Low dose bisphenol A impairs spermatogenesis by suppressing reproductive hormone production and promoting germ cell apoptosis in adult rats

Abstract

Bisphenol A (BPA), an estrogenic chemical, has been shown to reduce sperm count; however, the underlying mechanisms remain unknown. Herein, we show that oral administration of BPA (2 µg/kg) for consecutive 14 days in adult rats (BPA rats) significantly reduced the sperm count and the number of germ cells compared to controls. The serum levels of testosterone and follicle-stimulating hormone (FSH), as well as the level of GnRH mRNA in BPA rats were lower than those of control rats. Testosterone treatment could partially rescue the reduction of germ cells in BPA rats. Notably, the number of apoptotic germ cells was significantly increased in BPA rats, which was insensitive to testosterone. Furthermore, the levels of Fas, FasL and caspase-3 mRNA in the testicle of BPA rats were increased in comparison with controls. These results indicate that exposure to a low dose of BPA impairs spermatogenesis through decreasing reproductive hormones and activating the Fas/FasL signaling pathway.

Keywords: apoptosis; bisphenol A (BPA); spermatogenesis; testosterone.

Fig. 1. Influence of low dose BPA on spermatogenesis in adult rats.

A: Mean value of both testicular weights in control and BPA rats. B: Sperm count in the epididymis. Bar graphs show the average number of sperm heads. **P < 0.01. C: Numbers of spermatogonia and spermatocytes at stage VII of the spermatogenic cycle in 100 seminiferous tubules. *P < 0.05, **P < 0.01. Type A spermatogonia (Asg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7Sd) are indicated by white arrows, respectively. Scale bar=10 µm.

Fig. 2. Influence of BPA-reduced testosterone on spermatogenesis.

A and B: Levels of testosterone in serum and testicle. *P < 0.05. C: Level of AR mRNA in testes of control and BPA rats. D: Effects of testosterone replacement on reduction of sperm count in BPA rats. *P < 0.05 and ** P < 0.01 vs. control rats; ^#P < 0.05 vs. BPA rats treated with vehicle by ANOVA analysis followed by Bonferroni's post-hoc test. E: Effects of testosterone replacement on the reduction of spermatogonia and spermatocytes in BPA rats. Bar graph shows relative values of type A spermatogonia (Asg), preleptotene spermatocytes (pLSc), mid-pachytene spermatocytes (mPSc) and step 7 spermatids (7Sd) in 100 seminiferous tubules. *P < 0.05 and **P < 0.01.

Fig. 3. Effects of BPA on gonadotropic hormones and GnRH neurons in adult rats.

A and B: Levels of serum LH and FSH. *P < 0.05. C: Following testosterone replacement, the level of LH was lower in the BPA rats than that in the control rats. *P < 0.05. D: Photographs of GnRH- immunohistochemistry in preoptic area (POA). Arrows indicate GnRH+ cells. Scale bar=50 µm. Bar graph shows mean number of GnRH+ cells. *P < 0.05. E: Relative level of GnRH mRNA in POA. *P < 0.05.

Fig. 4. Effects of BPA on apoptosis of germ cells.

A: Quantitative analysis of apoptotic cells in the testicle. The apoptotic index is expressed as percentage of apoptotic seminiferous tubule (containing more than three apoptotic germ cells). **P < 0.01. Arrows indicate TUNEL positive cells. Scale bar=100 µm. B-D: Expression of Fas, FasL, and caspase-3 in the testes. Bar graphs show the relative levels of Fas mRNA (B), FasL mRNA (C) and the caspase-3 mRNA (D) in the testes of the control rats and BPA rats. **P < 0.01.