Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane–derived noncollagenous 1 domain peptide

Abstract

Spermatogenesis takes place in the epithelium of the seminiferous tubules of the testes, producing millions of spermatozoa per day in an adult male in rodents and humans. Thus, multiple cellular events that are regulated by an array of signaling molecules and pathways are tightly coordinated to support spermatogenesis. Here, we report findings of a local regulatory axis between the basement membrane (BM), the blood-testis barrier (BTB), and the apical ectoplasmic specialization (apical ES; a testis-specific, actin-rich adherens junction at the Sertoli cell-spermatid interface) to coordinate cellular events across the seminiferous epithelium during the epithelial cycle. In short, a biologically active fragment, noncollagenous 1 (NC1) domain that is derived from collagen chains in the BM, was found to modulate cell junction dynamics at the BTB and apical ES. NC1 domain from the collagen a3(IV) chain was cloned into a mammalian expression vector, pCI-neo, with and without a collagen signal peptide. We also prepared a specific Ab against the purified recombinant NC1 domain peptide. These reagents were used to examine whether overexpression of NC1 domain with high transfection efficacy would perturb spermatogenesis, in particular, spermatid adhesion (i.e., inducing apical ES degeneration) and BTB function (i.e., basal ES and tight junction disruption, making the barrier leaky), in the testis in vivo. We report our findings that NC1 domain derived from collagen a3(IV) chain-a major structural component of the BM-was capable of inducing BTB remodeling, making the BTB leaky in studies in vivo. Furthermore, NC1 domain peptide was transported across the epithelium via a microtubule-dependent mechanism and is capable of inducing apical ES degeneration, which leads to germ cell exfoliation from the seminiferous epithelium. Of more importance, we show that NC1 domain peptide exerted its regulatory effect by disorganizing actin microfilaments and microtubules in Sertoli cells so that they failed to support cell adhesion and transport of germ cells and organelles (e.g., residual bodies, phagosomes) across the seminiferous epithelium. This local regulatory axis between the BM, BTB, and the apical ES thus coordinates cellular events that take place across the seminiferous epithelium during the epithelial cycle of spermatogenesis.-Chen, H., Mruk, D. D., Lee, W. M., Cheng, C. Y. Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane-derived noncollagenous 1 domain peptide. FASEB J. 31, 3587-3607 (2017). www.fasebj.orgIn the mammalian testis, basement membrane (BM), a modified form of the extracellular matrix and a product of Sertoli and peritubular myoid cells (1, 2), is mostly constituted by type IV collagen, laminins (e.g., laminin a1, a2, b1, b2, g1), heparin sulfate proteoglycan, and nidogen (formerly called entactin) (3-5). As BM is in direct contact with Sertoli cells in the seminiferous epithelium, it is likely that there are crosstalks between Sertoli cells and the B...

NC1‐peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1

“…15 These studies thus illustrate that the NC1-peptide exerts its modulating effects to support spermatogenesis are mediated through changes in the cytoskeletal organization of both actin-and MT-cytoskeletons, in particular the organization of actin filament bundles and MTs across the Sertoli cell at the ultrastructures known as the basal ectoplasmic specialization (ES) or apical ES at the Sertoli cell-cell or Sertoli-spermatid interface, respectively. 14,15 Nonetheless, the underlying mechanism by which the NC1-peptide induces remodeling of actin filaments and MTs at the basal and apical ES remains unknown and unexplored. Herein, we provide compelling evidence that the MT plus (+)-end tracking protein (+TIP) EB1 (end-binding protein 1) earlier shown to be a key regulator of cytoskeletal dynamics in the testis, 16 is likely working with other MT regulating proteins, in particular MARK4, Dia1 and dynein 1, and possibly involving other cell polarity proteins (eg, Par6 and Crb3) and also planar cell polarity (PCP) protein Dishevelled (Dvl3) to support NC1-peptide to modulate spermatogenic function.…”

“…[10][11][12][13] Studies have shown that NC1-peptide was also generated in the testis, capable of modulating Sertoli cell tight junction (TJ)-permeability barrier function, 14 and its cloning into the mammalian expression vector pCI-neo to be used for its overexpression in Sertoli cells in vitro as well as in the testis in vivo was shown to perturb the Sertoli cell BTB function both in vitro and in vivo. 15 These studies thus illustrate that the NC1-peptide exerts its modulating effects to support spermatogenesis are mediated through changes in the cytoskeletal organization of both actin-and MT-cytoskeletons, in particular the organization of actin filament bundles and MTs across the Sertoli cell at the ultrastructures known as the basal ectoplasmic specialization (ES) or apical ES at the Sertoli cell-cell or Sertoli-spermatid interface, respectively. 14,15 Nonetheless, the underlying mechanism by which the NC1-peptide induces remodeling of actin filaments and MTs at the basal and apical ES remains unknown and unexplored.…”

“…24,25 It was also noted that transfection with empty vector alone (pCI-neo/Ctrl) also led to mild epithelial damage, but limited to ~8% of the tubules as noted herein, and consistent with earlier reports. 15,22…”

“…It has been shown that overexpression of NC1-peptide cloned into mammalian expression vector pCI-neo in the testis using Polyplus in vivo-jetPEI transfection reagent with a 70% transfection efficiency rapidly perturbed spermatogenic function and BTB integrity by days 3 and 7, respectively. 15 Herein, we expanded this earlier study by investigating changes in spermatogenic function within hours using the regimen shown in Figure 1A, following a single transfection at time 0, in order to provide mechanistic insights underlying NC1-peptide-mediated spermatogenic and BTB disruption. Overexpression of NC1-peptide was confirmed by RT-PCR (left panel, Figure 1B) and qPCR (right panel, Figure 1B) using primer pair specific to NC1-peptide (Table 3).…”

Bisphenol A and Male Reproductive System

“…The aim of spermatogenesis is to produce a unique male gamete that can fertilize an ovum (31,32). Recently, several studies have focused on the effects of BPA, but the experimental data are rather controversial, and there is no general agreement about the effects of BPA on spermatogenesis and sperm parameters (e.g., sperm count, motility, viability, density and morphology).…”