N-WASP Is Required for Structural Integrity of the Blood-Testis Barrier

During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties. Here we demonstrate that the actin nucleation promoting factor Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) provides an essential function necessary for BTB restructuring, and for maintaining spermatogenesis. Our data suggests that the N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling. These arrays are proposed to mediate the restructuring process through endocytic recycling of BTB components. Disruption of N-WASP in Sertoli cells results in major structural abnormalities to the BTB, including mis-localization of critical junctional and cytoskeletal elements, and leads to disruption of barrier function. These impairments result in a complete arrest of spermatogenesis, underscoring the critical involvement of the somatic compartment of the seminiferous tubules in germ cell maturation

The SCAR and WASp nucleation-promoting factors act sequentially to mediate Drosophila myoblast fusion

The actin nucleation-promoting factors SCAR/WAVE and WASp, together with associated elements, mediate the formation of muscle fibres through myoblast fusion during Drosophila embryogenesis. Our phenotypic analysis, following the disruption of these two pathways, suggests that they function in a sequential manner. Suppressor of cyclic AMP receptor (SCAR) activity is required before the formation of pores in the membranes of fusing cells, whereas Wiskott–Aldrich syndrome protein (WASp) promotes the expansion of nascent pores and completion of the fusion process. Genetic epistasis experiments are consistent with this step-wise temporal progression. Our observations further imply a separate, Rac-dependent role for the SCAR complex in promoting myoblast migration. In keeping with the sequential utilization of the two systems, we observe abnormal accumulations of filamentous actin at the fusion sites when both pathways are disrupted, resembling those present when only SCAR-complex function is impaired. This observation further suggests that actin-filament accumulation at the fusion sites might not depend on Arp2/3 activity altogether

Sertoli cell-specific knockout of N-WASP leads to disorganization of F-actin in the seminiferous epithelium.

<p>(<b>A</b>) Immunofluorescence analysis of sectioned seminiferous tubules from 8W (week)-old control (WT) and N-WASP^SC-cKO testes, showing the expression and localization patterns of N-WASP (green fluorescence, left) and F-actin (FITC-phalloidin, green fluorescence, right). Yellow arrowheads in the control panels point to the localization of either N-WASP or F-actin at the site of the BTB near the basement membrane of the tunica propria, which is annotated by a broken white-line. Cell nuclei were visualized by DAPI staining. Scale bars: 80 µm in the first two rows, and 240 µm in the third row. (<b>B</b>) A similar analysis was performed on testes from younger, 35D (day)-old mice. Actin microfilament organization in N-WASP^SC-cKO tubules was strongly disrupted at this stage as well. Scale bar: 80 µm.</p

A model illustrating the suggested roles of N-WASP and branched-actin polymerization in BTB dynamics.

<p>(Left) The BTB, composed of junctional and cytoskeletal structural and regulatory elements, provides a seal between the basal (down) and adluminal (up) compartments of the epithelium in seminiferous tubules. (Middle) At stage VIII of the epithelial cycle, the BTB undergoes restructuring, involving near simultaneous dismantling of the “old” BTB via endocytosis, to accommodate the apically directed transport of preleptotene spermatocytes between neighboring Sertoli cells, and assembly of a “new” BTB, to maintain the barrier. These events are mediated by the branched actin regulators drebrin E and Arp3, which localize to the BTB, and the nucleation promoting factor N-WASP, which activates branched-actin polymerization. The proposed role for the branched-actin arrays is to facilitate endocytic vesicle-mediated protein trafficking, so that integral membrane proteins at the “old” BTB can be recycled to the basal region and contribute to assembly of a “new” BTB. (Right) In N-WASP^SC-cKO testes lacking Sertoli-cell N-WASP function, the branched-actin polymerization machinery can no longer mediate proper endocytic vesicle-mediated protein recycling, and the BTB, once dismantled, cannot be regenerated.</p

The branched microfilament regulators Arp3 and drebrin E are properly localized to the BTB in N-WASP^SC-cKO tubules.

<p>(<b>A</b>) Immunoblotting data using lysates of testes from N-WASP^SC-cKO and age-matched control mice demonstrate up-regulation in Arp3 and drebrin E expression in mutant mouse testes <i>vs.</i> the age-matched control testes with β-actin served as a protein loading control. Histogram generated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen-1004447-g001" target="_blank">Figure 1</a>. **, <i>P</i><0.01. (<b>B</b>) Immunofluorescence analysis of Arp3 (green fluorescence, left) and drebrin E (green fluorescence, right) in the seminiferous epithelium of control (WT) and N-WASP^SC-cKO mice. Yellow arrowheads mark the localization of either Arp3 or drebrin E at the BTB, which is near the basement membrane of the tunica propria (annotated by a broken white-line). Unlike basal ES proteins (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen-1004447-g004" target="_blank">Figure 4</a>) and apical ES proteins (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen-1004447-g005" target="_blank">Figure 5</a>), which were mis-localized in the seminiferous epithelium of mutant testes, these branched actin regulatory proteins remained properly localized to the damaged BTB. Cell nuclei were visualized by DAPI staining. Scale bars: 80 µm in the first two rows, and 240 µm in the third row.</p

Spermatogenetic arrest and abnormal actin microfilament organization in the seminiferous epithelium of N-WASP^SC-cKO mouse testes.

<p>(<b>A</b>) Lysates (∼50 µg protein) from 8-wk-old testes of N-WASP^SC-cKO mice (<i>Dhh</i>-Cre; <i>N-WASP</i>^flox/<i>N-WASP</i>⁻) and age-matched wild-type (WT) control mice (<i>N-WASP</i>^flox/<i>NWASP</i>⁻) were used for immunoblotting, to assess changes in the expression of N-WASP, with GAPDH serving as a protein loading control. Histograms in this and subsequent figures are composites of quantified immunoblot data (mean ± SD) from <i>n</i> = 4 mice, normalized for the loading control. WT protein levels in these graphs were arbitrarily set at 1, against which statistical comparison was performed. **, <i>P</i><0.01. (<b>B</b>) <b>(Left panels)</b> Hematoxylin and eosin (H&E) staining of paraffin sections, illustrating that specific disruption of N-WASP in Sertoli cells led to major defects in spermiogenesis. N-WASP^SC-cKO tubules (bottom) were shrunk in diameter and lacked the normal spermatid-filled seminiferous epithelium seen in control (WT) mouse testes (top). Magnified images of mutant tubules (bottom center and right panels) demonstrate the presence of meiotic round spermatids (red arrowheads) and rare step 11 or 12 spermatids (green arrowhead). Scale bars: 240 µm (left column), and 60 µm (magnified columns). <b>(Right panels)</b> Laminin-γ3 chain (green fluorescence), a specific marker of step 13–16 spermatids at the apical ES <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen.1004447-Koch1" target="_blank">[20]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen.1004447-Yan1" target="_blank">[21]</a>, was not detected in the seminiferous epithelium of N-WASP^SC-cKO mouse testes. Absence of advanced-stage spermatids is indicative of a full failure of spermiogenesis in these mutant mice. Scale bar: 50 µm, which applies to other micrographs. (<b>C</b>) Histological analysis of age-matched control (WT) and N-WASP^SC-cKO tubules from 21- and 35-D (day)-old mice. At 21D and 35D, round spermatids (annotated by blue arrowheads) were found in some tubules from N-WASP^SC-cKO testes, illustrating the occurrence of meiosis. However, abnormal multinucleated spermatocytes (annotated by yellow arrowheads) derived from normal spermatocytes (green arrows), were found in 21D N-WASP^SC-cKO-testes, presumably giving rise to the degenerated spermatocytes (red arrow). Degenerating round spermatids were also noted (blue arrow) in 21D N-WASP^SC-cKO-testes. In 35D N-WASP^SC-cKO-testes, elongating spermatids were occasionally found (red arrowhead) and also abnormal multinucleated round spermatids as annotated by blue arrowheads in the yellow boxed area. The lack of elongating spermatids at different stages supports the notion of a full failure of spermiogenesis in N-WASP^SC-cKO-testes. Scale bars: 120 µm, and 40 µm in insets.</p

BTB integrity is compromised in testes of N-WASP^SC-cKO mice.

<p>Images show localization within seminiferous tubules of FITC-inulin (green), a small fluorescent molecule injected into the blood system of control (WT) and N-WASP^SC-cKO mice (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#s4" target="_blank"><i>Materials and Methods</i></a> for detailed experimental protocol). In control mouse testes, the functional BTB blocked FITC-inulin from entering into the apical (adluminal) compartment, and the distance traveled by the marker was limited to the basal compartment (annotated by a white bracket). The lower magnification inset shows four seminiferous tubules (white asterisks) that are devoid of green fluorescence inside the epithelium. In N-WASP^SC-cKO mouse tubules, the marker penetrated deep inside the seminiferous epithelium (yellow arrows), reaching the tubule lumen (annotated by yellow bracket). The lower magnification inset shows five such tubules (yellow asterisks), demonstrating that disruption of BTB integrity is a common feature of N-WASP^SC-cKO mouse testes. Scale bars: 50 µm (magnified image), and 200 µm in insets. Data from BTB integrity assays were semi-quantified and are shown in the bar graph, which displays the distance traveled by FITC-inulin <i>vs.</i> the tubule radius for <i>n</i> = 5 mice (8–14 weeks old) in each group. *, <i>P</i><0.01.</p

Abnormal localization and expression patterns of apical ES proteins in the seminiferous epithelium of N-WASP^SC-cKO mouse testes.

<p>(<b>A</b>) Immunoblotting data using lysates of testes from N-WASP^SC-cKO and age-matched control mice, to examine changes in the expression of the apical ES integral membrane proteins nectin-3, β1-integrin, and ICAM-2. β-Actin served as a protein loading control. Histogram generated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen-1004447-g001" target="_blank">Figure 1</a>. **, <i>P</i><0.01. N.D., not detectable. (<b>B</b>) Immunofluorescence analysis of nectin-3 (green fluorescence, left), β1-integrin (green fluorescence, middle) and ICAM-2 (green fluorescence, right) in the seminiferous epithelium of control (WT) and N-WASP^SC-cKO mice. Boxed areas are magnified and shown in insets. Nectin-3, β1-integrin and ICAM-2 were associated with apical ES in control (WT) testes. In N-WASP^SC-cKO mouse testes, nectin-3 was considerably diminished to a level almost undetectable in the seminiferous epithelium, consistent with the immunoblotting data shown in (<b>A</b>). While β1-integrin and ICAM-2 were detectable in the mutant testes, they were mis-localized and were both found near the disrupted BTB (see yellow arrowheads that annotate ICAM-2 in the mutant testes). Relative location of the basement membrane is annotated by a white broken-line). Cell nuclei were visualized by DAPI staining. Scale bars: 80 µm in the first two rows, 240 µm in the third row, and 15 µm in insets.</p

Antibodies used for different experiments in this report.

<p>Abbreviations used: IB, immunoblotting; IHC, immunohistochemistry; IF, immunofluorescence analysis.</p><p>*, the number bracketed represents the corresponding Tyr residue of p-FAK in the rat testis.</p

Modifications in the localization and expression patterns of the ES regulatory protein FAK and its phosphorylated forms in N-WASP^SC-cKO tubules.

<p>(<b>A</b>) Immunoblotting data using lysates of testes from N-WASP^SC-cKO and age-matched control mice, to examine changes in the expression of the non-receptor protein tyrosine kinase FAK and its phosphorylated/activated forms p-FAK-Tyr⁴³⁸ and -Tyr⁶¹⁴. β-Actin served as a protein loading control. Histogram generated as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen-1004447-g001" target="_blank">Figure 1</a>. **, <i>P</i><0.01. N.D., not detectable. (<b>B</b>) Immunofluorescence analysis of p-FAK-Tyr⁴³⁸ (green fluorescence, top) and p-FAK-Tyr⁶¹⁴ (green fluorescence, bottom), in the seminiferous epithelium of mouse testes in both animal groups. Yellow arrowheads annotate p-FAK-Tyr⁴³⁸ that was normally localized to the BTB, which was located near the basement membrane at the tunica propria (annotated by white broken-line). Unlike p-FAK-Tyr⁴³⁸ which was localized both at the BTB and the apical ES, p-FAK-Tyr⁶¹⁴ was restrictively expressed at the apical ES, analogous to p-FAK-Tyr⁵⁷⁶ in the rat testis <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004447#pgen.1004447-Siu3" target="_blank">[42]</a>. Boxed areas were magnified and shown in insets, illustrating that p-FAK-Tyr⁴³⁸ and -Tyr⁶¹⁴ were normally associated with the apical ES at the elongating/elongated spermatid/Sertoli cell interface. The expression of p-FAK-Tyr⁴³⁸ was considerably diminished in the seminiferous epithelium of N-WASP^SC-cKO mouse testes, consistent with the immunoblot data shown in (<b>A</b>). While the expression p-FAK-Tyr⁶¹⁴ was considerably down-regulated, yet it remained detectable in the seminiferous epithelium of N-WASP^SC-cKO mouse testes, and its spatiotemporal expression was considerably altered. For instance, p-FAK-Tyr⁶¹⁴ was no longer restricted to the apical ES at the elongating/elongated spermatids but associated with round spermatids and spermatocytes and also at the damaged BTB. Cell nuclei were visualized by DAPI staining. Scale bars: 80 µm, and 15 µm in insets.</p