Characterizating the function of coxsackievirus and adenovirus receptor (CXADR) in the testis

Abstract

Coxsackievirus and adenovirus receptor (CXADR) is constitutively expressed in Sertoli cells (SCs) and all types of germ cells (GCs). CXADR is localized at the blood-testis barrier (BTB) constituted by tight junctions and basal ectoplasmic specializations (basal ES) between adjacent SCs. CXADR is also found at the interface between SC and GC at the apical ES. Earlier in vitro studies indicated that CXADR plays role in BTB function and GC migration, as well as conferring phosphorylation of occludin. However, conventional knockout of CXADR leads to embryonic lethality, making the study of the physiological importance of CXADR during testicular development impossible. To evaluate the functions of CXADR on spermatogenesis in vivo, SC-specific Cxadr deletion mice (SC-Cxadr-/-) were generated using the Cre/loxP system. Functional investigations, RNA sequencing, proteomics on SC-Cxadr-/- testes and in vitro CXADR-depleted cell culture model were employed to identify and unravel the regulatory mechanisms involving in male reproductive impairment. The Cre excision of Cxadr in SCs was achieved under the control of SC-specific Amh gene. The deletion of CXADR was confirmed by PCR and immunohistochemistry. Adult SC-Cxadr-/- mice exhibited a 44% reduction in fertility efficacy, as well as the reduced ratio of testes/body weight observed from 120 days old onwards due to GC apoptosis and loss. Compromised BTB function also has been observed in SC-Cxadr-/- testes. An array of BTB components including occludin/ZO-1 complex and β-catenin were down-regulated or mislocalized in SC-Cxadr-/- seminiferous epithelia. Up-regulation of non-phosphorylated β-catenin and inhibition of Cdc42 were further found in SC-Cxadr-/- seminiferous epithelia with disorganized F-actin at the apical ES. Transcriptomic and proteomic analyses of SC-Cxadr-/- testes revealed that the enriched gene ontology (GO) terms of biological process indeed relate to reproduction. Up-regulation of several Wnt targets were confirmed in Cxadr-depleted mouse testes or Sertoli cell line, including Wnt5a, Wnt6 and Wnt9a. Combining the genome-wide data and results from functional analyses of SC-Cxadr-/- testes, the Rap1/Wnt/Hippo signaling network was identified and predicted to cause fertility impairment in the knockout mice. Apart from being a structural protein at the BTB, CXADR apparently also served as the signaling mediator to trigger the downstream effects on junction disruption and actin reorganization via non-phosphorylation of β-catenin and Cdc42 inhibition. Interestingly, overexpression of constitutively active Cdc42 in Cxadr-depleted Sertoli cell line (MSC-1) model could partially recover the CXADR-mediated disorganization of F-actin filaments in vitro. Taken together, CXADR in SCs affects BTB function and F-actin organization via crosstalk of CXADR/β-catenin/Cdc42 and CXADR plays an indispensable role in spermatogenesis.