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. 2006 Jun;8(6):499–509. doi: 10.1593/neo.05847

Figure 4.

Figure 4

In seminiferous tubules showing normal spermatogenesis, a strong immunoreactivity at the level of the blood-testis barrier, apical to spermatogonia, basal to primary spermatocytes, along the Sertoli cell junctional complexes and between interstitial Leydig cells (black arrow, A) was observed. The expression pattern of Cx43 protein in CIS-only tubules was found to be heterogeneous. In very few tubules, a disorganized immunoreactive signal was present at the level of the blood-testis barrier (CIS cells: transparent arrows, B). Most of these tubules were found completely immunonegative for Cx43 protein (C). Leydig cells displayed a strong and persistent Cx43 immunoreactivity (black arrows, B and C). In seminoma, only between cells of single cell clusters within connective tissue (black arrow, D) and between endothelial cells of small blood vessels (arrowheads, E) an immunostaining for Cx43 could be detected independent of staging. Double immunostaining for Cx43 and inhibin-α clearly colocalized both proteins to the same cell type and identified these cells as Leydig cells (arrow, D). Cx43 protein (blue/pink) was imunolocalized to the plasma membranes of Leydig cells, whereas inhibin-α immunostaining (brown) was detectable in the cytoplasm of the same cell(s) (D). The tumor cells showed no immunoreaction for Cx43 at all (D and E). Scale bars = 50 µm. Representative Western blot analysis (F) from normal testicular tissue revealed three immunoreactive bands between 41 and 45 kDa (lane 2). Incubation with CIP changed the electrophoretic profiles of Cx43. Observed bands at 45 (Cx43-P2) and 43 kDa (Cx43-P1) were lost after CIP treatment, resulting in one strong band at 41 kDa (Cx43-P0, lane 3) indicating that these two former bands represent double- and single phosphorylated Cx43 isoforms. In seminoma, only a weak immunoreactive band at 45 kDa (Cx43-P2) could be demonstrated (lane 4). In addition, this band was lost after CIP treatment, resulting in the Cx43-P0 isoform (lane 5). In negative controls, no immunoreactive bands have been observed (lane 6). Lane 1: protein marker.