Peripubertal DEHP exposure impairs reproductive function in adult male mice by  inducing damage to Sertoli cells and disrupting the blood-testis barrier

XU Guiyong; ZHANG Lu; SUN Fengqiong; YANG Rui; ZHOU Ziyuan; YANG Guanghong

doi:10.16016/j.2097-0927.202503059

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Peripubertal DEHP exposure impairs reproductive function in adult male mice by inducing damage to Sertoli cells and disrupting the blood-testis barrier

Monographic Reports | 更新时间：2025-07-27

- Peripubertal DEHP exposure impairs reproductive function in adult male mice by inducing damage to Sertoli cells and disrupting the blood-testis barrier
- Journal of Army Medical University Vol. 47, Issue 15, Pages: 1695-1707(2025)
- 作者机构：
  
  1.贵州医科大学公共卫生与健康学院，环境污染与疾病监控教育部重点实验室，贵阳
  2.陆军军医大学（第三军医大学）军事预防医学系军队环境卫生学教研室，重庆
- 作者简介：
  
  ZHOU Ziyuan， E-mail： ziyuanzhou@tmmu.edu.cn；
  YANG Guanghong， E-mail： 280446859@qq.com
- 基金信息：
  
  the National Key Research and Development Program of China(2022YFC2702902);the Key Project of Joint Regional Fund of National Natural Science Foundation of China(U23A20494)
- DOI：10.16016/j.2097-0927.202503059
  CLC： R339.21;R994.6;TQ323.4
- Received：19 March 2025，
  
  Revised：03 April 2025，
  
  Accepted：20 May 2025，
  
  Published：15 August 2025
- 稿件说明：
移动端阅览
徐桂勇，张露，孙凤琼，等.围青春期DEHP暴露通过损害Sertoli细胞和血睾屏障致小鼠生殖损伤［J］.陆军军医大学学报，2025，47（15）：1695-1707.DOI：10.16016/j.2097-0927.202503059.

XU G Y，ZHANG L，SUN F Q，et al.Peripubertal DEHP exposure impairs reproductive function in adult male mice by inducing damage to Sertoli cells and disrupting the blood-testis barrier［J］.J Army Med Univ，2025，47（15）：1695-1707.DOI：10.16016/j.2097-0927.202503059.
徐桂勇，张露，孙凤琼，等.围青春期DEHP暴露通过损害Sertoli细胞和血睾屏障致小鼠生殖损伤［J］.陆军军医大学学报，2025，47（15）：1695-1707.DOI：10.16016/j.2097-0927.202503059. DOI：

XU G Y，ZHANG L，SUN F Q，et al.Peripubertal DEHP exposure impairs reproductive function in adult male mice by inducing damage to Sertoli cells and disrupting the blood-testis barrier［J］.J Army Med Univ，2025，47（15）：1695-1707.DOI：10.16016/j.2097-0927.202503059. DOI：

摘要

目的

探究围青春期邻苯二甲酸二（2-乙基己基）酯（di-2-ethylhexyl phthalate，DEHP）暴露通过损害成年雄性小鼠Sertoli细胞功能及血睾屏障，致其生殖损伤的作用机制。

方法

180只3周龄雄性C57BL/6J小鼠通过随机数字表法分为（

=30）：玉米油组（对照组）、5 mg/kg DEHP暴露组（5 mg/kg 组）、25 mg/kg DEHP暴露组（25 mg/kg组）、125 mg/kg DEHP暴露组（125 mg/kg组）、250 mg/kg DEHP暴露组（250 mg/kg组）、500 mg/kg DEHP暴露组（500 mg/kg 组），连续灌胃5周。首次取材于9周龄进行，每组取10只。各组剩余F0代小鼠于14周龄按雄雌比1∶2与未暴露成年雌性小鼠合笼交配2周，第二次取材于16周龄完成，每组取20只。采用计算机辅助分析系统检测9周龄和16周龄雄性小鼠精子密度与活力；记录F1代仔鼠平均出生体质量、每窝4 d存活率、每窝产仔数及每窝内雌雄性别比。ELISA检测血清中激素水平；伊红染色观察精子畸形；HE染色观察睾丸组织病理变化；免疫荧光检测睾丸组织中血睾屏障相关的紧密连接蛋白1（zonula occludens-1，ZO-1）、缝隙连接蛋白43（connexin43，Cx43）以及细胞凋亡相关分子Cleaved-Caspase3的蛋白表达；免疫组化检测睾丸组织中Sertoli细胞标志物SOX9蛋白的表达；TUNEL检测睾丸组织细胞凋亡情况。通过比较毒理基因组学数据库（comparative toxicogenomics database，CTD）筛选邻苯二甲酸单（2-乙基己基）酯（mono-2-ethylhexyl phthalate，MEHP）致生殖损伤相关基因集，并结合GO/KEGG和Reactome富集分析预测可能信号通路。以MEHP 0、50、100、200和400 μmol/L 处理TM4细胞株建立体外暴露模型；检测不同剂量MEHP处理组中TM4细胞活力；采用流式细胞术检测细胞凋亡；通过Western blot检测细胞凋亡相关蛋白B细胞淋巴瘤蛋白2（B-cell lymphoma 2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax），及血睾屏障相关蛋白ZO-1和Cx43蛋白的表达水平。

结果

与对照组相比，500 mg/kg组雄性小鼠使雌性小鼠的怀孕率从80.0%降低到52.5%，总产仔数从212只减少到125只。与对照组相比，9周龄DEHP暴露组小鼠精子密度和活力显著降低，精子总畸形率显著增加（

0.05）；16周龄小鼠的精子活力仍然呈剂量依赖性下降趋势（

0.05）。25、125、250、500 mg/kg组小鼠曲细精管生精上皮高度显著下降，细胞空泡化增加，上皮萎缩和退化明显。免疫荧光结果显示，与对照组比较，250、500 mg/kg睾丸组织中的细胞凋亡增加，而血睾屏障相关的ZO-1和Cx43蛋白表达减少。血清中雄激素结合蛋白、促性腺激素释放激素和睾酮水平呈剂量依赖性降低（

0.05），而黄体生成素和血清中促卵泡激素的水平均呈剂量依赖性升高（

0.05）。生物信息学分析显示，通过CTD分别筛选出与MEHP相关的男性系统疾病基因为153个。基于153个基因结合GO富集分析、KEGG通路和Reactome富集分析，凋亡信号通路明显富集。CCK-8结果显示，与对照组相比较，MEHP处理组TM4细胞活力呈剂量依赖性降低（

0.05）；Western blot结果显示，与对照组相比，MEHP处理组中Bcl-2、ZO-1和Cx43蛋白的表达水平明显降低（

0.05），而Bax蛋白的表达水平明显增加（

0.05）。

结论

围青春期DEHP暴露可能通过破坏血睾屏障和激活Sertoli细胞凋亡而导致成年雄性小鼠的精子质量降低及生殖力损伤。

Abstract

Objective‍　‍To investi

gate the mechanism by which peripubertal exposure to di-2-ethylhexyl phthalate （DEHP） induces reproductive injury in adult male mice through impairment of Sertoli cell function and the blood-testis barrier. Methods‍　‍A total of 180 male C57BL/6J mice （3 weeks old） were randomly divided into corn oil group （control group）， and 5， 25， 125， 250 and 500 mg/kg DEHP exposure groups. All groups received daily oral gavage for 5 consecutive weeks. Ten mice from each group were sacrificed for first material collection when they were 9 weeks old. The remaining F0 generation mice were mated with unexposed adult female mice at a male-to-female ratio of 1∶2 at 14 weeks of age for a period of 2 weeks. The second material collection was completed at 16 weeks of age， with 20 mice taken from each group. A computer-assisted analysis system was used to detect sperm density and motility in 9-week-old and 16-week-old male mice. The average birth body weight of F1 generation pups， survival rate of pups at 4 d after birth per litter， number of pups per litter， and male-to-female sex ratio within the litter were recorded. ELISA was used to detect hormone levels in the serum. Sperm abnormalities were observed using eosin staining. HE staining was used to observe pathological changes in testicular tissue. Immunofluorescence assay was utilized to detect the protein expression of tight junction protein 1 （zonula occludens-1， ZO-1） and gap junction protein 43 （connexin43， Cx43） related to the blood-testis barrier in testicular tissue， as well as the protein expression of apoptosis-related molecule Cleaved-Caspase3. Immunohistochemistry was used to detect the expression of Sertoli cell marker SOX9 protein in testicular tissue. TUNEL was used to detect apoptosis in testicular tissue. Comparative toxicogenomics database （CTD） was used to screen for mono-2-ethylhexyl phthalate （MEHP） induced reproductive damage-associated genes and combined with GO/KEGG and Reactome enrichment analysis to predict possible signaling pathways. An

in vitro

exposure model was established by treating TM4 cells with MEHP at concentrations of 0， 50， 100， 200 and 400 μmol/L， respectively. CCK-8 assay， flow cytometry and Western blotting were applied to detect the cell viability， apoptosis， and protein expression of apoptosis-related proteins B-cell lymphoma 2 （Bcl-2） and Bcl-2-associated X protein （Bax）， and blood-testis barrier-related proteins ZO-1 and Cx43. Results‍　‍Compared with the control group， the pregnancy rate was decreased from 80.0% to 52.5%， and the total number of pups was decreased from 212 to 125 in the female mice mating with the male from the 500 mg/kg group. Compared with the control group， the sperm density and motility were significantly reduced， and the total rate of sperm abnormalities was obviously increased in the 9-week-old DEHP-exposed mice （

0.05）， and even in the 16-week-old mice， the sperm motility was still in a dose-dependent downward trend （

0.05）. In the 25， 125， 250 and 500 mg/kg DEHP exposure groups， the thickness of the seminiferous epithelium was obviously decreased， with more cellular vacuolization and notable epithelial atrophy and degeneration. Cell apoptosis in the testicular tissue was enhanced， while the expression of ZO-1 and Cx43 were decreased in the 250 and 500 mg/kg exposure groups. The levels of androgen-binding protein， gonadotropin-releasing hormone， and testosterone in the serum were decreased in a dose-dependent manner （

0.05）， while those of luteinizing hormone and follicle-stimulating hormone showed a dose-dependent increase （

0.05）. Bioinformatics analysis showed that 153 genes related to male system diseases were screened out by CTD for MEHP. GO enrichment， KEGG pathway， and Reactome enrichment analyses displayed that the apoptosis signaling pathway was significantly enriched. CCK-8 assay showed that TM4 cell viability was decreased in a dose-dependent manner after MEHP treatment when compared with the control group （

0.05）. Western blotting indicated that MEHP treatment reduced the expression levels of Bcl-2， ZO-1 and Cx43 （

0.05）， and enhanced that of Bax in comparison with the control group （

0.05）. Conclusion‍　‍Peripubertal DEHP exposure may lead to reduced sperm quality and reproductive damage in adult male mice by disrupting the blood-testis barrier and activating Sertoli cell apoptosis.

关键词

Keywords

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