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American Journal of Physiology - Endocrinology and Metabolism logoLink to American Journal of Physiology - Endocrinology and Metabolism
. 2014 Aug 12;307(7):E553–E562. doi: 10.1152/ajpendo.00176.2014

Differential effects of c-Src and c-Yes on the endocytic vesicle-mediated trafficking events at the Sertoli cell blood-testis barrier: an in vitro study

Xiang Xiao 1, Dolores D Mruk 1, Elissa W P Wong 1, Will M Lee 2, Daishu Han 3, Chris K C Wong 4, C Yan Cheng 1,
PMCID: PMC4187029  PMID: 25117412

Abstract

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in the mammalian body. However, it undergoes cyclic restructuring during the epithelial cycle of spermatogenesis in which the “old” BTB located above the preleptotene spermatocytes being transported across the immunological barrier is “disassembled,” whereas the “new” BTB found behind these germ cells is rapidly “reassembled,” i.e., mediated by endocytic vesicle-mediated protein trafficking events. Thus, the immunological barrier is maintained when preleptotene spermatocytes connected in clones via intercellular bridges are transported across the BTB. Yet the underlying mechanism(s) in particular the involving regulatory molecules that coordinate these events remains unknown. We hypothesized that c-Src and c-Yes might work in contrasting roles in endocytic vesicle-mediated trafficking, serving as molecular switches, to effectively disassemble and reassemble the old and the new BTB, respectively, to facilitate preleptotene spermatocyte transport across the BTB. Following siRNA-mediated specific knockdown of c-Src or c-Yes in Sertoli cells, we utilized biochemical assays to assess the changes in protein endocytosis, recycling, degradation and phagocytosis. c-Yes was found to promote endocytosed integral membrane BTB proteins to the pathway of transcytosis and recycling so that internalized proteins could be effectively used to assemble new BTB from the disassembling old BTB, whereas c-Src promotes endocytosed Sertoli cell BTB proteins to endosome-mediated protein degradation for the degeneration of the old BTB. By using fluorescence beads mimicking apoptotic germ cells, Sertoli cells were found to engulf beads via c-Src-mediated phagocytosis. A hypothetical model that serves as the framework for future investigation is thus proposed.

Keywords: testis, spermatogenesis, seminiferous epithelial cycle, blood-testis barrier, endosome, transcytosis, recycling, ectoplasmic specialization, tight junction, c-Yes, c-Src


studies have shown that endocytic vesicle-mediated protein-trafficking events that take place in the seminiferous epithelium during the epithelial cycle are crucial to spermatogenesis (7, 40, 41), similar to their role in regulating cellular events in other tissue barriers, such as membrane traffic (37), autophagy (27), and intracellular cargo trafficking (8). For instance, protein endocytosis, transcytosis, and recycling are necessary for the maintenance of the blood-testis barrier (BTB) integrity during the transport of preleptotene spermatocytes at the BTB that takes places at stage VIII of the epithelial cycle (36, 45, 46, 52). In brief, proteins at the “old” BTB site located above the preleptotene spermatocytes that are connected in clones via intercellular bridges are endocytosed, transcytosed, and recycled to assemble the “new” BTB located beneath these cells (52). Thus, the integrity of the BTB can be maintained during the transport of preleptotene spermatocytes across the barrier without the immunological barrier being compromised (36, 52). At the same time, extensive endocytosis also occurs near the tubule lumen when Sertoli cells phagocytose residual bodies that contain unwanted cytosolic/nuclear materials and debris of maturing spermatids so that residual bodies can become phagosomes and be digested by lysosomal enzymes in Sertoli cells.

Recently, we have used biochemical assays to demonstrate the occurrence of these events in the Sertoli cell epithelium with an established physiological tight junction (TJ)-permeability barrier that mimics the Sertoli cell BTB in vivo plus the presence of the ultrastructures of TJ, basal ectoplasmic specialization (ES), gap junction, and desmosome (4446, 52). We showed that the testosterone- and cytokine-mediated (e.g., TGFβ2) enhancement in protein endocytosis led to an increase in protein recycling and endosome-mediated protein degradation, respectively (38, 52). In short, testosterone (23, 43) promotes whereas cytokines (45, 52) perturb BTB integrity, and as such, these biomolecules are working in concert in the microenvironment of the BTB to maintain its homeostasis. However, in these earlier studies, the underlying molecular mechanism(s) that affects targeting of the endocytic vesicles to the pathways of transcytosis and/or recycling vs. degradation remains unclear. Recently, multiple studies have shown that both of the closely related members of the Src family kinases, namely c-Src and c-Yes, are involved in endocytic vesicle-mediated trafficking of proteins in mammalian cells possibly via their regulations of the actin cytoskeleton (9, 34), but the evidence was obscure as to how much they would perform overlapping functions in these events (49). We hypothesized that the Sertoli cell BTB utilized c-Yes and c-Src as the molecular “switches” in which c-Yes favors the targeting of the biotinylated and endocytosed BTB proteins for recycling vs. c-Src targeting the biotinylated and endocytosed proteins for intracellular degradation so that these two nonreceptor tyrosine kinases that work together provide a novel mechanism to regulate preleptotene spermatocyte transport across the BTB at stage VIII of the epithelial cycle.

MATERIALS AND METHODS

Animals.

The use of Sprague-Dawley rats (Charles River Laboratories, Kingston, NY) was approved by The Rockefeller University Institutional Animal Care and Use Committee (with protocol nos. 09-016 and 12-506). Adult males were housed in groups of two, and unweaned pups in groups of 10 with a foster mother were placed in individual cages under a 12:12-h light-dark cycle in a temperature- (21 ± 1°C) and humidity-controlled environment. All animals had free access to standard rat chow and water ad libitum. At specified time points, animals were euthanized by slow (20–30%/min) displacement of chamber air with compressed carbon dioxide via a CO2 tank with a regulator. Following unconsciousness/euthanization, testes were removed, and if necessary, the animals were subjected to cervical dislocation as a secondary means to ensure euthanization.

Primary Sertoli cell cultures and knockdown of c-Src and c-Yes in Sertoli cells in vitro by RNAi.

Sertoli cells were isolated from testes of 20-day-old rats and maintained in DMEM-F-12 (Sigma-Aldrich, St. Louis, MO) supplemented with growth factors at 35°C with 95% air-5% CO2 (vol/vol) in a humidified atmosphere, as described (24). Cells were seeded at a density of 0.5 × 106 cells/cm2 in six-well plates (5 ml/well DMEM-F-12) for different assays and lysate preparation or at a density of 0.03–0.05 × 106 cells/cm2 on round glass coverslips (18 mm; Thomas Scientific, Swedesboro, NJ) in 12-well plates (2 ml/well DMEM-F-12) for immunofluorescent staining. All culture dishes were coated with Matrigel basement membrane matrix (diluted 1:7 with DMEM-F-12; BD Bioscience, San Jose, CA), as described earlier (48, 50). About 36 h after plating cells, cultures were treated with a hypotonic buffer (20 mM Tris, pH 7.4, at 22°C) to lyse contaminating germ cells (24), thereby yielding Sertoli cells with a purity of >98%. Cells were rinsed twice and cultured in fresh DMEM-F-12 medium with supplements (24) overnight. Two days after isolation, when a functional TJ-permeability barrier was established when monitored by assessing transepithelial electrical resistance across the cell epithelium (48, 50), Sertoli cells were used for c-Src or c-Yes knockdown by RNAi, as described (48). Besides the presence of a functional TJ barrier, ultrastructures of TJ, basal ES, gap junction, and desmosome that mimic the BTB in vivo were found in these cell cultures. Approximately 100 and 50 nM siRNA was used for biochemical assay/lysate preparation (for immunoblotting) and cell staining (for immunofluorescence analysis), respectively. Nontargeting control siRNA duplexes (catalog no. D-001810-10; Thermo Fisher Scientific, Lafayette, CO), ON-TARGETplus siRNA duplexes for rat c-Src (SMARTpool of 4 duplexes: UCAUAGAGGACAACGAAUA, CAAGAUCACUAGACGGGAA, GGACAGACCGGUUACAUCC, and CACGAGGGUUGCCAUCAAA; catalog no. L-080144-02), and rat c-Yes (GAUCAAUUGCUACCGGAAA; catalog no. J-085484-11) were used for cell transfection for 24-h using the TransIT-X2 Dynamic Delivery System (Mirus Bio, Madison, WI) according to the manufacturer's instructions. Some experiments were also verified by using RiboJuice (EMD Millipore, Billerica, MA) as the siRNA transfection reagent (48). Pilot experiments illustrate that similar phenotypes and efficacy were obtained by using either transfection reagent. On the following day (i.e., 24 h after transfection), cells were washed twice to remove silencing duplexes and transfection medium, and this time point was designated as day 1 after transfection. On day 2.5 (i.e., 1.5 days after transfection was completed and 4.5 days after cells were plated on Matrigel-coated dishes or coverslips), cells were used for different assays, immunofluorescence experiments, or immunoblot analysis as reported herein.

Endocytosis assay.

2.5 days after siRNA transfection (i.e., 4.5 days in culture), endocytosis assay was performed as described previously (45, 52). Cells were kept on ice (or at 4°C) during the whole process, unless stated otherwise, to minimize unwanted endocytosis. Cell surface proteins were biotinylated using 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (a membrane impermeable cross-linker which biotinylates only cell surface proteins; Thermo Scientific, Rockford, IL) for 30 min at 4°C in PBS-calcium and magnesium (CM) (0.15 M NaCl, pH 7.4, at 22°C containing 10 mM NaH2PO4, 0.9 mM CaCl2, and 0.33 mM MgCl2). Thereafter, excess/nonreacted (i.e., free) biotin was quenched with 50 mM NH4Cl in PBS-CM. Endocytosis was then initiated by incubation of cells in DMEM-F-12 at 35°C and terminated at specified time points by placing cells on ice at 4°C. Biotins on uninternalized biotinylated cell surface proteins were subsequently removed by using a stripping buffer (100 mM Tris·HCl, pH 8.6, at 22°C containing 50 mM MESNA, 100 mM NaCl, and 2.5 mM CaCl2) and quenched with 5 mg/ml iodoacetamide in PBS-CM. Cells were lysed with RIPA buffer [50 mM Tris·HCl, pH 8.0, at 22°C containing 150 mM NaCl, 5 mM EGTA, 0.2% SDS, 1% Triton X-100 (vol/vol), 1% sodium deoxycholate, and 2 mM N-ethylmaleimide; protease inhibitors (2 mM PMSF, 1 μg/ml aprotinin, and 10 µM leupeptin) were added immediately before use], and biotinylated proteins were recovered by using NeutrAvidin UltraLink Resin (Thermo Scientific) and subjected to immunoblotting analysis using the corresponding specific antibodies (see Table 1).

Table 1.

Antibodies used for different experiments in this report

Antibody Host Species Vendor Catalog No. Application(s)/Dilution(s)
c-Src Mouse EMD Millipore 05-184 IB (1:500)
Mouse Santa Cruz Biotechnology sc-8056 IB (1:200), IHC (1:100)
c-Yes Mouse BD Transduction Laboratories 610375 IB (1:1000)
FAK Rabbit EMD Millipore 06-543 IB (1:1000)
JAM-A Rabbit Life Technologies 36-1700 IB (1:300), IF (1:100)
CAR Rabbit Santa Cruz Biotechnology sc-15405 IB (1:200)
N-cadherin Rabbit Santa Cruz Biotechnology sc-7939 IB (1:200)
Actin Goat Santa Cruz Biotechnology sc-1616 IB (1:200)
EEA-1 Mouse BD Transduction Laboratories 610457 IF (1:100)

IB, immunoblotting; IHC, immunohistochemistry; IF, immunofluorescence microscopy; EEA-1, early endosome antigen-1. All antibodies used herein cross-reacted with their corresponding protein in the rat.

Kinetics of disappearance of endocytosed Sertoli cell surface proteins.

Disappearance of biotinylated Sertoli cell surface proteins after endocytosis from the cell cytosol was assessed on basis of the aforementioned endocytosis assay (52), with modifications. On day 2.5 after siRNA transfection, Sertoli cell surface proteins were biotinylated with 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin and incubated in DMEM-F-12 at 18°C for 2 h to allow the accumulation of endocytosed biotinylated proteins in early endosomes since no delivery of internalized cell surface proteins to lysosomes occurs at this temperature (13, 45). Biotins on uninternalized biotinylated cell surface proteins were removed and quenched as mentioned above. Thereafter, cells were incubated in DMEM-F-12 at 35°C for different time points to initiate protein endocytosis/recycling/degradation and then terminated at specified time points by placing cells on ice, and biotins on newly recycled biotinylated cell surface proteins were removed and quenched afterward. Cells were then terminated in RIPA buffer, and biotinylated proteins retained in cell cytosol were recovered with NeutrAvidin UltraLink Resin and subjected to immunoblotting analysis.

Recycling assay.

The kinetics of recycling of endocytosed Sertoli cell surface proteins was monitored as described previously (45, 52) with modifications. On day 2.5 after siRNA transfection, Sertoli cell surface proteins were biotinylated with 0.5 mg/ml EZ-Link Sulfo-NHS-SS-Biotin for 30 min at 4°C and incubated in DMEM-F-12 at 18°C for 2 h, after which biotins on uninternalized biotinylated cell surface proteins were removed and quenched. Cells were then incubated in DMEM-F-12 at 35°C to allow protein transcytosis, recycling, and/or degradation. At specified time points, cells were taken out from the incubator, and DMEM-F-12 was replaced with a hypotonic Tris buffer [20 mM Tris, pH 7.4, at 22°C containing 2 mM EGTA; protease inhibitors (2 mM PMSF, 1 μg/ml aprotinin, and 10 µM leupeptin) were added immediately before use] and incubated at room temperature for ∼20 min. Cells were scraped off in the Tris buffer and sonicated (twice for ∼1 s; Cole Parmer 4710 Series Ultrasonic Homogenizer) to release the biotinylated proteins in cell cytosol, and broken cells with recycled biotinylated surface proteins in membranes were pelleted at 20,817 g for 15 min at 4°C. Cell pellet was lysed using RIPA buffer, sonicated, and centrifuged at 14,000 g for 10 min at 4°C, and the supernant was taken as the membrane fraction. Thereafter, biotinylated proteins were recovered with NeutrAvidin UltraLink Resin and subjected to immunoblotting analysis. In some experiments, the membrane fraction was collected by treating cells with 0.01% trypsin in Ca2+-free PBS (0.15 M NaCl, pH 7.4, at 22°C containing 10 mM NaH2PO4) for 20 min to extract the recycled cell surface biotinylated proteins, to be followed by adding 1% soybean trypsin inhibitor (wt/vol) to prevent further unwanted trypsinization, as described previously (52), in which similar results were observed. In selected experiments, 60-mm dishes were used for recycling assay so that a sufficient protein amount was obtained to assess the kinetics of recycling of multiple BTB proteins.

Phagocytosis assay.

On day 2.5 after siRNA transfection, negatively charged 2.0-μm diameter FluoSpheres fluorescent microspheres (Life Technologies, Grand Island, NY) that mimicked the apoptotic germ cells (30) were added to the Sertoli cells (5-μl beads in 1 ml of DMEM-F-12) cultured at a density of 0.5 × 106 cells/cm2 on Matrigel-coated 12-well plates (1 ml/well medium) with an established functional TJ-permeability barrier. Cells treated with either LPS (100 μg/ml, a known phagocytosis inducer) (31) or BLT-1 (block lipid transport-1; 50 μM, a known inhibitor of phagocytosis) (26) instead of plain medium were used as positive and negative controls for phagocytosis assay, respectively. Cells with or without treatment were incubated with fluorescent beads for 3–5 h at 35°C with 95% air-5% CO2 (vol/vol) in a humidified atmosphere. For analysis of phagocytosis assay, Sertoli cells were stripped from the culture substrate with trypsin (0.05%)-EDTA (0.02%) solution (Sigma-Aldrich) at 35°C for ∼10 min with mild agitation, and the reaction was stopped by adding 0.05–1% (wt/vol, final concentration) soybean trypsin inhibitor. Cell aggregates were dispersed by pipetting up and down >10 times. Cells were collected by low-speed centrifugation at 100 g for 5 min and washed with DMEM-F-12 twice. This step was found to efficiently clear the uninternalized beads. Cells were transferred to wells of new 12-well plates (1 ml/well medium), and the fluorescence intensity was quantified via bottom reading using a FilterMax F5 Multi-Mode Microplate Reader and the Multi-Mode Analysis Software (Molecular Devices, Sunnyvale, CA), with an excitation filter at 535 nm and an emission filter at 595 nm. Each treatment group had triplicate wells, including blanks and all controls. For immunofluorescence microscopy, Sertoli cells cultured on Matrigel-coated coverslips were washed extensively, fixed with 4% paraformaldehyde in PBS, and mounted with ProLong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole.

Degradation assay.

To examine the changes in kinetics of protein degradation after c-Src/c-Yes knockdown, total Sertoli cell proteins (both cell surface and intracellular proteins) were irreversibly biotinylated with 2 mM EZ-Link NHS-LC-Biotin (a membrane permeable chemical cross-linker which biotinylates both cell surface and intracellular proteins; Thermo Scientific) for 30 min at 4°C, using Sertoli cells on day 2.5 after siRNA transfection, as described earlier (3). Excess/nonreacted (i.e., free) biotin was quenched with 50 mM NH4Cl in PBS-CM. Thereafter, cells were incubated in DMEM-F-12 at 35°C for specified time points and terminated in RIPA buffer. Biotinylated proteins were recovered with NeutrAvidin UltraLink Resin and subjected to immunoblotting analysis.

General methods.

Total protein concentration was determined with the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA), using BSA as a standard. For immunoblotting, cell lysate was used at 15–25 μg protein/lane. In biochemical assays, each experimental set for an endocytosis, recycling, or degradation assay had duplicate or triplicate samples, and all samples were run in a single immunoblot analysis experimental session to eliminate interexperimental variations, and each experiment was repeated with at least n = 3 experiments. When a target protein (e.g., JAM-A) at a specified time point had an artefact, such as an uneven/fuzzy protein band due to the presence of an air bubble during transfer or a smear appearance, it was replaced with data from a duplicate (or triplicate) blot, and a black line was inserted to annotate such replacement. Since there were two β-actin blots for two target proteins shown in Figs. 14 here, the spliced location of the actin blot might not match one of the target protein blots. Immunohistochemistry and immunofluorescence staining were performed as described previously (4850) by using the AEC Substrate Kit or reagents from Molecular Probes (Life Technologies, Eugene, OR). Images were captured by using either a color (DS-Fi1-U2) or a monochromatic (Ds-Qi1Mc-U2) camera attached to a Nikon 90i motorized microscope and NIS-Elements AR software (version 3.2; Nikon Instruments, Melville, NY). Images were compiled in Adobe Photoshop CS3 (version 10.0.1; Adobe Systems, San Jose, CA) to obtain merged images. Table 1 lists the antibodies and conditions that were used for immunoblotting, immunohistochemistry, and immunofluorescence experiments, including dual-labeled immunofluorescence analysis reported herein. Electron microscopy used to visualize specialized junctions and also endocytic vesicles at the Sertoli cell-cell interface in primary cultures of Sertoli cells in vitro was performed at the Rockefeller University BioImaging Core Facility essentially as described (19, 47).

Fig. 1.

Fig. 1.

Effects of c-Src knockdown by RNAi on the kinetics of endocytosis of integral membrane proteins at the Sertoli cell blood-testis barrier (BTB) in vitro. A: Sertoli cells (SC) cultured in vitro for 4 days were processed for electron microscopy (EM). Left: specialized junctions that constitute the BTB were found at the cell-cell interface when cells were cultured on Matrigel-coated bicameral units (annotated by green arrowheads); the apical compartment (AC) was noted, and typical SC nucleus was also detected. Right: in these SC cultures, endocytic vesicle-mediated trafficking was also detected and annotated by yellow and red arrowheads, illustrating that the endocytic vesicle (also known as basal tubulobulbar complex in the testis) remained attached or had just been detached and released from the SC plasma membrane, respectively, confirming the presence of endocytic vesicle-mediated trafficking. *Microvillus that is a typical ultrastructure when SC are cultured in vitro. Scale bar, 1 μm on left and 0.2 μm on right. B: SC were cultured for 2 days with an established tight junction (TJ)-permeability barrier before they were transfected with RNAi duplexes for 24 h. About 36 h thereafter, cells were harvested for immunoblot analysis. It was noted that c-Src was knocked down by ≥60% (left) without apparent off-target effects in potentially related signaling pathways since the levels of c-Yes, FAK, and other BTB proteins JAM-A, CAR, and N-cadherin were not perturbed following c-Src silencing. These findings were representative data of 3 independent experiments. β-Actin served as the protein loading control. Each bar in the histogram (right) is the mean ± SD of n = 3 experiments. C: when the kinetics of protein endocytosis in cells transfected with nontargeting control duplexes vs. specific c-Src duplexes was compared, the knockdown of c-Src by RNAi was found to decelerate the kinetics of endocytosis of JAM-A and CAR significantly. Each data point is the mean ± SD of n = 4 experiments. D: effects of c-Src silencing that impeded the endocytosis of BTB integral membrane proteins JAM-A and CAR were corroborated in this dual-labeled immunofluorescence analysis in which cells transfected with nontargeting control duplexes, endocytosed JAM-A protein partially colocalized with EEA-1 [early endosome antigen-1; a endocytic marker (see yellow arrowheads)], and fewer endocytosed proteins, such as JAM-A, were found to colocalize with EEA-1 following c-Src knockdown, since more EEA-1 was found in SC cytosol instead of near the cell surface as in control cells. These micrographs are representative findings of 3 experiments. Bar, 20 μm (which applies to other micrographs). In B and C, *P < 0.05 and **P < 0.01. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 4.

Fig. 4.

c-Src is involved in Sertoli cell phagocytosis and endocytic vesicle-mediated protein degradation at the Sertoli cell BTB in vitro. A: Sertoli cells after being transfected with either c-Src or c-Yes siRNA duplexes vs. control duplexes were assessed on their phagocytic activity using FluoSpheres fluorescent microspheres. Phagocytosis of the microspheres was induced following treatment with LPS, a phagocytosis inducer, but blocked by block lipid transport-1 (BLT-1), a phagocytosis inhibitor. Knockdown of c-Yes had no effects on Sertoli cell phagocytosis activity, but the knockdown of c-Src impeded Sertoli cell phagocytosis. Both BLT-1 treatment and c-Src and c-Yes RNAi were also found to induce actin microfilament truncation but not LPS treatment. Scale bar, 45 μm (which applies to other micrographs). B: each bar is the mean ± SD of n = 6 experiments; enlarged images highlight representative findings of changes in phagocytic activity of Sertoli cells on the fluorescent microspheres. C: immunohistochemical localization of c-Src in the seminiferous epithelium of a stage VIII tubule, illustrating the intense localization of immunoreactive c-Src in residual bodies. Boxed green area is magnified and shown below the micrograph. Scale bar, 25 μm (12.5 μm in magnified image). D: kinetics of degradation was estimated in a biochemical assay in which the disappearance of total cell surface and intracellular biotinylated/endocytosed JAM-A or N-cadherin was assessed following the knockdown of c-Src or c-Yes vs. nontargeting control in Sertoli cell BTB in vitro. β-Actin served as protein loading control. Each bar in the line graph is the mean ± SD of n = 4 experiments. The knockdown of c-Src, but not c-Yes, was found to impede the degradation of endocytosed cell surface JAM-A and N-cadherin. *P < 0.05; **P < 0.01.

Statistical analyses.

Comparisons involving an experimental group vs. its corresponding control were performed by paired Student's t-test, whereas statistical comparisons between multiple experimental groups were performed by two-way ANOVA and post hoc Dunnett's test. GB-STAT software (version 7.0; Dynamic Microsystems, Silver Spring, MD) was used for statistical analyses. Each in vitro experiment was repeated three to five times using different batches of Sertoli cells, and within each in vitro experiment, each treatment point consisted of Sertoli cells in duplicate or triplicate cultures.

RESULTS

Knockdown of c-Src by RNAi retards protein endocytosis at the Sertoli cell BTB in vitro.

Sertoli cells cultured for 2 days were found to establish a functional TJ-permeability barrier and with ultrastructures of TJ, basal ES, gap junction, and desmosome, which also mimicked the Sertoli cell BTB in vivo, as earlier reported by our laboratory (19, 24, 35, 44, 48, 50). Besides our laboratory, this in vitro system has been widely used by investigators to study Sertoli cell BTB regulation (4, 12, 17, 25, 29, 32). As shown in Fig. 1A, left, specialized junctions, including TJ, basal ES, gap junction, and desmosome, were detected at the Sertoli cell-cell interface when cells were cultured on Matrigel-coated dishes for 2 days. Furthermore, endocytic vesicles that either remained attached or had just detached from the Sertoli cell plasma membrane at the BTB were also detected (Fig. 1A, right). In short, these cells with an intact epithelium and a functional TJ-permeability barrier on day 2 (designated as time 0) were then transfected with the c-Src-specific siRNA duplexes vs. the nontargeting control duplexes for 24 h, and 1.5 days thereafter (i.e., on day 2.5) these cells were either harvested for immunoblot analysis, as shown in Fig. 1B, or used for endocytosis assay and dual-labeled immunofluorescence analysis, as shown in Fig. 1, C and D, respectively. When c-Src was knocked down by ∼60% (Fig. 1B), no apparent off-target effects were detected in potentially related signaling pathways since the steady-state levels of c-Yes and other regulatory (e.g., FAK) and integral membrane proteins of TJ (e.g., JAM-A and CAR) and basal ES (e.g., N-cadherin) were not affected (Fig. 1B), illustrating the specificity of this silencing experiment. When these cells were subjected to the biochemical endocytosis assay to monitor the kinetics of internalization of biotinylated cell surface proteins JAM-A and CAR, the knockdown of c-Src was found to decelerate the endocytosis of JAM-A and CAR (Fig. 1C). The disruptive effect that retarded JAM-A endocytosis was more mild vs. CAR, illustrating that different integral membrane proteins at the BTB might respond differently to c-Src knockdown. The findings shown in Fig. 1C were collaborated in Fig. 1D, since JAM-A localized at the Sertoli cell-cell interface was found to have lessened internalization and reduced association with EEA-1 (early endosome antigen-1; a marker of endocytic vesicle) following c-Src knockdown vs. controls in which internalized JAM-A was associated with EEA-1 (see yellow arrowheads in Fig. 1D).

Knockdown of c-Yes by RNAi accelerates protein endocytosis at the Sertoli cell BTB in vitro.

In contrast to c-Src, the knockdown of c-Yes in Sertoli cells with an established TJ-permeability barrier that mimicked the Sertoli cell BTB in vivo by ∼70% without any apparent off-target effects in potentially related signaling pathways (Fig. 2A) was found to accelerate endocytosis of the biotinylated cell surface proteins JAM-A and CAR (Fig. 2B). These findings were corroborated in an immunofluorescence analysis experiment in which JAM-A was found to be redistributed with more JAM-A relocalized from the cell surface to the cell cytosol and associated with EEA-1 (Fig. 2C).

Fig. 2.

Fig. 2.

Effects of specific c-Yes knockdown by RNAi on the kinetics of endocytosis of integral membrane proteins at the Sertoli cell BTB in vitro. A: when c-Yes was knocked down by ≥70%, no off-target effects in potentially related signaling pathways were detected, including the steady-state level of c-Src in which β-actin served as the protein loading control. Each bar is the mean ± SD of 3 independent experiments. B: results of protein endocytosis assay (top) in which cultures at specified time points of the assay were terminated for lysate preparation to assess the kinetics of protein endocytosis. Each data point (bottom) is the mean ± SD of n = 4 independent experiments. *P < 0.05; **P < 0.01. C: effect of c-Yes silencing that impeded the endocytosis of BTB integral membrane proteins JAM-A and CAR was corroborated in this dual-labeled immunofluorescence analysis in which cells transfected with nontargeting control duplexes, endocytosed JAM-A protein colocalized with EEA-1, and more JAM-A were found to be internalized and to colocalize with EEA-1. These micrographs are representative findings of an experiment that was repeated 3 times with similar results. Bar, 20 μm.

c-Yes regulates recycling of protein back to the cell surface at the Sertoli cell BTB in vitro.

We next examined the fate of the biotinylated and endocytosed JAM-A and CAR in Sertoli cells with an established TJ-permeability barrier by tracking down the disappearance of internalized/biotinylated proteins from the cell cytosol (Fig. 3A). Whereas the silencing of either c-Src or c-Yes was found to have antagonistic effects on the rate of protein endocytosis with c-Src retarding and c-Yes accelerating internalization of biotinylated protein, the knockdown of either c-Src or c-Yes delayed the disappearance of biotinylated/endocytosed protein from the cell cytosol (Fig. 3A), illustrating that each may target the internalized protein similarly or differently. We first examined the impact of c-Yes RNAi on the recycling of endocytosed protein back to the Sertoli cell surface by tracking down the reappearance of biotinylated/internalized protein on the plasma membrane (Fig. 3B). It showed that the knockdown of c-Yes decelerated the kinetics of protein reappearance on the Sertoli cell plasma membrane via recycling for both JAM-A and CAR (Fig. 3B). These findings thus illustrate that c-Yes is likely involved in protein transcytosis and/or recycling at the Sertoli cell BTB.

Fig. 3.

Fig. 3.

Differential fate of endocytosed protein following specific knockdown of c-Yes and c-Src vs. nontargeting control at the Sertoli cell BTB in vitro. A: following knockdown of c-Yes or c-Src by ∼60 or ∼70%, respectively, the kinetics of disappearance of endocytosed/biotinylated protein from Sertoli cell cytosol was estimated. Knockdown of c-Yes delayed the disappearance of endocytosed/biotinylated JAM-A and CAR, which was analogous to a knockdown of c-Src that also delayed the disappearance of CAR from the cell cytosol. This could be the result of changes in recycling. B: knockdown of c-Yes delayed the recycling of endocytosed/biotinylated JAM-A and CAR back to the Sertoli cell plasma membrane. The data for the silencing of c-Src were not shown since consistent data were not obtained in multiple experiments. These findings illustrate that c-Yes is involved in protein recycling. In both A and B, β-actin served as protein loading control. Immunoblot data shown here were representative findings of an experiment that was repeated 4 times and yielded similar results. Each data point in the line graph is the mean ± SD of n = 4 experiments. *P < 0.05; **P < 0.01.

c-Src regulates phagocytosis and endosome-mediated protein degradation at the Sertoli cell BTB in vitro.

Since we failed to track down the recycling of proteins back to the Sertoli cell surface following c-Src silencing, which yielded inconsistent findings based on the recycling assay, we next examined whether the knockdown of c-Src impeded endocytic vesicle-mediated protein degradation by using two approaches. We first examined whether c-Src knockdown vs. c-Yes impeded Sertoli cell phagocytic activity using a fluorescence-based phagocytosis assay (Fig. 4, A and B), since c-Src was found to be localized with residual body of step 19 spermatids in stage VIII tubules (Fig. 4C), implicating its likely involvement in engulfment and digestion of spermatid remnants and furthermore, intracellular protein degradation. Second, we used a degradation assay (3) to monitor the degradation of biotinylated JAM-A and N-cadherin, both on the cell surface and in cell cytosol, following c-Src vs. c-Yes knockdown and nontargeting siRNA control (Fig. 4D). For the phagocytosis assay, Sertoli cells treated with LPS [lipopolysaccharide; a known inducer of Sertoli cell phagocytosis (10, 31)] served as a positive control, whereas BLT-1 [2-hexyl-1-cyclopentanone thiosemicarbazone, an inhibitor of phagocytosis (26)] served as a negative control. Sertoli cells are known phagocytic cells (10, 15, 31), serving as scavengers to lyse unwanted or defective germ cells. Thus, when Sertoli cells were exposed to FluoSpheres fluorescent microspheres (an analog representation of apoptotic germ cells) at 35°C, phagocytosis took place, and this process was enhanced and blocked by LPS and BLT-1, respectively (Fig. 4, A an B). Whereas the knockdown of c-Yes had no effect on Sertoli cell phagocytic activity, the silencing of c-Src by ∼60% impeded Sertoli cell phagocytosis (Fig. 4, A and B). These findings were further verified in a degradation assay in which the knockdown of c-Src, but not c-Yes, impeded the degradation of both JAM-A and N-cadherin (Fig. 4D). These findings thus support the notion that c-Src is involved in endocytic vesicle-mediated protein degradation pathway, unlike c-Yes, which is involved in protein transcytosis and recycling.

DISCUSSION

Each man and male rat produces as many as ∼400 and 50 million sperm, respectively, per day after puberty, which lasts the entirety of adulthood (1, 2, 22). Thus, extensive restructuring takes place at the Sertoli cell-cell and Sertoli-germ cell interface in the seminiferous epithelium to transport developing germ cells across the epithelium during spermatogenesis. To support this enormous cellular output, efficient endocytic vesicle-mediated trafficking is necessary to internalize, transcytose, and recycle integral membrane proteins during spermatogenesis so that de novo synthesis of proteins can be reduced to a minimum to reserve the pool of amino acids and proteins. Thus, it is envisioned that cyclic restructuring of the BTB during the epithelial cycle of spermatogenesis involves rapid endocytosis, transcytosis, and recycling of integral membrane proteins at the “old” and “disassembling” BTB above the preleptotene spermatocytes for the “assembly” of the “new” BTB behind spermatocytes that are being transported across the immunological barrier (Fig. 5). This concept is also supported by morphological findings that illustrate the presence of endocytic vesicles also known as basal tubulobulbar complexes in the mammalian testis (33), as shown in Fig. 1B and depicted in Fig. 5. In fact, recent studies have supported the notion that protein endocytosis is involved in the transport of preleptotene spermatocytes across the BTB and the maintenance of the immunological barrier (38, 45, 51, 52). However, in these earlier reports, the downstream signaling molecules crucial to regulating endosomal sorting for the eventual recycling and/or degradation of endocytosed proteins remain unknown. Studies in c-Src and c-Yes, two members of the nonreceptor protein kinases, have illustrated their differential functions in cell physiology, in particular their roles in endocytic vesicular transport of proteins in other mammalian cells (34, 39, 49). For instance, c-Yes is monopalmitoylated, and it is transported from the Golgi pool of caveolin to plasma membrane to mediate endosome trafficking, illustrating its involvement in protein transcytosis; but c-Src is nonpalmitoylated, and it is shuffled between plasma membrane and late endosomes/lysosomes, suggesting that it may be intimately related to endosome-mediated degradation (11, 34, 39). To corroborate this, c-Yes-knockout mice, although fertile, have defects in pIgA-pIgR transcytosis (21, 39), whereas in c-Src-knockout mice, which are infertile (14), VEGF, which is known to promote blood-brain barrier leakage (53), fails to compromise the blood-brain barrier (14). Herein, using various biochemical assays that track the fate of cell surface-labeled proteins with biotin (e.g., sulfo-NHS-SS-biotin) via endocytosis and recycling assays or total biotinylated proteins (e.g., NHS-LC-biotin) via degradation assay and phagocytosis assay, which coupled with dual-labeled immunofluorescence analysis, c-Yes, and c-Src were found to play differential roles in endocytic vesicle-mediated protein trafficking, c-Yes promotes endocytosed BTB integral membrane proteins for transcytosis and recycling, whereas c-Src promotes endosome-mediated protein degradation, such as via proteasome, for the removal of residual bodies. On the other hand, based on our previous findings, c-Yes, preceding redistribution of occludin and N-cadherin from the Sertoli cell-cell interface by relocating these proteins from cell surface into the cytosol, was downregulated in cultured Sertoli cells in response to TGFβ3 treatment, which is known to induce endosome-mediated protein degradation (50, 52). Also, because a knockdown of c-Yes led to protein mislocalization at the BTB both in vitro and in vivo (48), we propose that c-Yes may stabilize integral membrane proteins from leaving the BTB, but once the protein is endocytosed, it helps to transcytose to be reused via recycling.

Fig. 5.

Fig. 5.

A hypothetical model illustrating the differential roles of c-Yes and c-Src in endocytic vesicle-mediated trafficking that maintain BTB homeostasis during the epithelial cycle. This hypothetic model regarding the differential roles of c-Yes and c-Src in endocytic vesicle-mediated protein trafficking was prepared on the basis of findings reported herein. As shown at top during the degeneration of apical ectoplasmic specialization (ES) to facilitate the release of step 19 spermatids from the seminiferous epithelium once they are transformed to spermatozoa, c-Src assists the formation and/or degradation of residual body derived from spermiogenesis. On the other hand, c-Yes assists endocytic vesicle-mediated protein endocytosis, transcytosis, and/or recycling so that proteins at the “old” apical ES (top) or old BTB (bottom) can be recycled to assemble “new” apical ES or new BTB in stage VIII of the cycle.

It is likely that c-Src or c-Yes mediates its effects via changes in the phosphorylation status of BTB components, such as the integral membrane proteins and/or their adaptors at the Sertoli cell BTB, or actin filament regulatory proteins, such as the actin-bundling proteins palladin and Eps8 and branching/debundling-inducing protein Arp3, which in turn modulates its fate regarding endocytosis, transcytosis, recycling, or degradation. For instance, c-Yes structurally interacts with occludin in the Madin-Darby canine kidney cell line and T84 (human intestinal cell line) cells, and a disruption of c-Yes-occludin association leads to dephosphorylation and TJ-barrier disruption (5, 28). In fact, the phosphorylation status of integral membrane proteins (e.g., occludin, N-cadherin) in tissue barriers plays a crucial role in defining their localization either at the cell-cell interface or in the basolateral domain (6, 16). Furthermore, at the Sertoli cell BTB, c-Yes was shown to structurally interact with occludin and N-cadherin (50), and c-Src interacted with CAR (42), connexin 43 (18), and desmoglein-2 (20). c-Yes was also shown to physically interact with an actin-barded end capping and bundling protein, Eps8, at the Sertoli cell BTB, and c-Yes knockdown facilitates actin polymerizaiton in vitro (48). Additionally, knockdown of both c-Src and c-Yes was shown to perturb the F-actin network, as shown herein. Thus, these two nonreceptor protein kinases likely regulate the fate of integral membrane proteins by altering the phosphorylation status of critical BTB components and/or actin dynamics during the epithelial cycle of spermatogenesis.

In summary, we have demonstrated that c-Yes and c-Src are playing differential roles in regulating endocytic vesicle-mediated trafficking at the Sertoli cell BTB in which c-Yes promotes endocytosed proteins to the transcytosis and recycling pathway, whereas c-Src targets endocytosed proteins to endosome-mediated degradation and promotes phagocytosis. These findings also illustrate that this protein kinase pair serves as the crucial molecular “switches” at the BTB microenvironment during the cyclic events of spermatogenesis to regulate barrier function efficiently.

GRANTS

This work was supported by grants from the National Institute of Child Health and Human Development (U54-HD-029990, Project 5 to C. Y. Cheng; R01-HD-056034 to C. Y. Cheng), the Hong Kong Research Grants Council General Research Fund (771513 to W. M. Lee), the National Science Foundation of China (NSFC 31371176 to X. Xiao; NSFC 3126110491 to D. Han; NSFC/RGC Joint Research Scheme N_HKU 717/12 to W. M. Lee), and the Committee on Research and Conference Grants (University of Hong Kong) seed funding (to W. M. Lee).

Present address of X. Xiao: Department of Reproductive Physiology, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013, China.

DISCLOSURES

The authors have nothing to disclose.

AUTHOR CONTRIBUTIONS

X.X., E.W.W., and C.Y.C. performed experiments; X.X., D.D.M., E.W.W., and C.Y.C. analyzed data; X.X., D.D.M., E.W.W., W.M.L., D.H., C.K.W., and C.Y.C. interpreted results of experiments; X.X. and C.Y.C. prepared figures; X.X., D.D.M., E.W.W., W.M.L., D.H., C.K.W., and C.Y.C. approved final version of manuscript; C.Y.C. conception and design of research; C.Y.C. drafted manuscript; C.Y.C. edited and revised manuscript.

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