Single-cell transcriptome dissection of the toxic impact of Di (2-ethylhexyl) phthalate on primordial follicle assembly.

Rationale: Accumulated evidence indicates that environmental plasticizers are a threat to human and animal fertility. Di (2-ethylhexyl) phthalate (DEHP), a plasticizer to which humans are exposed daily, can trigger reproductive toxicity by acting as an endocrine-disrupting chemical. In mammals, the female primordial follicle pool forms the lifetime available ovarian reserve, which does not undergo regeneration once it is established during the fetal and neonatal period. It is therefore critical to examine the toxicity of DEHP regarding the establishment of the ovarian reserve as it has not been well investigated. Methods: The ovarian cells of postnatal pups, following maternal DEHP exposure, were prepared for single cell-RNA sequencing, and the effects of DEHP on primordial follicle formation were revealed using gene differential expression analysis and single-cell developmental trajectory. In addition, further biochemical experiments, including immunohistochemical staining, apoptosis detection, and Western blotting, were performed to verify the dataset results. Results: Using single-cell RNA sequencing, we revealed the gene expression dynamics of female germ cells and granulosa cells following exposure to DEHP in mice. Regarding germ cells: DEHP impeded the progression of follicle assembly and interfered with their developmental status, while key genes such as Lhx8, Figla, and others, strongly evidenced the reduction. As for granulosa cells: DEHP likely inhibited their proliferative activity, and activated the regulation of cell death. Furthermore, the interaction between ovarian cells mediated by transforming growth factor-beta signaling, was disrupted by DEHP exposure, since the expression of GDF9, BMPR1A, and SMAD3 was affected. In addition, DNA damage and apoptosis were elevated in germ cells and/or somatic cells. Conclusion: These findings offer substantial novel insights into the reproductive toxicity of DEHP exposure during murine germ cell cyst breakdown and primordial follicle formation. These results may enhance the understanding of DEHP exposure on reproductive health.

Impairment of ovaries by 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat.

2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero had been shown to affect ovarian development and functions. However, its mechanism remained unknown. In this study, to investigate the effect of maternal exposure to TCDD on ovaries, the pregnant Sprague Dawley (SD) rats were treated with TCDD (100 ng/kg or 500 ng/kg) or only vehicle and corn oil on the day 8-14 of gestation through the gavage with a stainless-steel feeding needle (once a day). The vaginal opening and estrous cycle of female offspring rats (F1) were monitored twice a day. The ovarian histology, follicle counts, real-time PCR, western blotting and DNA methylation analysis about Gdf9 and Bmp15 were carried out in F1 rats. The results showed that exposure to TCDD (especially the dose of 500 ng/kg) in utero on GD8-14 might change the ovary weight, the concentration of E2 and FSH, the estrous cycles and the numbers of primordial and secondary follicles of the offspring rats. In addition, the mRNA and protein expression of GDF9 and BMP15 was down-regulated, while the methylation patterns of Gdf9 and Bmp15 were not affected. In conclusion, maternal exposure to TCDD could affect the ovary development and functions which were possibly associated with down-regulation of mRNA and protein expression of GDF9 and BMP15. However, the down-regulation was not related to the pattern of methylation of Gdf9 and Bmp15.