title: Epidermal growth factor regulates connexin 43 in the human epididymis: role of gap junctions in azoospermia.
abstract: BACKGROUND
  Gap junctions (GJs) allow for direct communication between adjacent cells. They are composed of connexons consisting of transmembrane proteins, connexins (Cxs). The objectives of this study were to determine if GJ proteins GJA1 (Cx43), GJB1 (Cx32) and GJB2 (Cx26) are present in the epididymis of men with a normal epididymis, to assess whether or not Cx expression and localization are altered in azoospermic patients, and to determine if epidermal growth factor (EGF) regulates GJA1 expression.
  
  
  METHODS
  Epididymides were obtained from men with localized testis cancer with active spermatogenesis and histologically normal epididymal tubule (group 1), men with non-obstructive azoospermia secondary to Sertoli-cell only syndrome (group 2) and from azoospermic men with normal spermatogenesis and epididymal obstruction (group 3). Epididymides were subdivided into three segments: caput, corpus and cauda. Quantitative real-time RT-PCR was performed to assess GJA1, GJB1, GJB2 and EGF receptor (EGFR) mRNA levels in epididymides from patients from each group (all n=3, except n=1 for caput blockage). A human caput epididymal cell line was then used to determine the role of EGFR signaling on the regulation of human epididymal GJA1.
  
  
  RESULTS
  Real-time RT-PCR analysis revealed that GJA1, GJB1, GJB2 and EGFR were expressed along the human epididymis. In the cauda epididymidis of group 2 and 3 men, we observed a significant decrease in GJA1 (P=0.0456 and P=0.0465, respectively) and GJB1 (P=0.0450 and P=0.0497, respectively) mRNA levels when compared with group 1 men. We also observed a decrease in EGFR mRNA levels (P=0.0358) in the cauda epididymidis of group 3 men when compared with group 1. Immunocytochemistry revealed that in the epididymis, GJA1 and EGFR were localized between basal and principal cells and between adjacent principal cells. In group 2 and 3 patients, however, we noted a dramatic increase in cytosolic immunostaining for both GJA1 and EGFR in both principal and basal cells. Using a human caput epididymal cell line derived from fertile men, we demonstrated that changes in GJA1 phosphorylation could be regulated by EGF (P=0.015) and the extracellular regulated kinase 1/2 signaling pathway (P=0.03). Furthermore, while the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway did not alter GJA1 phosphorylation, treatment with PI3K/AKT inhibitor LY294002 significantly (P=0.024) inhibited the EGF-stimulated increase in GJA1 total protein levels at 24 h. Immunolocalization indicated that loss of PI3K/AKT signaling was associated with increased cytosolic localization of Cx43 in this cell line.
  
  
  CONCLUSIONS
  Together, these data suggest that in azoospermic men decreased expression of EGFR may be responsible for decreasing GJA1 levels and increasing its cytosolic localization via the PI3K/AKT signaling pathway.
authors.affiliation: INRS-Institut Armand Frappier, Université du Québec, 531 Boulevard des Prairies, Laval, QC,  Canada H7V 1B7.
authors.forename: Evemie
authors.initials: E
authors.lastname: Dubé
authors.forename: Julie
authors.initials: J
authors.lastname: Dufresne
authors.forename: Peter T K
authors.initials: PT
authors.lastname: Chan
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Cyr
mesh_terms: D000328:Adult; D017209:Apoptosis; D053713:Azoospermia / Q000378:metabolism*; D000072259:Connexin 26; D018031:Connexin 43 / Q000378:metabolism*; D017630:Connexins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004822:Epididymis / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008954:Models, Biological; D012333:RNA, Messenger / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism; D013091:Spermatogenesis; D013737:Testis / Q000378:metabolism; D000097002:Gap Junction beta-1 Protein
chemical_list: D000072259:Connexin 26; D018031:Connexin 43; D017630:Connexins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D012333:RNA, Messenger; D000097002:Gap Junction beta-1 Protein; C500216:GJA1 protein, human; C000607042:GJB2 protein, human