Adelina Zuasti

Early hematopoiesis and developing lymphoid organs in the zebrafish

In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5‐somite stage, about 11.5 hours post‐fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early blood‐forming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf. Dev Dyn 1999;214:323–336. 

Sex Differences in the Renal Changes Elicited by Angiotensin II Blockade During the Nephrogenic Period

The renin–angiotensin system plays an important role in renal development. However, it is unknown whether reduction in angiotensin II effects during the nephrogenic period leads to different renal alterations in males and females during the adult age. The aim of this study was to evaluate whether the role of angiotensin II on renal development is sex dependent and whether there are sex differences in blood pressure, renal hemodynamics, and severity of renal damage during adult life when nephrogenesis is altered by blocking angiotensin II effects. Newborn Sprague–Dawley rats were treated with an angiotensin II type 1 receptor antagonist (L-158.809; 7 mg/kg per day) during the first 2 weeks of life. At 3 months of age, changes in blood pressure, albuminuria, and renal hemodynamics were assessed, and stereological and histopathologic studies were performed. Blood pressure increased (127±0.5 versus 115±0.7 mm Hg in control rats; P<0.05) and nephron number decreased (37%; P<0.05) similarly in treated males and females. However, only males had an elevation in albuminuria (5.92±1.65 versus 0.33±0.09 mg per day in control rats; P<0.05), a fall in glomerular filtration rate (12.6%; P<0.05), and a significant decrease in papillary volume (42%; P<0.05). Mean glomerular volume, glomerulosclerosis, arteriolar hypertrophy, and tubulointerstitial damage in cortex and medulla were also higher (P<0.05) in angiotensin II type 1 receptor antagonist–treated males than in treated females. The results of this study suggest that females seem to be more protected than males to the renal consequences of reducing angiotensin II effects during renal development.

Granulopoiesis in the Head Kidney of Sparus auratus

The head kidney of Sparus auratus (teleost) is comprised of erythropoietic, granulopoietic and lymphopoietic cells. This study describes the ultrastructure of the granulopoietic series of cells. Heterophils, eosinophils and basophils were found at various stages of development. The most numerous cells are the neutrophils, the cells of the basophilic series being very scarce. The most outstanding characteristics of the neutrophil series are the eccentric and slightly segmented nucleus and cytoplasm with numerous, homogeneously dense granules. The eosinophils show a lobed nucleus and two types of granules in the cytoplasm. In the basophils, the cytoplasmic granules are large with fibrillar contents.

Transforming growth factor β1 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.

Proliferation and Apoptosis in Aged and Photoregressed Mammalian Seminiferous Epithelium, with Particular Attention to Rodents and Humans

Imbalances in the proliferation and apoptosis processes are involved in numerous epithelial alterations. In the seminiferous epithelium, normal spermatogenesis is regulated by spermatogonia proliferation and germ cell apoptosis, and both processes are involved in diverse pathological alterations of the seminiferous epithelium. Other physiological phenomena including aging and short photoperiod, in which apoptosis and proliferation seem to play important roles, cause testicular changes. Aging is accompanied by diminished proliferation and increased apoptosis, the latter occurring in specific states of the seminiferous cycle and considered the cause of epithelium involution. However, there is no clear evidence concerning whether proliferation decreases in the spermatogonia themselves or is due to an alteration in the cell microenvironment that surrounds them. As regards the factors that regulate the process, the data are scant, but it is considered that the diminution of c-kit expression in the spermatagonia, together with the diminution in antiapoptotic factors (Bcl-x(L))) of the intrinsic molecular pathway of apoptosis play a part in epithelial regression. A short photoperiod, especially in rodents, produces a gradual involution of the seminiferous epithelium, which is related with increased apoptosis during the regression phase and a diminution of apoptosis during recrudescence. Proliferative activity varies, especially during the total regression phase, when it usually increases in the undifferentiated spermatogonia. In other species showing seasonal reproduction, however, decreased proliferation is considered the main factor in the regression of the seminiferous epithelium. Little is known about how both phenomena are regulated, although data in rodents suggest that both the intrinsic and extrinsic pathways of apoptosis contribute to the increase in this process. In conclusion, regression of the seminiferous epithelium in physiological situations, as in many pathological situations, is a result of alterations in equilibrium between the proliferation and apoptosis of germinal cell types. However, both physiological phenomena showed important differences as regard proliferation/apoptosis and their regulation pathways, probably as a result of their irreversible or reversible character.

Characterization of corpora amylacea glycoconjugates in normal and hyperplastic glands of human prostate

SummaryIt has been shown that there are sugars in corpora amylacea, but little attention has been focused on the expression of glycoconjugates in corpora amylacea of normal and hyperplastic prostatic glands. The present study characterizes and compares the expression of glycoconjugates in corpora amylacea of normal and hyperplastic prostatic glands of elderly men by using alcian blue (AB) stain and lectin histochemistry. Corpora amylacea were larger and more numerous in hyperplastic glands compared to normal glands. The stain with AB revealed the presence of sulfated and carboxyl components in corpora amylacea. In hyperplastic prostatic glands the sulfur and acid contents of corpora amylacea were increased. Lectin affinities of corpora amylacea from normal prostatic glands demonstrated the presence of fucose, mannose, sialic acid, N-acetyl galactosamine and N-acetyl glucosamine residues. In the hyperplastic glands the lectin binding pattern of corpora amylacea was qualitatively similar to normal glands, but an increase in GalNAc, sialic acid, mannose and fucose residues was observed. Normal prostatic glands showed a weak to moderate content of mannose residues, and in contrast a strong GNA and Con-A staining was observed in hyperplastic glands. MAA and SNA affinities indicated that the content of sialic acid residues was higher in hyperplastic glands compared with normal prostatic glands. Also NAcGal residues were increased in hyperplastic glands. Luminal secretion, secretory cells and apical border of epithelium showed a similar although more intense Lectin-binding pattern as compared with corpora amylacea both in normal and hyperplastic prostatic glands. Lectin histochemistry shows that the glycoconjugates expressed in the glandular epithelium are similar to those found in corpora amylacea both in normal and hyperplastic glands. In addition, in hyperplastic glands, where the corpora amylacea are higher in size and more numerous, the reaction to lectins is more intense especially with mannose and sialic acid residues. The results suggest that corpora amylacea are originated at least in part from prostatic secretion.

Effect of ageing on the proliferation and apoptosis of testicular germ cells in the Syrian hamster Mesocricetus auratus.

The cellular mechanisms implicated in the atrophy of seminiferous epithelium in ageing are currently under debate, although recent reports suggest that apoptosis may be the primary mechanism implicated in aged germ cell loss. Other investigators have suggested that changes in spermatogonial proliferation are also involved. In the present work, the changes in proliferation and apoptosis in the seminiferous epithelium of aged (24 months) Syrian hamsters were examined in concert and compared with those in young (6 months) animals. Proliferation of germ cells was studied by bromodeoxyuridine labelling and apoptosis was assessed by transmission electron microscopy and in situ TUNEL labelling. Aged animals showed a significant decrease in the numbers of total and proliferating spermatogonia plus preleptotene spermatocytes per unit volume and per testis and in the proliferative index (24.8 +/- 1.6%) compared with young animals (30.8 +/- 1.2%) (P < 0.05). The number of apoptotic spermatogonia plus spermatocytes per unit volume and the apoptotic index were significantly higher in aged animals (1.51 +/- 0.23% v. 0.77 +/- 0.04%; P < 0.05). Apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. In aged hamsters, tubular degeneration could be classified into several categories, showing an increase of apoptotic cells in tubular cross-sections characterized by maturation arrest in comparison with all other types. Spermatogonial proliferation was also diminished as seen in tubular cross-sections showing hypospermatogenesis, sloughing off of germ cells and maturation arrest. The results obtained in the present study suggest that the decrease in the proliferation of spermatogonia and the increase in apoptosis constitute two consecutive mechanisms correlated with the ageing of the seminiferous epithelium.

Characterization of glycoside residues of porcine zona pellucida and ooplasm during follicular development and atresia

The glycoside residues (glycoconjugates, GC) of the zona pellucida (ZP) glycoproteins are important during the first phases of fecundation. Our aim in this work was to determine the lectin affinity pattern of porcine ZP in order to analyze the changes that take place during: (a) preantral folliculogenesis, (b) the follicular atresia process, and (c) antral growth. Several prepubertal and adult pig ovaries and different sized antral follicles were used. Conventional carbohydrate histochemical techniques and peroxidase and digoxigenin (DIG) lectins were used to reveal the acid groups and the glycosidic residues of the ZP. It was seen that the ZP forms in the preantral follicles throughout their growth period. In primordial and primary follicles, ZP in the process of formation showed neutral GC. SBA, RCA‐I, MAA, WGA lectins, and AAA after methylation‐saponification (MS) were positive in the ZP of primordial and primary follicles. The affinity for SBA, RCA‐I, MAA, and WGA increased in the multilaminar‐primary follicles and new affinities for UEA‐I and LFA were observed. After MS, AAA, SNA, PNA, and SBA reactivity was observed. The ZP of antral follicle oocytes of different sizes showed the same lectin pattern as multilaminar‐primary follicles. The oocyte ooplasm and the follicular fluid of large antral follicles showed less affinity for WGA and LFA lectins and less intensive staining with AB (pH 2.5). Atresia did not change the antral or preantral follicle oocyte ZP lectin pattern. In conclusion, the follicles showed substantial changes in their ZP glycosidic composition as they developed, especially, during the change from primary to multilaminar‐primary follicles. The ZP glycosidic composition showed no significant change during the growth of antral follicles and follicular atresia in our study. Mol. Reprod. Dev. 75: 1473–1483, 2008.