Abstract
Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer which is widely used in the manufacture of plastics. As a common environmental contaminant and recognized endocrine disrupting chemical, DEHP is able to deregulate the functions of a variety of tissues, including the reproductive system both in males and females. In order to investigate the possible effects of DEHP on the first wave of folliculogenesis, occurring in the mouse ovary postnatally, mice were administered 20 or 40 μg/kg DEHP through intraperitoneal injection at days 5, 10 and 15 post partum (dpp). Following DEHP treatment the gene expression profile of control and exposed ovaries was compared by microarray analyses at 20 dpp. We found that in the exposed ovaries DEHP significantly altered the transcript levels of several immune response and steroidogenesis associated genes. In particular, DEHP significantly decreased the expression of genes essential for androgen synthesis by theca cells including Lhcgr, Cyp17a1, Star and Ldlr. Immunohistochemistry and immune flow cytometry confirmed reduced expression of LHCGR and CYP17A1 proteins in the exposed theca cells. These effects were associated to a significant reduction in ovarian concentrations of progesterone, 17β-estradiol and androstenedione along with a reduction of LH in the serum. Although we did not find a significant reduction of the number of primary, secondary or antral follicles in the DEHP exposed ovaries when compared to controls, we did observe that theca cells showed an altered structure of the nuclear envelope, fewer mitochondria, and mitochondria with a reduced number of cristae. Collectively, these results demonstrate a deleterious effect of DEHP exposure on ovarian steroidogenesis during the first wave of folliculogenesis that could potentially affect the correct establishment of the hypothalamic-pituitary-ovarian axis and the onset of puberty.
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This work was supported by National Basic Research Program of China (973 Program, 2012CB944401), and National Nature Science Foundation of China (31572225 and 31471346).
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Fang-Nong Lai and Jing-Cai Liu are co-first authors.
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Fig. S1
Protocol of mice DEHP injections and experimental procedures. Three treatment groups, control, 20, and 40 μg/kg DEHP were artificially setted. The flow diagram showed the process of the experiment performed. (PDF 161 kb)
Fig. S2
Venn diagrams and heat maps of microarrays of differential expressed genes (DEGs) in ovaries obtained from prepuberal mice exposed to DEHP. (A) Venn diagrams of up-and downregulated genes in ovaries exposed to 20 and 40 μg/kg DEHP vs control. (B) Heat maps of up-and downregulated genes in ovaries exposed to 20 and 40 μg/kg DEHP vs control. (PDF 280 kb)
Fig. S3
Protein–protein interaction (PPI) networks of differentially expressed genes (DEGs) in DEHP-exposed ovaries. Blue and red points indicate down-and upregulated genes, respectively. (A) 20 μg/kg DEHP-treatment vs control. (B) 40 μg/kg DEHP-treatment vs control. (PDF 2301 kb)
Fig. S4
Protein–protein interaction (PPI) networks of DEGs in ovaries obtained from prepuberal mice exposed to DEHP. Blue and red points indicate down- and upregulated DEG encoded proteins in 20 and 40 μg/kg DEHP-treated ovaries vs control. (PDF 977 kb)
Fig. S5
Effect of DEHP on the mRNA levels of immune response genes. Green bars represent mRNA fold change between control and DEHP-exposed ovaries assessed by microarray, red bars represent relative mRNA levels assessed by RT-qPCR and normalized by beta-actin. Data are expressed as mean ± SD. Independent experiments were repeated at least three times (* < 0.05; ** P < 0.01). (PDF 61 kb)
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Lai, FN., Liu, JC., Li, L. et al. Di (2-ethylhexyl) phthalate impairs steroidogenesis in ovarian follicular cells of prepuberal mice. Arch Toxicol 91, 1279–1292 (2017). https://doi.org/10.1007/s00204-016-1790-z
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DOI: https://doi.org/10.1007/s00204-016-1790-z