Ralph Brehm

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

Reference Gene Validation for RT-qPCR, a Note on Different Available Software Packages

Background An appropriate normalization strategy is crucial for data analysis from real time reverse transcription polymerase chain reactions (RT-qPCR). It is widely supported to identify and validate stable reference genes, since no single biological gene is stably expressed between cell types or within cells under different conditions. Different algorithms exist to validate optimal reference genes for normalization. Applying human cells, we here compare the three main methods to the online available RefFinder tool that integrates these algorithms along with R-based software packages which include the NormFinder and GeNorm algorithms. Results 14 candidate reference genes were assessed by RT-qPCR in two sample sets, i.e. a set of samples of human testicular tissue containing carcinoma in situ (CIS), and a set of samples from the human adult Sertoli cell line (FS1) either cultured alone or in co-culture with the seminoma like cell line (TCam-2) or with equine bone marrow derived mesenchymal stem cells (eBM-MSC). Expression stabilities of the reference genes were evaluated using geNorm, NormFinder, and BestKeeper. Similar results were obtained by the three approaches for the most and least stably expressed genes. The R-based packages NormqPCR, SLqPCR and the NormFinder for R script gave identical gene rankings. Interestingly, different outputs were obtained between the original software packages and the RefFinder tool, which is based on raw Cq values for input. When the raw data were reanalysed assuming 100% efficiency for all genes, then the outputs of the original software packages were similar to the RefFinder software, indicating that RefFinder outputs may be biased because PCR efficiencies are not taken into account. Conclusions This report shows that assay efficiency is an important parameter for reference gene validation. New software tools that incorporate these algorithms should be carefully validated prior to use.

Altered expression of connexins 26 and 43 in Sertoli cells in seminiferous tubules infiltrated with carcinoma‐in‐situ or seminoma

The expression of connexins (cx) 26 and 43 in testis infiltrated with carcinoma‐in‐situ (CIS) or seminoma was examined to gain insight into the relationship between aberrant gap junctional communication and spermatogenic impairment in the neoplastic testis. In uninvolved tubules with normal spermatogenesis, cx43 immunostaining was localized to the Sertoli–Sertoli junctional complex and cx26 was absent. In contrast, infiltrated tubules with spermatogonial arrest or CIS‐only were negative for cx43, but displayed strong intracytoplasmic Sertoli cell staining for cx26. The Sertoli cells in these tubules re‐expressed cytokeratin 18 (ck18), signifying a reversion to a less differentiated state. Western blot analysis for cx43 revealed a single immunoreactive band at 43 kD (normal spermatogenesis) and three bands at 43, 41, and 39 kD (impaired spermatogenesis with CIS or seminoma). For cx26, a doublet band at 26/28 kD (normal spermatogenesis) and an additional doublet band at 52/54 kD (impaired spermatogenesis with CIS or seminoma) were observed. The altered expression of cx26 and cx43 in Sertoli cells in testes infiltrated with CIS or seminoma suggests that a derangement in intercellular communication between Sertoli cells and between Sertoli cells and germ cells may play a role in the resulting spermatogenic impairment and possibly in the proliferation and neoplastic progression of CIS cells. Copyright

DNMT1 and HDAC1 gene expression in impaired spermatogenesis and testicular cancer

DNA methylation catalyzed by DNA methyltransferases (DNMTs) and histone deacetylation catalyzed by histone deacetylases (HDACs) play an important role for the regulation of gene expression during carcinogenesis and spermatogenesis. We therefore studied the cell-specific expression of DNMT1 and HDAC1 for the first time in human testicular cancer and impaired human spermatogenesis. During normal spermatogenesis, DNMT1 and HDAC1 were colocalized in nuclei of spermatogonia. While HDAC1 was additionally present in nuclei of Sertoli cells, DNMT1 was restricted to germ cells exhibiting a different expression pattern of mRNA (in pachytene spermatocytes and round spermatids) and protein (in round spermatids). Interestingly, in infertile patients revealing round spermatid maturation arrest, round spermatids lack DNMT1 protein, while pachytene spermatocytes became immunopositive for DNMT1. In contrast, no changes in the expression pattern could be observed for HDAC1. This holds true also in testicular tumors, where HDAC1 has been demonstrated in embryonal carcinoma, seminoma and teratoma. Interestingly, DNMT1 was not expressed in seminoma, but upregulated in embryonal carcinoma.

Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4

Abstract.Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in normal testes. Transcripts of maternally expressed 3 transcripts were expressed in seminoma without correlation with delta-like 1 homologue expression indicating an impaired imprinting status in seminoma. Interestingly, the transcripts of bromodomain-containing 2 and nuclear autoantigenic sperm protein associated with spermatogenesis were significantly upregulated in progressing tumour stages. Transcription factors TEA domain family member 4 and ETS variant gene 4 (ETV4), weakly expressed in normal testis, were strongly augmented during tumourigenesis. For ETV4 expression, a significant correlation with the increased expression of matrix metalloproteinase 2 and a disintegrin and metalloproteinase domain 15 was determined. The ETV4 protein was localised to nuclei of spermatogonia and revealed an intense staining in seminoma cells. Taken together, we characterised additional transcription factors and spermatogenesis-associated genes involved in the progression of seminoma.

Role of connexin 43 in Sertoli cells of testis.

Abstract: Connexin 43 (CX43, also known as GJA1) is the predominant testicular gap‐junction protein. In the seminiferous epithelium, gap junctions occur between adjacent Sertoli cells and between Sertoli and germ cells. The specific role of CX43 in Sertoli cell has been difficult to study because of the neonatal lethality of the Cx43 global knockout. Recently two laboratories have independently developed a Sertoli cell conditional Cx43 knockout using Cre‐loxP methodology. These have allowed the role of CX43 in Sertoli cells to be determined while circumventing lethal abnormalities in organs such as the heart seen in Cx43 global knockouts. In this review the focus is on the insights into the role of CX43 in Sertoli cells derived from these new model systems. Results indicate that CX43 is essential for cessation of proliferation and normal maturation in Sertoli cells. In addition, CX43 in Sertoli cells is required for spermatogenesis, but not early germ cell proliferation. These model systems show that CX43 is important in Sertoli cell development, and will be useful in further studies of its role in Sertoli cell and testis biology.

Claudin-11 is over-expressed and dislocated from the blood–testis barrier in Sertoli cells associated with testicular intraepithelial neoplasia in men

In mouse testis, claudin-11 is responsible for the formation of specific parallel TJ strands of the blood–testis barrier (BTB). Concerning the human BTB, there is no information about the transmembrane TJ proteins. We recently demonstrated the loss of functional integrity of the BTB in testicular intraepithelial neoplasia (TIN), associated with a dislocation of the peripheral TJ proteins ZO-1 and ZO-2. Here, we determined the expression and distribution of claudin-11 at the human BTB in seminiferous tubules with normal spermatogenesis (NSP) and TIN. Immunostaining of claudin-11 revealed intense signals at the basal BTB region in seminiferous epithelium with NSP. Within TIN tubules, claudin-11 immunostaining became diffuse and cytoplasmic. Double immunogold labeling demonstrated a co-localization of claudin-11 and ZO-1 at the inter-Sertoli cell junctions. Real-time RT-PCR of laser microdissected tubules showed an up-regulation of claudin-11 mRNA in TIN. Additionally, increased claudin-11 protein was observed by Western blot. We conclude that claudin-11 constitutes a TJ protein at the human BTB. In TIN tubules, claudin-11 is up-regulated and dislocated from the BTB. Therefore, the disruption of the BTB is related to a dysfunction of claudin-11 and not to a failure of its expression.

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-α, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.

Successful matrix guided tissue regeneration of decellularized pulmonary heart valve allografts in elderly sheep

In vivo repopulation of decellularized allografts with recipient cells leads to a positive remodeling of the graft matrix in juvenile sheep. In light of the increasing number of heart valve replacements among older patients (>65 years), this study focused on the potential for matrix-guided tissue regeneration in elderly sheep. Pulmonary valve replacement was performed in seven-year old sheep using decellularized (DV), decellularized and CCN1-coated (RV), or decellularized and in vitro reendothelialized pulmonary allografts (REV) (n=6, each group). CCN1 coating was applied to support re-endothelialization. In vitro re-endothelialization was conducted with endothelial-like cells derived from peripheral blood. Echocardiograms of all grafts showed adequate graft function after implantation and at explantation 3 or 6 months later. All explants were macroscopically free of thrombi at explantation, and revealed repopulation of the allografts on the adventitial side of valvular walls and proximal in the cusps. Engrafted cells expressed vimentin, sm α-actin, and myosin heavy chain 2, while luminal cell lining was positive for vWF and eNOS. Cellular repopulation of valvular matrix demonstrates the capacity for matrix-guided regeneration even in elderly sheep but is not improved by in vitro endothelialization, confirming the suitability of decellularized matrix for heart valve replacement in older individuals.

From carcinoma in situ to testicular germ cell tumour.

The mechanisms of invasive tumour development from pre‐invasive CIS are unknown. We examined changes in functional parameters of the tubular wall according to the increase in CIS cells and tubular size. Immunohistochemistry was performed on 37 testicular specimens from 25 patients with carcinoma in situ and/or malignant germ cell tumour for the detection of actin/myosin in myocytes, and laminin/integrin α6 in the basement membrane of seminiferous tubules. Tumour cells were detected by PlAP, Sertoli cells by inhibin alpha and vimentin and by cytokeratin 18/connexin 26 immunoreactivity, which is selectively expressed together with CIS. Areas showing clusters of tumour cells surrounded by a fibrous sheet could be identified as enlarged tubules because of focal Sertoli cell‐specific co‐expression of inhibin alpha, vimentin, cytokeratin 18, and connexin 26 immunoreaction. These clusters exhibited an intact basement membrane shown by a persistent laminin/integrin α6 immunoreactivity, but myocytes had lost their contractility indicated by the loss of myosin/actin immunoreactivity. They often showed septa originating from the fibrous sheet containing numerous capillaries. Focal areas of syncytiotrophoblastic cells within classical seminoma also expressing inhibin alpha, cytokeratin 18, and connexin 26 could be differentiated from single Sertoli cells within tumor cell clusters by typical hCG but absence of vimentin immunoreactivity. In contrast to the current concept of CIS cells passing the tubular wall, these data provide evidence for an additional theory, i.e. that the switch from pre‐invasive CIS to invasive tumour takes place in situ by tubular enlargement due to tumour cell proliferation followed by Sertoli cell degeneration and conversion of the tubular wall into connective tissue.