Abstract：
Male fertility depends on normal pubertal development. Di-(2-ethylhexyl) phthalate (DEHP) is a potent antiandrogen chemical, and exposure to DEHP during peripuberty can damage the developing male reproductive system, especially the testis. However, the specific cellular targets and differentiation processes affected by DEHP, which lead to testicular toxicity, remain poorly defined. Herein, we presented the first single-cell transcriptomic profile of the pubertal mouse testis following DEHP exposure. To carry out the experiment, 2 groups (n = 8 each) of 3-week-old male mice were orally administered 0.5% carboxymethylcellulose sodium salt or 100 mg/kg body weight DEHP daily from postnatal day 21-48, respectively. Using single-cell RNA sequencing, a total of 31 distinct cell populations were identified, notably, Sertoli and Leydig cells emerged as important targets of DEHP. DEHP exposure significantly decreased the proportions of Sertoli cell clusters expressing mature Sertoli markers (Sox9 and Ar), and selectively reduced the expression of testosterone synthesis genes in fetal Leydig cells. Through cell-cell interaction analyses, we observed changed numbers of interactions in Sertoli cells 1 (SCs1), Leydig cells 1 (LCs1), and interstitial macrophages, and we also identified cell-specific ligand gene expressions in these clusters, such as Inha, Fyn, Vcam1, and Apoe. Complementary in vitro assays confirmed that DEHP directly reduced the expression of genes related to Sertoli cell adhesion and intercellular communication. In conclusion, peripubertal DEHP exposure reduced the number of mature Sertoli cells and may disrupt testicular steroidogenesis by affecting the testosterone synthesis genes in fetal Leydig cells rather than adult Leydig cells.
摘要：
男性生育能力取决于正常的青春期发育。邻苯二甲酸二（2-乙基己基）酯（DEHP）是一种有效的抗雄激素化学物质，青春期暴露于DEHP会损害发育中的男性生殖系统，尤其是睾丸。然而,受DEHP影响的特定细胞靶标和分化过程，导致睾丸毒性，仍然定义不清。在这里，我们介绍了DEHP暴露后青春期小鼠睾丸的第一个单细胞转录组学图谱。为了进行实验，两组(每组8只)3周龄雄性小鼠,分别从出生后第21天至第48天每天口服0.5%羧甲基纤维素钠盐或100mg/kg体重DEHP。使用单细胞RNA测序，总共鉴定了31个不同的细胞群，特别是,Sertoli和Leydig细胞成为DEHP的重要靶标。DEHP暴露显着降低了表达成熟Sertoli标记（Sox9和Ar）的Sertoli细胞簇的比例，选择性降低胎儿睾丸间质细胞中睾酮合成基因的表达。通过细胞-细胞相互作用分析，我们观察到支持细胞1（SCs1）中相互作用的数量变化，睾丸间质细胞1（LCs1）和间质巨噬细胞（ITM），我们还确定了这些簇中的细胞特异性配体基因表达，比如Inha,Fyn,Vcam1和Apoe.补充体外试验证实，DEHP直接降低了与支持细胞粘附和细胞间通讯相关的基因的表达。总之,青春期周围的DEHP暴露会减少成熟的睾丸支持细胞的数量，并可能通过影响胎儿睾丸间质细胞而不是成年睾丸间质细胞中的睾丸激素合成基因来破坏睾丸类固醇生成。
