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

Abstract

are contributed equally to the completion of this work.Abbreviations: +TIP, microtubule plus (+)-end tracking protein; AJ, adherens junction; aPKC, atypical protein kinase C; Arp3, actin-related protein 3; BM, basement membrane; BTB, blood-testis barrier; Crb3, Crumbs homolog-3; DAPI, 4', 6-diamidino-2-phenylindole; Dia1, Diaphanous-related formin-1; Dlg1, Discs large 1; Dvl3, Disheveled 3; EB1, end-binding protein 1; Eps8, epidermal growth factor receptor pathway substrate 8; ES, ectoplasmic specialization; Fzd3, Frizzled 3; F12/DMEM, Ham's F12 nutrient mixture/Dulbecco's modified Eagle's medium (1:1, vol/vol); GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GJ, gap junction; H&E, hematoxylin and eosin staining; IF, immunofluorescence microscopy; IgG, immunoglobulin; IP, immunoprecipitation; MAP1a, microtubule-associated protein 1a; MARK, microtubule affinity-regulatory kinase 4; MT, microtubule; mTOR, mammalian target of rapamycin; NC1 domain, non-collagenous domain 1; Pals1, protein associated with Lin-7 1; PatJ, Pals1-associated tight junction protein; Par6, partitioning-defective (Par) protein 6; rpS6, ribosomal protein S6; TJ, tight junction; ZO-1, zonula occludens-1. AbstractDuring the epithelial cycle of spermatogenesis, different sets of cellular events take place across the seminiferous epithelium in the testis. For instance, remodeling of the blood-testis barrier (BTB) that facilitates the transport of preleptotene spermatocytes across the immunological barrier and the release of sperms at spermiation take place at the opposite ends of the epithelium simultaneously at stage VIII of the epithelial cycle. These cellular events are tightly coordinated via locally produced regulatory biomolecules. Studies have shown that collagen α3 (IV) chains, a major constituent component of the basement membrane, release the non-collagenous (NC) 1 domain, a 28-kDa peptide, designated NC1-peptide, from the C-terminal region, via the action of MMP-9 (matrix metalloproteinase 9). NC1-peptide was found to be capable of inducing BTB remodeling and spermatid release across the epithelium. As such, the NC1-peptide is an endogenously produced biologically active peptide which coordinates these cellular events across the epithelium in stage VIII tubules. Herein, we used an animal model, wherein NC1-peptide cloned into the pCI-neo mammalian expression vector was overexpressed in the testis, to better understanding the molecular mechanism by which NC1-peptide regulated spermatogenic function. It was shown that NC1-peptide induced considerable downregulation on a number of cell polarity and planar cell polarity (PCP) proteins, and studies have shown these polarity and PCP proteins modulate spermatid polarity and adhesion via their effects on microtubule (MT) and F-actin cytoskeletal organization across the epithelium. More important, NC1-peptide exerted its effects by downregulating the expression of microtubule (MT) plus-end tracking protein (+TIP) called EB1 (end-binding protein 1). We cloned the full-length EB1 cDNA for its ov...

Busulfan impairs blood–testis barrier and spermatogenesis by increasing noncollagenous 1 domain peptide via matrix metalloproteinase 9

“…Actually, these previous studies have shown the extensive disruption of the F‐actin and MT‐based cytoskeletal tracks, being shortened, truncated, and mis‐aligned across the seminiferous epithelium following NC1 overexpression 28 . The polymerization behaviors of tubulins and β‐actin that detected by biochemical‐based spin‐down assays were also shown to be affected by increased NC1 level 30,59 . In this sense, the phenotypes of spermatogenesis and cytoskeleton disturbance that we observed herein after one‐dose busulfan exposure closely resemble those following NC1 overexpression.…”

“…28 The polymerization behaviors of tubulins and βactin that detected by biochemical-based spin-down assays were also shown to be affected by increased NC1 level. 30,59 In this sense, the phenotypes of spermatogenesis and cytoskeleton disturbance that we observed herein after one-dose busulfan exposure closely resemble those following NC1 overexpression. And this prompted the possibility that NC1 could be a key segment for BU-induced BTB injury.…”

Cell-Cell Interaction-Mediated Signaling in the Testis Induces Reproductive Dysfunction—Lesson from the Toxicant/Pharmaceutical Models

“…Studies have shown that NC1 peptide is released from the structural collagen α3 (IV) chains in the basement membrane, which in turn, serves as a biologically active peptide to induce Sertoli cell BTB remodeling, thereby facilitating the transport of preleptotene spermatocytes across the BTB at stage VIII-early stage IX of the epithelial cycle [ 124 ]. In brief, the NC1-peptide that induces Sertoli cell BTB remodeling, such as by perturbing the Sertoli cell-TJ permeability barrier function following its overexpression in Sertoli cells, is mediated through changes in the organization of both actin and microtubule cytoskeletons [ 124 , 125 ]. Additionally, these effects on cytoskeletal organization are mediated through activation of Cdc42, but not RhoA [ 88 ].…”