Essential role of clusterin in pancreas regeneration.

Based on our previous observations that clusterin induction accompanies pancreas regeneration in the rat, we sought to determine if regeneration might be impaired in mice that lacked clusterin. We studied the impact of absent clusterin on morphogenic and functional features of regenerating pancreas. Clusterin induction was accompanied in the regenerating pancreas by a robust development of new lobules with ductules, acini, and endocrine islets in wild type after partial pancreatectomy. In clusterin knock-out mice, however, pancreatectomy resulted in a poor formation of regenerating lobule. In particular, regeneration of beta-cells was also significantly reduced and was associated with persistent hyperglycemia. Duct cells obtained from pancreatectomized clusterin knock-out mice exhibited impaired beta-cell formation in vitro; this was restored by administration of exogenous clusterin. We suggest that clusterin plays a critical role to promote both exocrine and endocrine regeneration following pancreas injury, as well as for in vitro beta-cell regeneration.

COVID-19 and andrology: Recommendations of the French-speaking society of andrology (Société d'Andrologie de langue Française SALF).

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) metamorphosed our medical practice. In early June 2020, more than 6,400,000 COVID-19 (coronavirus-19 disease) cases were diagnosed across the world and more than 380,000 deaths were linked to COVID-19. Many medical symptoms of COVID-19 were reported. We will focus, here, on potential impacts of COVID-19 on men's andrological health. Our society (French-speaking society of andrology, SALF) also emitted some recommendations in the andrological management of men infected by SARS-CoV-2. First, considering the fever and the potential presence of SARS-CoV2 in semen, SALF recommends waiting for 3 months (duration of one spermatogenesis cycle and epididymal transit) before re-starting ART in the case of men diagnosed COVID-19 positive. Whatever the nature of testosterone and COVID-19 relationships, we recommend an andrological examination, sperm parameters, and hormonal evaluation at the time of the COVID-19 is diagnosed, and several months later. Furthermore, we are concerned by the potential morbid-mortality of the COVID-19, which mainly affects men. This "andrological bias", if proven, must be reduced by specific andrological diagnosis, therapeutic and prophylactic measures. Research in this direction must be substantiated and financially supported over the next few months (years).

Le SRAS-CoV-2 (nouveau coronavirus ou coronavirus numéro 2 responsable du syndrome respiratoire aigu sévère) a métamorphosé notre pratique médicale. Début juin 2020, plus de 6,400,000 cas de COVID-19 (maladie à coronavirus 2019) ont été diagnostiqués dans le monde et plus de 380,000 décès ont été reliés à cette maladie. De nombreux symptômes médicaux de cette infection virale ont été signalés. Nous nous concentrerons, ici, sur les impacts potentiels de COVID-19 sur la santé andrologique des hommes. Notre société (Société d'andrologie de langue Française, SALF) émet ici quelques recommandations dans la prise en charge andrologique des hommes infectés par le SRAS-CoV-2. Tout d'abord, compte tenu de la fièvre et de la présence potentielle du SRAS-CoV2 dans le sperme, la SALF recommande d'attendre 3 mois (durée d'un cycle de spermatogenèse et transit épididymaire) avant de recommencer les techniques d'assistance médicale à la procréation pour les hommes diagnostiqués COVID-19 positifs. Quelle que soit la nature des relations entre la testostérone et l'infection à SARS-CoV-2, nous recommandons un examen andrologique, un examen des paramètres du sperme et une évaluation hormonale au moment du diagnostic de l'infection, ainsi qu'à distance (3­6 mois plus tard). De plus, nous sommes préoccupés par la morbidité et la mortalité potentielles de l'infection COVID-19, qui touche principalement les hommes. Ce "biais andrologique", s'il est. prouvé, doit être réduit par un diagnostic andrologique spécifique et des mesures thérapeutiques et prophylactiques. La recherche dans ce sens doit être étayée et soutenue financièrement au cours des prochains mois (années).

Ontogenesis of glomerular basement membrane: structural and functional properties.

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.

Transendothelial transport of serum albumin: a quantitative immunocytochemical study.

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, plasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68% of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNP-tagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells.

The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well-developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post-Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.

The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments.

The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH-terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.

[Environment and lifestyle: Impacts on male fertility?] / Toxiques, mode de vie, environnement : quels impacts sur la fertilité masculine ?

In this last century, an increase of men infertility has been registered. It has been suggested that environmental factors could a negative impact over sperm quality. Among these factors, impact of environmental toxicant has been spread by media. In this review of scientific literature, we identify several environmental factors that could impact men fertility in a negative way. These factors are tobacco, marijuana, weight, body mass index, heat, nutritional state, electromagnetic waves and altitude. For each of these factors, the impact over men fertility, their mechanism, as well their influence over the use of Assisted Reproductive Technics are reported.

Hyperglycemia and microangiopathy in the eel.

When female eels, fasting and sexually mature, were progressively adapted to cold water (2-4 degrees C), their blood sugar concentration rose to values averaging 600 mg/dl. Control eels, kept in warm water (18-20 degrees C), had a mean blood sugar concentration of 100 mg/dl. After a period of 5-6 mo, the blood capillaries of the rete mirabile in the swimbladder were examined in both control, low blood sugar eels, and in cold-adapted, high blood sugar eels. In the latter, the basal laminae of the capillaries were thickened; their amino acids composition was altered and the in vitro glucose carbon incorporation into basal laminae glycoproteins was increased over a wide range of medium glucose concentrations. Furthermore, the diffusion capacity of the rete, as measured with tracer molecules during steady-state conditions in a countercurrent perfusion system, was increased in the hyperglycemic eel. It is concluded that chronic hyperglycemia in the cold-adapted eel is associated with a microangiopathy characterized by morphologic, biochemical, and functional alterations.

Morphometrical and immunocytochemical characterization of peri-insular and tele-insular acinar cells in the rat pancreas.

Morphometrical and immunocytochemical techniques have been applied in order to characterize the pancreatic acinar cells located in peri-insular and tele-insular regions of the pancreas. The results obtained, have shown that the acinar cells of the peri-insular regions are twice as large as those of the tele-insular. On the other hand, the volume density of all organelles, except that of the zymogen granules, remains constant implying that the larger the cell, the larger are its organelles. For the zymogen granules however, their volume density was found to be higher in peri-insular acinar cells. The immunofluorescence technique applied for the demonstration of amylase and chymotrypsinogen has confirmed the presence of an inhomogeneity in the staining. Acinar cells in peri-insular regions show a brighter fluorescent staining. At the electron microscope level, both amylase and chymotrypsinogen were demonstrated in all organelles of acinar cells involved in protein secretion. Quantitative evaluations demonstrate no major differences in the intensity of labeling per micron2 between organelles of peri-insular and tele-insular cells. These results put together demonstrate that peri-insular acinar cells contain higher amounts of secretory proteins because their organelles are larger and their zymogen granules are more numerous. The partition of the exocrine pancreas into peri- and tele-insular regions, confirmed herein through morphometrical and cytochemical techniques, is discussed in relation to the possible influence of the endocrine secretion arising from the islets of Langerhans on the surrounding acinar cells.

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches.

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracisternal crystals in pancreatic acinar cells: failure in the distinct aggregation of secretory proteins.

Mechanisms leading to the formation of crystalline inclusions in the cisternal space of the rough endoplasmic reticulum are poorly understood. This phenomenon was investigated in pancreatic acinar cells using two different experimental models: 1) Intraperitoneal injection of DL-p-chlorophenylalanine methyl ester, and 2) culture of isolated acinar cells within the Matrigel basement membrane in the presence of 2% dimethyl sulfoxide. Features and composition of induced crystals were analyzed by protein A-gold and lectin-gold cytochemistry, electron microscope autoradiography, electron energy loss spectroscopic imaging and energy dispersive X-ray analysis. Crystal formation occurred in ribosome partially free rough endoplasmic reticulum (RER) regions and was similar in both experimental protocols. The protein A-gold revealed the presence of nine major pancreatic enzymes in the crystals. However, the labeling intensities varied among enzymes with higher concentrations of amylase than chymotrypsinogen when compared to the secretory granules. Concanavalin A and Helix pomatia labelings were weak over the crystals and did not correspond to those of RER or secretory granules. Sulfur contents in crystals were lower than phosphorus and their ratio was opposite to the one found in secretory granules. Electron microscope autoradiography demonstrated incorporation of radiolabeled leucine and presence of newly synthesized proteins in the crystals. Furthermore, cells containing both crystals and secretory granules displayed silver grains in most of the cellular compartments involved in secretion. Thus, failure in the normal concentration and sorting process of secretory proteins leading to crystal formation includes changes in protein glycosylation and decrease of disulfide bond formation while retaining secretory capabilities.

Introduction of a high-resolution cytochemical method for studying the distribution of phospholipids in biological tissues.

A novel cytochemical method for the in situ, ultrastructural localization of phospholipids in biological tissues is reported. The method is based on the enzyme-gold approach (M. Bendayan: J. Histochem. Cytochem. 29, 531, 1981). Phospholipase A2 from bee venom was adsorbed on colloidal gold particles (PLA2-gold) and applied for the specific labeling of its substrate, sn3-glycerophospholipids. The binding and enzymic competence of the PLA2-gold complex were confirmed by in vitro, preembedding experiments with erythrocytes and a crude lung surfactant preparation. The substrate specificity of the probe was assessed by labeling Epon thin sections of pure phospholipids. To test the potential applications of the PLA2-gold complex, lung and pancreatic tissues were fixed with glutaraldehyde-osmium and embedded in Epon for transmission electron microscopy (TEM). They were also prepared for critical-point-drying fracture-label (CPD-FL) replicas and thin-section fracture-label (TS-FL) specimens. On TEM thin sections incubated with PLA2-gold, all cellular membranes were labeled. The labeling density over each membrane compartment, as quantitated in lung type II pneumocytes, was classified in order of magnitude as follows: a) nuclear membranes; b) outer mitochondrial membrane and rough endoplasmic reticulm (RER); and c) Golgi complex, mitochondrial cristae and plasma membranes. In lung alveoli, the phospholipid-rich surfactant material was intensely labeled. Labeling of lung thin sections from chlorphentermine-treated rats (phospholipidosis-inducing drug) further demonstrates the reliability of PLA2-gold to label phospholipids. CPD-FL replicas and TS-FL specimens further extended the TEM observations: nuclear membranes and RER were more intensely labeled than plasma membranes. In exocrine pancreatic cells, two distinct labeling patterns were found for secretory granule membranes: sparse and dense. The specificity and reliability of the labeling were confirmed through several control experiments. The studies performed thus demonstrate the great potential of the PLA2-gold technique as a new approach to the high-resolution study of phospholipid distribution and density among biological structures.

Studies on pancreatic acinar cells in tissue culture: basal lamina (basement membrane matrix promotes three-dimensional reorganization.

Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma.

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.

Immunocytochemical localization of a non-vitellogenin, estrogen-induced plasma protein in the hepatocyte of the American bullfrog, Rana catesbeiana.

Recently, a non-vitellogenin, estrogen-induced frog plasma protein of unknown function and site of synthesis, which has been given the temporary name, protein-RcX, was isolated and partially characterized by Mitchell et al. In the present study, the protein A-gold immunocytochemical technique was applied to investigate its site of secretion; this was found to be the hepatocyte of the estradiol-17 beta-treated adult, male American bullfrog, Rana catesbeiana. Specific immunolabeling for protein-RcX was present over those intracellular compartments involved in protein secretion, i.e., the rough endoplasmic reticulum, Golgi apparatus and secretory granules, in addition, lysosomes were also labeled. No specific labeling for this protein was observed on hepatocytes of normal, non-estrogen treated, adult, male bullfrogs. Further, the labeling was abolished when plasma containing protein-RcX was added to the antibody prior to incubation but remained when purified vitellogenin was added. These observations support the hypothesis that protein-RcX is a non-vitellogenin, estrogen-induced plasma protein which is synthesized and secreted in parallel with vitellogenin by the hepatocyte of the estrogen-treated frog.

Application of the protein A-gold technique for electron microscopic demonstration of polypeptide hormones.

The protein A-gold (pAg) technique enables the electron microscopic detection of antigenic material on thin sections of aldehyde-fixed and Epon-embedded tissue. Application of this technique to the endocrine pancreas and gastrointestinal mucosa using antisera against insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin, and neurotensin allowed these polypeptides to be localized in the secretory granules of the corresponding specific cell types. The small size of marker gold particles permitting the precise identification of the labeled organelles coupled to the high specificity of the binding represent further evidence that the protein A-gold technique can be used as a general cytochemical reagent for the visualization of antigen-bound antibodies on thin sections.