Impact of bisphenol-A on the spliceosome and meiosis of sperm in the testis of adolescent mice

Abstract

Background: Bisphenol-A (BPA) has estrogenic activity and adversely affects humans and animals' reproductive systems and functions. There has been a disagreement with the safety of BPA exposure at Tolerable daily intake (TDI) (0.05 mg/kg/d) value and non-observed adverse effect level (5 mg/kg/d). The current study investigated the effects of BPA exposure at various doses starting from Tolerable daily intake (0.05 mg/kg/d) to the lowest observed adverse effect level (50 mg/kg/d) on the testis development in male mice offspring. The BPA exposure lasted for 63 days from pregnancy day 0 of the dams to post-natal day (PND) 45 of the offspring.

Results: The results showed that BPA exposure significantly increased testis (BPA ≥ 20 mg/kg/d) and serum (BPA ≥ 10 mg/kg/d) BPA contents of PND 45 mice. The spermatogenic cells became loose, and the lumen of seminiferous tubules enlarged when BPA exposure at 0.05 mg/kg/d TDI. BPA exposure at a low dose (0.05 mg/kg/d) significantly reduced the expression of Scp3 proteins and elevated sperm abnormality. The significant decrease in Scp3 suggested that BPA inhibits the transformation of spermatogonia into spermatozoa in the testis. The RNA-seq proved that the spliceosome was significantly inhibited in the testes of mice exposed to BPA. According to the RT-qPCR, BPA exposure significantly reduced the expression of Snrpc (BPA ≥ 20 mg/kg/d) and Hnrnpu (BPA ≥ 0.5 mg/kg/d).

Conclusions: This study indicated that long-term BPA exposure at Tolerable daily intake (0.05 mg/kg/d) is not safe because low-dose long-term exposure to BPA inhibits spermatogonial meiosis in mice testis impairs reproductive function in male offspring.

Keywords: Bisphenol-A; DNA damage; RNA-seq; Spliceosome; Synaptonemal complex protein 3 (SCP3); Testis.

Fig. 1

Testicular histopathological changes. Note: A the Control group; showing normal morphology of closely packed seminiferous tubules. B BPA 0.05 mg/kg /d group; C BPA 0.5 mg/kg/d group; D BPA 5 mg/kg/d group; E BPA 10 mg/kg/d group; F BPA 20 mg/kg/d group; G BPA 50 mg/kg/d group. 1, indicates the basal lamina; 2, the spermatogonia; 3, spaces between the basal lamina and the spermatogonia; 4, abnormal spaces in-between spermatogenic epithelium spermatogonia; 5, Lumen; 6, incomplete basal lamina. n = 13

Fig. 2

Scp3 expression in pachytene spermatocytes of different BPA groups. Note: Groups A was the control group. Groups B-G were 0.05, 0.5, 5, 10, 20, 50 mg/kg/d BPA groups. The arrows indicate positive staining of Scp3 protein. n = 3. The data is the average gray value of positive staining in the testis Scp 3 immunohistochemical results measured by Image J software, and the results are expressed as the mean ± standard error. Different superscript letters on the top of each bar (a, b, c) indicate significant differences (P < 0.05)

Fig. 3

Effect of BPA on mice spermatogonia DNA damage. Note: Group A was the control group, groups B-G were 0.05, 0.5, 5, 10, 20, 50 mg/kg/d BPA groups, respectively. Arrow 1 refers to the nucleus, and arrow 2 refers to the DNA fragments produced by apoptosis. n = 200. The results are expressed as the mean ± standard error. Different superscript letters on the top of each bar (a, b, c, d, e) indicate significant differences (P < 0.05)

Fig. 4

Sperm abnormalities at different BPA doses. Note: Group A was the control group, groups B-G were 0.05, 0.5, 5, 10, 20, 50 mg/kg/d BPA groups, respectively. The sperm count is the number of sperm in the whole field of microscope at 400X magnification after the sperm smear is finished. 1, head malformation; 2, mid-piece deletion; 3, acrosome deletion; 4, tail deletion. The results are expressed as the mean ± standard error. Different superscript letters (a, b, c) or * indicate significant differences (P < 0.05)

Fig. 5

Differential genes GO terms functional annotation clustering. Note: The vertical axis represents the name of the GO categories, and the horizontal axis represents the number or p-value of the genes in the GO categories

Fig. 6

Differential genes KEGG enrichment. Note: The vertical axis represents the name of the metabolic pathway, and the horizontal axis represents the degree of enrichment. The size of the dot indicates the number of differentially expressed genes in this pathway, and the color of the dots corresponds to a different q-value. (n = 3)

Fig. 7

Relative expression levels of Snrpc and Hnrnpu genes. Note: The ordinate is the differential fold change of Snrpc and Hnrnpu gene relative to the house-keeping genes, and the abscissa is the seven groups established according to the exposure doses of the BPA in the experiment. The results are expressed as the mean ± standard error. “ *” indicate significant differences (P < 0.05). n = 3