linked oligosaccharides of mucous cells and the terminal disaccharide Gal (beta-1,4) GlcNAc in the N-linked oligosaccharide chains of serous cells suggests the existence of complex type sugar chains N-glycosidically linked to the peptide region of the glycoproteins. The binding pattern of the DSA and the neuraminidase-DSA sequence provides evidence for the existence of sialyltransferase activity in the forming mucous granules of mucous bronchial cells.

Cytochemical characterization of sulfo- and sialoglycoconjugates of human laryngeal glandular cells.

The composition and distribution of sulfo- and sialoglycoconjugates in human laryngeal glands have been investigated at light and electron microscopic levels by use of peroxidase-, digoxigenin-, and colloidal gold-conjugated lectins in combination with several chemical and enzymatic deglycosylation procedures. The present study reveals a variety of terminal oligosaccharide sequences in serous and mucous glands. Serous cells contained glycoconjugates with terminal Neu5Ac (alpha 2-3) Gal (beta 1-4) GlcNAc, Neu5Ac (alpha 2-6) Gal/GalNAc, Neu5Ac (alpha 2-3/6) Gal (beta 1-3 GalNAc, GlcNAc, and Gal (beta 1-4) GlcNAc sequences. Scarce SO4Gal(beta 1-3)GalNAc terminal oligosaccharide chans were detected. Serous cells show wide morphological variability of secretory granules (electron lucent, electron dense, and bizonal) with different lectin affinities. Glycoconjugates in human laryngeal mucous glands contained a variety of terminal oligosaccharide sequences including SO4Gal(beta 1-4)GlcNAc, SO4Gal(beta 1-3) GalNAc, SO4GalNAc, and Neu5Ac(alpha 2-3)GalNAc.

Planimetric and histological study of the aortae in atherosclerotic chickens treated with nifedipine, verapamil and diltiazem.

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.

A light and electron microscopic study of the epithelium of the extrapulmonary airways of Mauremys caspica and Lacerta lepida (Reptilia).

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.

Effects of atorvastatin on progression-regression of renal injury in hyperlipidemic chickens.

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.

Cytochemical characterization of oligosaccharide side chains of the glycoproteins of rat zona pellucida: an ultrastructural study.

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.

Characterization of glycoconjugates in developing rat respiratory system by means of conventional and lectin histochemistry.

The glycoconjugates of the respiratory system of rats from 15 days of gestation through the adult period have been characterized by means of both conventional and lectin histochemistry. The main changes occurred at 20-21 days of gestation immediately before birth. An increase of acidic groups in the glycoproteins of the lung and airway epithelium was observed by conventional mucin histochemistry. The combined use of neuraminidase digestion and lectin histochemistry demonstrated an increase of sialic acid residues at the terminal position of the glucidic moieties of the glycoproteins. The sialic acid residues were linked alpha (2-3, 6) to D-galactose (beta 1-3)-N-acetylgalactosamine, thus masking the PNA-reactivity detected on the luminal surface of Clara cells and pneumonocytes before birth. In the adult period, alpha-L-fucose residues, detected by UEA-I, were localized in the glycoproteins contained in goblet cells and periciliary layer of the rat airway epithelium. The modifications observed in the lung of developing rats are similar to those previously described in human fetal and neonatal lungs. This suggests that the rat represents a useful model to study the glycoprotein synthesis during lung development.

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.

Influence of sulphate groups in the binding of peanut agglutinin. Histochemical demonstration with light- and electron-microscopy.

The influence of sulphation of mucus glycoproteins in the binding of peanut agglutinin (PNA) to tissue sections has been investigated by means of histochemical techniques at the light- and electron-microscopic level. A sequential methylation-saponification procedure was applied for the desulphation of tissue samples. Labelling by peroxidase- and colloidal gold-conjugated PNA was compared in control and desulphated samples of rat intestinal mucosa. The high-iron-diamine (HID) technique was used as a control for the effectiveness of the desulphation technique, and the Alcian Blue, pH 2.5 (AB 2.5), PAS and phosphotungstic acid-HCl (acid-PTA) techniques served as controls for the integrity of the oligosaccharide chains, respectively. In general, a marked increase of PNA reactivity was observed in desulphated samples when compared with control sections. These findings indicate that sulphation of galactose inhibits the binding of PNA to carbohydrate moieties in tissue sections. Staining patterns obtained with HID, PNA and the desulphation-PNA sequence in the goblet cells of the large intestine suggest a modification of the secretory product stored in these cells as the cell matures and moves from the lower crypt region toward the luminal surface. These modifications were not detected in the small intestine. Ultrastructural detection of PNA-binding sites suggests that galactose residues are incorporated into the oligosaccharide chains of O-linked glycoproteins at the medial cisternae of the Golgi apparatus. However, sulphation occurs at the trans side of the Golgi complex and the trans Golgi network. In conclusion, desulphation procedures are useful for revealing PNA-binding sites.

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments.

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase-PNA/SBA and galactose oxidase-neuraminidase-PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins.

Modifications of the lectin binding pattern in the rat zona pellucida after in vivo fertilization.

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).

Modifications of carbohydrate residues and ZP2 and ZP3 glycoproteins in the mouse zona pellucida after fertilization.

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.

Hypokalemia alters sex hormone and gonadotropin levels: evidence that FSH may be required for luteinization.

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.

Cytochemical and western blot analysis of the subcompartmentalization of the acrosome in rodents using soybean lectin.

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.