Actin cross-linking protein palladin and spermatogenesis

Abstract

In the seminiferous epithelium of the mammalian testis, the most distinctive ultrastructure is the extensive bundles of actin filaments that lie near the Sertoli-spermatid interface and the Sertoli-Sertoli cell interface known as the apical ectoplasmic specialization (apical ES) and the basal ES, respectively. These actin filament bundles not only confer strong adhesion at these sites, they are uniquely found in the testis. Recent studies have shown that ES also confers spermatid and Sertoli cell polarity in the seminiferous epithelium during the epithelial cycle. While these junctions were first described in the 1970s, there are few functional studies in the literature to examine the regulation of these actin filament bundles. It is conceivable that these actin filament bundles at the ES undergo extensive re-organization to accommodate changes in location of developing spermatids during spermiogenesis as spermatids are transported across the seminiferous epithelium. Additionally, these actin filaments are rapidly reorganized during BTB restructuring to accommodate the transit of preleptotene spermatocytes across the barrier at stage VIII of the epithelial cycle. Thus, actin binding and regulatory proteins are likely involved in these events to confer changes in F-actin organization at these sites. Interestingly, there are no reports in the field to study these regulatory proteins until recently. Herein, we summarize some of the latest findings in the field regarding a novel actin cross-linker and actin-bundling protein called palladin. We also discuss in this opinion article the likely role of palladin in regulating actin filament bundles at the ES during spermatogenesis, highlighting the significant of palladin and how this protein is plausibly working in concert with other actin-binding/regulatory proteins and components of polarity proteins to regulate the cyclic events of actin organization and re-organization during the epithelial cycle of spermatogenesis. We also propose a hypothetic model by which palladin regulates ES restructuring during the epithelial cycle of spermatogenesis.

Keywords: F-actin; Sertoli cells; blood-testis barrier; ectoplasmic specialization; seminiferous epithelial cycle; spermatogenesis; testis; tight junction.

Figure 1. A schematic drawing illustrating the functional domains of a 90 kDa isoform of palladin polypeptide. In the testis, the predominant form of palladin detected by immunoblotting is the 90 kDa isoform, which is composed of a protein-rich region (PR region) for protein-protein interactions near its N terminus, and three IgC2 domains close to its C terminus for binding to F-actin to confer its F-actin cross-linking and F-actin bundling activity.

Figure 2. Stage-specific localization and expression of palladin in the seminiferous epithelium of adult rat testes. Palladin (red fluorescence) was localized to the tunica propria, associated with peritubular myoid cells and also near the basement membrane, consistent with its localization at the BTB. (A) Most notably, palladin was localized at the apical ES, surrounding the head of elongating spermatids, in step 15–19 spermatids at stage I‒VIII of the epithelial cycle. More importantly, its localization at the apical ES altered during the progression of spermiogenesis. For instance, at stage I‒V, palladin was found to surround the entire spermatid head, but it shifted mostly to the tip of the spermatid head beginning at stage VII, and most obvious at stage VIII, illustrating that palladin is no longer needed to confer the actin filament bundles at stage VIII in preparation for the release of sperm at spermiation. Immunofluorescence microscopy was performed as described in reference using a rabbit anti-palladin antibody (Protein Tech, Cat. # 10853–1-AP, working dilution, 1:100), spermatid nuclei were visualized by DAPI (4’,6-diamidino-2-phenylindole) staining. For each staged tubule, the “yellow” boxed area on the left was magnified and shown on the right, scale bar = 50 μm in the micrograph on the left, and scale bar = 10 μm in the micrograph enlarged on the right, which apply to corresponding micrographs in all other stages. These observations support the notion that palladin is involved in conferring changes in the organization of F-actin filaments from their “bundles” and “de-bundled” configuration to facilitate spermatid transport across the seminiferous epithelium as well as changes in the shape/morphology of the spermatid head, as well as spermatid polarity during spermiogenesis. (B) Palladin (red fluorescence) was also found to co-localize with F-actin at the BTB besides the tunica propriate (F-actin was detected by using phalloidin-FITC) (Sigma-Aldrich, Cat. # P5282; working dilution, 1:70). Bar = 10 μm in the micrograph on the left, which apply to corresponding micrographs in this panel.

Figure 3. A schematic drawing illustrating the mechanism by which palladin regulates ES restructuring during the epithelial cycle of spermatogenesis. Left panel shows the apical ES (upper) and the basal ES at the BTB (lower) at stage VII of the epithelial cycle when the ES is intact, maintained by adhesion proteins at the apical ES and basal ES (and also proteins of the TJ, GJ and desmosome at the BTB) to confer spermatid adhesion and the integrity of the BTB, respectively, since these proteins are anchored to the underlying actin filament bundles at both sites. The ES integrity is made possible in which c-Src and FAK maintains the proper phosphorylation status on palladin, which exerts its actin cross-linking and actin-bundling activity; also, palladin recruits Eps8 to the apical ES and the basal ES to maintain and strengthen the integrity of the actin filament bundles. On the right panel is the seminiferous epithelium at stage VIII of the epithelial cycle in which both the apical ES (upper) and the basal ES/BTB (lower) undergo restructuring to facilitate the release of spermatzoa at spermiation and the transit of preleptotene spermatocytes across the BTB. This is made possible in which c-Src and FAK phosphorylate palladin to inactivate its actin cross-linking and bundling intrinsic activity, such changes in phosphorylation status also reduce the binding affinity of palladin to Eps8, instead, more Arp3 is recruited to the apical and the basal ES, which induce branched actin polymerization, destabilizing the ES at both sites to facilitate spermiation and the transit of preleptotene spermatocytes at the BTB. The segregation of laminin chains from the integrin receptors at the apical ES (note: laminin- α3β3γ3-α6β1-integrin is an adhesion protein complex at the apical ES) also induces the generation of biologically active laminin fragments mediated via the action of MMP-2, which can further potentiate BTB restructuring as reported earlier.^, Changes in actin filament organization at the ES at both sites from the “bundled” to the “de-bundled” configuration also favor endocytic vesicle-medicated protein endocytosis, transcytosis and recycling to assemble “new” apical ES and “new” basal ES/BTB during disassembly of the “old” apical ES and the “old” basal ES/BTB. However, “aged” proteins undergo endosome-/ubiquitin-mediated intracellular degradation instead of being recycled. In short, this model illustrates the physiological significance of palladin in the restructuring of the apical and the basal ES during the epithelial cycle of spermatogenesis via its interaction with Eps8, Arp3 and c-Src.