Transport of iron and transferrin synthesis by the seminiferous epithelium of the rat in vivo

The transport of radioactive iron across the seminiferous tubules was analyzed in vivo by light-microscope quantitative radioautography. At 5 min after a single intratesticular injection of 55Fe-transferrin, a strong labeling of the basal aspect of the seminiferous epithelium was observed. Between 30 min and 2 h, the labeling on the basal aspect of the seminiferous epithelium decreased. This decrease was accompanied by a substantial increase of the radioautographic reaction over the cellular elements in the adluminal compartment. These results were consistent with the demonstration of 59Fe associated with meiotic spermatocytes and differentiating spermatids isolated by velocity sedimentation from testes injected with 59Fe-transferrin. Furthermore, after a single intratesticular injection of 59Fe-labeled human transferrin, radiolabeled rat transferrin was immunoprecipitated from homogenates of isolated tubules with a specific antibody and appeared as a single radioactive band on fluorographs of urea/polyacrylamide gels. Similarly, 59Fe-labeled rat transferrin but not 125I-transferrin was immunoprecipitated from rete testis fluids of testes infused with either 59Fe- or 125I-labeled human transferrin. Finally, the synthesis of testicular transferrin in vivo was demonstrated in fluorographs of immunoprecipitated transferrin after an intratesticular injection of 35S-methionine in rats whose livers were excluded from the general circulation by ligation of both the hepatic artery and the portal vein. Thus, our results demonstrated a unidirectional system of iron transport from the basal compartment of the seminiferous epithelium to the germ cells in the adluminal compartment involving two distinct transferrins, i.e., a serum transferrin and a testicular transferrin synthesized by the seminiferous epithelium.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.     

Kinetics of transferrin endocytosis and iron uptake by intact isolated rat seminiferous tubules and Sertoli cells in culture

The receptor-mediated endocytotic cycle of rat and human transferrin has been studied in intact, isolated rat seminiferous tubules and Sertoli cells in culture. Double-labeled [( 59Fe125I]) transferrin has been used to study the fate of transferrin and iron. Diferric transferrin binds to the tubules and the cultured Sertoli cells and is internalized. The iron remains inside, while the transferrin recycles and is released into the medium. Although, as reported before (Wauben-Penris et al., 1986), "extra" binding sites for human transferrin exist as compared to rat transferrin, this does not result in extra uptake of transferrin or iron. Both rat and human transferrin transport iron into the cells and recycle back to the surface, and do so with identical kinetics. A striking difference has been found between the mean efficient recycling times of the transferrin receptors in intact tubules (90 min) and in Sertoli cells in culture (21 min). Possible explanations of this difference are discussed. Light-microscopic autoradiography of [125 I]-labeled transferrin has revealed that the transferrin protein is excluded from the adluminal compartment, even after 21 h of incubation. This indicates that externally added transferrin itself does not deliver iron to the postmeiotic germ cells in intact, isolated rat seminiferous tubules.     

Intracellular pathways of endocytosed transferrin and non-specific tracers in epithelial cells lining the rete testis of the rat

Summary The uptake and pathway of different markers and ligands for fluid-phase, adsorptive and receptor mediated endocytosis were analyzed in the epithelial cells lining the rete testis after their infusion into the lumen of these anastomotic channels. At 2 min after injection, diferric transferrin bound to colloidal gold was seen attached to the apical plasma membrane and to the membrane of endocytic coated and uncoated pits and vesicles. The injection of transferrin-gold in the presence of a 100-fold excess of unconjugated diferric transferrin revealed no binding or internalization of transferrin-gold. Similarly, apotransferrin-gold was neither bound to the apical plasma membrane nor internalized by these cells. These results thus indicate the presence of specific binding sites for diferric transferrin. At 5 min, internalized diferric transferrin-gold reached endosomes. At 15 and 30 min, the endosomes were still labeled but at these time intervals the transferrin-gold also appeared in tubular elements connected to or associated with these bodies or seen in close proximity to the apical plasma membrane. At 60 and 90 min, most of the transferrin-gold was no longer present in these organelles and was seen only exceptionally in secondary lysosomes. These results thus suggest that the tubular elements may be involved in the recycling of transferrin back to the lumen of the rete testis. The coinjection of transferrin-gold and the fluid-phase marker native ferritin revealed that both proteins were often internalized in the same endocytic pit and vesicle and shared the same endosome. However, unlike transferrin, native ferritin at the late time intervals appeared in dense multivesicular bodies and secondary lysosomes. When the adsorptive marker cationic ferritin and the fluid-phase marker albumin-gold were coinjected, again both proteins often shared the same endocytic pit and vesicle, endosome, pale and dense multivesicular body and secondary lysosomes. However, several endocytic vesicles labeled only with cationic ferritin appeared to bypass the endosomal and lysosomal compartments and to reach the lateral intercellular space and areas of the basement membrane. The rete epithelial cells, therefore, appear to be internalizing proteins and ligands by receptor-mediated and non-specific endocytosis which, after having shared the same endocytic vesicle and endosome, appear to be capable of being segregated and routed to different destinations.     

Receptor-mediated and adsorptive endocytosis by male germ cells of different mammalian species

Summary The routes for adsorptive and receptor-mediated endocytosis were studied in vivo after microinjection of tracers into the lumen of the seminiferous tubules, and in vitro in isolated germ cells of different mammals. Cationic ferritin was located on the plasma membrane, in vesicles, in tubules, in multivesicular bodies and in lysosome-like granules of mouse spermatocytes. In these cells the number of multivesicular bodies varied during spermatogenesis. Spermatids and to a lesser extent residual bodies also performed adsorptive endocytosis. In the rat and monkey (Macaca fascicularis) diferric transferrin was specifically taken up by germ cells via receptor-mediated endocytosis. The labelling was observed subsequently in membrane pits, vesicles, endosome-like bodies and pale multivesicular bodies. A progressive decrease in the frequency of the labelling of the germ cells by transferrin-gold particles was observed from spermatogonia to spermatocytes and to early spermatids, which could indicate that iron is particularly required by germ cells during the mitotic and meiotic processes. Adsorptive and receptor-mediated endocytosis therefore occurs in all classes of germ cells. These endocytic processes are most probably required for germ cell division, differentiation and metabolism.     

Transferrin receptors and cation-independent mannose-6-phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes

The distribution of transferrin receptors (Tf-R) was determined in Clone 9 hepatocytes and compared to that of 215 kDa, cation-independent mannose-6-phosphate receptors (M6P-R) by double labeling. Cells were allowed to take up exogenous human transferrin (Tf) for 5 to 30 min, after which Tf, Tf-R, and M6P-R were localized by immunofluorescence using specific antibodies. All these proteins were found to be concentrated in the juxtanuclear or Golgi region. When Clone 9 cells were treated with NH4Cl to trap M6P-R in endosomes (Brown, W. J., J. Goodhouse, M. G. Farquhar: J. Cell Biol. 103, 1235-1247 (1986)), the distribution of the two receptors differed: Tf-R remained the same as in controls, but M6P-R were localized in large vacuolated endosomes. To carry out double labeling experiments at the electron microscope level, transferrin gold conjugates (Tf-Au) were prepared, and M6P-R were detected by immunoperoxidase labeling. Tf-Au binding to the cell surface was specific as it was reduced approximately 70 to 79% in the presence of excess native Tf. When Clone 9 cells were incubated with Tf-Au at 37 degrees C for 5 to 30 min, or binding of Tf-Au was carried out at 4 degrees C followed by warming to 37 degrees C, Tf-Au was found within a peripheral tubulovesicular network and within multivesicular endosomes that were not labeled with anti-M6P-R. Other multivesicular endosomes of similar size and morphology were heavily labeled for M6P-R but contained little or no Tf-Au. Tf-Au and M6P-R were also found in separate endosomes in cells treated with NH4Cl. Native Tf was localized in the same compartments as Tf-Au by immunoperoxidase labeling of both Clone 9 cells and mouse myeloma cells. We conclude that in Clone 9 hepatocytes, Tf/Tf-R internalized from the cell surface and M6P-R bearing newly synthesized lysosomal enzymes from the Golgi deliver their ligands to two different subpopulations of multivesicular endosomes. The endosomal subpopulation visited by Tf/Tf-R is known to correspond kinetically to early endosomes. The endosomal subpopulation heavily labeled for M6P-R presumably represent a later endosomal compartment which serves as the junction point where endocytosed ligands and newly synthesized lysosomal enzymes enroute to lysosomes meet.     

Newly synthesized proteins in seminiferous intertubular and intratubular compartments of the rat testis

Two-dimensional gel electrophoresis combined with autoradiography and Western blot procedures have been used to characterize newly synthesized proteins in testicular intertubular fluid (TIF) and seminiferous tubular fluid (SNF). Fluids were collected following in vivo and in vitro intratesticular injection of [35S]methionine into control and hypophysectomized adult rats. A discrete number of [35S]methionine-labeled proteins were detected within TIF and SNF. Their presence and relative abundance varied according to in vivo and in vitro labeling conditions. While two major blood plasma proteins, albumin and transferrin, were radioactively labeled after in vivo labeling, these two proteins were insignificantly labeled in samples collected after in vitro labeling. Three acidic proteins, possibly secreted by Sertoli cells (Mr = 72,000, 45,000 and 35,000), were more abundant in TIF samples collected after in vitro [35S]methionine labeling than after in vivo labeling. Incubated seminiferous tubules and TIF of hypophysectomized rats showed a decrease in [35S]methionine-labeling intensity of the Mr = 72,000 acidic protein, possibly reflecting changes in the seminiferous epithelium caused by pituitary hormonal deprivation. Autoradiographs of TIF and most remarkably, of SNF, showed many protein spots that suggested cell breakage and leakage during sample collection. Results of this study suggest that most albumin and transferrin found in TIF and SNF have an extratesticular origin and that proteins secreted by the Sertoli cell can gain access to both TIF and SNF.     

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.     

Endosomes and Golgi vesicles in adsorptive and fluid phase endocytosis

We studied with morphometric methods the endocytosis by pheochromocytoma cells of a conjugate of wheat germ agglutinin with ferritin (WGA-Ft) and of horseradish peroxidase (HRP). Quantitative studies indicated that WGA-Ft was cleared slowly from cell surfaces and that it was not recycled to the surface. Cells labeled with WGA-Ft for 15 min at room temperature were washed and incubated in medium containing HRP for 15 or 30 min at 37 degrees C. The greatest proportion of labeled vesicles and tubules contained only WGA-Ft (83.4% at 15 min and 85.3% at 30 min). A very small fraction of labeled vesicles and tubules contained only HRP (0.2% at 15 min and 0.9% at 30 min). Vesicles and tubules at the Golgi apparatus were labeled almost exclusively with WGA-Ft (97% at 15 min and 30 min); the rest had both labels. Most labeled lysosomes contained both labels (80.1% at 15 min and 80.8% at 30 min). Of the remainder more contained WGA-Ft alone (20% at 15 min and 10.9% at 30 min), then HRP alone (none at 15 min and 8.2% at 30 min). In contrast to the various and varying patterns of labeling with WGA-Ft and HRP of the other organelles studied, the vast majority of endosomes contained both markers (94.1% at 15 min and 100% at 30 min); the rest contained WGA-Ft only. These results demonstrate that endosomes are recipients of both fluid phase and adsorptive endocytosis markers; these findings are consistent with the hypothesis that endosomes mediate the sorting out and subsequent intracellular traffic of membrane bound and fluid phase markers. Cisterns of the Golgi apparatus did not contain WGA-Ft; in sharp contrast, when WGA-HRP was used, the cisterns of the Golgi apparatus consistently contained HRP.     

Role of sialic acid in the endocytosis of prosaposin by the nonciliated cells of the rat efferent ducts

The present study examines the mechanism of endocytosis of testicular prosaposin by the nonciliated cells of the efferent ducts. Testicular prosaposin is secreted by Sertoli cells into the lumen of the seminiferous tubules as a 70 kDa isomer where it binds to the tail of spermatozoa. In the efferent ducts, after dissociating from the plasma membrane of the spermatozoa, prosaposin is endocytosed by the nonciliated cells, presumably by receptor-mediated endocytosis. The initial step of receptor-mediated endocytosis usually results from the binding of a ligand's terminal oligosaccharide to a receptor on the cell surface. Thus, in the present study, several monosaccharides were injected in the lumen of the efferent ducts to compete with the binding and endocytosis of prosaposin. A quantitative electron microscopic approach was utilized and the number of gold particles, indicating anti-prosaposin immunoreactive sites, were scored over the various cell compartments including the plasma membrane, endocytic vesicles, early endosomes, and late endosomes. The length of the plasma membrane and the areas of endocytic vesicles, early endosomes, and late endosomes were measured with an image analyzer and the number of grains expressed per μm (plasma membrane) and μm² (endocytic vesicles/endosomes) respectively. The quantitative analysis was performed in untreated animals (controls) and animals treated with various sugars (i.e., glucose, galactose, mannose, mannose 6-phosphate, N-acetylglucosamine and N-acetylgalactosamine) injected into the lumen of the efferent ducts at a concentration of 20 mM. Sialic acid caused the greatest decrease in the labeling density of the endocytic elements. Mannose 6-phosphate also caused a decrease in labeling but to a lesser extent. Various amounts of sialic acid (0.02 mM, 0.2 mM, 2 mM, 20 mM, and 200 mM) showed that most of these concentrations produced a significant decrease in the labeling density of endocytic vesicles and endosomes. Moreover, Western blots of prosaposin isolated from seminiferous tubular fluids followed by glycan analysis with Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), revealed that this protein has sialic acid residues that are terminally linked to galactose and/or N-acetylgalactosamine ( α-NeuNAc-[2->6]-Gal and α-NeuNAc-[2->6]-GalNAc). These data indicate that testicular prosaposin is removed from the lumen of the efferent ducts by the noncialiated cells via a receptor that recognizes prosaposin's terminal sialic acid residues. Mol. Reprod. Dev. 51:156–166, 1998. © 1998 Wiley-Liss, Inc.     

Brefeldin A enhances receptor-mediated transcytosis of transferrin in filter-grown Madin-Darby canine kidney cells.

The effects of brefeldin A (BFA) on transferrin (Tf) transcellular transport, Tf receptor (TfR) distribution, and TfR-mediated endocytosis in filter-grown Madin-Darby canine kidney (MDCK) cells were studied. BFA (1.6 micrograms/ml) markedly enhanced the transcytosis of 125I-labeled Tf (125I-Tf) in both apical-to-basal and basal-to-apical directions; yet, BFA did not enhance the transcytosis of either native horseradish peroxidase (HRP) or membrane-bound HRP-poly(L-lysine) conjugates. Furthermore, this enhanced transcytosis of 125I-Tf was abolished either by competition with excess unlabeled Tf or by incubation at temperatures less than or equal to 25 degrees C. In addition, BFA treatment to MDCK cells: (a) increased 125I-Tf specific binding to the apical membrane and decreased 125I-Tf specific binding to the basal membrane; (b) decreased TfR recycling at the basolateral membrane; (c) altered the apical/basolateral distribution of TfRs in favor of the apical side; and (d) markedly increased 59Fe extraction, but not transcytosis, from apically endocytosed 59Fe-loaded Tf. These effects are consistent with a model in which BFA alters the traffic pattern of internalized Tf by decreasing basolateral TfR recycling, while diverting the nonrecycled fraction to the apical side of the cell. Our results indicate that, unlike the reported inhibition of polymeric IgA transcytosis (Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617-627), BFA can enhance the transcytosis of Tf in MDCK cells. Thus, by altering the intracellular traffic of ligand-receptor complexes, BFA can elicit either a decrease or an increase in transcytosis depending on the nature of the intracellular receptor processing.     

Fine structure of seminiferous tubules from prenatally irradiated rats

Summary The ultrastructure of the seminiferous tubules was studied in rats that had been subjected to whole body irradiation on the 19th day of gestation. The seminiferous tubules from 3 months-old irradiated animals are devoid of germ cells and contain only Sertoli cells. Compared with controls of the same age, the seminiferous tubule basal membrane is thickened and multilayered and several alterations are observed in the Sertoli cells. The most characteristic of these alterations are: (a) an abnormal number of nuclear heterochromatin clumps, (b) the presence of numerous cytoplasmic vacuoles and various sized lipid droplets, (c) elaborate interdigitations and junctions between adjacent cells, and (d) the presence of anomalous ectoplasmic specializations disposed perpendicularly to the Sertoli cell membrane.     

Laminin ultrastructural immunolocalization in rat testis during ontogenesis

The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18-20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages. Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.     

Sertoli cell function in albino rats treated with etoposide during prepubertal phase

Sertoli cell plays a key role in spermatogenesis. Many studies refer that this cell is not harmed by the majority of anticancer treatments known to cause damage to the testis. However, in the previous study we observed that etoposide, an efficient chemotherapeutic drug, provokes an increase in numerical density of the Sertoli cells. This phenomenon suggests that this cell was harmed by etoposide. Thus, we decided to investigate a possible direct action of etoposide on Sertoli cells analyzing the function of this cell and relating it with the integrity and damage of the seminiferous epithelium. Prepubertal albino rats received 5 mg/kg of etoposide for eight consecutive days and were sacrificed in different ages. The control groups received 0.9% saline solution. The testes were fixed in Bouin’s liquid for transferrin immunolabeling and testicular labeled tissue volume density measurement. Except for the younger rats, all the etoposide-treated rats showed diminution of transferrin immunolabeling in the seminiferous epithelium, and consequently, of total labeled testicular tissue volume density. We concluded that the diminution of transferrin labeling in the seminiferous epithelium was not associated with germ cell absence such as commonly reported. The results suggest etoposide impairs Sertoli cell function.     

Endocytosis in nonciliated epithelial cells of the ductuli efferentes in the rat

The nonciliated cells lining the ductuli efferentes presented three distinct cytoplasmic regions. The apical region contained, in addition to cisternae of endoplasmic reticulum and mitochondria, two distinct membranous elements. The tubulovesicular system consisted of dilated tubules connected to the apical plasma membrane and subjacent distended vesicular profiles. The apical tubules, not connected to the cell surface, consisted of numerous densely stained tubules of small size which contain a compact, finely granulated material. The supranuclear region, in addition to a Golgi apparatus and ER cisternae, contained dilated vacuoles, pale and dense multivesicular bodies, as well as numerous dense granules identified cytochemically as lysosomes. The basal region contained the nucleus and many lipid droplets. The endocytic activity of these cells was investigated using cationic ferritin (CF) and concanavalin-A-ferritin (Con-A-ferritin) as markers of adsorptive endocytosis; and native ferritin (NF), concanavalin-A-ferritin in the presence of alpha-methyl mannoside, and horseradish peroxidase or albumin bound to colloidal gold for demonstrating fluid-phase endocytosis. These tracers were injected separately into the rete testis, and animals were sacrificed at various time intervals after injection. At 1 min, CF or Con-A-ferritin were seen bound to the apical plasma membrane, to the membrane of microvilli, and to the membrane delimiting elements of the tubulovesicular system. Between 2 and 5 min, these tracers accumulated in the densely stained apical tubules and at 15 min in the dilated vacuoles. Between 30 min and 1 hr, the tracers appeared in multivesicular bodies of progressively increasing density, whereas at 2 hr and later time intervals, many dense lysosomal elements became labeled. The tracers for fluid-phase endocytosis showed a distribution similar to that for CF or Con-A-ferritin except that they did not bind to the apical plasma membrane, microvilli, or membrane delimiting the tubulovesicular system. At no time interval were any of the tracers observed in the abluminal spaces. Thus, the nonciliated epithelial cells of the ductuli efferentes are actively involved in fluid-phase and adsorptive endocytosis, both of which result in the sequestration of endocytosed material within the lysosomal apparatus of the cell.     

Endocytosis and intracellular transport of transferrin across the lactating rabbit mammary epithelial cell.

To study the transcytosis and segregation of ligand in the mammary epithelial cell, endocytosis and intracellular transit of human blood transferrin were followed in lactating rabbit mammary epithelial cells. Human transferrin labeled with biotin added to an incubation medium was bound to the basal membrane of mammary epithelial cells and carried across the cell to the lumen of the acini within 5-60 min. At the same time, biotinylated human transferrin accumulated at the apex of the cell. After incubation with human transferrin labeled with colloidal gold, label was detected inside endosome-like structures, vesicles and saccules of the Golgi apparatus, and inside the lumen within 2-5 min. A significant label accumulated at the apex of the cell after 30-60 min. Biotin labeling did not modify the time of transit of human transferrin, as attested by comparison with the time of transit of native transferrin. Human transferrin was never detected inside vesicles containing casein micelles. In contrast, rabbit milk transferrin was immunocytochemically detected inside vesicles containing casein micelles. These results indicate that transcytosis of human transferrin follows a pathway different from vesicles that carry casein micelles.     

Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half- life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR- gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome- like elements. Extracting ATR-gold complexes with Triton X after a 30- min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.     

In situ demonstration of both TUNEL-labeled germ cell and Sertoli cell in the cimetidine-treated rats

Cimetidine has caused dysfunction in the male reproductive system. In the rat testis, intratubular alterations and loss of peritubular tissue due to peritubular myoid cell death by apoptosis have been recently shown. Thus, the aim of this study is to evaluate which cells of the seminiferous epithelium have been affected and/or died by apoptosis after the treatment with cimetidine. For this purpose, an experimental group containing five male albino Wistar rats received intraperitoneal injections of cimetidine (50 mg/kg body weight) during 52 days. The testes were fixed with 4% buffered formaldehyde and were embedded in paraffin. For detection of DNA breaks (apoptosis) in the cells of the seminiferous epithelium, the testicular sections were treated by the TUNEL method (Apop-Tag Plus Peroxidase Kit). In the tubules affected by cimetidine, altered peritubular tissue, including the presence of TUNEL labeling in the myoid peritubular cells, were usually found. In these tubules, the seminiferous epithelium exhibited low density of germ cells and TUNEL-positive labeling in the germ cells of the basal compartment. The concomitant staining in both germ cells of the basal compartment and late spermatids suggest a sensitivity of these cells in the damaged tubules. Besides germ cells, TUNEL-positive Sertoli cells were also found in the injured seminiferous tubules. Thus, a relationship between dying germ cells and Sertoli cell damage and/or death must be considered in tubules where peritubular tissue has been affected by toxicants.     

Laminin ultrastructural immunolocalization in rat testis during ontogenesis

Summary The synthesis of one of the main glycoproteins of the basement membrane, the laminin, was demonstrated by ultrastructural immunolocalization during rat foetal (16th day to 20th day of gestation) and postnatal development of the testis. The lamina densa, part of seminiferous tubular basement membrane, is labeled uniformly at all studied stages. The lamina lucida is not well defined before the postnatal stages, at which times discrete immunostaining extends from the lamina densa to the adjacent seminiferous epithelial cells (spermatogonia and Sertoli cells). The extracellular matrix around the peritubular cells is not labeled before birth. Intracellular immunostaining was detected as early as the 16th day of gestation in both Sertoli cells and cells around the seminiferous tubules which will transform later into peritubular cells. It was located in rough endoplasmic reticulum (RER) cisternae and secretory vesicles. After 18–20 days of postnatal life, the immunostaining faints progressively. Some positive material is seen in the RER of the gonocytes at all studied stages.Sertoli cells and peritubular cells are the main producing cells of laminin after the 16th of gestation. The laminin secreted by gonocytes may play an important role in adhesion of gonocytes to the lamina densa and adjacent Sertoli cells before their transition from basal compartment to adluminal compartment.     

Ultrastructural study of the Sertoli cell and the limiting membrane in the seminiferous tubule of the adult cryptorchid rat

Cryptorchidism was simulated in 13-15-day-old rats by severing the gubernaculum testis and fixing the testis to the abdominal wall. Ultrastructural examination of the testis was made 100 days after birth when a number of modifications to the seminiferous tubules were noted. Germ cells were scanty, with only occasional spermatogonia and primary spermatocytes persisting. The nuclei of Sertoli cells were regular and oval or indented in shape. Their cytoplasm was characterized by a rich smooth endoplasmic reticulum, lipid inclusions and mitochondria with tubulo-vesicular cristae indicative of stero?dogenic activity. The decrease in the number of the germ cells induced a membrane rearrangement with numerous tight junctions and interdigitations between the Sertoli cells. Sertoli cell-specific junctional complexes were very extensive. The lamina propria of the seminiferous tubule appeared thickened and folded and the multilayered basal lamina had complex folds. After fixation with glutaraldehyde containing lanthanum, the latter substance was identified in the basal intercellular spaces of the seminiferous tubules indicating that the blood-testis barrier remains functional in the intra-abdominal testis.