Expression pattern of SOAT, OATP6A1, OATP1C1 and OSCP1 in human tissues.

<p>Tissue expression of the indicated carriers was analyzed by quantitative real-time PCR. Relative expression depicted at the y-axis represents x-fold higher expression in the respective tissue compared to the tissue with the overall lowest expression among the tissue panel (set as calibrator). The values represent means ± SEM of triplicate determinations.</p

SOAT expression in stably transfected SOAT-HEK293 cells.

<p>In the SOAT-HEK293 cells SOAT expression was induced by pre-treatment with tetracycline (+Tet). Control cells were untreated with tetracycline (−Tet) or represent non-transfected HEK293 cells (contr.). (<b>A</b>) Cell lysates were processed for WB analysis with the SOAT_311–377 antibody and revealed an apparent molecular weight of 49–55 kDa, likely representing different glycosylation states of the SOAT protein. (<b>B</b>) SOAT expression was directed to the plasma membrane of HEK293 cells by immunofluorescence analysis with the SOAT_311–377 antibody (green fluorescence). Nuclear staining with DAPI (blue fluorescence). Scale bar: 25 µm.</p

Immunohistological localization of SOAT in normal spermatogenesis after stage-dependent analysis.

<p>IHC was performed using the Soat_329–344 antibody with subsequent AEC staining and hematoxylin counterstain. The larger pictures show a whole segment of the seminiferous epithelium in different stages (I–VI) of spermatogenesis (primary magnification ×40), whereas insets show a detail of the respective stage (primary magnification ×100 oil). SOAT expression is also schematically indicated by red labeling on a drawing of spermatogenic stages (modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062638#pone.0062638-Bergmann1" target="_blank">[34]</a>). (<b>A</b>) Within stage I of spermatogenesis, the SOAT protein was detected in primary pachytene spermatocytes (P, black arrowheads) and round spermatids (step 1, white arrowheads). (<b>B</b>) SOAT immunoreactivity was detected in stage II of spermatogenesis within primary pachytene spermatocytes (P, black arrowheads) as well as in round spermatids (step 2, white arrowheads). (<b>C</b>) In stage III, only primary pachytene spermatocytes were stained with the Soat_329–344 antibody (P, black arrowheads), whereas round spermatids were negative (step 3, white circles). (<b>D</b>) Within stage IV of spermatogenesis, a similar staining pattern was detected, showing SOAT protein in primary pachytene spermatocytes (P, black arrowheads), but not in round spermatids (step 4, white circles). Note the newly formed acrosomal cap in step 4 spermatids. (<b>E</b>) In stage V, primary zygotene spermatocytes (Z, white arrows) as well as pachytene spermatocytes (P, black arrowheads) were stained. Elongating spermatids, showing a distinct acrosomal cap, were not stained (step 5, white circles). (<b>F</b>) Stage VI is characterized by the first meiotic cleavage (white cross). Positive staining for SOAT protein was detected in primary zygotene spermatocytes (Z, white arrows) as well as in secondary spermatocytes (SII, black arrows), which can be hardly distinguished from round spermatids.</p

Primer sequences for qualitative RT-PCR.

<p>Primer sequences for qualitative RT-PCR.</p

Qualitative mRNA expression analysis of SOAT, OATP6A1 and OSCP1 in seminiferous tubules (Tub) and interstitial tissue (Int) of human testes biopsies showing normal or impaired spermatogenesis following LACP.

<p>(<b>A</b>) Expression of all three carriers was detected in testis homogenate showing normal spermatogenesis. (<b>B</b>) SOAT, OATP6A1, and OSCP1 were only detected in seminiferous tubules from testis biopsies showing nsp, but not in the tubules from patients with SCO. No carrier mRNA was detected in interstitial tissue of the biopsies regardless their spermatogenic status. M, marker.</p