Table 1.
Antibodies used for different experiments in this report*.
Table 2.
Primers used for PCR.
Table 3.
Changes in the status of spermatogenesis in the rat testis following a knockdown of Rai14 by RNAi*.
Figure 1.
Rai14 is an actin-binding protein in the rat testis.
(A) A study by RT-PCR to confirm the expression of Rai14 in adult rat testis, Sertoli cells (SC, isolated from 20-day-old rat testes and cultured for 4-day), and germ cells (GC, isolated from adult rat testes and cultured for 16 hr). (B) Immunoblotting also confirmed the expression of Rai14 in the rat testis, Sertoli and germ cells, and the relative expression of Rai14 in SC vs. GC was shown in the histogram with n = 3 experiments in which the relative expression level of Rai14 in the testis was arbitrarily set at 1 so that the relative expression level between these samples can be compared. (C) The specificity of the anti-Rai14 antibody (Table 1) was assessed by immunoblotting using lysates of GC (20 µg protein). (D) Using the specific anti-Rai14 antibody, Rai14 was shown to be an actin-binding protein by co-immunoprecipitation (Co-IP); however, Rai14 did not structurally interact with any of the BTB-associated proteins including several actin-binding and regulatory proteins (e.g., Arp3, drebrin E, Eps8) and vimentin (an intermediate filament-based constituent protein). However, Rai14 was found to structurally interact with an actin cross-linking protein palladin which is known to be involved in conferring actin filament bundles in other mammalian cells [48]. (E) Rai14 (red) was also shown to be an actin-binding protein by dual-labeled immunofluorescence analysis in which it co-localized with F-actin (green) in Sertoli cells. Cell nuclei (blue) were visualized by DAPI. Scale ba = 20 µm, which applies to all other micrographs.
Figure 2.
Stage-specific expression of Rai14 and its association with the F-actin-rich ectoplasmic specialization (ES) in the seminiferous epithelium of adult rat testes.
Dual-labeled immunofluorescence analysis was performed using frozen cross-sections of testes from adult rat testes to examine co-localization of Rai14 (red) and F-actin (green) in the seminiferous epithelium. At stage VI, Rai14 was weakly detected at the apical ES at the Sertoli-spermatid interface, and its localization to the BTB was also weakly detectable. However, at stage VII-early stage VIII, the expression of Rai14 was the strongest. Rai14 was intensely localized to the apical ES, most abundantly at the front-end of the spermatid head, and co-localized with F-actin at the site (“yellow” in merged image of the apical ES), and its localization at the BTB remained not clearly visible. At stage VIII, Rai14 expression at the apical ES was still considerably strong, but it no longer restricted to the front-end of the spermatid head, instead, Rai14 was scattered around the spermatid head at the apical ES, but not tightly co-localized with the F-actin when compared to late stage VII tubule. In late stage VIII tubules, Rai14 also considerably expressed near the basement membrane (annotated by “white” broken line), consistent with its localization at the BTB (see “white” arrowheads), but not tightly co-localized with F-actin. At stage XI-XII, Rai14 remained localized to the apical ES, but it also shifted to the front-end of the spermatid head, partially co-localized with F-actin. “Yellow” and “green” boxed areas were magnified and shown in corresponding micrographs to better illustrate the localization and/or co-localization of Rai14 and/or Rai14/F-actin at the apical ES. Scale bar = 50 µm or 10 µm in the micrograph or inset, respectively, which apply to all other micrographs and insets.
Figure 3.
Rai14 is an apical and basal ES protein in the rat testis.
Dual-labeled immunofluorescence analysis was used to assess co-localization of Rai14 (red) with constituent protein laminin-γ3 chain (green), actin regulatory protein Arp3 (green), actin binding protein drebrin E (green) and actin filament cross-linking protein palladin (green) at the apical ES in stage VII tubules when these proteins were all highly expressed. It was found that Rai14 indeed partially co-localized with each of these apical ES proteins (see “orange yellow” in merged images) as shown in (A). Rai14 (red) also partially co-localized with basal ES/TJ protein ZO-1 (see “yellow” arrowheads), and to a lesser extent with N-cadherin (see “white” arrowheads) in stage IV-V tubules when these proteins were highly expressed as shown in (B). Bar = 10 µm in (A) or (B), which applies all other images in (A) and (B).
Figure 4.
Down-regulation in the expression of Rai14 and a loss of association between Rai14 and F-actin during adjudin-induced apical ES disruption and spermatid loss from the seminiferous epithelium.
Adult rats were treated with a single dose of adjudin (50 mg/kg b.w., by gavage) which is known to induce spermatid loss from the seminiferous epithelium since the apical ES, in particular the actin filament bundles at the site, is one of the primary targets of adjudin [7], [24]. Rats (n = 3 rats per time point) at specified time points were terminated and used for immunoblotting (A) which illustrated a rapid down-regulation of Rai14 expression following adjudin treatment, and the histogram shown in (B) summarized these findings. Each bar in (B) is a mean±SD of n = 3 using immunoblots such as those shown in (A) and normalized again β-actin which served as a protein loading control, and the relative Rai14 level in rats at time 0 (control) was arbitrarily set at 1. *, P<0.05. (C) Frozen sections from the testes of these rats were also obtained for dual-labeled immunofluorescence analysis. In control rat testes (0 hr), Rai14 (red) was intensely localized to the apical ES in a late stage VII tubule, localized to the front-end of the spermatid head and co-localized with F-actin (green), and the relative location of the basement membrane was annotated (see “white” broken line). Within 12 hr after adjudin treatment, Rai14 staining at the apical ES in a similar staged tubules was diminished and spermatids failed to migrate across the epithelium, losing their polarity (see “white” arrowhead that annotates a mis-oriented spermatid with its head no longer pointing to the basement membrane and it was “trapped” near the basement membrane without any Rai14 staining); more important, Rai14 no longer co-localized with F-actin, and this trend of diminishing Rai14 expression and loss of co-localization with F-actin persisted through 48 hr (“white” arrowhead also annotates a mis-oriented spermatid and was trapped in the basement membrane) and 96 hr. At 96 hr, expression of Rai14 near the basement membrane, consistent with its localization at the BTB was up-regulated, however, it was not co-localized with the F-actin. Insets in these micrographs are the magnified view of the corresponding “yellow” boxed areas. Bar = 50 µm or 10 µm in the micrograph or in the inset, which applies to all remaining micrographs and insets. (D) A loss of co-localization between Rai14 and F-actin at 12- and 48-hr following adjudin treatment as shown in (C) was further confirmed by Co-IP using testis lysates (800 µg protein) from rats at 0, 12-hr and 48-hr with anti-Rai14 IgG as the precipitating antibody, and the immunoblot was visualized using an anti-actin antibody to assess Rai14-actin interaction (the lower panel is IB only without Co-IP to serve as protein loading control). Data shown in (D) were summarized in (E) with each bar graph = a mean±SD of n = 3 experiments, a loss in Rai and actin protein-protein interaction was detected by 12-hr (∼35% reduction) and by 48-hr, a loss of ∼90% was detected. *, P<0.05; **, P<0.01.
Figure 5.
Knockdown of Rai14 in the Sertoli cell epithelium with an established TJ-permeability barrier in vitro by RNAi disrupts actin filament organization and the TJ barrier.
(A) Sertoli cells cultured alone on Matrigel-coated 12-well dishes for 2-day with an established TJ-permeability barrier were transfected with Rai14 siRNA duplexes (Rai14 RNAi) versus non-targeting control duplexes (Ctrl RNAi) at 100 nM using Ribojuice transfection medium for 24 hr, thereafter, cells were washed twice and cultured in F12/DMEM for 12 hr to allow recovery. Thereafter, cells were transfected again under the same conditions for another 24 hr. Thereafter, cells were rinsed with fresh F12/DMEM and cultured for an additional 12 hr before termination, and used to prepare lysates for immunoblotting using antibodies against several BTB-associated constituent or regulatory proteins. A knockdown of Rai14 by ∼50% was noted in which the control was arbitrarily set at 1 against which statistical comparison was performed (B) without any apparent off-target effects (A). The findings shown herein are the results of 3 independent experiments excluding pilot experiments which were used to establish optimal experimental conditions, such as different concentrations of siRNA duplexes and Ribojuice. It was noted that we achieved only ∼50–60% knockdown of Rai14 in several pilot experiments, unlike other target genes [e.g., Scribble, β1-integrin, and P-glycoprotein] wherein we could silence the target gene expression by as much as ∼70–90%. **, P<0.01. (C) In parallel experiment, Sertoli cells cultured for 2.5-day on Matrigel-coated bicameral units with an established TJ-permeability barrier (manifested by a stable TER across the cell epithelium) were transfected with the Rai14 siRNA duplexes versus non-targeting control siRNA duplexes for 36 h, and cells were washed twice and replaced with fresh F12/DMEM. TER was monitored across the Sertoli cell epithelium to assess changes in the TJ-permeability barrier function. Each data point is a mean±SD of triplicate bicameral units, and this experiment was repeated three times using different batches of Sertoli cells and yielded similar results. *, P<0.01. (D) Effects of Rai14 on F-actin organization and protein distribution at the Sertoli cell-cell interface were also assessed by immunofluorescence microscopy. The knockdown of Rai14 in Sertoli cells was found to reduce Rai14 staining considerably, consistent with data shown in (A, B). Interestingly, Rai14 knockdown also induced changes in F-actin organization in which the actin filaments were truncated and defragmented in the Sertoli cell cytosol (see asterisks). Furthermore, β- and α-catenin, adaptor proteins at the BTB, were also found to be mis-localized, which no longer localized to the cell-cell interface, but moved into the cell cytosol. Sertoli cells were co-transfected with 1 nM siGLO red transfection indicator to illustrate successful transfection. Scale bar = 20 µm, which applies to all remaining micrographs.
Figure 6.
Effects of Rai14 knockdown in the testis in vivo on the spermatid polarity.
(A) Rat testes were transfected with Rai14 specific siRNA duplexes (Rai14 RNAi) vs. control duplexes (Ctrl RNAi) to knockdown Rai14. A considerable decline in the Rai14 signals in the seminiferous epithelium in this late stage VII tubule, in particular at the apical ES, was noted as shown in (B) with ∼30% knockdown. Each bar in (B) is a mean±SD of n = 80 tubules at stage VII–VIII randomly selected from 4 rats. **, P<0.01. It is noted that Rai14 is an actin cross-linking and bundling protein, and actin filament bundles are the major constitute component of the apical ES, and apical ES is crucial to confer spermatid polarity and adhesion, the findings shown in (C) are consistent with the function of Rai14 at the apical ES. Following the knockdown of Rai14 in the testis, spermatids were detected in the tubule lumen of this stage VII tubule as a result of premature release of spermatids due to defects in the apical ES adhesion function (see “white” arrowheads). Furthermore, some spermatids were embedded in the epithelium (see “purple” and “blue” boxed areas) that failed to be transported to near the tubule lumen to prepare for spermiation at late stage VIII of the cycle. Additionally, spermatids that displayed a loss of polarity were seen (see “red” and “yellow” boxed areas and mis-oriented spermatids were annotated by “white” arrowheads). Scale bar = 100 µm in (A), 100 µm in (C), and 10 µm in inset in (C), which apply to corresponding micrographs in the same panel.
Figure 7.
Effects of Rai14 knockdown in the testis on F-actin organization and protein distribution at the apical ES.
Following a knockdown of Rai14 in the testis, a considerable loss of Rai14 signals (red fluorescence) were detected in the seminiferous epithelium of stage VII tubules as illustrated in this tubule here, in particular at the apical ES site. As shown in the Rai14 RNAi stage VII tubule, Rai14 was virtually not detected at the apical ES surrounding the heads of these step 19 spermatids, and polarity was disrupted in many of these spermatids lacking Rai14 since mis-oriented spermatids no longer pointing toward the basement membrane (see “white” arrowheads), perhaps due to the loss of actin filament bundles at the apical ES, which likely required Rai14 for their maintenance. This notion was strengthened when F-actin (green fluorescence) was visualized by FITC-conjugated phalloidin in Rai14 RNAi vs. control group. In control testes, at stage VII, F-actin was restricted to the tip of the spermatid head, mostly to the concave side of the head. After Rai14 knockdown, F-actin no longer restricted to the concave side of the spermatid head, instead considerable F-actin in many step 19 spermatids was found on the convex side of the spermatid head (see “yellow” arrowheads). This mis-localization of F-actin suggests that actin filament bundles in these spermatids were disrupted. Furthermore, while the signals of palladin (red fluorescence, an actin cross-linking and bundling protein), in the seminiferous epithelium in Rai14 knockdown group were not diminished vs. the control group, palladin was also found to become mis-localized, no longer found at the tips of the spermatid heads, but mis-localized and diffused away from spermatid head. Interestingly, the localization of laminin-γ3 chain (red fluorescence), an apical ES component of the integrin-laminin adhesion complex at the apical ES, was found not to be disrupted including mis-oriented spermatids with a loss of polarity. Scale bar = 20 µm, and scale bar in inset = 10 µm, which apply to other micrographs in the corresponding panel.