LAUSR

Abstract This study aimed to clarify the functional role of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2)-signaling pathway in the expression and localization of connexin 43 (Cx43). Mice were treated with the mitogen-activated protein kinase kinase (MEK1/2) inhibitor, PD325901, which induced a progressive decrease in ERK1/2 phosphorylation (pERK) in the proximal epididymis of the mice, without affecting total ERK level. Cx43 staining with punctuated reactive sites was observed in the basolateral membranes in the initial segment (IS) of mouse epididymis. However, PD325901 induced a significant decrease in Cx43 labeling in the basolateral membranes. Interestingly, Cx43, which was undetectable in the apical region of epididymis under control conditions, showed a significant increase in the apical region after PD 325901 treatment. To confirm whether Cx43 was present in tight junctions (TJs) after PD 325901 treatment, PD325901-treated epididymis samples were double-labeled with Cx43 and zonula occludens (ZO)-1 (a TJ protein marker). Thereafter, confocal microscopy showed the colocalization of Cx43 and ZO-1 in the epididymis after PD325901 treatment. Collectively, our results indicated that PD325901 treatment induced a significant increase in Cx43 localization on TJs, where it was colocalized with ZO-1. Therefore, the study suggested that ERK phosphorylation is essential for the proper expression and localization of the gap junction (GJ) protein, and that the relationship between GJs and TJs could play an important role in establishing and maintaining microenvironmental homeostasis for sperm maturation in the IS of mouse epididymis. Summary Sentence The MAPK/ERK signaling pathway is essential to regulate the expression and localization of Cx43 in the mouse proximal epididymis. Graphical Abstract