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.

中文翻译：

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claudin-11 在犬青春期前睾丸和犬成年睾丸中的表达，显示正常的精子发生、精子发生受损或睾丸肿瘤

血睾屏障 (BTB) 由不同的细胞间连接组成，包括紧密连接蛋白，如 claudin-11 (CLDN11)。对于狗，一般只公布了有限的数据来处理这些蛋白质。因此，评估了它们的生理相关性、它们的出生后表达以及它们在病理状况中的分布模式，例如在改变的精子发生和睾丸瘤形成中。对来自常规阉割的犬睾丸以及出于诊断目的送入的犬睾丸进行了调查。根据形态学评估，将狗和睾丸分为几组：（1）精子发生正常的狗，（2）青春期前四个月大的狗，（3）管内精原细胞瘤，（4）弥漫性精原细胞瘤，（5）支持细胞瘤（ SCT)、(6) 间质细胞瘤 (LCT) 和 (7) 精子发生受损（例如混合性萎缩）的狗。为了检查 BTB 成分的可能变化，使用针对 CLDN11 的商业抗体进行了免疫组织化学 (IHC) 和免疫荧光。还使用 IHC 评估了支持细胞 (SC) 细胞核 (SOX9) 和管周肌样细胞 (平滑肌肌动蛋白, SMA)。此外，还对 CLDN11 进行了半定量蛋白质印迹 (WB) 和 RT-PCR 分析。在具有正常精子发生的小管中，CLDN11 的 IHC 显示 BTB 定位处的基底外侧染色。在青春期前的脐带中，CLDN11 沿着 SCs 的细胞质延伸区广泛表达，假设 BTB 既没有建立也没有功能。可以检测到从管内精原细胞中残留的 SCs 之间/中的弱表达 CLDN11 转变为弥漫性精原细胞瘤中剩余的 SOX9 阳性 SCs 的细胞质中只有小的 CLDN11 免疫阳性染色点。使用 RT-PCR 和 WB 分析证实了弥漫性精原细胞中 CLDN11 表达的减少甚至丧失，从而表明在精原细胞中，CLDN11 在转录水平上被下调并完全失去了其封闭功能。SCT 中的基底 SCs 仍显示 CLDN11/SOX9 共定位，表明管腔肿瘤性 SCs 在肿瘤进展过程中发生去分化。在 LCT 中，没有检测到 CLDN11。患有混合萎缩症的狗在 mRNA 和蛋白质水平上显示出小管中 CLDN11 的上调与生精停滞，得出的结论是，在这些小管内，调节机制失去了平衡。CLDN11 在青春期前犬睾丸、精子发生受损、管内精原细胞瘤中的空间表达以及在弥漫性精原细胞瘤和 LCT 中的缺失首次显示。由于改变的 CLDN11 水平可能是修改 BTB 完整性的适应性机制的一部分，因此需要进行进一步的功能研究来表征犬 BTB。