The mediastinum of the bovine testis

Summary The bovine testis has a central mediastinum consisting of longitudinally oriented rete channels and spacious lymph vessels, embedded in the mediastinal stroma. The latter represents a contractile-elastic unit and is composed of myofibroblasts, collagen bundles and accumulations of elastin, connecting the myofibroblasts. The dimension of the mediastinum varies in cross sections at different levels between 3.5 and 31.8 mm². In one cross section 30 rete channels and 30 openings of straight testicular tubules are encountered. Nearly 25% of the area is occupied by thinwalled, valveless lymph vessels. Arterial convolutes, interpolated between straight centripetal and straight centrifugal branches of the testicular artery flank the rete on all sides. It is concluded that the pulsation within these convolutes together with the contractile-elastic stroma promotes lymph and rete content in a caudo-cranial direction. Chordae retis as described by Roosen-Runge and Holstein (1978) for the human testis are a common feature in the bovine mediastinum testis. The rete channels are lined by a simple cuboidal or columnar epithelium. Short intraepithelial crypts are present and function as epithelial reserve for dilatation and expansion of the rate. The inventory of organelles is rather inconspicuous in the rete epithelium. The apical border bears short microvilli and gives a strong reaction for alkaline phosphatase. The basal cytoplasm contains many small to medium-sized electron-dense bodies and is site of a strong acid phosphatase reaction. The rete epithelium as a whole reacts strongly with leucine aminopeptidase, the marker enzyme of the testicular excurrent duct system. Many free mononuclear cells, mostly macrophages, are observed in the basal half of the rete epithelium.     

Three-dimensional organization of the plasmalemmal vesicular system in directly frozen capillaries of the rete mirabile in the swim bladder of the eel

Summary Several recent studies comparing chemically fixed and cryofixed endothelium have indicated that glutaraldehyde fixation may result in increases in the population of vesicles in the cytoplasm. Other reports based on ultrathin serial-section reconstruction of chemically fixed endothelium have revealed that the vesicular system is comprised of interconnected membranous compartments, which are ultimately continuous with either cell surface but do not extend across the endothelial cell. In this study, we have investigated the three-dimensional organization of the vesicular system in directly frozen, freeze-substituted capillaries of the rete mirabile from the swim bladder of the eel, specifically using the same block of embedded capillaries in which frozen capillaries had previously been found to contain less vesicles than chemically fixed capillaries. The results show that essentially all vesicles remain inter-connected with each other and are part of two separate sets of invaginations from the luminal and abluminal cell surface like in chemically fixed tissue. Any increase in vesicle number resulting from glutaraldehyde fixation does not affect the overall three-dimensional organization of the vesicular system in these endothelial cells.     

Structure of the Rete Mirabile in the Kidney of the Rat as Seen with the Electron Microscope

Electron micrographs of the rete mirabile in the medulla of the rat have revealed that the endothelium of the afferent and efferent vessels are markedly different in fine structure. The venous capillaries returning blood from the papilla are lined with a fenestrated endothelium much like that in the peritubular capillaries of the kidney. The arterial capillaries delivering blood to the papilla have an unperforated lining of overlapping endothelial cells with extremely irregular tapered margins. It is pointed out that the organization of particularly the latter vessels suggests that the functional capabilities of these retia go beyond those of a simple diffusion countercurrent exchanger.     

Loose connective tissue of rat rete testis

Summary Fine structure, postnatal development and reaction to efferent duct ligation of the loose connective tissue of the rat rete testis were studied by light and electron microscopy.The loose connective tissue of adult rats consists of elongate fibroblasts in a homogenous ground substance, together with some Leydig cells, lymphocytes, macrophages and mast cells. During postnatal development this tissue increases in amount, while the interstitial areolar tissue decreases. The looseness of the tissue becomes more evident between days 22 and 27, and may reflect an increase in hydration.Efferent duct ligation for 15 min to five days has no effect on the histological appearance of the tissue.     

High level of endostatin in epididymal epithelium: protection against primary malignancies in this organ?

Rete testis and epididymis are rare locations for primary tumors or metastasis. Assuming that this may be related to expression level of angiogenic inhibitors, we focused our study on the expression pattern of collagen 18/endostatin. In situ hybridization and immunohistochemistry for collagen 18 and endostatin were carried out on sections of human rete testis and epididymis as well as on epididymal adenoma and human testicular tissue with or without carcinoma in situ (CIS). In situ hybridization revealed strong expression of collagen 18 mRNA in rete testis, efferent ducts and epididymal duct. Immunostaining showed collagen 18 in epithelium and basement membrane as well as in blood vessels of rete testis. Further, in both efferent ducts and epididymal duct, collagen 18 was mainly localized in the basement membrane of these ducts and of the blood vessel wall. Endostatin immunostaining was localized in the epithelium of rete testis, efferent ducts and epididymal duct. This pattern of endostatin staining was absent in epididymal adenoma tissue while tumor associated blood vessels exhibited strong endostatin staining. No endostatin staining was detectable in normal germinal epithelium and CIS cells while Leydig cells exhibited strong endostatin staining. High endostatin expression in epididymis may protect this organ against tumor development. Gene therapeutic strategies providing high expression of endostatin in normal epithelia may be useful to prevent tumor development.     

Spatial topography of the excurrent duct system in the bovine testis

Summary The rete testis of the bull is situated within an axial mediastinum and consists of approximately 30 longitudinally arranged, anastomosing rete channels. At the cranial testicular pole all rete channels empty into a common space, the area confluens reds, which is subdivided by small septa and narrow chordae retis. The area confluens always contains numerous spermatozoa and is connected with the bulbous initial portions of the efferent ductules by short, often tortuous rete tubules. Since the connection between rete and efferent ductules is situated within the tunica albuginea, the bovine excurrent duct system is not provided with an extratesticular rete as in many other mammals.Straight testicular tubules merge from all directions to connect with superficial rete channels, but the inlets are not evenly distributed. In the periphery each straight tubule begins with a cup-like structure followed by a narrow stalk region and a heavily folded portion opening either immediately into a rete channel or into a tube-like lateral rete extension.In close contiguity to the rete testis lie extremely coiled arterial portions connecting the centripetal and the centrifugal branches of the testicular artery. Since intrinsic musculature is scarcely developed in the mediastinum, and transport of rete content relies primarily on massage due to external pressure changes, the pulsatile blood flow through these coiled arteries may influence conveyance processes within the rete testis.An intimate spatial association between area confluens reds and adjacent large, thin-walled lymph vessels may facilitate a transfer of androgens into the fluid of the rete testis.Supported by the Stiftung zur Förderung der wissenschaftlichen Forschung an der Universität Bern     

Changes in the Peritubular Tissue of Rat Testis after Cadmium Treatment

The present study demonstrates histological and immunohistochemical changes in the peritubular testicular tissue of rat testis after application of cadmium chloride. After 5-day cadmium exposure, advanced deterioration of the boundary testicular tissue, mainly oedema, disarrangement of collagen fibres and peritubular cells, dilatation and thrombosis of blood vessels were observed. Changes in the boundary tissue were accompanied with desquamation of the germinal epithelium. Immunohistochemically, positive reaction for α-smooth muscle actin and desmin in tunica media of large testicular blood vessels basically was not affected. No reaction for vimentin was seen in endothelial cells of blood capillaries, whereas positive reaction presented only these cells in large blood vessels. The myofibroblasts positively reacting for desmin and α-smooth muscle actin form a single incomplete layer in the lamina propria of seminiferous tubules. Vimentin reactivity in the myofibroblasts and in the supporting Sertoli cells as well as Leydig cells in damaged testicular tissue was not observed. An increase in fibroblasts and free inflammatory cells positive for vimentin in the peritubular space on the peripheric area of the testis was observed.     

Visualization of Intrapulmonary Lymph Vessels in Healthy and Inflamed Murine Lung Using CD90/Thy-1 as a Marker

Background

Lymphatic vessels play a pivotal role in fluid drainage and egress of immune cells from the lung. However, examining murine lung lymphatics is hampered by the expression of classical lymph endothelial markers on other cell types, which hinders the unambiguous identification of lymphatics. The expression of CD90/Thy-1 on lymph endothelium was recently described and we therefore examined its suitability to identify murine pulmonary lymph vessels under healthy and inflammatory conditions.

Methodology/Principal Findings

Immunohistochemistry with a monoclonal antibody against CD90.2/Thy-1.2 on 200 µm thick precision cut lung slices labeled a vascular network that was distinct from blood vessels. Preembedding immunostaining and electron microscopy verified that the anti-CD90.2/Thy-1.2 antibody labeled lymphatic endothelium. Absence of staining in CD90.1/Thy-1.1 expressing FVB mice indicated that CD90/Thy-1 was expressed on lymph endothelium and labeling was not due to antibody cross reactivity. Double-labeling immunohistochemistry for CD90/Thy-1 and α-smooth muscle actin identified two routes for lymph vessel exit from the murine lung. One started in the parenchyma or around veins and left via venous blood vessels. The other began in the space around airways or in the space between airways and pulmonary arteries and left via the main bronchi. As expected from the pulmonary distribution of lymph vessels, intranasal application of house dust mite led to accumulation of T cells around veins and in the connective tissue between airways and pulmonary arteries. Surprisingly, increased numbers of T cells were also detected around intraacinar arteries that lack lymph vessels. This arterial T cell sheath extended to the pulmonary arteries where lymph vessels were located.

Conclusions/Significance

These results indicate that CD90/Thy-1 is expressed on lymphatic endothelial cells and represents a suitable marker for murine lung lymph vessels. Combining CD90/Thy-1 labeling with precision cut lung slices allows visualizing the anatomy of the lymphatic system in normal and inflamed conditions.     

Rete testis and the adjacent seminiferous tubules during postembryonic development in mice

Testicular compartment that includes rete testis and the adjacent transitional zone (TZ) of seminiferous tubules has been examined only by light and electron microscopy until now. However, recent data suggest that adult Sertoli cells (SCs) located in this compartment are capable to commence active proliferation both in vitro and in vivo, and hence, are not completely differentiated. The present study is first to investigate mouse rete testis and TZ during the postembryonic development and is intended to determine new protein markers for cells of this compartment, the state of their differentiation, and also their proliferative activity. It was demonstrated that rete testis cells were stained for SC marker Wt1 transiently, until day 25 of postembryonic development, then the staining disappeared. Another SC marker Dmrt1 that involved in the process of SC differentiation was not expressed in the rete testis cells during the postnatal development and in the adult state. One more feature that distinguished rete testis cells from SCs was lower proliferative activity of rete testis cells in 2–6 days old mice. SCs from TZ expressed Wt1 at all ages examined. However, at earlier ages, they were heterogeneous on Dmrt1 expression, and only by day 25, Dmrt1 expression was completely disappeared from TZ SCs. It is interesting that on day 18 when SCs in seminiferous tubules complete differentiation and exit from cell cycle proliferation of TZ SCs was at significantly higher level. It is also showed that in 3D culture, Wt1+ cells isolated from rete testis and TZ of 60 days old GFP male mice were capable to form seminiferous tubules de novo in cooperation with testicular cells from 6 days old mice.     

The fine structure of the capillaries and their surroundings in the cerebral hemispheres of Myxine glutinosa (L.)

Summary The fine structure of blood capillaries and their surroundings in the hemispheres of the hagfish, Myxine glutinosa, is described. As in other species, one or more endothelial cells delinate the lumen. Where such cells meet, they show complicated interdigitations. At some places tight junctions (zonulae occludentes) are found.The cytoplasm pressents at places many glycogen particles, ergastoplasmic membranes and free ribosomes in addition to the usual organelles. In addition, the luminal surface gives origin to deep invaginations from which channels penetrate into the cytoplasm. Numerous, presumably pinocytotic vesicles ranging from 450–1000 Å in diameter are present in the endothelial cell cytoplasm. A special type of cytoplasmic tubules is also present. The basement membrane is very thick, and measures 1000 Å at the narrowest place.Only one type of glial cell is present. This cell has certain characteristics in common with astrocytes of mammals but is for certain reasons considered to be a primitive glial cell. All capillaries have a complete lining of glial elements. The glial processes in contact with the vessels vary in shape and size. Glia cell bodies also participate. Closed contacts are present between the glial cells, but also desmosomal attachments are observed.The finding that capillaries in the hemispheres of Myxine are entirely surrounded by processes or bodies of glial cells shows that even at the most primitive vertebrate level glial cells are essential for the transport of material from the blood into the nervous tissue. Addendum. After the paper was submitted for publication, Dewey and Barr have considered the possible role of the zonulae occludentes in electronic coupling between cells [J. Cell Biol. 23, 553–585 (1964)]. The reader is referred to this paper.     

Morphological study by scanning electron microscopy of the lymphatic vessels of the guinea pig

The purpose of this study was to describe the morphology of the whole lymphatic way: from capillaries to thoracic duct including cisterna chili using scanning electron microscopy and Evan's technique. We observed the lymph vascular wall that is: the endothelial surface, the muscular layer and the adventitial one. All these vessels were covered by an endothelial surface, with raised nuclei and long cell axes oriented parallel to the direction of flow. The borders between adjacent endothelial cell were often seen and open junctions were noted in lymphatic capillaries. The technique we used, permitted the removal of connective tissue by HC1 hydrolysis, so that smooth muscle cells could be examined. The latter showed a great variety of aspects and a very irregular course. The adventitial layer was thin in capillaries and became complex in thoracic duct where collagen fibers and connective elements were seen.     

Vascularization and glomerular ultrastructure in the pig mesonephros

Summary Vascularization of the pig mesonephros was investigated in embryos 5–8 cm in length. Vascular injections with microfil were cleared and dissected; corrosion casts were studied under the scanning electron microscope (SEM). Perfusion-fixed tissue was used for SEM and transmission electron microscope (TEM) studies, including freeze-fracture specimens.The branches of one mesonephric artery carry up to 15 glomeruli. Several glomeruli occupy the same arterial branch, with very short afferent arterioles proper. The efferent vessels, frequently 2–5, leave the extensive vascular pole opposite the entering arteriole and split into peritubular capillaries radiating towards the superficial veins. These capillaries form vascular regions in the shape of flattened pyramids. Along its course, one nephron is supplied by vessels derived from 4–7 glomeruli. The nephrons have less vascular contact than in the definitive kidney.The ultrastructure of the single mesonephric vessels matches the metanephric counterparts. Epithelioid cells with renin granules are common in afferent arterioles, larger arteries, and efferent vessels. The lobulated glomeruli are up to 750 m long and flattened, showing the usual features of podocytes, mesangial cells, and an attenuated endothelium with fenestrations between 50 and 250 m. It partially retains its own basement membrane. There is no proximal mesangium.Supported by Deutsche Forschungsgemeinschaft.     

Studies on the testis of the camel (Camelus dromedarius). III. Histochemical observations

Synopsis The histochemical localization of carbohydrates and lipids and some oxidative, hydrolytic and steroid-linked enzymes has been studied in the testis of the camel with particular reference to the effect of the season on the distribution of these substances. PAS-positive, but diastase-resistant, material was seen mainly in the wall of blood vessels and in the boundary tissues of the seminiferous tubuli recti and rete testis. Clear cyclical changes were seen for glycogen in the lining epithelium of the seminiferous tubules. Glycogen was most abundant in early stages and very scanty or absent in the late stages of the cycle of the seminiferous epithelium. Numerous small lipid droplets were seen in the interstitial cells and towards the lumen of the seminiferous tubules that contain elongate spermatids or spermatozoa. Large lipid droplets were also demonstrable in the basal layer of the seminiferous epithelium and in the cytoplasmic debri. Alkaline phosphatase was demonstrated in the boundary tissues of the seminiferous tubules, tubuli recti and reti testis and in the cells bordering the lumen of the seminiferous tubules. Succinate and lactic dehydrogenases showed similar patterns of distribution in the interstitial elements and intratubularly.⁵-3 hydroxysteroid dehydrogenase was exclusively demonstrated in the interstitial cells. 17-hydroxysteroid dehydrogenase could not be demonstrated. The season seems to have no effect on the distribution of all these substances. The possible significance of all these findings is discussed.     

Statins enhance clonal growth of late outgrowth endothelial progenitors and increase myocardial capillary density in the chronically ischemic heart

Background

Coronary artery disease and ischemic heart disease are leading causes of heart failure and death. Reduced blood flow to heart tissue leads to decreased heart function and symptoms of heart failure. Therapies to improve heart function in chronic coronary artery disease are important to identify. HMG-CoA reductase inhibitors (statins) are an important therapy for prevention of coronary artery disease, but also have non-cholesterol lowering effects. Our prior work showed that pravastatin improves contractile function in the chronically ischemic heart in pigs. Endothelial progenitor cells are a potential source of new blood vessels in ischemic tissues. While statins are known to increase the number of early outgrowth endothelial progenitor cells, their effects on late outgrowth endothelial progenitor cells (LOEPCs) and capillary density in ischemic heart tissue are not known. We hypothesized that statins exert positive effects on the mobilization and growth of late outgrowth EPCs, and capillary density in ischemic heart tissue.

Methodology/Principal Findings

We determined the effects of statins on the mobilization and growth of late outgrowth endothelial progenitor cells from pigs. We also determined the density of capillaries in myocardial tissue in pigs with chronic myocardial ischemia with or without treatment with pravastatin. Pravastatin therapy resulted in greater than two-fold increase in CD31+ LOEPCs versus untreated animals. Addition of pravastatin or simvastatin to blood mononuclear cells increased the number of LOEPCs greater than three fold in culture. Finally, in animals with chronic myocardial ischemia, pravastatin increased capillary density 46%.

Conclusions

Statins promote the derivation, mobilization, and clonal growth of LOEPCs. Pravastatin therapy in vivo increases myocardial capillary density in chronically ischemic myocardium, providing an in vivo correlate for the effects of statins on LOEPC growth in vitro. Our findings provide evidence that statin therapy can increase the density of capillaries in the chronically ischemic heart.     

In situ labelling of vascular endothelium with fluorescent acetylated low density lipoprotein

Summary Acetylated low density lipoprotein is metabolized by a receptor-mediated process in endothelial cells. We have used the lipoprotein labelled with the fluorescent probe 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate to localize endothelial cells lining blood vessels. Following intravenous injection of the labelled lipoprotein, the vascular sinusoids and all other hepatic blood vessels were clearly labelled in cryostat sections of mouse liver. The endothelium of other organs such as brain, kidney, and testis was also brightly labelled. In addition, the lipoprotein was used to label the endothelium of bovine aorta, the vasculature in the chick chorioallantoic membrane and the vessels in a growing murine melanoma. These results demonstrate that the fluorescent labelled lipoprotein can be used forin situ labelling of the endothelium from large as well as small blood vessels in a variety of species.     

Isolation and culture of ram rete testis epithelial cells: structural and biochemical characteristics

Cultures of rete testis epithelial cell-enriched preparations from testes of adult rams have been investigated, and some of their properties have been determined. In monolayers, the cells form mosaic-like borders, and retain many ultrastructural features characteristic of rete epithelial cells in situ, including an indented nucleus with prominent heterochromatin clumps, short rod-shaped or round mitochondria that are easily distinguished from the elongated mitochondria of Sertoli cells, the presence of desmosomes, and few if any lipid droplets or vacuoles. Unlike Sertoli cell-enriched aggregates in culture, rete testis epithelial cell preparations do not form cytoplasmic extensions, and no associated germ cells are present. Rete cells in culture express cytokeratin and vimentin in the cytoskeleton, whereas Sertoli cells prepared from testes of adult rams contain vimentin but not cytokeratin. Both rete cells and Sertoli cells stain positively for laminin but not for fibronectin, Collagen Type I, or Collagen Type III. The rete cells synthesize and secrete several proteins into the culture medium, evident in gel electrophoresis patterns of radiolabeled proteins. This pattern is similar, but not identical, to that secreted by Sertoli cell-enriched preparations. Rete cells in culture in the presence of serum continue to undergo mitotic division, but Sertoli cells do not. A variety of criteria were employed to estimate the relative numbers of Sertoli cells present in the rete testis epithelial cell-enriched preparations from testes of adult rams, including morphological and ultrastructural differences between the two cell types, and the presence of desmosomal proteins and cytokeratin in rete cells but not in Sertoli cells. The relative number of fibroblast-like cells was determined by measuring the expression of fibronectin and Collagen Type I, and an immunocytochemical probe for the detection of Factor VIII was used to estimate the degree of contamination by vascular endothelial cells. Using these markers, we determined that the rete testis epithelial cell-enriched preparations were about 93% pure. Primary cultures under defined conditions contained relatively few Sertoli cells (0.4%), but were contaminated to a larger extent by fibroblast-like cells (approximately 4%) and by endothelial cells (about 3%). The possible functions of rete testis epithelial cells are discussed herein.     

Physiologie de la Barrière Sang-Testicule

There is evidence for the existence of a barrier between the blood and the lumina of the seminiferous tubules, from the uneven coloration of the testis after injection of some dyes, from the distribution of some radioactive markers, from the composition of the fluids from the rete testis and the seminiferous tubules, from the rate of penetration of various substances into these fluids, and from the presence of specialized junctions between the Sertoli cells, which block the penetration of lanthanum and other electron-opaque markers into the tubules. This barrier develops only at the time of puberty. However, the endothelial cells in the testis share certain characteristics with the endothelial cells of the brain, which form the blood-brain barrier. Also, the peritubular tissue has a specific transport system for urea, and these two tissues may also regulate the entry of substances into the testis. The barrier remains effective in some circumstances where spermatogenesis is disrupted, but it is less effective outside the breeding season in seasonal breeders. There are also some treatments which break down the barrier and disrupt spermatogenesis. Spermatogonia injected into the rete must pass through the barrier to re-establish spermatogenesis in infertile testes, but leukaemic cells injected into the rete can also pass from the lumen of the tubules into the interstitium, where the disease then recurs.     

Dynamic Changes in Endothelial Cell Adhesion Molecule Nepmucin/CD300LG Expression under Physiological and Pathological Conditions

Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4⁺ T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.     

The ultrastructure of lymphatic valves in the adult rabbit lung

Summary An electron microscopic investigation has revealed that the pulmonary lymphatic valves of adult rabbits are not simple duplicatures of the lymphatic vessel wall. They consist of an uninterrupted central connective tissue core, covered on both sides with a single layer of flattened endothelial cells. Near their insertion in the lymphatic vessel wall, the connective tissue core reveals a distinct thickening being composed mainly of collagen bundles. In the other parts it contains mainly elastic fibers and fine filaments, enclosing also some rather peculiar connective tissue cells. Nervous and muscular elements were not observed. The endothelium is continuous and exhibits no open junctions. The valvular basement membrane is better developed than in lymphatic capillaries. The endothelial cells contain numerous cytoplasmic filaments which might be endowed with contractile properties. The nuclei of the endothelial and the connective tissue cells are irregularly spaced and frequently clustered near the free edge of the valve.These ultrastructural features suggest that the function of the lymphatic valves is mainly passive. They are firmly inserted in the lymphatic vessel wall by collagen fibers and their moving parts are slender and elastic. Their endothelium appears relatively impermeable and is firmly attached to the subjacent connective tissue.This study has been supported by a grant from The Council for Tobacco Research—U.S.A.. We thank Professor Robert C. Rosan (Saint Louis University—U.S.A.) for expert advice, R. Janssens for technical, G. Pison and St. Ons for photographic and N. Tyberghien for secretarial assistance.