Question
Do BPA analogs have similar adverse effects as BPA on the structure and function of the developing mammalian ovary in vivo?
Introduction
Endocrine disrupting chemicals (EDCs) are compounds that can mimic or block the action of endogenous hormones in the body. These natural or synthetic chemicals can have major effects on development, fertility, and may be associated with cancers of the reproductive tract and mammary glands. Developmental exposures seem to have a larger effect than exposures in adulthood (Rogers et al 2013). Bisphenol A (BPA) is an estrogen mimicking EDC that is commonly found in cans, plastic food and drink containers, dental materials, and receipts (Maffini et al 2006). Recently, increasing numbers of animal studies have found that even at low levels BPA has detrimental effects on meiotic events and follicle formation in the developing ovary in both mouse and primate models (Hunt & Hassold, 2008; Hunt et al 2012). This may lead to a decrease in reproductive lifetime of the offspring due to malformation and miss-packaging of ovarian follicles (Hunt et al 2012).
BPA has been found to bind to estrogen receptors (ERα and ERβ) leading to dysfunction in the normal regulation of genes affected by these receptors. These receptors act as transcription factors that travel into the nucleus and cause changes to gene expression. ERα is expressed in theca cells (support cells for each follicle) and in the ovarian stroma during ovary development and in adulthood (Sar & Welsch, 1999). ERβ is expressed in granulosa cells of developing follicles (Sharma et al 1999). Knockouts of ERα (ERKO) and ERβ (ERBKO) have very different phenotypes. ERKO shows a phenotype where follicles appear to develop normally but then never ovulate or produce the correct outer covering, the presence of large hemorrhagic cysts, and stromal hyperplasia, and an excess of other circulating hormones like luteinizing hormone, testosterone, and estrogen (Abbot et al 2006). This shows that ERα is important in regulation of the effect of other hormones in the ovary (Couse & Korach, 2001). ERBKO exhibits a much milder phenotype where individuals are able to ovulate and produce pups but have fewer ovulation events and smaller litter sizes (Abbot et al 2006). This shows that ERβ is very important in the mediation of estrogen’s effect on the ovary in ways related to maturation of follicles (Emmen et al 2005). BPA’s effect on these receptors leads to changes in gene expression and regulation in the ovary. BPA has an agonist effect on ERβ and has both an agonist and an antagonist effect on ERα (Kuiper et al 1998; Hiroi et al 1999). In terms of the phenotype of BPA exposed individuals, activation of ERβ would be expected to cause miss-regulation of follicle maturation and formation, and variable action on ERα would be expected to cause variation in stromal proliferation, and the potential presence of cysts and abnormally small oocytes.
Due to an increase in our understanding of the detrimental effects of BPA, analogs of BPA are beginning to be used instead. Less is known about these derivatives of BPA, though recent studies have determined that many of these compounds have similar effects on the estrogen receptors (Stossi et al 2014). Two of the many BPA analogs that are commonly used are Tetrachlorobisphenol A (TCBPA) and Bisphenol S (BPS). TCBPA is an ERα agonist and BPS has agonistic effects on both receptors, with a higher activity in binding to ERβ (Li et al 2010; Molina-Molina et al 2013). As these compounds also have estrogenic effects, they will likely produce a phenotype in the developing ovary.
| EDC | Effect on ERα | Effect on ERβ |
| BPA | +/- | + |
| TCBPA | + | none |
| BPS | – | – |
Table 1. Effect of BPA and two analogs on estrogen receptors ERα and ERβ. (+ signifies agonist effect and – signifies antagonist effect)
Hypotheses and Predictions
I hypothesize that both TCBPA and BPS will have abnormal phenotypes that are different than the phenotype shown by BPA exposed developing ovaries due to their different action. TCBPA will likely have little effect on the formation of follicles but will cause decreases in stromal cells and an increase in the number of small oocytes. BPS will likely cause changes in follicle maturation and decrease stromal proliferation and increase the number of small oocytes.
Experimental approach
In order to investigate this question I will use a mouse model system. While this species is not as applicable to the effect of BPA and its analogs on humans as a primate model, mice are easier to house, less expensive, reproduce much more quickly, and will produce multiple offspring per pregnancy. I will have four treatment groups: control (no treatment), BPA, TCBPA, and BPS. Female mice will have a device that releases a continuous low level dose of either BPA, TCBPA, or BPS implanted subcutaneously (protocol by Hunt et al 2012). A pilot study will be conducted to make sure that dosage levels are close to the normal serum concentrations found in humans (Hunt et al 2012). Control females will have the device implanted but without the chemical additives. Dosage will continue throughout pregnancy; this method is the most similar to normal human exposure and may give the most realistic results (Hunt et al 2012). The mice will then be mated and embryos will be harvested at mid and late gestation, the beginning of meiotic division and follicle formation respectively.
Female embryos will studied to determine the effect of BPA analogs on chromosome behavior during meiosis and follicle formation in the developing ovary. Immunofluorescence tagging of synaptonemal complex proteins will allow relatively easy quantification of meiotic defects. Chromosome associations will also be observed using immunofluorescence and the numbers compared to normal associations in the control group. Follicle formation will be investigated by taking histological sections through the ovary and observing/counting any follicular abnormalities that result. These methods are a good way to observe chromosome behavior and ovarian structures after in vivo exposure. Protocols are described in Hunt et al 2012.
Possible outcomes
There are many possible outcomes for this experiment:
- TCBPA and BPS may not show gross phenotypic changes in the developing ovary. Further study may then be needed to identify if gene expression is affected without causing large outward changes.
- TCBPA and BPS may show novel phenotypes in the developing ovary due to their different effects on the ERs.
- The severity of the novel phenotypes may range from severe to mild and may not indicate that these chemicals will adversely affect future generation reproduction.
- TCBPA and BPS may show phenotypes in the developing ovary that are similar to those of BPA but the severity of the phenotype may differ due to different levels of activity when binding to the ERs.
Lay person summary
Hormones are very important molecules in the normal growth and function of male and female reproductive tracts. Natural and synthetic chemicals can mimic the function of hormones in the body by interacting with normal hormone pathways; these chemicals are called endocrine disrupting chemicals (EDCs). BPA is an EDC that is used to make plastics found in food and drink containers, the inside of cans, and receipts. BPA mimics the function of estrogen and as such has detrimental effects on the developing ovary, where the correct levels of estrogen is important for proper growth. Recently, chemicals derived from BPA (BPA analogs) have started to be used instead of BPA, due to the increasing evidence for health concern; however, little is known about the effects of the analogs on development. I will perform an experiment on mice that will expose female mice to continuous low levels of BPA and two analogs (TCBPA and BPS) throughout pregnancy. The resulting fetuses will be harvested at different time points during development and then I will observe the structure of the ovary. This topic is interesting because these compounds could have detrimental effects on human reproduction and fertility in the future, as the next generation is produced by mothers that have been exposed to BPA, TCBPA, and BPS.
Literature cited
– Abbott, D. H., Padmanabhan, V., & Dumesic, D. A. (2006). Contributions of androgen and estrogen to fetal programming of ovarian dysfunction. Reprod Biol Endocrinol, 4(17), i0006-3363.
– Couse, J. F., & Korach, K. S. (2001). Contrasting phenotypes in reproductive tissues of female estrogen receptor null mice. Annals of the New York Academy of Sciences, 948(1), 1-8.
– Emmen, J. M., Couse, J. F., Elmore, S. A., Yates, M. M., Kissling, G. E., & Korach, K. S. (2005). In vitro growth and ovulation of follicles from ovaries of estrogen receptor (ER) α and ERβ null mice indicate a role for ERβ in follicular maturation. Endocrinology, 146(6), 2817-2826.
– Hiroi, H., Tsutsumi, O., Momoeda, M., Takai, Y., Osuga, Y., & Taketani, Y. (1999). Differential interactions of Bisphenol A and 17β-estradiol with estrogen receptor α (ERα) and ERβ. Endocrine journal, 46(6), 773-778.
– Hunt, P. A., & Hassold, T. J. (2008). Human female meiosis: what makes a good egg go bad?. Trends in Genetics, 24(2), 86-93.
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– Kuiper, G. G., Lemmen, J. G., Carlsson, B. O., Corton, J. C., Safe, S. H., van der Saag, P. T., … & Gustafsson, J. A. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor β. Endocrinology, 139(10), 4252-4263.
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– Sar, M., & Welsch, F. (1999). Differential expression of estrogen receptor-β and estrogen receptor-α in the rat ovary. Endocrinology, 140(2), 963-971.
– Sharma, S. C., Clemens, J. W., Pisarska, M. D., & Richards, J. S. (1999). Expression and Function of Estrogen Receptor Subtypes in Granulosa Cells: Regulation by Estradiol and Forskolin 1. Endocrinology, 140(9), 4320-4334.
– Stossi, F., Bolt, M. J., Ashcroft, F. J., Lamerdin, J. E., Melnick, J. S., Powell, R. T., … & Mancini, M. A. (2014). Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays. Chemistry & biology, 21(6), 743-753.