Marina C. Rodríguez

Ultrastructural observations on nucleoli and related structures during human spermatogenesis

SummaryThe ultrastructural study of nucleoli and ribonucleoprotein-containing structures in human seminiferous tubules revealed that the nucleoli of spermatogonia, spermatocytes and Sertoli cells exhibited a tripartite structure consisting of: (1) a fibrillar center, (2) a compact granular portion, and (3) a reticular portion containing both pars fibrosa and pars granulosa. The nucleoli of primary spermatocytes showed a developed reticular portion. At pachytene, the compact granular portion enlarged and lost its connection with the fibrillar center and the reticular portion which decreased in size. This suggests a nucleolar segregation similar to that of ovocytes in many species. Two similar developmental stages of nucleoli were observed in spermatogonia. In addition to nucleoli, there were other ribonucleoprotein-containing structures such as intranuclear closely-packed granules in Ap spermatogonia, coarse granules in the chromatin rarefaction zone of Ad spermatogonia, the nuage and Lubarsch crystals of spermatogonia, the chromatoid body of spermatids, the annulate lamellae of both spermatids and Sertoli cells, and many structures of the spermatid neck region.

Comparison of changes in erythrocyte and platelet phospholipid and fatty acid composition and protein oxidation in chronic obstructive pulmonary disease and asthma

Objective: To analyse and compare the phospholipid and fatty acid composition of total lipids and the occurrence of lipid peroxidation and protein oxidation directly in erythrocytes or platelets from chronic obstructive pulmonary disease (COPD) and asthma patients. Patients: Fifteen consecutive outpatients with COPD (all smokers) and asthma (non-smokers) recruited during a moderate-to-severe (COPD) or moderate (asthma) exacerbation. Fifteen subjects with smoking habits similar to those of COPD patients were studied as a control group. Methods: Phospholipid and total fatty acid compositions were analysed by two-dimensional thin layer chromatography or gas chromatography–mass spectrometry, respectively. The lipid fluorescence of lipid extracts was measured by spectrofluorimetry. Protein carbonyl contents and profiles were measured by immunoblot detection. Results: No differences were found either in erythrocyte or platelet cholesterol or phospholipid levels. Only a decrease in the content of phosphatidylserine + phosphatidylinositol (P < 0.003) was detected in platelets from the asthma patients. In erythrocytes, the fatty acid profile changed in both lung pathologies, especially as regards polyunsaturated fatty acids (decreases in arachidonic and 22:4 fatty acid contents). Other observed changes were: COPD, an increase in palmitic fatty acid; asthma, an increase in oleic and decreases in eicosapentaenoic and 22:6 + 24:1 fatty acids. In platelets, the fatty acid profiles revealed many differences between both lung pathologies: COPD, a decrease in 18:1 and increases in 20:5 and 22:5 + 24:0; asthma, a decrease in 20:4 and increase in 22:6 + 24:1. In COPD vs. asthma patients, fatty acid changes were mainly detected in platelets, especially in 18-carbon species, with decreases in stearic and 18:1 fatty acids in the COPD patients. Protein oxidation levels were increased in both lung pathologies in both erythrocytes and platelets. Conclusions: COPD and asthma are associated with common or specific changes in the lipid composition of erythrocytes and/or platelets. The data point to lipid peroxidation and protein oxidation phenomena in both types of blood cell, although platelets would be more susceptible to stress.

Quantification of cell types throughout the cycle of the human seminiferous epithelium and their DNA content

SummaryThe numbers of each different cell type in the human seminiferous epithelium were determined throughout the 6 stages of the cycle in both semithin and ultrathin sections obtained from 15 young adult men with normal testicular histology. Up to 4 types of A spermatogonia (Ad, Ap, Al and Ac) were distinguished. In addition, the DNA nuclear content of seminiferous epithelium cells was determined on Feulgen-stained sections. Both Ad and Ap spermatogonia showed a 2c DNA content and were present in the 6 stages of the cycle, though their numbers decreased in stages III–V. Both Al and Ac spermatogonia showed a DNA content varying from 2c to 4c. Al spermatogonia were observed in stages III–V; their numbers plus those of Ad spermatogonia in these stages were similar to the numbers of Ad spermatogonia in the other stages lacking in Al spermatogonia. Ac spermatogonia appeared in stages III–VI and their numbers plus those of Ap spermatogonia in stages III–V were similar to the numbers of Ap spermatogonia in the other stages lacking in Ac spermatogonia. The results suggest that Ad spermatogonia are the stem cells. Some of them replicate their DNA; during this replication they appeared as Al spermatogonia. Al spermatogonia divide, giving rise to both Ad and Ap spermatogonia. Some Ap spermatogonia replicate their DNA; during this process they are transformed into Ac spermatogonia which divide, giving rise to B spermatogonia.

Effects of cocaine on testicular structure in the rat

The effects of hourly injections of moderate doses of cocaine hydrochloride (0.5 and 10 mg/kg body weight) over 5 h on testicular structure and testosterone levels were studied in male Wistar rats. Cocaine produced a rapid disruption of spermatogenesis; the number of normal seminiferous tubules declined to 50% (low dose) and 40% (high dose), and regressive tubules (tubules with cellular degeneration, cell sloughing, or abnormal cell structures) increased to 50% (low dose) and 60% (high dose) after treatment with cocaine. The mean tubular diameter, the surface occupied by the tubules, and the volume of seminiferous tubules per pair of testes were significantly reduced (P less than 0.01) after both doses of cocaine. Cocaine produced ultrastructural changes in the cells of the seminiferous epithelium (spermatogonia, spermatids, and Sertoli cells) including vacuoles, abundant lipid droplets, and giant mitochondria. Lower doses of cocaine increased serum testosterone levels (P less than 0.025) while higher doses did not. These findings indicate an acute effect of cocaine on the structure of the rat testis.

Changes in surface area and number of Leydig cells in relation to the 6 stages of the cycle of the human seminiferous epithelium

SummaryIn order to evaluate the occurrence of a Leydig cell cycle related to the cycle of the seminiferous epithelium in man, the numbers of peritubular Leydig cells and surface area of these cells along 1 mm of tubular basement membrane at each stage of the cycle were calculated on histological sections of young adult testes. The Leydig cells that were located separated from the tubules (perivascular Leydig cells) were also classified according to the stage of the cycle shown by the nearest seminiferous tubule; the surface area and number of these cells were also calculated. The total surface area and numbers of Leydig cells (peritubular plus perivascular) along 1 mm of tubular basement membrane did not change during the cycle of the seminiferous epithelium. Both the surface area and the numbers of peritubular Leydig cells were greater in stages I and II of the cycle, when spermatozoa are released; they decreased in stages III and IV and increased again in stages V and VI, whereas the contrary occurred in perivascular Leydig cells. The average surface area of each Leydig cell type remained constant throughout the stages of the cycle.

Platelet linoleic acid is a potential biomarker of advanced non-small cell lung cancer

New parameters that could be used as tumor markers for lung cancer would be valuable. Our aim was to analyze the fatty acid profiles of total lipids from erythrocytes and platelets from patients with advanced non-small cell lung cancer (NSCLC), chronic obstructive pulmonary disease (COPD) and asthma to reveal the fatty acids that could be used as NSCLC biomarkers. In our study, 50, 15 and 15 patients with advanced NSCLC, COPD and asthma and 50 healthy subjects were enrolled. Fatty acid profiles were investigated using gas chromatography/mass spectrometry followed by ROC (receiver operating characteristics) curves analysis to gain information about biomarkers. Sialic acid (SA) and cytokeratins were measured by the thiobarbituric acid and immunoradiometric methods respectively. Useful fatty acid markers were as follows: erythrocytes, 22:0 and linoleic acid (LA, 18:2n6); platelets, 16:0, 18:0, and LA. At the cutoff value to obtain maximum accuracy, the best biomarker was platelet LA, with higher diagnostic yields than the commonly used markers SA or cytokeratins (100%, 76%, 75% and 86% sensitivity, specificity, positive predictive value and accuracy, respectively). These findings suggest that platelet LA might be used as a biomarker of NSCLC in relation to different aspects of the disease process that now needs to be explored.

Erythrocyte and Platelet Phospholipid Fatty Acids as Markers of Advanced Non-Small Cell Lung Cancer: Comparison with Serum Levels of Sialic Acid, TPS and Cyfra 21-1

The phospholipid fatty acid profiles of erythrocytes and platelets from fifty patients with advanced non-small cell lung cancer were investigated using gas chromatography/mass spectrometry, followed by “ROC” curves analysis to gain novel biomarker information. Sialic acid and cytokeratins were also examined. Potentially useful fatty acid markers: Erythrocytes: phosphatidylcholine, 18:2n6 and 20:4n6; phosphatidylethanolamine, 22:4n6 and 22:6n3 + 24:1n9. Platelets: phosphatidylcholine, 22.0; phosphatidylethanolamine, 22:5n3 + 24:0. At the cut-off value to obtain maximum accuracy, the best biomarkers were found in platelets: phosphatidylserine + phosphatidylinositol (PS + PI), 21:0; sphyngomyelin: 20:1n9 and 22:1n9. All these fatty acids showed similar/higher diagnostic yields than the commonly used markers sialic acid or cytokeratins.

Amphiphilic and hydrophilic nature of sheep and human platelet phosphotyrosine phosphatase forms

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40-35%, respectively) and -poor phases (60-65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPalpha (mouse anti-human PTPase MoAbs) showed that RPTPalpha was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPalpha, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.

Comparison of Changes in Erythrocyte and Platelet Fatty Acid Composition and Protein Oxidation in Advanced Non-Small Cell Lung Cancer

The formation of free radicals and lipid peroxidation products is linked both to carcinogenesis and tumor behavior. Blood samples from 50 patients with advanced (Stages III–IV) non-small cell lung cancer (NSCLC), and from 50 healthy volunteers were used for plasma β-thromboglobulin (β-TG) measurements, red blood cell (RBC) and platelet lipid analyses, and lipid fluorescence determinations. Samples from 15 randomly selected patients and 15 controls also were used for analysis of the expression of oxidized proteins. We observed: (a) higher levels of plasma β-TG in patients, (b) alterations in membrane fatty acids. The RBC fatty acid profile changed especially in the 18-carbon species (increases in stearic and oleic and a decrease in linoleic fatty acids), and in arachidonic acid, which also decreased significantly. The platelet fatty acid profile mainly showed a decrease in arachidonic acid and a parallel increase in palmitic fatty acid; (c) the loss of polyunsaturated fatty acids (PUFA) in RBC and platelets could be correlated with changes in lipid extract fluorescence only for platelets; (d) protein oxidation levels were increased also only in the case of platelets. The changes detected point to platelet activation and lipid peroxidation processes associated with NSCLC. The oxidative stress affected RBC and platelets differently, although changes in PUFA might still have important physiological consequences in both types of cells.

Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4°C

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.