BPA-Induced Deregulation Of Epigenetic Patterns: Effects On Female Zebrafish Reproduction

Abstract

Bisphenol A (BPA) is one of the commonest Endocrine Disruptor Compounds worldwide. It interferes with vertebrate reproduction, possibly by inducing deregulation of epigenetic mechanisms. To determine its effects on female reproductive physiology and investigate whether changes in the expression levels of genes related to reproduction are caused by histone modifications, BPA concentrations consistent with environmental exposure were administered to zebrafish for three weeks. Effects on oocyte growth and maturation, autophagy and apoptosis processes, histone modifications, and DNA methylation were assessed by Real-Time PCR (qPCR), histology, and chromatin immunoprecipitation combined with qPCR analysis (ChIP-qPCR). The results showed that 5 μg/L BPA down-regulated oocyte maturation-promoting signals, likely through changes in the chromatin structure mediated by histone modifications, and promoted apoptosis in mature follicles. These data indicate that the negative effects of BPA on the female reproductive system may be due to its upstream ability to deregulate epigenetic mechanism.

Figure 1. Transcription profiles of genes involved in steroidogenesis and oocyte growth.

Letters above each column indicate statistical differences among groups (p < 0.05 vs. untreated controls; ANOVA followed by Tukey’s multiple comparison test). (A) star: steroidogenic acute regulatory protein (F = 6.710; P = 0.0049); (B) cyp11a: cytochrome P450, family 11, subfamily (F = 4.608; P = 0.0178); (C) esr1: estrogen receptor 1 (F = 5.192; P = 0.0099); (D) esr2a: estrogen receptor 2a (F = 5.350; P = 0.0115); (E) esr2b: estrogen receptor 2b (F = 5.890; P = 0.0066); (F) fshr: follicle stimulating hormone receptor (F = 4.586; P = 0.0257). Data were generated in duplicate from five biological replicates.

Figure 2. Transcription profiles of genes involved in oocyte maturation and germinal vesicle breakdown.

Letters above each column indicate statistical differences among groups (p < 0.05 vs. untreated controls; ANOVA followed by Tukey’s multiple comparison test). (A) lhcgr: luteinizing hormone/choriogonadotropin receptor (F = 29.18; P = 0.0001); (B) pgrmc1: progesterone membrane receptor component 1 (F = 3.382; P = 0.0462); (C) pgrmc2: progesterone membrane receptor component 2 (F = 0.1755; P = 0.9114). Data were generated in duplicate from five biological replicates.

Figure 3

Histological analysis of ovaries from control fish (A) and fish exposed (B) to 5 μg/L BPA. Ovarian sections show different follicular stages and atretic follicles. (C) A follicle with the morphological markers of atresia: ZR*: Zona radiata breakdown; YR*: Yolk resorption; FC*: Follicular cell proliferation. (D) Percentage of atretic follicles in ovary from control fish (CTRL) and fish exposed (BPA) to 5 μg/L BPA.

Figure 4

ChIP-qPCR analysis of H3K4me3 and H3K27me3 trimethylation of lysine 4 (K4) and 27 (K27) in the amino terminal of histone 3 (H3) enrichment. (A) ChIP-qPCR results for star (steroidogenic acute regulatory protein) gene associated with H3K4me3 and H3K27me. (B) ChIP-qPCR results for fshr (follicle stimulating hormone receptor) gene associated with H3K4me3 and H3K27me. (C) ChIP-qPCR results for lhcgr (luteinizing hormone/choriogonadotropin receptor) gene associated with H3K4me3 and H3K27me.