Isolation and characterization of epididymal epithelial cell plasma membranes

Sperm maturation and storage occur in a unique milieu created in large part by the epididymal epithelium. To learn more about the interaction of the epididymal epithelial cell with both luminal and systemic environments, we now report on the preparation and characterization of epididymal epithelial cell plasma membranes. A preparation enriched for epididymal epithelial cell plasma membranes was isolated from collagenase-digested epididymal tubule fragments by hand-Dounce homogenization, differential centrifugation, and sucrose gradient centrifugation. The final membrane fraction was enriched 11-fold for the plasma membrane marker 5'-nucleotidase; 2.6-fold for the lysosomal marker acid phosphatase, and 3-fold for the Golgi marker thiamine pyrophosphatase. No enrichment was observed for mitochondrial or endoplasmic reticulum enzyme markers. Specific and saturable transferrin-binding activity was also detected in the final preparation. Electron microscopy revealed the presence of vesicles and sheets of membranes as well as an occasional Golgi apparatus. The plasma membrane fraction was used to generate monoclonal antibodies. Of 102 wells exhibiting growth, 12 were positive by immunofluorescent staining of frozen sections. Ten of these recognized determinants in epithelial cells, and 2 stained peritubular smooth muscle cells. Most of the epithelial cell-specific antibodies stained brush border alone or in combination with the basolateral plasma membrane. Three antibodies stained the Golgi apparatus. Most antibodies were specific for particular epididymal regions, 3 also recognized determinants in the kidney, and 1 stained residual bodies in the testis.     

Steroidogenic characteristics of in vitro cultured epididymal epithelial cells of the rat

The paper presents the steroidogenic features of cultured epithelial cells of rat epididymis and their ability to synthesize steroid hormones. The cytoplasm of epididymal epithelial cells accumulated lipid droplets and contained active enzymes of steroidogenesis. Numerous mitochondria with lamellar cristae occurred near lipid droplets. Frequently, mitochondria formed a direct contact with lipid droplets and smooth endoplasmic reticulum. The hormone assay showed that the epididymal epithelial cells cultured without dihydrotestosterone synthesized and released the following steroids: dehydroepiandrosterone (DHEA), testosterone (T) and 17beta-estradiol (E). The levels of DHEA and T were very low. The concentration of E detected in media of cultured epididymal epithelial cells exceeded many times the concentration of E in control media. The cytoplasmic presence of organelles and enzymes that participated in the steroid synthesis indicated their similarity to steroidogenic cells. Epididymal epithelial cells were capable of moderate in vitro synthesis of androgens. It cannot be excluded that steroidogenesis in the cultured epididymal epithelial cells is maintained to sustain 17beta-estradiol synthesis pathways.     

In vitro culture of epithelial cells from the caput, corpus, and cauda epididymis of Sus domesticus

This work describes a protocol to culture epididymal epithelial cells from the caput, corpus, and cauda regions of Sus domesticus. Epididymal epithelial fragments were obtained by dissection and enzymatic digestion with collagenase. About 30 epididymal fragments from each epididymal region were cultured in 24-well culture plates with supplemented RPMI-1640 medium at 37 degrees C, 5% CO2 in air, and 100% humidity. A confluent monolayer of polygonal and tightly packed epithelioid cells from the three epididymal regions was obtained after 12-16 days in culture and maintained in vitro for more than 60 days. The proportion of epididymal epithelial cells in these cultures was assessed by immunofluorescent staining for cytokeratins. Throughout the 2 months of culture, about 80% of the cells were cytokeratin-positive. Electron microscopy observations indicated that cultured cells from caput, corpus, and cauda epididymal regions were tightly adhered to each other by junctional complexes and that stereocilia were present in their apical membranes. Moreover, the presence of an extensive rough endoplasmic reticulum, Golgi apparatus and numerous vesicles in the cytoplasm suggested that cultured cells maintained secretory and absorptive activities. These results show that the epididymal epithelial cells in culture from S. domesticus retain some fundamental features that characterize the epididymal epithelium in the intact organ. This system might be a valuable tool for studying the mechanism of sperm maturation in vitro, including epididymal cell secretions and the analysis of regional differences.     

Trichloroethylene exposure elicits damage in epididymal epithelium and spermatozoa in mice

We have investigated the toxic effects of trichloroethylene (TCE) on the epididymis and epididymal sperm in mice. Mice were exposed to TCE (1000 ppm) by inhalation for 6 h/day for 5 days/week for 1 to 4 weeks. Segments of the epididymis (caput, corpus and cauda) were examined by light and electron microscopy. At the light microscopic level, degeneration and sloughing of epithelial cells were evident as early as 1 week after TCE exposure, and were most pronounced after 4 weeks. Such epithelial damage was observed in the caput, corpus and cauda regions of the epididymis. Ultrastructural observations revealed vesiculation in the cytoplasm, disintegration of basolateral cell membranes, and sloughing of epithelial cells. Sperm were found in situ in the cytoplasm of degenerated epididymal cells. Additionally, a large number of sperm in the epididymal lumen exhibited abnormalities including malformation of head and tail components. Our results demonstrated that exposure to TCE by inhalation causes damage to the epididymal epithelium and sperm.     

Structural and functional aspects of cultured epididymal epithelial cells isolated from pubertal rats

Acidic epididymal glycoprotein (AEG) and androgen-binding protein (ABP) antisera were used to study functional activities of primary cell cultures of the epididymal epithelium of 20--23-day-old rats. Extensive AEG immunoreactivity was associated with almost all epithelial cells of the distal caput, corpus and cauda epididymidis. ABP immunoreactivity was solely confined to some epithelial cells of the caput epididymidis. AEG and ABP immunoreactive cells were identified as principal cells. Morphological studies of enzymically dispersed aggregates of the epididymal epithelial cells showed that stromal cells were satisfactorily removed and that cell aggregates consisted of a predominant population for cells displaying the morphological characteristics of principal cells. Scanning and transmission electron microscopic studies of cultured epididymal epithelial cells in monolayers demonstrated that microvilli and pit-like invaginations of the cell surface were preserved during the first 7--10 days of culture and then gradually disappeared. Other characteristic subcellular structures such as Golgi apparatus and rough endoplasmic reticulum cisterna were preserved. Electrophoretic analysis of [35S]methionine-labelled secretory polypeptides released by epididymal epithelial cells into the culture medium demonstrated a distinct protein band pattern which differed from that observed in the medium of cultured rat Sertoli cells. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually immature rats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell function.     

Electron microscopic observations of young rat liver

Summary Electron microscopic observations on normal liver tissue of four-day-old rats reveal the presence of numerous lamellar structures (lamellar bodies). These can be contained within parenchymal cells or in bile canaliculi, Disse's space, and in the lumen of blood sinusoids. Such bodies can also be found in Kupffer and endothelial cells.The lamellar bodies within hepatic cells are generally seen in very intimate relation to glycogen particles and lipide droplets, but in some to agranular endoplasmic reticulum and Golgi membranes as well.On the basis of this intimate relation to intracellular glycogen granules and lipide droplets, it is presumed that lamellar bodies represent a special intermediate stage in carbohydrate and lipide metabolism.Discontinuities in the endothelial layer of intrahepatic sinusoids are described.This work was supported in part by a N.A.T.O. research fellowship of the Consiglio Nazionale delle Ricerche, Roma.Assistant Professor in the Department of Veterinary Anatomy, Histology and Embryology (Dir.: Prof. A. de Girolamo), University of Naples, Naples, Italy.     

Morphological and morphometric changes and epithelial apoptosis are induced in rat epididymis by long-term letrozole administration

The epididymis is an organ that plays a key role in sperm maturation. The aim of this study was to examine the association between the chronic treatment of mature male rats with letrozole and morphological evaluation and morphometric values of epididymis as well as changes in the number of apoptotic cells in epididymal epithelium. Adult rats were treated with letrozole for 6 months and the epididymis weight, morphology, morphometric values and the number of apoptotic cells in the epithelium were examined. Long-term aromatase inhibition resulted in presence of intraepithelial clear vacuoles, hyperplasia of clear cells and a hyperplastic alteration in the epithelium known as a cribriform change. Moreover, changes in diameters of the epididymal duct and the epididymal lumen and changes in the epididymal epithelium height were observed. The number of apoptotic epithelial cells was increased in letrozole-treated group. It can be indicated that chronic treatment with letrozole can affect morphology, morphometric values and apoptosis in the epididymis of adult male rats. Observed changes are similar to that observed in the aging processes and may also be important for patients treated with aromatase inhibitors.Key words: Estrogen, aromatase, letrozole, epididymis, morphology, apoptosis     

Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication

In theepididymis, Cx43 forms gap junctions between principal and basal cellsbut not between adjacent principal cells. Cx30.3, 31.1, and 32 wereidentified in adult rat epididymis by RT-PCR, whereas Cx26 was presentin young rats. Postnatal development studies indicate that Cx26 mRNAwas detectable only in the caput-corpus region of the epididymis andthat levels increased by fivefold during the first 4 wk postnatally,when epithelial cells differentiate, and decrease to nondetectablelevels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout theepididymis in young rats and began to increase in the second and thirdweeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32were localized to the lateral plasma membranes between adjacentepithelial cells of the epididymis. Colocalization studies indicatethat Cx26 and Cx32 exist either independently of one another or cancolocalize along the lateral plasma membrane of epithelial cells inyoung rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.

     

The cellular mechanism for the formation of lamellar bodies in type-II pneumocytes in rat lungs]

Pneumocyte type II cells from lungs of native rats and of rats that inspired a hypoxic mixture of gases were investigated by transmission electron microscopy. In cells of experimental rats, membrane structures were found that well compare with lamellar bodies. Experimental results and analysis of literature allowed to put forward a hypothesis about the cell mechanism of formation of lamellar bodies from the spiral twisted membranes of the endoplasmic reticulum.     

PHOTORECEPTOR-PIGMENT EPITHELIAL CELL RELATIONSHIPS IN RATS WITH INHERITED RETINAL DEGENERATION : Radioautographic and Electron Microscope Evidence for a Dual Source of Extra Lamellar Material

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H³ and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.     

The lipofuscin in neuroglial cells of the optic nerve of Formosan rock-monkey (Macaca cyclopis)

The ultrastructure of lipofuscin granules in neuroglial cells of the optic nerve of the Formosan Rock-Monkey was investigated by electron microscopy. In the cytoplasm of astroglial cells, numerous irregular lipofuscin granules were characterized by the presence of large lipid droplets, small electron-dense pigment granules, and some lamellar structures. The lipofuscin granules of the oligodendroglial cells were composed largely of dense, coarse pigment granules, multilinear structures, and a few small lipid droplets. The lipofuscin granules in microglial cells were characterized by numerous lipid droplets in various sizes, small electron-dense pigment granules, and prominent lamellar structures. It was reported that the lipofuscin granules are wear-and-tear materials and products from the cells in lower functional activity. However, our observations suggest that the presence of lipofuscin granules in the neuroglial cells of the optic nerve is likely a characteristic product of active phagocytosis.     

In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin.

Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.     

Disturbances of energetic metabolism in rat epididymal epithelial cells as a consequence of chronic lead intoxication

Adult male Wistar rats were intoxicated with 1% lead acetate (PbAc) administered in drinking water for nine months, which amounts to a period five times longer than the duration of one spermatogenesis. There were mitochondrial ultrastructure disorders of epididymal epithelial cells observed in PbAc-treated rats; also a significant lead-induced decrease in ATP concentration in epididymal epithelial cells (by 32%, P < 0.05), Adenylate Energy Charge value (AEC) (by 8%, P < 0.05) and an increase in ADP (28.5%, P < 0.05), AMP (27%, P < 0.05) and adenosine (by 56%, P < 0.05). The results were measured using high performance liquid chromatography (HPLC) and detected even at low lead concentrations in whole blood (M:7.03 μg/dL; Q1–Q3: 2.99–7.65). The function of mitochondria in cultured epididymal epithelial cells of control and PbAc-treated animals were evaluated using fluorophores: Mitotracker Green FM and JC-1. After incubation with Mitotracker Green FM, we observed active mitochondria producing bright green fluorescence in the cytoplasm of cultured epididymal epithelial cells, both in the control group and the Pb-treated animals. Incubation of cultured epididymal epithelial cells of animals from both groups produced red-orange fluorescence with the mitochondrial JC-1 probe indicating mitochondria with high membrane potential (ΔΨm > 80–100 mV) and green fluorescence in the mitochondria with low membrane potential (ΔΨm <80 mV). The results showed that a chronic low-level exposure to lead, even without severe clinical symptoms of contamination, disrupted the ultrastructure and energy metabolism of mitochondria in epididymal epithelial cells.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Inflammatory changes in the epididymis after vasectomy in the Lewis rat

The epididymides of Lewis rats were studied at intervals up to 7 months after vasectomy, vasectomy followed 3 months later by vasovasostomy, or sham operations. Epididymal histology was related to testicular alterations and to serum antisperm antibodies as determined with an enzyme-linked immunosorbent assay. In vasectomy and vasovasostomy groups. 13 of 33 rats had testicular alterations, which consisted mainly of pronounced depletion of germ cells. Over half of the rats with testicular alterations also had severe epididymal lesions that included interstitial changes characteristic of an inflammatory response. These consisted of aggregates of mononuclear cells, including lymphocytes, plasma cells, and macrophages. The lumina of epididymides with interstitial changes contained polymorphonuclear leukocytes and/or macrophages. All animals with altered testes had greatly decreased numbers of epididymal sperm. In many instances, the lumen of the proximal cauda epididymidis was collapsed, and columnar cells of the epididymal epithelium contained many very large lysosomes. The distal cauda epididymidis was distended with sperm and debris. None of the rats that lacked testicular alterations showed epididymal changes. Mean serum antisperm antibody levels were significantly higher for rats with epididymal interstitial changes than for animals without such epididymal alterations. Infiltrations of inflammatory cells into the epididymal interstitium and lumen are part of the constellation of changes that occurs after immunization with testicular homogenates to produce experimental allergic orchitis. The observations reported here support the hypothesis that reproductive tract alterations after vasectomy in this model have an immune basis.     

Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.     

Diversity in the surface morphology of adjacent epithelial cells of the choroid plexus: an ultrastructural analysis

It is generally known that the luminal surface of the choroidal epithelial cells is covered with a luxuriant coat of slender microvilli and cilia. However, extensive ultrastructural studies on the surface morphology of choroidal epithelial cells are lacking. This study, therefore, is focused on the detailed surface morphology of the choroid plexus of the lateral ventricle of adult Wistar rats using transmission and scanning electron microscopy. The animals were anesthetized, perfused with 0.9% oxygenated saline followed by 3% gluteraldehyde and the choroid plexus was processed for routine electron microscopy. The results of the ultrastructural observations presented in this study show that even the neighboring choroidal epithelial cells may express distinct morphology. In addition to the usually described morphology of choroidal epithelial cells, in this study, the presence of cells with uniform small blebs, crenulated or doughnut shaped structures, large mature blebs, or cells with an extensive network of fibers were observed. Although, dissimilar surface morphology of adjacent choroidal epithelial cells may indicate their distinct functional status, further studies are necessary to understand the physiological relevance of the varied surface morphology of choroidal epithelial cells.     

Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK)

Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.     

Protein transport to the epididymal lumen

Summary Experiments were performed to clarify the debate over the entry of circulating proteins into the epididymal lumen by use of the marker horseradish peroxidase (HRP). Epididymal tubules from the caput epididymidis of the rat were immersed in medium TC 199 containing HRP (3.5 mg/ ml) for 5 min to 3 h at 33° C. Sections were examined for the presence of tracer within the epithelial cells by electron microscopy. From 5 min to 3 h, vesicles containing peroxidase reaction products were found throughout the cytoplasm of the principal cells. Vesicles occurred close to both the basal and apical membranes, and many were found opening into the interstitial space and lumen, depending on the length of incubation. By 5 min labelled vesicles were infrequently found in the apical part of the cells. Reaction product was observed in the epididymal lumen adhering to the microvilli from 30 min of incubation onwards. At all periods of incubation peroxidase was present at the base of the epithelium and between the cells, but it was never found within the tight junctional complexes, and no reaction deposits were found within epithelial cells of tubules incubated in the absence of peroxidase. It is concluded that large molecules leaving the capillaries may enter the epididymal lumen in the caput by means of fluid-phase endocytosis.