Retinoic acid (RA) regulates all four major events in spermatogenesis; spermatogonial differentiation, meiotic entry, spermiogenesis, and spermiation. For the pre-meiotic phase, Sertoli cells are the source of RA and for the post-meiotic phase, pachytene spermatocytes are the source of RA. While the entire spermatogenic process is regulated by RA, how each of these phases is regulated by RA remains completely unknown. Homeobox B1 (Hoxb1) has two retinoic acid response elements (RARE) upstream and downstream of the gene. In this study, we investigated if RA facilitates spermatogenesis by its action on Hoxb1. The expressions of the Hoxb1 and Sonic hedgehog (Shh) genes were analyzed in the post-natal mouse testes and the testicular localizations of Hoxb1, Shh and Gli1 were analyzed by immunohistochemistry in the adult rat testis. To delineate the signaling mechanisms, Hoxb1 expression was altered in vitro and in vivo using retinoic acid and miR-361-3p. Finally, the levels of miR-361-3p and HOXB1 were analyzed in infertile human sperm samples. Hoxb1 and Shh gene expressions were found to be low in the testis of post-natal Swiss mice of 7, 14, 28, 35, and 60 days, after which the expressions of both spiked. Immunohistochemistry in the adult mouse testis showed the expressions of Hoxb1, Shh, and Gli1 in the elongating spermatids. Exposure of GC2 cells to RA and in vivo IP RA injection upregulated Hoxb1 and Shh signaling in the testis with increased expressions of Shh, Gli1, and Hdac1. Retinoic acid administration in Swiss mice compromised sperm production and reduced epididymal sperm count. The analysis of infertile human semen samples revealed an increased level of HOXB1 and a decreased level of miR-361-3p as compared to fertile controls. We conclude that retinoic acid regulates late stage of spermatogenesis (spermiogenesis) by affecting Hoxb1 and Shh signaling.

视黄酸 (RA) 调节精子发生的所有四个主要事件；精原细胞分化、减数分裂进入、精子发生和精子形成。对于减数分裂前阶段，支持细胞是 RA 的来源，对于减数分裂后阶段，粗线期精母细胞是 RA 的来源。虽然整个生精过程都受到 RA 的调节，但每个阶段是如何受 RA 调节的仍然完全未知。同源框 B1 ( Hoxb1 ) 在基因上游和下游有两个视黄酸反应元件 (RARE)。在这项研究中，我们研究了 RA 是否通过对 Hoxb1 的作用来促进精子发生。分析出生后小鼠睾丸中Hoxb1和Sonic hedgehog（ Shh）基因的表达，并通过免疫组织化学分析成年大鼠睾丸中Hoxb1、Shh和Gli1的睾丸定位。为了描述信号传导机制，使用视黄酸和 miR-361-3p 在体外和体内改变了Hoxb1表达。最后，分析了不育人类精子样本中 miR-361-3p 和HOXB1的水平。研究发现，出生后 7、14、28、35 和 60 天的瑞士小鼠的睾丸中Hoxb1和Shh基因表达较低，之后两者的表达均出现峰值。成年小鼠睾丸的免疫组织化学显示，伸长的精子细胞中表达了 Hoxb1、Shh 和 Gli1。 GC2 细胞暴露于 RA 和体内 IP RA 注射上调了睾丸中的 Hoxb1 和Shh信号传导，增加了Shh、Gli1和Hdac1的表达。给瑞士小鼠注射视黄酸会损害精子的产生并减少附睾精子的数量。 对不育人类精液样本的分析显示，与可育对照相比， HOXB1水平升高，而 miR-361-3p 水平降低。我们得出结论，视黄酸通过影响 Hoxb1 和 Shh 信号传导来调节精子发生（精子发生）的后期。