Molecular Mechanism Of Bisphenol A Induced Testicular Germ Cell Apoptosis In Mice

Objective Bisphenol A (BPA) is a potential endocrine disruptor and well-known testicular toxicant. The present study was to explore the effects of pubertal BPA exposure on germ cell apoptosis in testes and to clarify whether the mitochondrial signaling pathway and the Fas/FasL apoptotic pathway were involved in BPA-mediated germ cell apoptosis in testes.Methods Male mice were randomly divided into three groups, namely control group, low-dose BPA group and high-dose BPA group. Mice in BPA groups were administered with BPA (160 or 480 mg/kg) by gavage daily from PND35 to PND49. Mice in control group were treated with corn oil. Twelve hours after the last administration, all mice were killed. The testes were excised, dissected and weighed. The testes then divided in two parts: right one was immersed in modified Davidson's fluid (mDF) for 12–24 h for testicular histology and apoptosis analysis. The other part of the testes was kept at ?80℃for subsequent immunoblotting.Results The average body weight and the absolute weight of testes were significantly decreased in mice exposed to two different doses of BPA. However, pubertal BPA exposure had little effect on the relative weight of testes. Testicular histology showed that pubertal BPA exposure slightly reduced the layers of spermatogenic cells and disturbed the array of spermatogenic cells. No large vacuoles or complete spermatogenic failure were observed in testes of mice exposed to BPA during puberty. TUNEL analysis showed that the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII–VIII seminiferous tubules in testes. To explore whether the Fas/FasL pathway played a key role in BPA-mediated germ cell apoptosis in testes, we determined the relative protein expression in the Fas/FasL apoptotic pathway by immunoblotting. The present results indicated that a dose-dependent increase in the level of Fas and FasL was observed in testes of mice exposed to BPA (160 and 480 mg/kg) during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. To investigate whether the mitochondrial signaling pathway were involved in BPA-mediated germ cell apoptosis in testes, we analyzed the relative protein expression in the mitochondrial signaling pathway by immunoblotting. The current results showed that pubertal BPA exposure caused the translocation of cytochrome c (Cyt c) from mitochondria into cytosol. In addition, pubertal BPA exposure markedly upregulated the level of Bax and active caspase-9 in mouse testes. Further study indicated that pubertal BPA exposure significantly upregulated the expression of Bax in testes in a dose-dependent manner. Importantly, the level of Bax in mitochondrial fractions of testicular lysates was significantly increased in mice exposed to BPA during puberty. Unexpectedly, pubertal BPA exposure did not reduce protein level of the antiapoptotic member Bcl-2 in testes. Actually, the expression of Bcl-2 was slightly upregulated in testes of mice exposed to a high dose of BPA during puberty. However, the ratio of the proapoptotic member Bax vs the antiapoptotic member Bcl-2 was significantly increased in testes of mice exposed to BPA during puberty.Conclusion Pubertal BPA exposure induced germ cell apoptosis in testes of male mice. Apoptotic germ cells were observed mainly in stages VII–VIII seminiferous tubules in testes. In addition, BPA-mediated germ cell apoptosis in testes depends on not only the Fas/FasL signaling but also the mitochondrial apoptotic pathway.