Abstract
Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell–cell and Sertoli cell–spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood–testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell–cell interface, and the apical ES at the Sertoli–spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.
Highlights
In actively migrating mammalian cells such as macrophages and fibroblasts, they generate branched actin filament networks and parallel actin filament bundles in lamellipodia and filopodia, respectively, by engaging two entirely different actinWen et al Cell Death and Disease (2018)9:208neither Sertoli nor germ cells possess lamellipodia and filopodia in vivo to support active cell movement
Studies have shown that the testis-specific adherens junction (AJ) known as ectoplasmic specialization (ES) that are found at the Sertoli–spermatid interface is the only anchoring junction that supports spermatid transport during spermiogenesis; and ES is found at the Sertoli cell-cell interface, which is the crucial component of the blood–testis barrier (BTB) that supports preleptotene spermatocyte transport across the immunological barrier[7,8,14,15,16]
Spire 1 was expressed in the seminiferous epithelium, prominently associated with apical ES at the Sertoli-elongating/elongated spermatid interface such as in stages VI–VIII tubules and conspicuously found at the convex side of spermatid heads, co-localizing with F-actin; it expressed in the basal compartment near the basal ES/ BTB and partially co-localized with F-actin (Fig. 1e)
Summary
In actively migrating mammalian cells such as macrophages and fibroblasts, they generate branched (i.e., unbundled) actin filament networks and parallel actin filament bundles in lamellipodia and filopodia, respectively, by engaging two entirely different actinWen et al Cell Death and Disease (2018)9:208neither Sertoli nor germ cells possess lamellipodia and filopodia in vivo to support active cell movement. Studies have shown that Sertoli cells in the testis are utilizing the Arp2/3 (actin related protein 2/3)N-WASP (neural Wiskott-Aldrich syndrome protein) complex[19] and formin 120,21 to regulate F-actin organization at the apical and basal ES to support germ cell transport in the epithelium during the epithelial cycle. It remains to be investigated if Spire is expressed by Sertoli and/or germ cells and if it is involved in regulating F-actin organization in the testis
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