PAI-1 secretion of endometrial and endometriotic cells is Smad2/3- and ERK1/2-dependent and influences cell adhesion

In the endometrium transforming growth factor-betas (TGF-ßs) are involved mainly in menstruation and endometriosis. After binding of the ligands to the high-affinity receptors, TGF-ß receptors (TBR1 and TBR2), TGF-ßs activate Smad signaling to modulate gene expression and cellular functions. However, recently also Smad-independent pathways have been studied in more details. To evaluate both pathways, we have analyzed TGF-ß signaling in human endometrial and endometriotic cells. Although endometrial and endometriotic cells secrete TGF-ß1, secretion by stromal cells was higher compared to epithelial cells. In contrast, secretion of TGF-ß2 was higher in endometriotic stromal and endometriotic epithelial cells compared to normal endometrial cells. Treatment of endometrial and endometriotic stromal and epithelial cells with TGF-ß1 or TGF-ß2 increased Smad-dependent secretion of plasminogen activator inhibitor-1 (PAI-1) dramatically in all three cell lines. Of note, endometriotic cells secreted clearly higher levels of PAI-1 compared to endometrial cells. Whereas a TBR1 kinase inhibitor completely blocked the TGF-ß1 or TGF-ß2-induced PAI-1 secretion, an ERK1/2 inhibitor only partially reduced PAI-1 secretion. This inhibition was not dependent on epidermal growth factor receptor (EGFR) activation by phosphorylation but on kinase activity of the TBR1. Finally, treatment of endometrial and endometriotic cell lines with recombinant PAI-1 showed reduced cell adhesion, especially of the endometrial cells. In summary, our results demonstrate that both Smad-dependent and TBR1-dependent ERK1/2 pathways are necessary for TGF-ß-dependent high level secretion of PAI-1, which might increase cellular deadhesion

Investigation of the role of class I KNOX genes in the floral development of Eschscholzia californica

Four class I KNOX genes were isolated from the Eschscholzia californica, Papaveraceae. Phylogenetic analysis suggests that these four KNOX genes cover all three for the model Arabidopsis thaliana established clades of class I KNOX genes, STM (SHOOT APICAL MERISTEM-LESS), KNAT1/BP (Knotted-like Arabidopsis thaliana-1/BREVIPEDICELLUS) and KNAT2/6 (Knotted-like Arabidopsis thaliana-2/6). Two of these genes (EcSTM1 and EcSTM2) were identified as STM-like genes. Expression analysis in vegetative and floral shoots tips revealed a continuous expression of EcSTM1, EcKNAT1 and EcKNAT2, whereas EcSTM2 is exclusively expressed in early floral buds. EcSTM1-VIGS (Virus-induced gene silencing) resulted in the absence of the inner floral whorls. In flowers of EcSTM2-VIGS treated plants, the carpels were initiated but did not develop properly. Rarely, a floral reversion was observed. Combined silencing of EcSTM1 and EcSTM2 led to combinations of the phenotypes observed for EcSTM1-VIGS and EcSTM2-VIGS

Untersuchung der Rolle von Klasse I KNOX Genen in der Blütenentwicklung bei Eschscholzia californica

Four class I KNOX genes were isolated from the Eschscholzia californica, Papaveraceae. Phylogenetic analysis suggests that these four KNOX genes cover all three for the model Arabidopsis thaliana established clades of class I KNOX genes, STM (SHOOT APICAL MERISTEM-LESS), KNAT1/BP (Knotted-like Arabidopsis thaliana-1/BREVIPEDICELLUS) and KNAT2/6 (Knotted-like Arabidopsis thaliana-2/6). Two of these genes (EcSTM1 and EcSTM2) were identified as STM-like genes. Expression analysis in vegetative and floral shoots tips revealed a continuous expression of EcSTM1, EcKNAT1 and EcKNAT2, whereas EcSTM2 is exclusively expressed in early floral buds. EcSTM1-VIGS (Virus-induced gene silencing) resulted in the absence of the inner floral whorls. In flowers of EcSTM2-VIGS treated plants, the carpels were initiated but did not develop properly. Rarely, a floral reversion was observed. Combined silencing of EcSTM1 and EcSTM2 led to combinations of the phenotypes observed for EcSTM1-VIGS and EcSTM2-VIGS

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

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

Time-lapse imaging as a tool to investigate contractility of the epididymal duct--effects of cGMP signaling.

The well orchestrated function of epididymal smooth muscle cells ensures transit of spermatozoa through the epididymal duct during which spermatozoa acquire motility and fertilizing capacity. Relaxation of smooth muscle cells is mediated by cGMP signaling and components of this pathway are found within the male reproductive tract. Whereas contractile function of caudal parts of the rat epididymal duct can be examined in organ bath studies, caput and corpus regions are fragile and make it difficult to mount them in an organ bath. We developed an ex vivo time-lapse imaging-based approach to investigate the contractile pattern in these parts of the epididymal duct. Collagen-embedding allowed immobilization without impeding contractility or diffusion of drugs towards the duct and therefore facilitated subsequent movie analyses. The contractile pattern was made visible by placing virtual sections through the acquired image stack to track wall movements over time. By this, simultaneous evaluation of contractile activity at different positions of the observed duct segment was possible. With each contraction translating into a spike, drug-induced alterations in contraction frequency could be assessed easily. Peristaltic contractions were also detectable and throughout all regions in the proximal epididymis we found regular spontaneous contractile activity that elicited movement of intraluminal contents. Stimulating cGMP production by natriuretic peptide ANP or inhibiting degradation of cGMP by the phosphodiesterase 5 inhibitor sildenafil significantly reduced contractile frequency in isolated duct segments from caput and corpus. RT-PCR analysis after laser-capture microdissection localized the corresponding molecules to the smooth muscle layer of the duct. Our time-lapse imaging approach proved to be feasible to assess contractile function in all regions of the epididymal duct under near physiological conditions and provides a tool to evaluate acute (side) effects of drugs and to investigate various signaling pathways

Highly Conserved Testicular Localization of Claudin-11 in Normal and Impaired Spermatogenesis.

In this study we tested expression of tight junction proteins in human, mouse and rat and analyzed the localization of claudin-11 in testis of patients with normal and impaired spermatogenesis. Recent concepts generated in mice suggest that the stage-specifically expressed claudin-3 acts as a basal barrier, sealing the seminiferous epithelium during migration of spermatocytes. Corresponding mechanisms have never been demonstrated in humans. Testicular biopsies (n = 103) from five distinct groups were analyzed: normal spermatogenesis (NSP, n = 28), hypospermatogenesis (Hyp, n = 24), maturation arrest at the level of primary spermatocytes (MA, n = 24), Sertoli cell only syndrome (SCO, n = 19), and spermatogonial arrest (SGA, n = 8). Protein expression of claudin-3, -11 and occludin was analyzed. Human, mice and rat testis robustly express claudin-11 protein. Occludin was detected in mouse and rat and claudin-3 was found only in mice. Thus, we selected claudin-11 for further analysis of localization. In NSP, claudin-11 is located at Sertoli-Sertoli junctions and in Sertoli cell contacts towards spermatogonia. Typically, claudin-11 patches do not reach the basal membrane, unless flanked by the Sertoli cell body or patches between two Sertoli cell bodies. The amount of basal claudin-11 patches was found to be increased in impaired spermatogenesis. Only claudin-11 is expressed in all three species examined. The claudin-11 pattern is robust in man with impaired spermatogenesis, but the proportion of localization is altered in SCO and MA. We conclude that claudin-11 might represent the essential component of the BTB in human

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

<p>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.</p

Time-dependent effects of TGFβ pathway inhibitors on TER in the <i>in vitro</i> model.

<p>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 (_**).</p

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

<p>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.</p

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

<p>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.</p