Ultrastructural changes in the follicular epithelium of Ceratophrys cranwelli previtellogenic oocytes.

In this work we carried out ultrastructural, autoradiographic and biochemical analyses of the follicular epithelium during C. cranwelli previtellogenesis. This study revealed that the follicular epithelium in early previtellogenesis is constituted of a single layer of squamous homogeneous cells. During mid-previtellogenesis two types of cells develop: dark cells and clear cells. The follicular dark cells are actively involved in the synthesis of RNA, which is transferred to the oocyte through the interface. In late previtellogenesis the dark cells show apoptotic characteristics such as chromatin condensation, DNA fragmentation and cytoplasm shrinkage. This process forms apoptotic bodies that seem to be engulfed by the oocyte. Our results show evidence that, during mid- and late C. cranwelli previtellogenesis, the follicular epithelium undergoes remodelling processes interacting with the oocyte.

Implication of gap junction coupling in amphibian vitellogenin uptake.

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.

Renal extracellular matrix alterations in lead-treated rats.

Although the nephrotoxic effects of lead are well documented, the subcellular mechanisms of its action on the kidney remain unclear. The aim of the present work was to investigate the effects of chronic lead exposure on the expression of laminin-1 and fibronectin in the kidney of lead-treated rats. Western immunoblotting of the kidney extracts revealed that experimental exposure to lead resulted in a marked decrease in the intensity of the bands corresponding to laminin-1 and an increase in the intensity of the band corresponding to fibronectin. Immunohistochemical studies demonstrated a weak labelling to laminin-1 and a strong labelling to fibronectin in all renal basement membranes together with a decrease in their thickness. Other ultrastructural alterations found were a diminution in the amount of endothelial fenestrae, an increased fusion of foot processes in epithelial cells of the glomerulus and the presence of intranuclear inclusion bodies in the proximal tubule cells. Lead intoxication might be responsible for the above alterations in the renal extracellular matrix that could play an important role in the pathogenesis of lead nephropathy.

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats.

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.

Effect of gap junction uncoupling in full-grown Bufo arenarum ovarian follicles: participation of cAMP in meiotic arrest.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Comparative study of vitellogenesis in the anuran amphibians Ceratophrys cranwelli (Leptodactilidae) and Bufo arenarum (Bufonidae).

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.

Amphibian Bufo arenarum vitronectin-like protein: its localization during oogenesis.

In the present study, we analyzed the localization of vitronectin-like protein in oocytes during oogenesis as well as in the serum and liver tissue of the amphibian Bufo arenarum. Vitronectin-like protein was purified from serum by heparin-affinity chromatography and showed to have the two biological properties in common with most animal vitronectins (VN): heparin binding activity and an RGD-dependent cell-spreading activity. SDS-PAGE of vitronectin-like protein revealed that it consists of two bands of 64 kDa and 72 kDa, while immunoblotting analyses showed that this protein strongly cross-reacts with two monoclonal antibodies against human VN. No immunofluorescent staining of vitronectin-like protein was observed in previtellogenic oocytes (stages I and II). In vitellogenic oocytes (stages III, IV and V) fluorescence was observed in the cortical cytoplasm localized in yolk platelets, extending concomitantly with the vitellogenic process. When we examined the yolk platelet formation pathway by immunoelectron microscopy, gold particles indicated that vitronectin-like protein was located on the yolk platelet precursors: multivesicular bodies and primordial yolk platelets. Gold particles also were seen sparsely distributed in all oocyte investing layers. The mean serum vitronectin-like protein concentration in amphibian animals was 127.8 +/- 11.6 micrograms/ml in adult males and 181.5 +/- 14.3 micrograms/ml in adult females. Serum vitronectin-like protein of males and females was susceptible to hormonal stimulation (17-beta estradiol). These results suggest that vitronectin-like protein is stored in the yolk platelets and may be involved in the later events of amphibian development.