Expression of claudin-11 in canine prepubertal testes, and in canine adult testes showing normal spermatogenesis, impaired spermatogenesis, or testicular neoplasia

Abstract

The blood-testis barrier (BTB) consists of different cell-to-cell connections, including tight junction proteins like claudin-11 (CLDN11). For dogs, only limited data is published dealing with these proteins in general. Therefore, their physiological relevance, their postnatal expression, and their distribution pattern in pathological conditions, e.g. in altered spermatogenesis and testicular neoplasia were assessed. Canine testes from routine castrations, and those sent in for diagnostic purposes were investigated. Based on morphological evaluation, the dogs and testes were divided into groups: (1) dogs with normal spermatogenesis, (2) four months old prepubertal dogs, (3) intratubular seminoma, (4) diffuse seminoma, (5) Sertoli cell tumours (SCT), (6) Leydig cell tumours (LCT), and (7) dogs with impaired spermatogenesis (e.g. mixed atrophy). In order to examine possible alterations of the BTB components, immunohistochemistry (IHC) and immunofluorescence using a commercial antibody against CLDN11 was performed. Sertoli cell (SC) nuclei (SOX9) and peritubular myoid cells (smooth-muscle-actin, SMA) were also assessed using IHC. Additionally, semi-quantitative Western-blot (WB) and RT-PCR analyses of CLDN11 were conducted. In tubules with normal spermatogenesis, IHC of CLDN11 revealed a basolateral staining at BTB localisation. In prepubertal cords, CLDN11 was diffusely expressed along the cytoplasmic extensions of SCs supposing that the BTB was neither built up nor functional, yet. A shift from weakly expressed CLDN11 between/in residual SCs in intratubular seminoma to only small CLDN11 immunopositive stained spots in the cytoplasm of remaining SOX9-positive SCs in diffuse seminoma was detectable. Reduction or even loss of CLDN11 expression in diffuse seminoma was confirmed using RT-PCR and WB analyses, thus indicating that in seminoma, CLDN11 was downregulated at transcriptional level and completely lost its sealing function. Basal SCs in SCT still showed a CLDN11/SOX9 co-localisation, suggesting that luminal neoplastic SCs undergo de-differentiation during tumour progression. In LCT, no CLDN11 was detectable. Dogs with mixed atrophy showed an upregulation of CLDN11 in tubules with spermatogenic arrest on mRNA and protein level, leading to the conclusion that within these tubules regulatory mechanisms lost their equilibrium. For the first time, the spatial expression of CLDN11 in prepubertal canine testis, impaired spermatogenesis, intratubular seminoma and its absence in diffuse seminoma and LCT was shown. Since altered CLDN11 levels could be part of adaptive mechanisms to modify BTB integrity, further functional investigations to characterize the canine BTB need to be conducted.