Developmental programming: Impact of prenatal exposure to bisphenol-A and methoxychlor on steroid feedbacks in sheep

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Abstract

Bisphenol-A (BPA), a polymer used in plastics manufacturing, and methoxychlor (MXC), a pesticide, are endocrine disrupting compounds with estrogenic and anti-androgenic properties. Prenatal BPA or MXC treatment induces reproductive defects in sheep with BPA causing prepubertal luteinizing hormone (LH) hypersecretion and dampening of periovulatory LH surges and MXC lengthening follicular phase and delaying the LH surge. In this study, we addressed the underlying neuroendocrine defects by testing the following hypotheses: 1) prenatal BPA, but not MXC reduces sensitivity to estradiol and progesterone negative feedback, 2) prenatal BPA, but not MXC increases pituitary responsiveness to gonadotropin releasing hormone (GnRH), and 3) prenatal BPA dampens LH surge response to estradiol positive feedback challenge while prenatal MXC delays the timing of the LH surge. Pregnant sheep were treated with either 1) 5 mg/kg/day BPA (produces approximately twice the level found in human circulation, n = 8), 2) 5 mg/kg/day MXC (the lowest observed effect level stated in the EPA National Toxicology Program's Report; n = 6), or 3) vehicle (cotton seed oil: C: n = 6) from days 30 to 90 of gestation. Female offspring of these ewes were ovariectomized at 21 months of age and tested for progesterone negative, estradiol negative, estradiol positive feedback sensitivities and pituitary responsiveness to GnRH. Results revealed that sensitivity to all 3 feedbacks as well as pituitary responsiveness to GnRH were not altered by either of the prenatal treatments. These findings suggest that the postpubertal reproductive defects seen in these animals may have stemmed from ovarian defects and the steroidal signals emanating from them.

Highlights

► Prenatal BPA/MXC does not affect reproductive neuroendocrine steroid feedbacks. ► Prenatal BPA or MXC treatment failed to alter pituitary sensitivity to GnRH. ► LH excess in BPA-treated sheep may be due to reduced ovarian feedback signals.

Introduction

Endocrine disrupting compounds (EDCs) are substances that can mimic, modulate, or block the normal function of physiological systems (Diamanti-Kandarakis et al., 2009). In the last 50 years, the number of EDCs that humans are exposed to has increased dramatically (http://www.endocrinedisruption.com/endocrine.TEDXList.overview.php) posing a real threat to their wellbeing. Bisphenol-A (BPA) and methoxychlor (MXC) are two such EDCs widely studied. Annually, more than eight billion pounds of BPA are produced with more than 100 tons being diffused into the atmosphere (Vandenberg et al., 2012). It is used in the manufacture of plastics and has both estrogenic and anti-androgenic properties. BPA is released into the food chain from polycarbonate containers (baby food bottles and reusable food containers), epoxy resins used in inner coating of metallic food cans, and dental sealants (Vandenberg et al., 2012). BPA is also released from paints and lining of drinking water pipes (vom Saal and Hughes, 2005). A recent NHANES study reported that over 90% of subjects studied from different age categories have detectable levels of BPA in their urine (Calafat et al., 2008). Measurable levels of BPA have been reported in human maternal circulation (Padmanabhan et al., 2008, Schönfelder et al., 2002), cord blood (Kuroda et al., 2003), colostrum (Kuruto-Niwa et al., 2007), breast milk (Ye et al., 2008), and the placenta (Schönfelder et al., 2002).

Methoxychlor (MXC), which has anti-androgenic and estrogenic properties (Staub et al., 2002), is an organochlorine pesticide applied to fruits, vegetables, and animal feed (Reynolds et al., 1976, Agency for Toxic Substances and Disease Registry 2002 http://www.atsdr.cdc.gov/toxguides/toxguide-47.pdf). It has been found in the circulation of men and women (Botella et al., 2004, Carreño et al., 2007) and adipose tissue of women (Botella et al., 2004). MXC was banned in the European Union in 2002 (http://ec.europa.eu/sanco_pesticides/public/index.cfm?event=activesubstance.selection&a=1) and U.S.A. in 2003 (http://www.epa.gov/oppsrrd1/REDs/methoxychlor_red.htm). It is a persistent chemical (Howard, 1991) and is still found in the environment (Bempah and Donkor, 2011).

Studies in rats and mice have provided evidence that EDCs can alter reproductive function in both males (reviewed in Wong and Cheng, 2011) and females (mice, rabbits, and cattle; reviewed in Fowler et al., 2012). There is growing interest in EDCs with steroidogenic potential due to their ability to induce cancers (breast Fenton, 2006, prostate Muir, 2005, testicular Garner et al., 2008), endometriosis (Missmer et al., 2004), and genital abnormalities in boys (Paulozzi et al., 1997). They have been implicated in lower sperm quality in human (Dallinga et al., 2002) as well as pubertal advancement in girls (Roy et al., 2009). A growing body of evidence suggests that perinatal exposure to EDCs leads to adult reproductive dysfunction (reviewed in Crain et al., 2008). For example, prenatal exposure to BPA was found to advance puberty in mouse (Howdeshell et al., 1999) and trigger sex reversal in crocodilian reptile (Stoker et al., 2003). Similarly, perinatal MXC exposure was found to negatively impact reproductive function in rats (Suzuki et al., 2004).

In female sheep, prenatal exposure to BPA and MXC induces reproductive defects (Savabieasfahani et al., 2006) during postpubertal life. While prenatal BPA treatment was found to induce LH excess early in life and disruption of the periovulatory LH surge manifested as absent or reduced LH surge amplitude (Savabieasfahani et al., 2006), prenatal MXC treatment delayed onset of LH surge without having an effect on the LH surge amplitude. LH excess in prenatal BPA-treated females may be the result of reduced estradiol/progesterone input from the ovary thus providing reduced negative feedback signal, reduced sensitivity of the hypothalamo-pituitary axis to steroid negative feedback, and/or increased pituitary responsiveness to GnRH. The reduced magnitude of LH surge or delay in timing of LH surge on the other hand may result from deficits in estradiol positive feedback mechanisms. In previous studies with prenatal testosterone-treated females, deficits in neuroendocrine feedback systems and increased pituitary sensitivity to GnRH were found to underlie LH excess and LH surge defects (Padmanabhan et al., 2010).

In this study, we addressed if prenatal BPA and MXC treatment have differential effects on neuroendocrine feedback systems (estradiol negative and positive, progesterone negative) and pituitary responsiveness to GnRH. Specifically, we tested the following hypotheses: 1) prenatal BPA, but not MXC treatment would reduce sensitivity to estradiol and progesterone negative feedback, 2) prenatal BPA, but not MXC would increase pituitary responsiveness to GnRH, and 3) prenatal BPA treatment would dampen the LH surge response to estradiol positive feedback challenge while prenatal MXC treatment would delay the timing of LH surge response to estradiol positive feedback challenge.

Section snippets

Animal breeding and prenatal treatment

All experimental procedures involving animals used in this study were approved by the Institutional Animal Care and Use Committee of the University of Michigan and are consistent with National Research Council's Guide for the Care and Use of Laboratory Animals. Adult Suffolk ewes of proven fertility were obtained from a local farmer and maintained at the University of Michigan Sheep Research Facility (Ann Arbor, MI, USA; 42° 18′ N). Beginning two to three weeks prior to breeding, ewes were

Estradiol negative feedback

Fig. 2 summarizes representative circulating patterns of LH (panel A) and summary statistics (panel B) during the estradiol negative feedback test. Prior to the estradiol treatment, number of pulses (7.0 ± 0.5), LH pulse amplitude (12.1 ± 2.3 ng/ml), and total LH released (566.5 ± 54.7 ng/ml) in prenatal BPA-treated females were similar to the controls (number of pulses: 6.3 ± 0.2; LH pulse amplitude: 11.7 ± 2.2 ng/ml; and total LH released: 527.2 ± 61.1 ng/ml) (P = 0.147, P = 0.996, P = 0.908 for the three

Discussion

The findings from this study fail to support the hypothesis that the LH excess seen in prenatal BPA-treated sheep is a function of altered sensitivity of neuroendocrine systems to steroid feedback and/or increased pituitary responsiveness to GnRH. Similarly, the LH surge defects seen in prenatal BPA- and MXC-treated females do not appear to stem from estradiol positive feedback disruptions. These findings are discussed in detail below.

The finding that prenatal BPA treatment fails to reduce

Declaration of interest

All authors have no conflict of interests to declare.

Funding

This work was supported by NIEHS grant ES016541 to VP and training grant support to BAS (T32DK071212).

Acknowledgments

We are indebted to Doug Doop and Gary McCalla for help with breeding and lambing and excellent animal care; Olga Astapova, David Han, Danielle Djoumbi, Jonathan Flak, Esther Aizenberg, Mozhgan Savabieasfahani and Mohan Manikkam for help with prenatal treatments, participation in feedback tests, and/or performance of LH/FSH assays; and Dr. Almudena Veiga-Lopez for her helpful edits and comments.

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    Present address: Extension and Outreach, University of Illinois, Urbana-Champaign, IL 61801, USA.

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