Ângela Alves

Caspase signalling pathways in human spermatogenesis

PurposeLittle is known about the apoptotic mechanisms involved in abnormal spermatogenesis. In order to describe the significance of apoptosis in azoospermia, testicular tissue from abnormal spermatogenesis was analysed.MethodsTesticular treatment biopsies were obtained from 27 men. Five presented oligozoospermia, 9 obstructive azoospermia (4 congenital bilateral absence of the vas deferens; 5 secondary azoospermia) and 13 non-obstructive azoospermia (5 hypospermatogenis; 3 maturation arrest; 5 Sertoli-cell-only syndrome). Immunohistochemical staining was performed for active caspases-3, −8 and −9. The presence of active caspases in Sertoli cells and germ cells was analyzed using stereological tools.ResultsIncreased active caspase-3 was found in Sertoli-cell-only syndrome. No significant differences were found in maturation arrest. In hypospermatogenesis, primary spermatocytes were the germ cells with higher active caspases. Oligozoospermia and secondary obstruction showed significant differences among germ cells for the presence of all active caspases. In oligozoospermia, spermatogonia presented significant increased active caspase-9 in relation to active caspase-8. In primary obstruction and hypospermatogenesis, germ cells presented significant increased active caspases-3 and −9.ConclusionsResults suggest that increased active caspase-3 might be involved in Sertoli-cell-only syndrome etiology. In cases of hypospermatogenesis, intrinsic lesions at the meiotic stage seem to be related to the pathology. In secondary obstruction apoptosis is suggested to be initiated due to extrinsic and intrinsic lesions, whereas in primary obstruction only the intrinsic apoptotic pathway seems to be present. Finally, in oligozoospermic patients spermatogonia death by mitochondrial damage additionally to meiosis malfunctioning, might be on the origin of the decreased sperm output.

Morphology and Phylogeny of Thelohanellus marginatus n. sp. (Myxozoa: Myxosporea), a Parasite Infecting the Gills of the Fish Hypophthalmus marginatus (Teleostei: Pimelodidae) in the Amazon River

Thelohanellus marginatus n. sp., a new myxosporean parasite infecting the primary gill filaments of the teleost fish Hypophthalmus marginatus (Pimelodidae) in the Amazon River, is described on the basis of microscopic and molecular procedures. The parasite forms whitish and ellipsoidal cysts up to 250 μm in diam. Myxospores ellipsoidal with a slightly more pointed anterior end, measuring 17.1 ± 0.6 μm in length, 6.9 ± 0.4 μm in width, and 5.1 ± 0.5 μm in thickness. A single pyriform polar capsule, 9.0 ± 0.3 μm long and 6.1 ± 0.4 μm wide, positioned slightly right to the medial plane in valvular view, contains a polar filament arranged in 4–5 coils. Molecular analysis of the SSU rRNA gene by Maximum Parsimony, Neighbor‐Joining, and Maximum Likelihood revealed the parasite clustering among other myxobolids, namely Henneguya and Myxobolus. Host affinity is supported as an important evolutionary signal for the phylogeny of myxobolids. The parasite here described represents the first record of the genus Thelohanellus Kudo, 1933 from the South American fauna.

Ultrastructural analysis of five patients with total sperm immotility

Asthenozoospermia has been related to structural defects of the sperm flagellum. However, few reports have studied in detail the ultrastructure of sperm with total immotility. We present an ultrastructural study of sperm from five patients with total sperm immotility, four due to dysplasia of the fibrous sheath (DFS) and one with situs-inversus. Of the four patients with DFS, three cases presented a hypertrophic and hyperplastic fibrous sheath that invaded the midpiece space, absence of the annulus, and a short midpiece containing a few disorganized and pale mitochondria. Of these cases, two presented absence of the central complex and radial spokes; another additionally presented absence of dynein arms and nexin bridges; and the other patient presented an intact annulus with a dysplastic fibrous sheath restricted to the principal piece with disorganized microtubule doublets. The patient with situs-inversus presented severe respiratory symptoms, with absence of dynein arms and nexin bridges. In conclusion, we present three cases with DFS associated with total sperm immotility, abnormal mitochondria, and absence of the annulus, central pair complex and radial spokes, of which one had in addition absence of dynein arms and nexin bridges. We also describe a patient, with total sperm immotility and a different presentation of DFS, as the annulus was present and the dysplastic fibrous sheath was restricted to the principal piece. These findings thus confirm the heterogeneity of the DFS condition. The changes observed in the patient with situs-inversus also further support previous observations.

Calcium Detection and Other Cellular Studies in the Esophagus and Crop of the Marine Slug Aglaja tricolorata (Euopisthobranchia, Cephalaspidea)

ABSTRACT To obtain more data about the digestive system of cephalaspideans, the esophagus and crop of Aglaja tricolorata were investigated using light and electron microscopy. In this cephalaspidean, the low epithelial cells of the anterior esophagus have a border of microvilli covered by a cuticle formed by acid polysaccharides. The esophageal pouches at the hind region of the anterior esophagus contain subepithelial secretory cells filled with electron-dense vesicles rich in neutral polysaccharides and proteins. A ciliated epithelium covers the base of the esophageal pouches. In the crop, the tall columnar epithelium is covered by a cuticle and forms many deep invaginations. The cuticle on the surface of the epithelium contains acid polysaccharides, but the material filling the lumen of the epithelial invaginations consists of neutral polysaccharides and proteins. Epithelial cells of the crop have a border of short microvilli and many mitochondria; lysosome-like bodies and some lipid droplets are present in the apical region. The elongated nucleus, numerous deep cell membrane invaginations and several hemidesmosomes are located at the basal region. Secretory cells are absent in the crop. Numerous large vacuolar cells are present in the connective tissue of the crop. With the pyroantimonate method, calcium was detected in the peripheral vesicles and central vacuole of these cells. WDS X-ray Intensity maps obtained by EPMA (Electron microprobe) confirmed the presence of calcium. This result supports the relationship between the vacuolar cells of aglajids and the calcium cells of pulmonate gastropods. In the posterior esophagus, epithelial cells are similar to the epithelial cells of the crop and secretory cells are also absent.

Morphological and Molecular Characterization of a Myxosporean Parasite Infecting the Skeletal Musculature of an Ornamental Fish Species from the Amazon River

1. Laboratory of Animal Pathology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, Porto, Portugal. 2. Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal. 3. Department of Sciences, High Institute of Health Sciences – North, CESPU, Gandra, Portugal. 4. Carlos Azevedo Research Laboratory, Federal Rural University of Amazonia, Belém, State of Pará, Brazil. 5. Edilson Matos Research Laboratory, Federal University of Pará, Belém, State of Pará, Brazil.

Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.

Histological, histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda, Cephalaspidea)

Due to its biological and systematic importance, the morphology and function of the male copulatory apparatus of Haminoea navicula, a Cephalaspidea gastropod mollusk, was investigated by light and electron microscopy. These systems are poorly understood in haminoids, but are often used in the taxonomy of the genus. In H. navicula, the male copulatory apparatus comprises the penis within a penial sheath, a seminal duct and the prostate with two lobes. The penis is a muscular structure with a tip covered by spikes formed by muscular cells. The penial sheath consists of muscular tissue folds lined by an epithelium. Below this epithelium, polysaccharide‐secreting cells and pigment cells were observed. A large number of vacuolar cells were found below the ciliated epithelium of the seminal duct. The proximal lobe of the prostate was formed by tubules that could be divided in basal, middle and apical zones, containing cells that secrete polysaccharides and proteins. The tubules of the prostate distal lobe contained a single type of secretory cells with vesicles that were stained by histochemical techniques for detection of polysaccharides and proteins. Ciliated cells were present along the tubules in both lobes of the prostate. This study revealed a complex prostate with five types of secretory cells, which secrete substances that should be involved in the maintenance and eventually also in the maturation of spermatozoa. A comparison with previous publications, shows that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non‐European species attributed to this genus.

Ultrastructure and molecular cytogenetics of human metaphase II oocytes with granular vacuoles

*Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar of University of Porto, Lg Prof Abel Salazar 2, 4009-003 Porto, Portugal **Centre for Reproductive Genetics Alberto Barros, Av. do Bessa 591 1o Dto. Frente, 4100-009 Porto, Portugal ***Department of Genetics, Faculty of Medicine of University of Porto, Alameda Prof. Hêrnani Monteiro, 4200-319 Porto, Portugal celia.cmsoares@gmail.com