TGFβs modulate permeability of the blood-epididymis barrier in an in vitro model

Abstract

The blood-epididymis barrier (BEB) is formed by epithelial tight junctions mediating selective permeability of the epididymal epithelium. Defective barrier function can disturb the balance of the epididymal milieu, which may result in infertility. The stroma of the epididymis contains high amounts of cytokines of the TGFβ family of unknown function. We screened possible effects of all three TGFβ isoforms on paracellular tightness in a BEB in vitro model based on the strongly polarized mouse epididymal epithelial MEPC5 cells in the transwell system. In this model we found a robust transepithelial electrical resistance (TER) of about 840 Ω x cm(2). Effects on the paracellular permeability were evaluated by two methods, TER and FITC-Dextran-based tracer diffusion assays. Both assays add up to corresponding results indicating a time-dependent disturbance of the BEB differentially for the three TGFβ isoforms (TGFβ3>TGFβ1>TGFβ2) in a TGFβ-receptor-1 kinase- and Smad-dependent manner. The tight junction protein claudin-1 was found to be reduced by the treatment with TGFβs, whereas occludin was not influenced. Epididymal epithelial cells are predominantly responsive to TGFβs from the basolateral side, suggesting that TGFβ may have an impact on the epididymal epithelium from the stroma in vivo. Our data show for the first time that TGFβs decrease paracellular tightness in epididymal epithelial cells, thus establishing a novel mechanism of regulation of BEB permeability, which is elementary for sperm maturation and male fertility.

Figure 1. Time-dependent TGFβ effects on TER in the BEB in vitro model.

TGFβs decreased epithelial barrier in a time-dependent manner. TGFβ3 decreased absolute values of TER (indicated by Ω x cm²) significantly within 4 h, 6 h and 24 h compared to untreated control each. TGFβ1 and TGFβ2 influenced the TER significantly after 6 h and 24 h. Data points represent mean values obtained from n = 6, generated by three independent repetitions performed in duplicate. SEM is indicated, p-values ≤0.05 (Mann-Whitney-test) were considered significant (_*), p≤0.005 highly significant (_**).

Figure 2. TGFβ effects on tracer diffusion in the BEB in vitro model.

Treatment with TGFβ3 increased the permeability coefficient (P_app) highly significant (p = 0.0002) compared to untreated control, whereas for TGFβ1 the level of significance (p = 0.0281) is lower. Each column represents a mean value obtained from n = 6, generated by three independent repetitions performed in duplicate. SEM is indicated, p-values ≤0.05 (Mann-Whitney-test) were considered significant (_*), p±0.005 highly significant (_**).

Figure 3. Cell morphology and claudin-1 immunostaining after treatment with TGFβs.

MEPC5 monolayers photographed in bright field (A–D), DAPI staining (E–H), and claudin-1 immunostaining (I–L). A: untreated MEPC5; B–D: MEPC5 cells treated with TGFβs for 24 h. No obvious changes in cell morphology are detectable. E: DAPI labeling of the nucleus in untreated MEPC5; F–H: MEPC5 cells treated with TGFβs for 24 h. No obvious changes are detectable. I: Immunostaining of claudin-1 in untreated MEPC5, DAPI labeling of the nucleus. The pattern of claudin-1 localization shows numerous cell clusters characterized by an intense immunofluorescence at the cell borders. J–L: MEPC5 cells treated with TGFβs for 24 h. After treatment with TGFβ3 (and to a lesser intent with TGFβ1) the staining was generally weaker, the striking immunofluorescence at cell borders was clearly reduced. Photos are examples from at least three independent experiments. Scale bar: 12.5 µm in A–D, 25 µm in E–L.

Figure 4. Western blot analysis of claudin-1 and occludin expression after treatment with TGFβs.

A: Western blot with lysates of MEPC5 after combined basolateral and apical stimulation with TGFβ1, TGFβ2 or TGFβ3 for 24 h compared to untreated cells. Lysates from three independent experiments (I–III) were used. TGFβ1 and especially TGFβ3 decreased the level of claudin-1 compared to the untreated sample. The levels of occludin were unaffected. Membrane (M) and cytosolic (C) protein fractions of mouse epididymis served as controls. B, C: Densitometric analysis of claudin-1 (B) and occludin (C) protein expression. Claudin-1 showed a significant reduction of expression by TGFβ3 by TGFβ1 treatment (p±0.05). All arbitrary units were normalized to the corresponding values of vinculin, serving as loading control. Columns represent mean values of three independent experiments (I–III) with SEM indicated. p-values compared to untreated controls according to Students t-test.

Figure 5. Time-dependent effects of TGFβ pathway inhibitors on TER in the in vitro model.

In case of inhibitor application, cells were incubated with inhibitors alone for 3βs + inhibitors for 2 h, 4 h and 6 h. Values prior to any treatment were taken as baseline (100%). A: The inhibition of TGFβ-R1 kinase activity by Ly364947 (Ly36) compared to untreated and vehicle-treated cells. B-D: TGFβs-affected TER values in the presence and absence of Ly364947. Effects observed for TGFβs are significantly inhibited by TGFβ-R1 inhibition. E: The inhibition of Smad3 by SiS3 compared to untreated and vehicle-treated cells. F–H: TGFβs-affected TER values in the presence and absence of SiS3. Inhibition of Smad3 resulted in attenuation of TGFβ effects. Data points represent mean values obtained from n = 6, generated by three independent repetitions performed in duplicate. SEM is indicated, p-values ±0.05 (Mann-Whitney-test) were considered significant (_*), p±0.005 highly significant (_**).

Figure 6. Dependency of TGFβ effects on TER from the side of stimulation.

TGFβ3 was applied into the upper compartment (apical) only (A), into the lower compartment (basolateral) only (B), or into both (apical + basolateral), (C). Basolateral application of TGFβ3, whether alone or together with apical stimulation decreased TER significantly at 4 h, 6 h and 24 h compared to untreated control, whereas apical stimulation had only slight effects. Due to increase of TER in the untreated control the effect of apical stimulation has to be considered significant after 6 h and 24 h. Data points represent mean values obtained from n = 6, generated by three independent repetitions performed in duplicate. Value prior to treatment was taken as baseline (100%). SEM is indicated, p-values ±0.05 (Mann-Whitney-test) were considered significant (_*), p±0.005 highly significant (_**).

Figure 7. Western blot analysis of claudin-1 and occludin expression after apical treatment with TGFβs.

A: Western blot with lysates of MEPC5 after apical stimulation with TGFβ1, TGFβ2 or TGFβ3 compared to untreated cells. TGFβ1 and TGFβ3 decreased the level of claudin-1 slightly compared to the untreated sample. The levels of occludin were unaffected. Membrane (M) and cytosolic (C) protein fractions of mouse epididymis served as controls. B, C: Densitometric analysis of claudin-1 (B) and occludin (C) protein expression. Claudin-1 showed a modest reduction of expression by TGFβ1 and TGFβ3 treatment which is not significant (p>0.05). All arbitrary units were normalized to the corresponding values of vinculin, serving as loading control. Columns represent mean values of three independent experiments with SEM indicated. p-values compared to untreated controls according to Students t-test.