Effects Of Bisphenol A (an Estrogenic Compound) And Estrogen On Male Reproductive System And Pain Modulation Via ERs Using ERKO Mice

Estrogen, transduced by binding to estrogen receptors (ERa and ERP), play a key role in the reproductive, endocrine and nervous system. Lacking ERs have indicated the importance role of ERs in the male reproductive function. In the nervous system estrogen exert protective effects on neuronal cells, and play a key role in pain modulation. Bisphenol A (BPA) has been demonstrated to act as an endocrine disrupting chemicals (EDCs) and an estrogen-mimicking chemical, exposure to BPA might have potential toxicological effects. Genetargeting techniques were used to generate mice lacking either functional ERa (aERKO), ERβ (βERKO) to provide a model for evaluating estrogen receptor action. In this report, we breed ERKO mice, and establish an animal experimental model for further study the important role of ERs (ERa and ERβ) in BPA induced male reproductive toxicology and pain modulation by estradiol.Part1 Breeding of ERs Knockout Mice and Gene Identification of Filial GenerationGenetargeting techniques were used to generate aERKO and βERKO mice to provide a unique tool to study the effects of estrogen in the context of the whole animal and to discern the role of each ERs in various tissues. Mice of the C57BL/6 strain that were heterozygous for genomic deletion of estrogen receptor types a and p (ERa+’- and ERp+/-) were purchased from the Jackson Laboratory. Inbreeding and appropriate backbreeding of the heterozygotes yielded homozygote knockout ERa (ERα-/-) and ERβ mice (ERβ-/-). PCR methods can identify the genotype of the ERα-/-and ERβ-/- mice precisely. It is feasible to breed ERKO mice, and establish an animal experimental model for further study the important role of ERs (ERa and ERβ) in BPA inducing male reproductive toxicology and pain modulation by estradiol.Part 2 BPA-induced Male Reproductive Toxicology via ERs Using ERKO MiceBisphenol A (BPA), an estrogenic compound, has been highlighted due to its endocrine disrupting actions on the human body, previous studies have shown that BPA may affect the male reproductive function. In this report, BPA at a dose of 100 μg/kg/day for 30 days via intragastric administration significantly decreased the epididymis weights, and increased the sperm abnormality rate, the histology of the testis showed unnormal. Similar to WT mice, the epididymis weights were decreased, the sperm abnormality rate was increased, and the histology of the testis showed unnormal in aERKO and βERKO mice after BPA treatment. Using real-time PCR, we observed that mRNA expression of ER a and ERβ was significantly changed in the testis and epididymis in BPA-treated animals, we also found that the BPA-induced mRNA expression of Cav3, RyRs and IP3Rs in testis and epididymis was dependent on ERs. Thus, it appears that BPA-induced male reproductive toxicology is mediated through the ERa and ERβ. However, the mode of action of BPA on the reproductive system and the potential mechanism of BPA affecting the male reproductive function require further investigation.Part 3 Effects of Bisphenol A and Estrogen on Pain Modulation via ERs Using ERKO MiceA number of studies have demonstrated that estrogens exert protective effects on neuronal cells, and estrogens, transduced by binding to ERa and ERβ, play a key role in pain modulation. In this study, the involvement of both receptors on nociceptive responses was measured in aERKO and βERKO female mice. Ovariectomies followed by estrogen replacement were performed in female groups to insure comparable sex hormone levels. To verify the involvement of ERa and ER β on acute and persistent pain mechanisms, tail-flick, hotplate and formalin tests were carried out. WT mice given E2 showed no changes in tail flick latency and hot plate latency. There were also no significant differences between WT and aERKO or βERKO mice. Estradiol significantly reduced the overall number of flinches during Phase II (not phase I) of the formalin nociceptive response. Using ERKO mice, we found that PERKO (not aERKO) females showed lower nociceptive responses compared to WT female mice during the phase II of the formalin test, but not during phase I, suggesting the pain modulation by ERβ.Action potential (AP) was successfully recorded in freshly isolated rat dorsal root ganglion (DRG) neurons using whole-cell patch clamp technique. We investigated the effects of 170-E2 on AP. The results showed that 17β-E2 could inhibit AP firing. The amplitude of AP was decreased and the half-width duration was increased, suggested that the excitability of DRG neurons was reduced. Action potentials are generated by special types of voltage-gated ion channels embedded in a cell’s plasma membrane. Voltage-gated Na+channels mediate a rapid and transient increase in Na+permeability in response to changes in membrane potential, thereby contributing to the generation and conduction of action potentials that serve as sensory signals from the periphery to the spinal cord through the primary afferent neurons.The whole-cell patch clamp technique was used to measure voltage-gated Na+ channels current in DRG neurons and determine the effects of E2 on Na+channels. TTX-S Na+ currents and TTX-R Na+currents were inhibited by 17P-E2 (not 17a-E2) in a concentration-dependent manner. We examined the effect of estradiol conjugated bovine serum albumin (E2-BSA) on Na+channels, E2-BSA produced a similar extent of inhibition of Na+channels currents, suggesting that the modulation of Na+ channels by 17β-E2 occurs via non-genomic pathways. Application of GO-6983 or H-89 did not alter the basal TTX-S Na+current and TTX-R Na+ current, but it prevented the decrease in Na+ currents induced by E2. These results confirmed that E2 modulated Na+channels in a PKC and PKA dependent pathway. Incubation with the selective ERs antagonist ICI182780, did not affect the inhibitory effect of E2 on Na+channels, ERa-selective agonist PPT or ERβ-selective agonist DPN also inhibited the Na+ currents, suggesting both ERα and ERβ appear to be involved in Na+ channels modulated by E2. Using ERKO mice, no differences in current densities of Na+ channels were found under control conditions in DRG neurons of WT, aERKO and PERKO mice. Application of E2 also reduced TTX-S Na+ current in DRG neurons of aERKO and βERKO mice. However, the E2 induced inhibiton of TTX-S Na+current in aERKO and βERKO mice was smaller than WT mice, suggested that the inhibition of TTX-S Na+current by E2 depended on both ERa and ERβ. E2 also inhibited TTX-R Na+current in aERKO and βERKO mice, compare to WT mice, the E2 induced inhibiton of TTX-R Na+current in PERKO was smaller, suggested that inhibition of TTX-R Na+ current by E2 depended on ERβ.Finally, we found that the extracellularly applied BPA inhibited TTX-S Na+ currents and TTX-R Na+currents via PKC and PKA-dependent signaling pathway. Moreover, several alterations in Na+ channel kinetics were also induced by BPA. Considering its complex modulatory effects on voltage-gated sodium channels, BPA might have potential toxicological effects on the nervous system and lead to change in excitability of nociceptive afferent fibers.