Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression

Abstract

Background: Epidemiologic studies suggested the association between prenatal di-(2-ethylhexyl) phthalate (DEHP) exposure and disorders of sex development (DSD), adult male disorders, and reproductive aging. Inhibiting testosterone synthesis by interfering with steroidogenic gene expression induces testicular toxicity, however, whether prenatal DEHP exposure induces testicular toxicity through this mechanism remains uncertain.

Methods: C57BL/6JGpt male mice underwent different doses (0, 100, 500, 1,000 mg/kg) of prenatal DEHP exposure during gestational day 10 to delivery day, the testicular toxicity (genital development, testosterone, semen quality, and morphology of testis tissue) in the neonatal, post-puberal and middle-aged stages was observed, and the steroidogenic gene (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd3b2) expression was analyzed by quantitative polymerase chain reaction (qPCR) and Western blot (WB). The interference of steroidogenic gene expression in TM3 cells after mono-(2-ethylhexyl) phthalate (MEHP) exposure was also explored for verification.

Results: Prenatal DEHP exposure induced immediate testicular injury in the neonatal stage [reduced anogenital distance (AGD) and intratesticular testosterone], DSD in the post-puberal stage (poor genital development), and reproductive aging in the middle-aged stage (obesity, reduced testosterone and semen quality, and atrophic seminiferous tubules), especially in the high dose. Prenatal DEHP exposure continuously interfered with steroidogenic gene expression (Hsd3b2, Hsd17b3) in RNA and protein levels. MEHP inhibited testosterone synthesis of TM3 cells by interfering with steroidogenic gene expression (Hsd3b2, Hsd17b3) in RNA and protein levels.

Conclusions: Prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression, thus indicating the association between prenatal exposure and DSD, adult male disorders, and reproductive aging.

Keywords: Disorders of sex development (DSD); di-(2-ethylhexyl) phthalate (DEHP); male disorders; sexual development; testis.

Figure 1

Mice treatment. PND, postnatal day; PNM, postnatal month.

Figure 2

Prenatal DEHP exposure induced immediate testicular injury in the neonatal stage by interfering with steroidogenic gene expression. (A-E) Phenotypes; prenatal DEHP exposure induced immediate testicular injury. (A) Birth weight (n=20); (B) testis weight (n=20); (C) penile length (n=20); (D) AGD (n=20); (E) intratesticular testosterone (n=5). (F-O) Prenatal DEHP exposure interfered with steroidogenic gene expression in the neonatal stage. (F-K) qPCR analysis of Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b2, and Hsd17b3 (n=5). (L-O) WB analysis of LHCGR, HSD3B2, and HSD17B3 (n=5). *, P<0.05; **, P<0.01; ***, P<0.001. DEHP, di-(2-ethylhexyl) phthalate; AGD, anogenital distance; mRNA, messenger RNA; LHCGR, luteinizing hormone/human chorionic gonadotropin receptor; qPCR, quantitative polymerase chain reaction; WB, Western blot.

Figure 3

Prenatal DEHP exposure induced DSD in the post-puberal stage by interfering with steroidogenic gene expression. (A-G) Prenatal DEHP exposure induced DSD (poor genital development, smaller genial organs). (A) Testis appearance; (B) genital appearance; (C) testis weight (n=10); (D) epididymis weight (n=10); (E) seminal vesicle weight (n=10); (F) penile length (n=10); (G) AGD (n=10). (H,I) Prenatal DEHP exposure induced adult male disorders. (H) Serum testosterone (n=10); (I) HE staining (100×) of the testis. The arrow showed the seminiferous tubules was hypogenetic in the 500 and 1,000 mg/kg groups (smaller tubular diameter and length). (J-N) mRNA (n=10; J,K) and protein expression (n=6; L-N) of Hsd3b2 and Hsd17b3; prenatal DEHP exposure interfered with steroidogenic gene expression in the post-puberal stage. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. DEHP, di-(2-ethylhexyl) phthalate; AGD, anogenital distance; mRNA, messenger RNA; DSD, disorders of sex development; HE, hematoxylin-eosin.

Figure 4

Prenatal DEHP exposure induced reproductive aging in the middle-aged stage by interfering with steroidogenic gene expression. (A-L) Prenatal DEHP exposure induced reproductive aging (obesity; increased epididymal fat weight, epididymis and seminal vesicle; reduced AGD, serum testosterone and semen quality). (A) General appearance; (B) genital appearance; (C) body weight (n=10); (D) epididymal fat weight (n=10); (E) epididymis weight (n=10); (F) seminal vesicle weight (n=10); (G) AGD (n=10); (H) serum testosterone (n=10); (I-K) semen quality (n=5; 400×); (L) HE staining of the testis (100×). The arrows show the seminiferous tubules was atrophic in the 1,000 mg/kg group (the seminiferous tubules were thin, and the lumen of seminiferous tubules was dilated). (M-Q) mRNA (n=5; M,N) and protein expression of Hsd3b2 and Hsd17b3 (n=5; O-Q); prenatal DEHP exposure interfered with steroidogenic gene expression in the middle-aged stage. *, P<0.05; **, P<0.01; ****, P<0.0001. DEHP, di-(2-ethylhexyl) phthalate; AGD, anogenital distance; mRNA, messenger RNA; HE, hematoxylin-eosin.

Figure 5

Results of MEHP exposure to TM3 cells in vitro. (A) MEHP inhibited testosterone synthesis (n=3); (B-G) MEHP interfered with mRNA expression of Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b2, and Hsd17b3 (n=3); (H-K) MEHP downregulated the expressions of LHCGR, HSD3B2, and HSD17B3 (n=3). *, P<0.05; **, P<0.01. MEHP, mono-(2-ethylhexyl) phthalate; mRNA, messenger RNA; LHCGR, luteinizing hormone/human chorionic gonadotropin receptor.