Table_1_Epididymal epithelial degeneration and lipid metabolism impairment account for male infertility in occludin knockout mice.docx

Occludin (OCLN) is a tight junction protein and Ocln deletion mutation causes male infertility in mice. However, the role of OCLN in male reproductive system remains unknown. In this study, we used an interdisciplinary approach to elucidate the underlying mechanism of male infertility in related to OCLN function, including Ocln knockout mice as well as a combined omics analysis and immunofluorescent labelling. Our results showed that the epididymis of Ocln-null mice displayed a phenomenon resembling epididymal sperm granuloma, which occurred especially in the junctional region between caput and corpus epididymidis. Sperm motility and fertilisation capacity were also impaired in these Ocln-null mice, accompanied by enlarged tubules in the proximal regions and degeneration in the distal regions of epididymis. Cellular localization analysis showed that OCLN immunofluorescence was enriched only in the apical junction of epithelial principal cells in the proximal regions of epididymis. Integrative omics analysis revealed the downregulation of gene clusters enriched in acid secretion and fatty acid metabolism in the Ocln-null epididymis, especially the enzymes related to the unsaturated arachidonic acid pathway. The number of proton-pump V-ATPase-expression clear cells, a key player of luminal acidification in the epididymis, declined drastically from prepubertal age before sperm arrival but not in the early postnatal age. This was accompanied by programmed cell death of clear cells and increased pH in the epididymal fluid of OCLN-deficient mice. The lipidomics results showed significantly increased levels of specific DAGs conjugated to unsaturated fatty acids in the Ocln-mutant. Immunofluorescent labelling showed that the arachidonic acid converting enzyme PTGDS and phospholipase PLA2g12a were prominently altered in the principal cells and luminal contents of the Ocln-mutant epididymis. Whereas the carboxylate ester lipase CES1, originally enriched in the WT basal cells, was found upregulated in the Ocln-mutant principal cells. Overall, this study demonstrates that OCLN is essential for maintaining caput-to-corpus epithelial integrity, survival of acid-secreting clear cells, and unsaturated fatty acid catabolism in the mouse epididymis, thereby ensuring sperm maturation and male fertility.

闭合蛋白（Occludin, OCLN）是一种紧密连接蛋白（tight junction protein），Ocln基因缺失突变可导致小鼠雄性不育。然而，OCLN在雄性生殖系统中的作用仍不明确。本研究采用跨学科方法，结合Ocln基因敲除（Ocln knockout）小鼠模型、整合组学分析与免疫荧光标记技术，阐明了与OCLN功能相关的雄性不育潜在机制。结果显示，Ocln全敲除（Ocln-null）小鼠的附睾呈现出类似附睾精子肉芽肿的现象，尤其好发于附睾头与附睾体（caput and corpus epididymidis）的连接区域。该类小鼠的精子活力与受精能力亦受损，伴随附睾近端小管扩张、远端小管退行性变。细胞定位分析表明，OCLN免疫荧光信号仅富集于附睾近端上皮主细胞（epithelial principal cells）的顶端连接区域。整合组学分析揭示，Ocln-null小鼠附睾中富集于酸分泌与脂肪酸代谢（fatty acid metabolism）的基因簇表达下调，尤其是与不饱和花生四烯酸通路（unsaturated arachidonic acid pathway）相关的酶类。作为附睾管腔酸化的关键效应细胞，表达质子泵V-ATPase（proton-pump V-ATPase）的亮细胞（clear cells）数量在精子出现前的青春期前即大幅减少，但出生后早期无明显变化。这一现象伴随亮细胞的程序性细胞死亡（programmed cell death），以及OCLN缺陷小鼠附睾液pH值升高。脂质组学（lipidomics）结果显示，Ocln突变小鼠体内与不饱和脂肪酸结合的特定二酰甘油（diacylglycerols, DAGs）水平显著升高。免疫荧光标记显示，花生四烯酸转化酶PTGDS（arachidonic acid converting enzyme PTGDS）与磷脂酶PLA2g12a（phospholipase PLA2g12a）在Ocln突变小鼠附睾的主细胞与管腔内容物中发生显著改变。而原本富集于野生型（wild type, WT）基底细胞（basal cells）的羧酸酯水解酶CES1（carboxylate ester lipase CES1），在Ocln突变小鼠的主细胞中表达上调。总体而言，本研究证实OCLN对于维持小鼠附睾头-体部上皮完整性、酸分泌亮细胞的存活以及不饱和脂肪酸分解代谢至关重要，从而保障精子成熟与雄性生育能力。