Myosin VIIa Supports Spermatid/Organelle Transport and Cell Adhesion During Spermatogenesis in the Rat Testis

Abstract

The biology of transport of spermatids and spermatid adhesion across the seminiferous epithelium during the epithelial cycle remains largely unexplored. Nonetheless, studies have implicated the role of motor proteins in these cellular events. In this article, we report findings to unravel the role of myosin VIIa, an F-actin-based barbed (+)-end-directed motor protein, to support cellular transport and adhesion in the testis. Using RNA interference to knock down myosin VIIa in Sertoli cells cultured in vitro as a study model was shown to perturb the Sertoli cell tight junction permeability barrier, mediated through disorganization of actin- or microtubule (MT)-based cytoskeletons owing to disruptive changes on the spatiotemporal expression of F-actin or MT-regulatory proteins. Consistent with these in vitro findings, knockdown of myosin VIIa in the testis in vivo also induced disorganization of the actin- and MT-based cytoskeletons across the seminiferous epithelium, mediated by disruptive changes in the spatiotemporal expression of actin- and MT-based regulatory proteins. More important, the transport of spermatids and organelles across the epithelium, as well as cell adhesion, was grossly disrupted. For instance, step 19 spermatids failed to be transported to the adluminal compartment near the tubule lumen to undergo spermiation; in this manner, step 19 spermatids were persistently detected in stage IX and XII tubules, intermingling with step 9 and 12 spermatids, respectively. Also, phagosomes were detected near the tubule lumen in stage I to III tubules when they should have been degraded near the base of the seminiferous epithelium via the lysosomal pathway. In summary, myosin VIIa motor protein was crucial to support cellular transport and adhesion during spermatogenesis.

Figure 1.

A study to assess the distribution of myosin VIIa in the seminiferous epithelium of adult rat testes. (A) A study by RT-PCR to illustrate the expression of the mRNA encoding Myo7a (myosin VIIa gene name) in the testis from adult rats (∼300 g body weight), most predominantly expressed by Sertoli cells (SCs; isolated from 20-d-old rat testes), almost none by total germ cells (GCs; from adult rat testes), with kidney (KN; also from adult rats) as a positive control and S16 serving as a PCR and loading control. The primer pairs specific to Myo7a and S16 and used for RT-PCR are listed in Table 3. (B) Using a rabbit anti–myosin VIIa antibody for IB analysis using lysates of Sertoli cells (∼40 µg of protein) (Table 1), only a prominent electrophoretic band corresponding to myosin VIIa at 240 kDa was detected, supporting the notion that this antibody was specific to myosin VIIa. Protein markers (Spectra multicolor high range protein ladder from Thermo Fisher Scientific) are shown on the left panel. (C) Myosin VIIa was also visualized in Sertoli cells cultured in vitro for ∼4 d when a functional TJ permeability barrier was established (68). Myosin VIIa was distributed in the Sertoli cell cytosol and associated with the actin microfilaments. Cell nuclei were visualized by DAPI. Scale bar, 40 µm. (D) Myosin VIIa (red fluorescence) was found to express prominently at the apical ES (also boxed in yellow insets) and also basal ES/BTB (boxed in green insets) in the seminiferous epithelium at the Sertoli cell–spermatid (step 8 to 19 spermatids) and Sertoli cell–cell interface, respectively, in all staged tubules. Myosin VIIa also colocalized with F-actin (green fluorescence) as noted in the enlarged yellow or green insets to illustrate the apical ES and basal ES/BTB, respectively. Cell nuclei were visualized by DAPI. Scale bars, 80 µm; 40 µm in insets.

Figure 2.

Myosin VIIa is an integrated component of the apical and basal ES in the seminiferous epithelium of adult rat testes. (A) Myosin VIIa (red fluorescence) appeared as bulblike structures at the concave side of spermatid heads in stage VII tubules and was found to associate with either Arp3 (green fluorescence), Formin 1 (green fluorescence), or Eps8 (green fluorescence) at the apical ES. Scale bar, 40 µm. (B) In stage VII tubules, myosin VIIa was noted to express most prominently at the concave side of spermatid heads as bulblike structures but also some on the convex side of spermatid heads. Thus, it colocalized with apical ES proteins β₁-integrin (specifically expressed by Sertoli cells at the apical ES, but on the convex side of spermatid heads) and also nectin 3 (specifically expressed by elongated spermatids at the apical ES, also on the convex side of spermatid heads). Scale bar, 40 µm. (C) Myosin VIIa also prominently expressed at the basal ES/BTB and colocalized with basal ES adhesion protein complex N-cadherin/β-catenin and also TJ adhesion protein complex occludin/ZO-1. Cell nuclei were visualized by DAPI. Scale bar, 40 µm.

Figure 3.

A study to assess the effects of myosin VIIa knockdown in Sertoli cells cultured in vitro on the TJ permeability barrier and distribution of BTB-associated proteins at the cell–cell interface. (A) Regimen used in this study to assess the effects of Sertoli cell–specific myosin VIIa knockdown on the: (i) steady-state mRNA levels of Myo7a by qPCR (n = 3 experiments), (ii) steady-state levels of myosin VIIa and BTB-associated proteins or their distribution at the cell–cell interface (n = 3 experiments), or (iii) TJ permeability barrier function (n = 4 experiments). Each experiment had triplicate culture wells and coverslips or quadruple bicameral units. (B) Immunoblots to illustrate the efficacy of myosin VIIa silencing by RNAi. It was noted that there was no detectable changes in the expression of a number of BTB-associated proteins following myosin VIIa knockdown in Sertoli cells by RNAi, with each lane containing 30 µg of total protein. These findings also supported the notion that there was no off-target effect following myosin VIIa knockdown. (C) A study using qPCR and IB analysis of n = 3 experiments showed that myosin VIIa expression was downregulated by ∼70% following its knockdown by RNAi. **P < 0.01 by Student t test. (D) A knockdown of Sertoli cell myosin VIIa by RNAi using the regimen shown in (A) with considerable perturbed Sertoli cell TJ barrier function. (E) Following myosin VIIa knockdown, the expression of myosin VIIa in Sertoli cells was considerably downregulated, as noted by IF analysis, and results of image analysis to assess these changes, as noted in the bar graph on the right panel, were consistent with data shown in (C), confirming a ∼70% knockdown. Myosin VIIa knockdown impeded distribution of the TJ proteins (e.g., CAR, ZO-1) and basal ES proteins (e.g., N-cadherin, β-catenin), as these cell adhesion protein complexes no longer tightly aligned at the cell cortical zone (see white brackets) as in control cells, but were diffusively located (see white brackets; see also histograms on the right panel following image analysis of 200 randomly selected cells of n = 3 independent experiments). Successful transfection was confirmed by siGLO. Cell nuclei were visualized by DAPI staining. Scale bar, 40 µm. **P < 0.01 by Student t test.

Figure 4.

A study to assess the effects of myosin VIIa knockdown on the organization of actin- and MT-based cytoskeletons in Sertoli cells cultured in vitro through changes on the spatial expression of actin and MT regulatory proteins. (A) Following myosin VIIa knockdown, F-actin (green fluorescence) organization across the Sertoli cell cytosol was notably perturbed. Actin microfilaments no longer stretched across the Sertoli cell as linear filaments but were grossly truncated after myosin VIIa RNAi vs control cells. This appeared to be the result of changes in the spatial expression of Arp3 and Eps8 wherein these proteins were internalized and moved closer to the cell nucleus (annotated by yellow arrowheads). These changes thus failed to support linear actin filaments to stretch across the cell cytosol. Also, the vimentin-based intermediate filaments no longer stretched across the Sertoli cells but retracted from cell cytosol to be closer to the cell nuclei after myosin VIIa RNAi. Cell nuclei were visualized by DAPI. Successful transfection was confirmed by siGLO. Scale bar, 40 µm. (B) A biochemical-based assay was used to assess the ability of the Sertoli cell lysate to induce actin filament bundling activity in cells following myosin VIIa knockdown vs controls (transfected with nontargeting negative control siRNA duplexes). A knockdown of myosin VIIa by ∼70% was found to considerably impede actin bundling activity in myosin VIIa silenced vs control cells. Bar graphs in the right panel are the composite data, with each bar representing a mean ± SD of n = 3 experiments with triplicate cultures in each experiment. **P < 0.01 by Student t test. (C) Following myosin VIIa knockdown, MTs [visualized by α-tubulin staining (green fluorescence), a building block of MTs] were also found to be considerably disorganized, in which the long stretches of MT protofilaments were retracted from cell cytosol and moved closer to the cell nuclei. Furthermore, the organization of the detyrosinated and acetylated α-tubulins (both are the most stable, i.e., less dynamic, form of MTs) and the tyrosinated tubulins (which promoted MT dynamics, making MTs less stable) was also perturbed in myosin VIIa silenced vs control cells wherein they no longer stretched across the cell cytosol but wrapped around the cell nuclei. Cell nuclei were visualized by DAPI. Scale bar, 40 µm. (D) A biochemical-based spin-down assay that assessed the overall MT polymerization activity, confirming that a knockdown of myosin VIIa by ∼70% considerably impeded the ability of Sertoli cells to induce MT polymerization. Composite data are shown in the bar graphs in the right panel, with each bar representing a mean ± SD of n = 3 experiments with triplicate cultures in each experiment. **P < 0.01 by Student t test.

Figure 5.

A study to assess the effects of myosin VIIa knockdown in the testis in vivo on the status of spermatogenesis in the seminiferous epithelium. (A) Regimen used to assess the effects of in vivo RNAi on the status of spermatogenesis by transfecting rat testes with myosin VIIa–specific siRNA duplexes (myosin VIIa RNAi) vs nontargeting negative control siRNA duplexes (Ctrl RNAi). (B) Using Polyplus in vivo-jetPEI as a transfection medium for RNAi with a transfection efficiency of ∼70% (see “Materials and Methods”), myosin VIIa expression was found to be downregulated by almost 70% (see composite data summarized in the bar graph below) in the testis following its knockdown. However, the steady-state levels of several BTB-associated proteins were not affected by myosin VIIa knockdown (each lane contained 80 µg of total protein), illustrating that there was no off-target effect using the myosin VIIa–specific siRNA duplexes for RNAi. The bar graph is a composite of data of the IB analysis, with each bar representing a mean ± SD of n = 3 rat testes. **P < 0.01 by Student t test. (C) Histological analysis using paraffin sections of testes (stained by hematoxylin and eosin). Representative images at different stages of the cycle are shown, and selected images encircled in black, yellow, or green boxes were enlarged. Following myosin VIIa knockdown, multiple defects on spermatogenesis were noted. First, in stage I to III tubules, phagosomes were noted in the epithelium near the tubule lumen (yellow arrowhead) where they should have been absent; also, step 15 to 16 spermatids had undergone spermiation except that a few remained embedded in the epithelium (green arrowhead). Second, in stage V tubules, most step 17 spermatids had undergone unwanted spermiation, except a few that were embedded in the epithelium (green arrowhead); some multinucleated round spermatids were noted (white arrowhead). Third, in stage VIII tubules, step 19 spermatids were persistently found deep inside the epithelium (green arrowhead), and some residual bodies (red arrowhead) were also found near the tubule base instead of near the tubule lumen. Fourth, in stage IX tubules, phagosomes remained near the tubule lumen (yellow arrowheads), and then they should have been transported to the base of the tubule (green box); also, step 19 spermatids were persistently found in the epithelium (yellow box). Fifth, in stage XII tubules, step 19 spermatids (annotated by green arrowhead) were found together with step 12 spermatids (blue arrowhead) (in yellow box); also, some step 12 spermatids were mislocalized (black arrowhead) with their heads pointed at least 90° away from the intended orientation of pointing to the basement membrane (in green box). As shown in the middle panel, spermatozoa in the epididymis had signs of abnormality following myosin VIIa knockdown, including persistent presence of residual bodies, sperm heads containing genetic materials detached from the mid-piece, and abnormalities of sperm heads. Bar graphs in the lower panel indicate the percentage of defective tubules and percentage of abnormal sperm in the epididymis by scoring ∼700 randomly selected tubules or epididymides per rat with ∼2000 tubules from three rats. Each bar is a mean ± SD of n = 3 rats. Upper panel: scale bars, 250 µm for the low-magnification micrograph; 80 µm, 40 µm, and 40 µm in insets boxed in black, yellow, and green, respectively. Middle panel: scale bar, 40 µm for the epididymal sperm. Each bar is a mean ± SD of n = 3 rats, with a total of ∼2000 tubules randomly scored. **P < 0.01 by Student t test.

Figure 6.

A study to assess the effects of myosin VIIa knockdown in the testis in vivo on the organization of actin-based cytoskeleton in the seminiferous epithelium. As noted in control testes transfected with nontargeting negative control siRNA duplexes (Ctrl RNAi), F-actin was very well organized. F-actin was prominently expressed at the apical ES (i.e., surrounding spermatid heads) and basal ES/BTB (located near the basement membrane, which is annotated by a dashed white line) to support the ES function, except in late stage VIII through IX when F-actin was converted to G-actin to facilitate the release of sperm at spermiation and the transport of preleptotene spermatocytes across the immunological barrier, respectively. Even in stage IX tubules, when step 8 spermatids appeared, F-actin had also wrapped around the developing step 8 spermatids when apical ES was first detected in control testes. Following myosin VIIa knockdown in the testis in vivo (myosin VIIa RNAi), F-actin was no longer restrictively expressed at the apical ES, tightly associated with the developing spermatids to support spermiogenesis, as noted in stage I to III, V, early VIII, and IX tubules. For instance, F-actin did not wrap around spermatid heads, and thus many spermatids had defects in polarity, with their heads pointed away from the basement membrane by as much as 90° to 180° (annotated by yellow arrowheads). Furthermore, F-actin at the basal ES/BTB near the basement membrane (annotated by a dashed white line) was diffusely expressed at the site (see white brackets in the myosin VIIa knockdown testes). Scale bars, 80 µm; 40 µm in enlarged images boxed in green or yellow.

Figure 7.

Myosin VIIa knockdown-induced disorganization of F-actin at the ES is mediated through changes in the spatiotemporal expression of actin regulatory proteins Arp3 and Eps8. In this study, we examined whether there were changes in the spatial expression of two actin regulatory proteins, Arp3 and Eps8. First, the efficacy of myosin VIIa knockdown was confirmed by IF analysis of myosin VIIa in the epithelium. Myosin VIIa knockdown in the testis reduced myosin VIIa expression at the apical and basal ES in the testis by as much as ∼70% (see also bar graph in the right panel). In stage VII tubules of control testes, Arp3 and Eps8 were highly expressed at the concave side of spermatid heads, appearing as bulblike structures to support extensive endocytic protein trafficking events at the site; both proteins were also expressed at the basal ES/BTB near the basement membrane (annotated by the dashed white line). However, after myosin VIIa knockdown, both Arp3 and Eps8 were considerably downregulated and mislocalized at the apical ES, and many elongated spermatids displayed defects in their polarity (yellow arrowheads). These changes thus impeded the localization of a spermatid-specific apical ES adhesion protein laminin γ3 chain (67, 83), because laminin γ3 was no longer highly expressed at the tip of elongated spermatids, failing to support spermatid adhesion. The bar graph in the right panel also illustrates a considerable reduction in the percentage of stage VII tubules and an increase in the percentage of stage VIII tubules due to defects in spermiation after myosin VIIa knockdown. This is due to the release of sperms that took place in stage VII tubules, thus making these tubules similar to stage VIII tubules when tubules were randomly scored. Scale bars, 80 µm; 40 µm in insets that are magnified images boxed in green or yellow.

Figure 8.

Myosin VIIa knockdown in the testis in vivo caused mislocalization of basal ES and TJ proteins at the BTB. Because myosin VIIa knockdown in the testis perturbed the organization of F-actin in the seminiferous epithelium that impeded apical ES protein distribution, we next examined whether there were any changes in the distribution of basal ES and TJ proteins at the basal ES that supported BTB function. As anticipated, basal ES proteins N-cadherin (an integral membrane protein) and β-catenin (an adaptor protein) and TJ proteins occludin (an integral membrane protein) and ZO-1 (an adaptor protein) no longer restrictively localized to the basal ES/BTB near the basement membrane (annotated by a dashed white line) following myosin VIIa RNAi vs control testes. These proteins were diffusely localized at the site (see yellow brackets in myosin VIIa RNAi testes vs white brackets in Ctrl RNAi testes) because they used F-actin for attachment. These changes were also semiquantitatively analyzed and are shown in the bar graphs below. Each bar is a mean ± SD of randomly scored tubules from n = 3 rats. Scale bar, 40 μm, which applies to all micrographs in this panel. **P < 0.01 by Student t test.

Figure 9.

Myosin VIIa knockdown in the testis in vivo induces disorganization of MT-based cytoskeleton through changes in the spatiotemporal expression of EB1. In control testes (Ctrl RNAi), MTs [visualized by α-tubulin staining (green fluorescence), which together with β-tubulin create the α-/β-tubulin dimers, serving as the building blocks of MTs) colocalized with EB1 [red fluorescence, a barbed (+)-end tracking protein (+TIP) known to stabilize MTs (85)]. MTs and EB1 appeared as tracklike structures that lay perpendicular to the basement membrane to support spermatid and organelle (e.g., residual bodies, phagosomes) transport in all stages of the epithelial cycle. However, following myosin VIIa–specific knockdown in the testis, these tracklike structures were either truncated, analogous to the disruption of “freeways” to support spermatid and organelle transport across the epithelium, thereby perturbing spermatogenesis. Scale bar, 80 µm.