Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Improvement of spermatogenesis in adult cryptorchid rat testis by intratesticular infusion of lactate.

In order to test the hypothesis that a lack of energy could be a cause of germ cell death at high temperatures, cryptorchid rats testes were infused with lactate, delivered by osmotic pumps over 3-15 days. In cryptorchid testes, the spermatids and spermatocytes were lost between 3 and 8 days. In cryptorchid testes supplemented with lactate, elongated spermatids persisted in a few seminiferous tubules at Day 15. Elimination of round spermatids occurred progressively between 3 and 15 days, mostly at stage VIII. The loss of spermatocytes increased after 8 days, and 30% of seminiferous tubules still contained meiotic or meiotic plus spermiogenetic cells at Day 15. After 8 days, the chromatin of step 8 round spermatids was abnormal and nuclear elongation did not commence. The Sertoli cell cytoplasm that was retracted toward the basal compartment of the seminiferous epithelium could not hold the germ cells of the adluminal compartment. Therefore, attachment of germ cells to Sertoli cells and the supply of lactate seem necessary for the development of germ cells at high temperatures. The improvement in spermatogenesis in cryptorchid supplemented testes for several days is a new finding.     

Interrelationships between Sertoli cells and germ cells in the Syrian hamster

The interrelationships of the Sertoli cells and germ cells in the Syrian hamster were examined using the electron microscope. Demosome-like junctions were observed attaching Sertoli cells to spermatogonia and spermatocytes. In the region of the junctions dense plaques lay on the cytoplasmic surfaces of the plasmalemma of the opposing cells. Sertoli cell cytoplasmic filaments converged in the area of the junctions and inserted into the subsurface densities. Filaments were not observed associated with the subsurface densities of the germ cells. In the region of the junctions a 15...20 nm gap, filled with an attenuate amorphous substance, separated the plasmalemmata. Another attachment device termed "junctional specialization" occurred between Sertoli cells, and preleptotene spermatocytes and all successive developmental steps in the germ cell line in the hamster. The junctional specializations consisted of a mantel of Sertoli cell cytoplasmic filament lying subjacent to the Sertoli cell plasmalemma and an opposed cisterna of the endoplasmic reticulum. In stages VII-VIII preleptotene supermatocytes were observed in transit from the basal compartment to the adluminal compartment. While Sertoli-Sertoli junctions adluminal to the spermatocytes remained intact, typical Sertoli-Sertoli junctions formed between opposed Sertoli cell processes basal to the spermatocytes. It is proposed that, during the passage of spermatocytes in to the adluminal compartment, junctional specializations associated with preleptotene spermatocytes in the basal compartment migrate basal to the spermatocytes and contribute to formation of Sertoli-Sertoli junctions. Treatment of seminiferous tubules with hypertonic media was used to demonstrate that the junctional specializations function in cell-to-cell adhesion. Data indicated that these junctions function to retain the developing spermatids within the seminiferous epithelijm until the time of spermiation. At spermination the junctional specializations disappear and the spermatids drift off into the tubule lumen.     

Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.     

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.     

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.     

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.     

Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.     

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.     

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non- transcytosing molecules.     

Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator

Endocytic motifs in the carboxyl terminus of cystic fibrosis transmembrane conductance regulator (CFTR) direct internalization from the plasma membrane by clathrin-mediated endocytosis. However, the fate of such internalized CFTR has remained unknown. Internalized membrane proteins can be either targeted for degradation or recycled back to the plasma membrane. Using cell surface biotinylation and antibody uptake studies, we show that CFTR undergoes constitutive endocytosis and recycling back to the plasma membrane. Expression of dominant negative Rme-1 (a protein that regulates exit from the endosomal recycling compartment) in CFTR-expressing cells results in the expansion of recycling compartments. Transferrin, a marker for the endosomal recycling compartment, and CFTR accumulate in these enlarged recycling endosomes. Such accumulation leads to a loss of cell surface CFTR because it is prevented from being recycled back to the cell surface. In contrast, traffic of the low-density lipoprotein (LDL) is unaffected by the expression of dominant negative Rme-1. In addition, chimeras containing the extracellular domain of the transferrin receptor and the carboxyl terminal tail of CFTR also enter Rme-1-regulated recycling compartments and accumulate in these compartments containing dominant negative Rme-1, suggesting that in addition to endocytic signals, the carboxyl terminal tail of CFTR also contains intracellular traffic information.     

Separation of Fe from transferrin in endocytosis: Role of the acidic endosome

NH₄Cl and monensin, two agents which neutralize intracellular acidic compartments, block the segregation of iron from transferrin after endocytosis, while neither of these reagents affects internalization of diferric transferrin into the cell. In conclusion the molecular separation of iron from transferrin inside the cell requires a non-lysosomal acidic compartment     

Coxsackie and adenovirus receptor (CAR) is a product of Sertoli and germ cells in rat testes which is localized at the Sertoli-Sertoli and Sertoli-germ cell interface

The coxsackie and adenovirus receptor (CAR), a putative cell-cell adhesion molecule, has attracted wide interest due to its importance in viral pathogenesis and in mediating adenoviral gene delivery. However, the distribution pattern and physiological function of CAR in the testis is still not clear. Here, we identified CAR in Sertoli cells and germ cells of rats. In vivo studies have shown that CAR resides at the blood-testis barrier as well as at the ectoplasmic specialization. The persistent expression of CAR in rat testes from neonatal period throughout adulthood implicates its role in spermatogenesis. Using primary Sertoli cell cultures, we observed a significant induction of CAR during the formation of Sertoli cell epithelium. Furthermore, CAR was seen to be concentrated at inter-Sertoli cell junctions, co-localizing with tight junction protein marker ZO-1 and adherens junction protein N-cadherin. CAR was also found to be associated with proteins of Src kinase family and its protein level declined after TNFα treatment in Sertoli cell cultures. Immunofluorescent staining of isolated germ cells has revealed the presence of CAR on spermatogonia, spermatocytes, round spermatids and elongate spermatids. Taken together, we propose that CAR functions as an adhesion molecule in maintaining the inter-Sertoli cell junctions at the basal compartment of the seminiferous epithelium. In addition, CAR may confer adhesion between Sertoli and germ cells at the Sertoli-germ cell interface. It is possible that the receptor utilized by viral pathogens to breakthrough the epithelial barrier was also employed by developing germ cells to migrate through the inter-Sertoli cell junctions.     

Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin.

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.     

Effects of alterations in cellular iron on biosynthesis of the transferrin receptor in K562 cells.

Treatment of K562 cells, a human erythroleukemia cell line, with desferrioxamine raised the levels of the receptor for transferrin (Tf) two- to threefold over that of the control cells. The levels of receptor were reduced by at least 50 and 35% of that of the control in cells treated with diferric Tf and ferric ammonium citrate, respectively. These changes were of total cellular receptors with no alteration in the proportion of receptors found on the cell surface. The half-lives of the receptor were identical in cells treated with desferrioxamine, diferric Tf, or ferric ammonium citrate. Cells metabolically labeled with [35S]methionine showed a 2.5-fold increase in the rate of receptor synthesis when treated with desferrioxamine and a 35 and 65% decrease when treated with ferric ammonium citrate and diferric Tf, respectively. In vitro translations of polyadenylated mRNA isolated from cells incubated with desferrioxamine showed a 2.5-fold increase in translatable mRNA for the receptor, whereas treatment of cells with ferric ammonium citrate and diferric Tf resulted in a 25 and 50% reduction, respectively, in translatable mRNA for this receptor.     

Modulation of receptor cycling by neuron-enriched endosomal protein of 21 kD

Although correct cycling of neuronal membrane proteins is essential for neurite outgrowth and synaptic plasticity, neuron-specific proteins of the implicated endosomes have not been characterized. Here we show that a previously cloned, developmentally regulated, neuronal protein of unknown function binds to syntaxin 13. We propose to name this protein neuron-enriched endosomal protein of 21 kD (NEEP21), because it is colocalized with transferrin receptors, internalized transferrin (Tf), and Rab4. In PC12 cells, NEEP21 overexpression accelerates Tf internalization and recycling, whereas its down-regulation strongly delays Tf recycling. In primary neurons, NEEP21 is localized to the somatodendritic compartment, and, upon N-methyl-d-aspartate (NMDA) stimulation, the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunit GluR2 is internalized into NEEP21-positive endosomes. NEEP21 down-regulation retards recycling of GluR1 to the cell surface after NMDA stimulation of hippocampal neurons. In summary, NEEP21 is a neuronal protein that is localized to the early endosomal pathway and is necessary for correct receptor recycling in neurons.     

Cholesterol-dependent retention of GPI-anchored proteins in endosomes.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the Cterminal end that serves as their sole means of membrane anchoring. Using fluorescently labeled ligands and digital fluorescence microscopy, we show that contrary to the potocytosis model, GPI-anchored proteins are internalized into endosomes that contain markers for both receptor-mediated uptake (e.g. transferrin) and fluid phase endocytosis (e.g. dextrans). This was confirmed by immunogold electron microscopy and the observation that a fluorescent folate derivative bound to the GPI-anchored folate receptor is internalized into the same compartment as co-internalized horseradish peroxidase-transferrin; the folate fluorescence was quenched when cells subsequently were incubated with diaminobenzidine and H2O2. Most of the GPI-anchored proteins are recycled back to the plasma membrane but at a rate that is at least 3-fold slower than C6-NBD-sphingomyelin or recycling receptors. This endocytic retention is regulated by the level of cholesterol in cell membranes; GPI-anchored proteins are recycled back to the cell surface at the same rate as recycling transferrin receptors and C6-NBD-sphingomyelin in cholesterol-depleted cells. Cholesterol-dependent endocytic sorting of GPI-anchored proteins is consistent with the involvement of specialized lipid domains or 'rafts' in endocytic sorting. These results provide an alternative explanation for GPI-requiring functions of some GPI-anchored proteins.     

Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat

The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.