MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

转录因子MAFB是调控多种器官与组织发育及分化的关键调节因子。既往研究显示，MAFB在胚胎及成年小鼠睾丸中均有表达，被推测为视黄酸（retinoic acid, RA）下游介导精子发生的靶标基因。然而，其确切的细胞定位与生物学功能仍未明确。本研究借助Mafb缺陷小鼠模型，对MAFB在胚胎及成年睾丸中的表达定位进行了检测，并系统分析了其基因功能。我们发现，MAFB与c-MAF是睾丸中仅有的两类大MAF家族转录因子，MAFA与NRL则无表达。在胚胎第18.5天（E18.5），MAFB定位于睾丸间质细胞与支持细胞；而在成年小鼠体内，MAFB则表达于间质细胞、支持细胞及粗线期精母细胞。与野生型对照小鼠相比，E18.5的Mafb缺陷小鼠睾丸生精小管结构完整，睾丸体细胞数量无异常改变，且与睾丸细胞发育及功能相关的基因表达水平在两种基因型小鼠间亦无显著差异。在成年小鼠中，支持细胞内的MAFB表达呈现阶段特异性，且可被视黄酸诱导上调。我们构建了他莫昔芬处理可激活Cre重组酶（Cre recombinase）的Mafbfl/fl CAG-CreER™（Mafb-cKO）小鼠，实验结果表明：新生期Mafb敲除小鼠在MAFB缺失后很快死亡，而成年条件性敲除小鼠则可正常存活。成年Mafb-cKO小鼠可维持正常生育能力，组织学分析、激素水平检测及生殖细胞阶段特异性标志物检测均证实其精子发生过程维持正常。此外，条件性敲除小鼠与对照小鼠的生精小管阶段比例亦无明显差异。然而，对Mafb-cKO小鼠支持细胞的RNA测序（RNA-Seq）分析显示，下调表达的基因主要与免疫功能及吞噬活性相关，而非精子发生相关通路。综上，尽管MAFB在多种睾丸细胞中均有显著表达，但本研究证实其对于小鼠胎儿睾丸形态发生及成年小鼠精子发生维持并非必需；不过，MAFB可能对支持细胞的吞噬活性至关重要。