Institute for Anatomy, University of Veterinary Medicine Hannover, Hannover, Germany.
Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Hannover, Germany.
PLoS One. 2018 Jun 1;13(6):e0198100. doi: 10.1371/journal.pone.0198100. eCollection 2018.
Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated.
间隙连接蛋白 43（Cx43）是睾丸中主要的间隙连接蛋白，在某些情况下，精子发生受损时，几种哺乳动物的 Cx43 表达已显示发生改变。除其他功能外，Cx43 被认为可以调节血睾屏障（BTB）的连接形成。本研究的目的是使用特异性 Cx43 敲除小鼠（SCCx43KO）来研究小鼠 BTB 中不同紧密连接（TJ）蛋白的表达模式。成年同型纯合雄性 SCCx43KO 小鼠（SCCx43KO-/-）主要表现为精子发生停滞和仅支持细胞的小管，这可能是由于 BTB 组装、组成或调节发生改变所致。使用免疫组织化学（IHC）和实时定量 PCR（qRT-PCR）检查成年野生型（WT）和 SCCx43KO-/-小鼠中的 TJ 分子 Claudin-3、-5 和 -11。在这种情况下，特别有趣的是研究 SCCx43KO-/-小鼠中具有残留精子发生的单个小管，以确定生殖细胞（GC）对 BTB 组成和动态的潜在 Cx43 无关影响。在没有残留精子发生的小管中，在突变小鼠中可以证明 Claudin-11 蛋白的细胞质弥散分布模式。尽管如此，Claudin-11 似乎形成了功能性 TJ。在那些小管的生精上皮中，无法用免疫组织化学检测到 Claudin-3 和 -5。相应地，Claudin-3 和 -5 mRNA 的表达减少，为成年 KO 小鼠中普遍受损的 BTB 动态提供了证据。对具有残留精子发生的小管的观察表明，GC 群体对 TJ 蛋白的调节与 Cx43 无关。为了确定青春期 SCCx43KO-/-小鼠中的初始 BTB 形成，对青春期 SCCx43KO-/-和 WT 小鼠进行 Claudin-11 的免疫组织化学染色和 qRT-PCR。此外，使用高渗葡萄糖固定剂对 BTB 完整性进行了功能分析。这些分析表明，SCCx43KO-/-小鼠在 WT 小鼠相同的时间内形成了完整的 BTB，但似乎加速了。
