0013-7227/06/$15.00/0 Endocrinology 147(12):5956–5966 Printed in U.S.A. Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2006-0805 Developmental Programming: Differential Effects of Prenatal Exposure to Bisphenol-A or Methoxychlor on Reproductive Function Mozhgan Savabieasfahani, Kurunthachalam Kannan, Olga Astapova, Neil P. Evans, and Vasantha Padmanabhan Downloaded from https://academic.oup.com/endo/article/147/12/5956/2501261 by guest on 27 September 2021 Departments of Pediatrics and the Reproductive Sciences Program (M.S., O.A., V.P.), University of Michigan, Ann Arbor, Michigan 48109; Wadsworth Center (K.K.), New York State Department of Health, and Department of Environmental Health Sciences, State University of New York at Albany, Albany, New York 12201; and Division of Cell Sciences (N.P.E.), University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom Increased occurrence of reproductive disorders has raised maternal MXC concentrations in fat tissue and BPA in blood ,concerns regarding the impact of endocrine-disrupting chem- averaged approximately 200 ␮g/g fat and 37.4 ؎ 3.3 ng/ml icals on reproductive health, especially when such exposure respectively. Birth weights of BPA offspring were lower (P < occurs during fetal life. Prenatal testosterone (T) treatment 0.05) relative to C. There was no difference in the time of leads to growth retardation, postnatal hypergonadotropism, puberty between groups. BPA females were hypergonado- compromised estradiol-positive feedback, polycystic ovaries, tropic during early postnatal life and ended their breeding and infertility in the adult. Prenatal dihydrotestosterone season later, compared with C. Characterization of cyclic treatment failed to affect ovarian morphology or estradiol- changes after synchronization with prostaglandin F2␣ in five positive feedback, suggesting that effects of prenatal T may be C, six MXC, and six BPA females found that the onset of the LH facilitated via conversion of T to estradiol, thus raising con- surge was delayed in MXC (P < 0.05) and the LH surge mag- cerns regarding fetal exposure to estrogenic endocrine-dis- nitude severely dampened (P < 0.05) in BPA sheep. These rupting chemicals. This study tested whether fetal exposure findings suggest that prenatal BPA and MXC exposure have to methoxychlor (MXC) or bisphenol A (BPA) would disrupt long-term differential effects on a variety of reproductive en- -docrine parameters that could impact fertility. (Endocrinol ؍ cyclicity in the ewe. Suffolk ewes were administered MXC (n (mg/kg⅐d sc in cotton seed oil) or the vehicle ogy 147: 5956–5966, 2006 5) (10 ؍ BPA (n ,(10 ,from d 30 to 90 of gestation. On d 60 of treatment (16 ؍ C; n) OCIETAL CONCERN HAS been mounting over the po- well established in human medicine that fetal exposure to the S tential deleterious effects on animal/human health of synthetic estrogen, diethylstilbesterol, has resulted in a wide environmental exposure to endocrine-disrupting com- variety of problems in the daughters of mothers prescribed pounds (EDCs). EDCs are hormonally active, synthetic, or diethylstilbesterol during pregnancy, including increased natural compounds that are present within our environment risk of cancers and infertility (9, 10). Exposure to estrogenic and food sources at concentrations that can interfere with the EDCs such as dicofol and DDT (dichloro-diphenyl-trichlor- normal activity of endocrine systems/tissues, most notably ethane), has also been reported to disrupt sexual differenti- the reproductive endocrine axis (1, 2). The controversy re- ation in other species including turtles (11) and birds (12). garding the deleterious effects of EDC exposure has mainly Exposure to methoxychlor (MXC) or bisphenol A (BPA), been fueled by studies that point to likely effects on human EDCs that do interact with estrogen receptors, was found to health, including the recent dramatic increases in the inci- masculinize the female brain (13), advance puberty (14), and dence of estrogen sensitive cancers (breast, prostate and tes- cause sex reversal (15). In addition to these specific effects of ticular) (3, 4), the decline in human sperm quality and quan- EDCs on the reproductive system, EDCs have the potential tity (5), a notable rise in endometriosis (2), an increase in to alter the endocrine status of the developing fetus and thus genital abnormality in boys (6), and early puberty (7) in girls. lead to adaptations that may predispose the fetus to obesity EDCs that can interact with estrogen receptors have re- and other metabolic/endocrine diseases in adulthood (16– ceived considerable attention because they can modulate 19). Based on current evidence, the U.S. Environmental Pro- signaling by native estrogen, a key regulator of several phys- tection Agency (EPA) has thus advocated that although iologic functions including reproduction (8). In addition, it is exposure to single estrogenic compounds, at current envi- ronmental levels, is insufficient to cause adverse effects in First Published Online August 31, 2006 adult humans, more information is needed to determine Abbreviations: BPA, Bisphenol A; C, control; CV, coefficient of vari- whether the same holds true for the human fetus and neo- ation; MXC, methoxychlor; P, progesterone; PG, prostaglandin; T, testosterone. nate, which lack some of the protective mechanisms found in the adult (20). Another deficiency of the current EDC Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the literature is the scarcity of information relative to levels of endocrine community. EDCs achieved in human. Low-dose studies carried out in 5956 Savabieasfahani et al. • Prenatal EDC Exposure and Reproductive Dysfunction Endocrinology, December 2006, 147(12):5956–5966 5957 recent years (21–23) target intake rather than levels achieved differentiation at d 30 (43, 44), development of hypophyseal portal in circulation or tissue load. Considering that animals may vasculature around d 50 (43), detection of LH and FSH in circulation metabolize, store, or respond to similar intake levels differ- around d 55 (45), and completion of primordial follicular differentiation by d 90 (44). EDC doses were selected on the basis of the lowest observed ently, it is essential to also have other reference points such effect level established in the EPA National Toxicology Program’s Re- as circulating and tissue concentrations of EDCs. port of the Endocrine Disruptors (20). Recent rodent studies have Whereas a link has been suspected between EDC exposure achieved effects with much lower intake levels (21–23). Controls (C; n ϭ and adverse effects on human and animal health, it is im- 16) received vehicle. portant to note that health risks have primarily been derived To determine concentrations of BPA achieved using this treatment regimen, blood samples were collected from both C and prenatal BPA- from epidemiological data and/or studies conducted in ro- treated animals (n ϭ 6/treatment group) at three different time points, dent models. Because the sensitivity to EDCs is likely to vary 50, 70, and 90 d of gestation (20, 40 and 60 d of BPA treatment). To avoid between species, studies in rodents need to be cross-vali- the stress associated with collection of biopsies from experimental ewes, dated using sensitive animal models with established peri- a preliminary estimate of MXC load was obtained after the collection of Downloaded from https://academic.oup.com/endo/article/147/12/5956/2501261 by guest on 27 September 2021 ods of developmental susceptibility. Sheep provide a pow- fat biopsies from one pregnant ewe and her twin lambs (a female and erful model system to investigate reproductive consequences a male) after the animals were killed on d 90 of gestation (d 60 of MXC treatment). Fat biopsies were also obtained on d 30 and 60 of MXC of in utero estrogenic/androgenic EDC exposures because a treatment from two nonpregnant sheep. Fat biopsies were obtained from large body of literature already exists documenting critical three untreated C. Choice of measurement of BPA levels in maternal periods of fetal susceptibility to native steroids (24–35) in the blood and MXC in maternal fat was based on previous studies, which sheep. For instance, prenatal exposure of female sheep to found that BPA accumulates in liver, blood, or proteinaceous tissues testosterone (T), an estrogen precursor, has been reported to (46), and MXC is lipophilic and accumulates in fatty tissues (47). lead to growth retardation (25, 26, 32) and neuroendocrine (24, 27–29) and ovarian (31–33) defects that culminate in a Maternal care, neonatal measures, and postnatal care progressive loss of ovarian cyclicity (33–35). Six weeks before lambing, when maximal fetal growth was under- To assess the effects of EDC exposure, we selected two way, pregnant ewes were group fed an additional 0.5–1 kg alfalfa hay environmentally relevant endocrine disrupting compounds, and 250 mg Aureomycin crumbles (chlortetracycline) per ewe daily. All MXC and BPA. The organochlorine insecticide, MXC, has lambs, the majority twins, were born between March 15 and April 20, been used extensively to control pests in agricultural, dairy, 2002. Birth dates, the number of offspring, and offspring gender were and domestic settings and is known to be moderately per- recorded. At birth each lamb was given oral vitamin E and selenium and injections for Clostridium perfringens types C and D and tetanus. New- sistent in the environment (36, 37). Indeed, measurable con- born weight and body dimensions were recorded from all male and centrations of MXC can be found in the body fat of humans female lambs the day after birth to allow adequate time for maternal and rats (38, 39). BPA is a plasticizer, the production of which bonding. These measures included weight, height (determined with the is estimated to be about 1.7 billion kg/yr (40), and again lambs standing), chest circumference, and anourethral, anonavel, and studies have found measurable concentrations of BPA in the anoscrotal (males) distances.