Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 by RNAi or an inactivation of dynein 1 by specific inhibitor ciliobrevin D in Sertoli cells cultured in vitro perturbs the tight junction (TJ)-permeability barrier function via changes in the distribution of TJ and basal ectoplasmic specialization (ES) proteins at the Sertoli cell-cell interface. A: treatment regimens by RNAi [Dync1h1-specific siRNA duplexes vs. non-targeting negative control siRNA duplexes as a control (Ctrl)] or ciliobrevin D (vs. DMSO serving as a vehicle Ctrl) to knockdown Dync1h1 or inactivate dynein 1 to obtain Sertoli cells for RT-PCR, qPCR, immunoblotting (IB), and immunofluorescence analysis (IF), and the number of independent experiments performed for each corresponding study. B: lysates from Sertoli cell cultures were used for IB following a knockdown of Dync1h1 or treatment with ciliobrevin D vs. the corresponding control cultures with the corresponding specific antibodies listed in Table 1. Collectively, these data (i.e., representative findings of n = 3 independent experiments) have shown that the steady-state protein level of Dync1h1 was considerably downregulated following Dync1h1 knockdown (but not treatment with the dynein 1 inhibitor nor any other functional groups of blood-testis barrier (BTB)-associated proteins examined herein), illustrating the knockdown of Dync1h1 is specific to dynein 1 expression without any apparent off-target effects. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and vimentin served as protein loading controls. C: steady-state mRNA or protein level of Dync1h1 was downregulated by ~70% when examined by qPCR (left) or IB (right), but only in the RNAi group, and dynein 1 inhibitor ciliobrevin D had no effects on Dync1h1 mRNA or protein expression. Each bar in the histogram is a mean ± SD of n = 3 independent experiments. **P < 0.01 when compared with the corresponding control by Student’s t-test. D: knockdown of Sertoli cell Dync1h1 or treatment of Sertoli cells with ciliobrevin D perturbed the Sertoli cell TJ-permeability barrier function. Each data point is a mean ± SD of quadruplicate bicameral units from a representative experiment. A total of n = 3 independent experiments were performed, which yielded similar results. *P < 0.05; **P < 0.01 when compared with the corresponding control by Student’s t-test. E: IF analysis was used to confirm considerable decline in Dync1h1 expression following RNAi but not treatment of Sertoli cells with ciliobrevin D (see top). Histogram (middle) summarized the relative fluorescence intensity of Dync1h1 in Dync1h1 RNAi group vs. the corresponding control cells, illustrating a ~70% knockdown. Sertoli cell Dync1h1 knockdown also perturbed the distribution of TJ proteins CAR and ZO-1 and basal ES proteins N-cadherin and β-catenin. In both control groups, TJ and basal ES proteins were tightly localized to the Sertoli cell-cell interface (see white brackets in control groups). After Dync1h1 knockdown or dynein 1 inactivation, these proteins were diffusively localized at the cell-cell interface (see yellow brackets in treatment groups). It is likely that these BTB-associated proteins were internalized and moved from the cell cortical zone into the cell cytosol. Co-transfection of siRNA duplexes with siGLO Red Transfection Indicator (red fluorescence; Dharmacon/GE/Thermo Fisher) used to illustrate successful transfection. Histograms (bottom) provide semiquantitative analysis regarding changes in the relative distribution of fluorescence at the cell-cell interface. Each bar is a mean ± SD of n = 3 independent experiments. **P < 0.01 when compared with corresponding control by Student’s t-test. Scale bar, 20 µm, which applies to all other micrographs. TER, transepithelial electrical resistance.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: Cell Culture, In Vitro, Permeability, Negative Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Functional Assay, Expressing, Fluorescence, Cotransfection, Transfection

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 by RNAi or an inactivation of dynein by inhibitor ciliobrevin D perturbs microtubule (MT) organization and MT polymerization in Sertoli cells in vitro. A: Dync1h1 knockdown or dynein 1 inactivation by inhibitor ciliobrevin D in Sertoli cells perturbed the organization of MTs since α-tubulin [Note: α- and β-tubulins are building blocks of MTs (86)] no longer stretched across the Sertoli cell cytosol as noted in cells from both control groups. Instead, MTs appeared to be largely truncated, surrounding the Sertoli cell nuclei, retracting from cell peripheries. On the other hand, tyrosinated tubulin (to promote MT dynamics), detyrosinated α-tubulin (to stabilize MTs), and acetylated α-tubulin (to stabilize MTs) were found to wrap around the Sertoli cell nucleus loosely, displaying a pattern similar to α-tubulin following Dync1h1 knockdown or dynein inactivation, unlike control Sertoli cells wherein they stretched across the cell cytosol. Furthermore, EB1, a +TIP protein known to stabilize MTs by promoting MT growth from the plus (+) end, also retracted from the Sertoli cell cytosol and localized closer to the Sertoli cell nucleus following Dync1h1 knockdown or dynein inactivation, unlike control cells wherein EB1 scattered along MTs that stretched across the entire Sertoli cell. Co-transfection of rhodamine-siGLO indicator (red fluorescence) with siRNA duplexes was used to illustrate successful transfection. Scale bar, 20 µm, which applies to other micrographs. B: MT spin-down assay was used to quantify the relative amount of polymerized MTs (in pellet) vs. free tubulins [in supernatant (S/N)] in Sertoli cell cytosol from these cultures. Dync1h1 knockdown or dynein inactivation in Sertoli cells perturbed MT dynamics in which the level of polymerized MTs quantified in Sertoli cells considerably reduced. Taxol (20 µM) and CaCl2 (2 mM) included in control Sertoli cell lysates in this assay served as the corresponding positive (+ve) and negative (-ve) control by stabilizing MTs and promoting MT depolymerization, respectively. GAPDH served as the protein loading control. Each bar in the histogram is a mean ± SD of n = 3 independent experiments. **P < 0.01. C: tubulin polymerization assay was used to assess the ability of Sertoli cell lysate to polymerize α- and β- tubulin oligomers in vitro after Dync1h1 knockdown. Rate of tubulin polymerization was noted on the y-axis during the assay period of 100 min. Kinetics of tubulin polymerization were further assessed during the initial 10 min (center) during the initial lag phase (see black boxed area, left) of polymerization, supporting the notion that Dync1h1 knockdown perturbed tubulin polymerization kinetics, consistent with the findings shown in B. *P < 0.05, by Student’s t-test.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vitro, Cotransfection, Fluorescence, Transfection, Spin Down Assay, Polymerization Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 by RNAi or an inactivation of dynein by inhibitor ciliobrevin D perturbs F-actin organization and actin polymerization in Sertoli cells in vitro. A: knockdown of Dync1h1 or an inactivation of dynein by inhibitor ciliobrevin D in Sertoli cells was found to induce extensive disorganization of F-actin network across the Sertoli cells, including truncation of actin microfilaments, and these filaments no longer stretched across the cell cytosol as noted in control cells to support the tight junction (TJ)-permeability barrier function. These changes were likely the result of disruptive changes in spatial expression of branched actin nucleation protein Arp3 and actin barbed end capping and bundling protein Eps8. For instance, these proteins no longer localized conspicuously at the Sertoli cell-cell interface as noted in control cells (see white arrowheads), but extensively internalized, moving from the cell cortical zone to cell cytosol, no longer at the cell-cell interface (see yellow arrowheads) in the treatment groups. These changes thus failed to support Sertoli cell TJ-barrier function as noted in Fig. 2D. It was noted that the organization of vimentin was also perturbed, since following Dync1h1 RNAi or ciliobrevin D treatment, vimentin no longer stretched across the Sertoli cell cytosol; instead, vimentin became wrapped around the Sertoli cell nuclei considerably. Co-transfection of rhodamine-siGLO indicator (red fluorescence) with siRNA duplexes illustrated successful transfection. Scale bar, 20 µm, which applies to other micrographs in the same panel. B: actin spin-down assay was performed as described in materials and methods, which separated filamentous (F; in pellet) actin from globular [G; in supernatant (S/N)] actin in Sertoli cell lysates after Dync1h1 knockdown or dynein inactivation by inhibitor ciliobrevin D. As noted herein (and also in composite data summarized in bar graph, right), Dync1h1 knockdown by ~70% or dynein inhibition by ciliobrevin D reduced the level of F-actin in Sertoli cells considerably. GAPDH served as a protein loading control. Phalloidin (0.1 µM) and urea (80 mM) were included in Sertoli cell lysates in the assay to serve as the corresponding positive (+ve) and negative (-ve) control. Each bar in the histogram is a mean ± SD of n = 3 independent experiments. **P < 0.01 by Student’s t-test.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vitro, Permeability, Expressing, Cotransfection, Fluorescence, Transfection, Spin Down Assay, Inhibition

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A study to assess the efficacy of in vivo knockdown of Dync1h1 by RNAi vs. inactivation of dynein by inhibitor ciliobrevin D in adult rat testis. A: regimens used for Dync1h1 knockdown or dynein inactivation by inhibitor ciliobrevin D (15 µM) in adult rat testes in vivo with n = 2 rats or n = 7 rats in 2 independent experiments for RNAi, and n = 7 rats for ciliobrevin D treatment. Since the phenotypes obtained from the experiments using two different regimens for RNAi were similar, these data were pooled for subsequent analysis. B: immunoblot (IB) analysis using lysates of testes showed that Dync1h1-specific knockdown induced a downregulation of Dync1h1 without perturbing the expression of multiple blood-testis barrier (BTB)-associated proteins, illustrating there was no apparent off-target effect following Dync1h1 knockdown. The use of ciliobrevin D to inactivate dynein did not alter Dync1h1 protein level, as well as all the BTB-associated proteins examined herein. GAPDH served as a protein loading control. C: a study by qPCR and IB illustrated a downregulation on the steady-state mRNA and protein level of Dync1h1 by at least 70% following Dync1h1 knockdown, but an inactivation of dynein had no apparent effects on Dync1h1 expression. For qPCR, Gapdh served as an internal control. For IB, GAPDH served as a protein loading control (see Fig. 2B). **P < 0.01, by Student’s t-test. D: efficacy of Dync1h1 knockdown in the testis in vivo was also assessed by immunofluorescence analysis using cross sections of testes and stained for Dync1h1 (red fluorescence). It was noted that Dync1h1 fluorescence signals were considerably diminished following transfection of testes with Dync1h1 siRNA duplexes vs. non-targeting negative control siRNA duplexes by as much as ~70% (see bar graph, bottom). For instance, Dync1h1 no longer expressed prominently along the MT-based tracklike structures (see yellow arrowheads) following Dync1h1 knockdown, unlike control testes where Dync1h1 localized closely with MT-based tracks (annotated by white arrowheads). However, following inactivation of dynein by inhibitor ciliobrevin A, although the Dync1h1 fluorescence signals remained unaffected (see bar graph, bottom), the distribution of Dync1h1 across the seminiferous epithelium was grossly disrupted as the tracklike structures conferred by Dync1h1 were notably truncated or misaligned [i.e., no longer lay perpendicular to the basement membrane (annotated by dashed white line; see green arrowheads)]. Each bar in the histogram is a mean ± SD of n = 4 rats. **P < 0.01 by Student’s t-test. ES, ectoplasmic specialization; TJ, tight junction.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Transfection, Negative Control

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 or an inactivation of dynein by inhibitor ciliobrevin D in adult rat testes in vivo perturbs spermatogenesis. A: histological analysis by hematoxylin-eosin staining using cross sections of testes at selected stages of the epithelial cycle following Dync1h1 knockdown or dynein inactivation by inhibitor ciliobrevin D showed extensive defects in spermatogenesis. First, spermatids had defects of polarity (black arrowheads) in which their heads no longer pointed toward the basement membrane, deviating by ~90°–180° from the basement membrane following Dync1h1 knockdown or dynein inactivation. Second, there was failure in the transport of phagosomes across the seminiferous epithelium. In control groups, phagosomes (yellow arrowheads) were found in the adluminal compartment near the tubule lumen, which were the residual bodies engulfed by the Sertoli cells at stage VIII at the time of spermiation. These phagosomes in stage VIII tubules were transported to the base of the epithelium at stage IX to prepare for their eventual lysosomal degradation. However, many phagosomes were found in the adluminal (apical) compartment in stage XI, XII, and XIII tubules (white arrowheads), which were not detected in similar staged tubules in controls. Third, there were defects in spermatid transport across the seminiferous epithelium. For instance, step 19 spermatids (green arrowheads) were consistently found in the seminiferous epithelium in stage IX, XI, XII, and XIII tubules, buried deep inside the epithelium, coexisting with step 9, 11, 12, and 13 spermatids (blue arrowheads) because of their failure to undergo spermiation in stage VIII tubules due to defects in microtubule organization. Boxed areas (insets) in testes of control groups (yellow) were magnified in corresponding yellow boxed rectangles, and insets in Dync1h1 RNAi testes (red) or ciliobrevin D treated testes (blue) were also magnified in corresponding red or blue boxed rectangles to better illustrate defects. Scale bar, 100 µm; inset, 40 µm. B: spermatozoa with structural defects were noted and summarized herein in Dync1h1 RNAi or ciliobrevin D treated group vs. the corresponding control cells, including 1) defective mid-piece so that sperm heads failed to anchor onto the tail, 2) persistent presence of residual body surrounding sperm heads, 3) malformed sperm heads, and 4) sperm heads without the tail. C: a threefold increase in defective tubules based on the criteria noted in A from n = 5 rats (left) and the percent of abnormal sperm found in the cauda epididymis from n = 5 rats (right). **P < 0.01, by Student’s t-test.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Staining

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 by RNAi in adult rat testes in vivo perturbs microtubule (MT) organization through changes in the distribution of EB1 in the seminiferous epithelium. A: in control testes, MTs (visualized by α-tubulin staining, green fluorescence, which together with β-tubulin created the α- and β-tubulin heterodimers to assemble the MT protofilaments) appeared as tracklike structures that lay perpendicular to the basement membrane. These tracklike structures in turn support the transport of spermatids and organelles (e.g., residual bodies, phagosomes) across the seminiferous epithelium as annotated by white arrowheads. EB1 (red fluorescence), a +TIP protein known to promote MT stabilization, also co-localized with α-tubulin to the tracklike structures of MTs in control testes as annotated by white arrowheads. However, following Dync1h1 knockdown, these tracklike structures of MTs and EB1 were extensively disorganized, either truncated (annotated by yellow arrowheads) or misaligned against the basement membrane (basement membrane annotated by a dashed white line). These disruptive changes were detected in virtually all stages of the tubules, and representative findings in stage I–III, V, VIII, IX, XI, and XIII tubules are shown herein. Scale bar, 40 µm, which applies to all other images. Using MT organization as a criterion to assess defective tubules, an ~fivefold increase in defective tubules was noted (see bar graph, bottom) with n = 5 rats. B: a biochemical-based MT spin-down assay was performed using lysates from these testes. Dync1h1 knockdown (confirmed by immunoblotting, see the fourth lane in the immunoblot and the accompanying histogram) or inactivation of dynein by inhibitor ciliobrevin D was found to considerably (last lane in the blot) reduce the ability of the testis lysate to induce MT polymerization (recovered in pellet). Taxol (20 µM) and CaCl2 (2 mM), known to stabilize MTs and promote MT depolymerization, served as the corresponding positive and negative control. Following Dync1h1 knockdown (bar graph, left), considerable reduction on the ability of testis lysate to support MT polymerization (bar graph, right) was noted. Each bar is a mean ± SD of n = 3 independent experiments. **P < 0.01, by Student’s t-test.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Staining, Fluorescence, Spin Down Assay, Western Blot, Negative Control

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: An inactivation of dynein by inhibitor ciliobrevin D in the testis in vivo perturbs microtubule (MT) organization through changes in the distribution of EB1 in the seminiferous epithelium. In control testes, MTs (green fluorescence, visualized by α-tubulin staining) appeared as tracklike structures that lay perpendicular to the basement membrane (annotated by a dashed white line) and stretched across the entire seminiferous epithelium (annotated by white arrowheads). EB1, a +TIP protein known to promote MT stabilization by binding to the fast growing plus end, also co-localized with MTs in the seminiferous epithelium in control testes (see white arrowheads). However, following dynein inactivation, these MT-based tracks were either truncated (yellow arrowheads) or misaligned (green arrowhead) against the basement membrane (basement membrane annotated by a dashed white line). These disruptive changes were detected in virtually all stages of the tubules, from I–XIV, and representative stage I–III, V, VIII, IX, XI, and XIII tubules were shown herein. Scale bar, 40 µm, which applies to other micrographs.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Fluorescence, Staining, Binding Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: Knockdown of Dync1h1 by RNAi in adult rat testes in vivo perturbs F-actin organization in the seminiferous epithelium. A: Dync1h1 was silenced in the testis by RNAi using Dync1h1-specific siRNA duplexes vs. control testes transfected with non-targeting negative control siRNA duplexes (Ctrl RNAi). F-actin (green fluorescence) was prominently organized at the apical ectoplasmic specialization (ES; around the Sertoli-spermatid interface, see yellow boxed areas) and at the basal ES (Sertoli cell-cell interface near the basement membrane, annotated by a dashed white line, see green boxed areas) in control testes. For instance, in stage I–III, V, and early VIII tubules in control testes, F-actin appeared as “bulblike” structures at the concave side of spermatid heads to support apical ES function except in late stage VIII tubules when spermiation took place, which was associated with a downregulation of F-actin expression to allow apical ES degeneration to facilitate sperm release at spermiation. Also, F-actin prominently localized at the blood-testis barrier (BTB) near the basement membrane, being used to support the immunological barrier function during the epithelial cycle. Furthermore, tracklike structures (annotated by white arrowheads) created by F-actin were noted in late stage VIII tubules, which were used to support phagosome and spermatid transport (step 8 spermatids first appeared in stage VIII tubules) across the seminiferous epithelium. However, after Dync1h1 knockdown, F-actin diffusely localized and grossly disorganized at the apical ES in stage I–III, V, and early VIII tubules, and no longer appeared as bulblike structures at the concave side of spermatid heads as noted in control testes. Additionally, F-actin no longer tightly localized at the basal ES to support BTB function; instead, F-actin appeared as truncated/branched network, diffusely localized at the site (see yellow brackets in Dync1h1 RNAi testes vs. white brackets in control testes). Also, many spermatids that had signs of defects in polarity were noted (annotated by yellow arrowheads) in which they no longer pointed toward the basement membrane but deviated by 90°–180° from the intended orientation. Some step 19 spermatids remained embedded deep inside the epithelium even in stage XIII tubules (green arrowheads) when they should have been released at stage VIII during spermiation. These disruptive changes were probably due to the absence of F-actin at the apical ES to support spermatid polarity and spermatid transport following Dync1h1 knockdown. Co-transfection of rhodamine-siGLO indicator (red fluorescence) illustrated successful transfection in the tubules. Scale bar, 40 µm; inset, 20 µm. B: when defective tubules were scored based on disruptive organization of F-actin in the seminiferous epithelium as noted in A, almost 30% of the tubules had signs of F-actin disorganization. **P < 0.01, by Student’s t-test. C: a biochemical-based actin spin-down assay was performed to assess the ability of testis lysates to polymerize actin monomers into microfilaments (i.e., F-actin, and recovered in pellet) to illustrate actin polymerization activity. It was noted that a knockdown of Dync1h1 or inactivation of dynein by inhibitor ciliobrevin D considerably impeded actin polymerization. Each bar in the histogram is a mean ± SD of n = 3 independent experiments. S/N, supernatant. **P < 0.01, by Student’s t-test.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Transfection, Negative Control, Fluorescence, Expressing, Cotransfection, Spin Down Assay, Activity Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: An inactivation of dynein 1 by inhibitor ciliobrevin D in adult rat testes in vivo perturbs F-actin organization in the seminiferous epithelium. Dynein 1 was inactivated by treatment of testes with ciliobrevin D vs. control testes treated with vehicle control (DMSO Ctrl). Dynein 1 inactivation by the inhibitor ciliobrevin D induced gross disruption of F-actin at the apical ectoplasmic specialization (ES). In control testes treated with DMSO only (Vehicle Ctrl), F-actin at the apical ES (enlarged in yellow boxed insets) appeared as “bulblike” structures at the concave side of spermatid heads in stage I–III, V, and VIII tubules. F-actin supported apical ES function until in late VIII of the cycle when the apical ES was degenerated to facilitate the release of sperm at spermiation. F-actin in late stage VIII tubules also appeared as tracklike structure (see white arrowheads) to support the transport of spermatids and organelles (e.g., phagosomes). Similarly, F-actin at the basal ES in control testes also supported the immunological barrier near the basement membrane (annotated by a dashed white line) by tightly localized at the blood-testis barrier (BTB; its relative position annotated by white brackets). However, following treatment of testes with ciliobrevin D to inactivate dynein, F-actin at the apical ES (enlarged in yellow boxed insets) was diffusively localized at the site, and no tracklike structures were noted across the epithelium. Furthermore, F-actin was not found in many elongating/elongated spermatids at the apical ES, causing defects of spermatid polarity with their heads pointed away from the basement membrane (annotated by yellow arrowheads) by 90°–180° from the intended orientation, considerably different from spermatids found in control testes. Also, many elongated spermatids were trapped deep inside the epithelium (annotated by green arrowheads) in stage VIII or even XIII tubules when they should have been emptied into the tubule lumen at spermiation because of defects in spermatid transport. Also, F-actin at the BTB near the basement membrane (annotated by a dashed white line) was diffusely localized (annotated by the yellow brackets) in treatment group because of disorganization of F-actin at the site when compared with control testes (white brackets). Scale bar, 40 µm; inset, 20 µm; these apply to other corresponding micrographs.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 or an inactivation of dynein by inhibitor ciliobrevin D in the testis in vivo perturbs distribution of basal ectoplasmic specialization (ES)- and tight junction (TJ)-associated proteins at the blood-testis barrier (BTB). A: in control testes, basal ES proteins N-cadherin and β-catenin and TJ proteins occludin and ZO-1 were tightly associated with the BTB (see white bracket) located near the basement membrane (annotated by a dashed white line) as noted in selected stage V tubules. However, following Dync1h1 knockdown or dynein 1 inactivation, these proteins were diffusely localized at the BTB by extending considerably away from BTB site (see yellow brackets), well beyond the basement membrane (annotated by a dashed white line). Co-transfection of rhodamine-siGLO indicator (red fluorescence) illustrated successful transfection. Histograms (bottom) summarized results of fluorescence analysis (top) regarding changes in the relative distribution of basal ES or TJ proteins in treatment vs. corresponding control groups. Each bar in the histogram is a mean ± SD of n = 4 rats, and 50 randomly selected cross sections of tubules in each testis were scored. **P < 0.01 by Student’s t-test. Scale bar, 40 µm. B: results of an in vivo BTB integrity assay in which the BTB found in control testes from the two control groups were similar to normal rat testes (-ve control group) and were capable of blocking the diffusion of membrane impermeable EZ-LinkSulfo-NHS-LC-Biotin across the immunological barrier. However, as noted in the positive control group (+ve) in which rats were treated with CdCl2 (3 mg/kg body weight, ip) for 5 days, known to disrupt the BTB function (94), biotin freely diffused across the immunological barrier to reach into the tubule lumen, which was visualized by Alexa Fluor 488-streptavidin (green fluorescence) with cell nuclei stained by DAPI. It was noted that the BTB became “leaky” in both Dync1h1 knockdown and dynein 1 inactivated testes. The histogram (bottom) summarized results of the BTB integrity assay in which each bar represents a mean ± SD of n = 3 rats that illustrated the ratio of the distance traveled by the biotin vs. the radius of a seminiferous tubule. For oblique sectioned tubules, the radius of the tubule was obtained by averaging the longest and shortest distance from the tubule lumen. **P < 0.01, by Student’s t-test. Scale bar, 350 µm and 80 µm (magnified micrograph), which apply to corresponding micrographs.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Cotransfection, Fluorescence, Transfection, Integrity Assay, Blocking Assay, Diffusion-based Assay, Positive Control, Staining

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

doi: 10.1152/ajpendo.00114.2018

Figure Lengend Snippet: A knockdown of Dync1h1 or an inactivation of dynein 1 by inhibitor ciliobrevin D in the testis in vivo perturbs the spatiotemporal expression of actin regulatory and apical ectoplasmic specialization (ES) proteins in the seminiferous epithelium of adult rat testes. A: in the testis of the two control groups [namely the Ctrl RNAi and DMSO (Ctrl)], the branched actin polymerization protein Arp3 and actin barbed end capping and bundling protein Eps8 were robustly expressed at the apical ES as “bulblike” structures on the concave side of spermatid heads to support extensive junction remodeling, including protein endocytosis and recycling in stage VII tubules. This thus supported the proper adhesion of elongated spermatids in early stage VIII tubules to prepare for their eventual release at spermiation at late stage VIII of the cycle as noted by laminin-γ3 chain staining, which is a spermatid-specific apical ES marker protein (41, 81, 97). Arp3 and Eps8 also tightly expressed at the basal ES/blood-testis barrier (BTB) to support BTB function. However, after either Dync1h1 knockdown (Dync1h1 RNAi) or inactivation of dynein 1 by ciliobrevin D, the spatiotemporal expression of either Apr3 or Eps8 were similar in both treatment groups: each of these actin-binding/regulatory proteins was grossly mis-localized (see yellow arrowheads), causing a disruption of the F-actin network at the apical ES as noted in Fig. 9A and Fig. 10. This, in turn, impeded the function of apical ES adhesion proteins as noted by changes in the distribution of laminin-γ3 chain, causing either premature release of spermatids or leading to defects in spermatid polarity (see white dotted lines). The yellow or green boxed areas illustrating the corresponding apical ES or basal ES region were magnified in insets. The basement membrane was annotated by a dashed white line. Co-transfection of rhodamine-siGLO indicator (red fluorescence) illustrated successful transfection. Scale bar, 40 µm; inset, 20 µm. B: these histograms illustrate a considerable increase in stage VII (based on Arp3 or Eps8 staining) or VIII (based on laminin-γ3 staining) when the corresponding marker proteins together with the relative location/presence of step 19 spermatids in the epithelium were used to define these stages. In control groups, the percent of stage VII and VIII was estimated to be ~20% and 7.5%, respectively, consistent with results of an earlier report (36). Each bar is a mean ± SD of n = 3 rats. For each testis, at least 50 stage VII or VIII tubules were randomly selected and scored for each group. **P < 0.01, by Student’s t-test.

Article Snippet: MT spin-down assay was performed as described ( 49 , 85 ) to estimate the relative level of polymerized MTs versus free tubulins in Sertoli cell or testis lysates following Dync1h1 RNAi or treatment with dynein inhibitor ciliobrevin D according to the manufacturer’s protocols (Cat No. BK-038, Cytoskeleton, Denver, CO).

Techniques: In Vivo, Expressing, Staining, Marker, Binding Assay, Cotransfection, Fluorescence, Transfection