Developmental Biology 238, 224–238 (2001) doi:10.1006/dbio.2001.0413, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Estrogen Receptor-␣ Knockout Mice Exhibit Resistance to the Developmental Effects of Neonatal Diethylstilbestrol Exposure on the Female Reproductive Tract John F. Couse,*,† Darlene Dixon,‡ Mariana Yates,* Alicia B. Moore,‡ Liang Ma,§,1 Richard Maas,§ and Kenneth S. Korach*,2 *Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; †Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695; ‡Comparative Pathobiology Section, Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and §Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115 Data indicate that estrogen-dependent and -independent pathways are involved in the teratogenic/carcinogenic syndrome that follows developmental exposure to 17␤-estradiol or diethylstilbestrol (DES), a synthetic estrogen. However, the exact role and extent to which each pathway contributes to the resulting pathology remain unknown. We employed the ␣ERKO mouse, which lacks estrogen receptor-␣ (ER␣), to discern the role of ER␣ and estrogen signaling in mediating the effects of neonatal DES exposure. The ␣ERKO provides the potential to expose DES actions mediated by the second known ER, ER␤, and those that are ER-independent. Wild-type and ␣ERKO females were treated with vehicle or DES (2 ␮g/pup/day for Days 1–5) and terminated after 5 days and 2, 4, 8, 12, and 20 months for biochemical and histomorphological analyses. Assays for uterine expression of the genes Hoxa10, Hoxa11, and Wnt7a shortly after treatment indicated significant decreases in DES-treated wild-type but no effect in the ␣ERKO. In contrast, the DES effect on uterine expression of Wnt4 and Wnt5a was preserved in both genotypes, suggesting a developmental role for ER␤. Adult ␣ERKO mice exhibited complete resistance to the chronic effects of neonatal DES exposure exhibited in treated wild-type animals, including atrophy, decreased weight, smooth muscle disorganization, and epithelial squamous metaplasia in the uterus; proliferative lesions of the oviduct; and persistent vaginal cornification. Therefore, the lack of DES effects on gene expression and tissue differentiation in the ␣ERKO provides unequivocal evidence of an obligatory role for ER␣ in mediating the detrimental actions of neonatal DES exposure in the murine reproductive tract. © 2001 Academic Press Key Words: estrogen receptor; diethylstilbestrol; teratogenesis; uterus; Hox; Wnt. INTRODUCTION dogenous and synthetic estrogens, respectively, as carcino- gens and teratogens in humans and laboratory animals Laboratory and epidemiological studies have clearly im- (IARC, 1979, 1987; Marselos and Tomatis, 1992, 1993). plicated 17␤-estradiol (E2) and diethylstilbestrol (DES), en- Beginning in the 1940s, DES was prescribed during preg- nancy to maintain placental steroid production and thereby 1 Present address: Tulane University, Department of Cell and Molecular Biology, 2000 Percival Stern Hall, 6400 Freret Street, New Orleans, LA 70118. 2 To whom correspondence should be addressed at National ogy, Receptor Biology Section, MD B3-02, P.O. Box 12233, Re- Institutes of Health, National Institute of Environmental Health search Triangle Park, NC 27709. Fax: (919) 541-0696. E-mail: Sciences, Laboratory of Reproductive and Developmental Toxicol- [email protected]. 0012-1606/01 $35.00 Copyright © 2001 by Academic Press 224 All rights of reproduction in any form reserved. Effects of DES in the ␣ERKO Female Mouse 225 lessen the risk of spontaneous abortion or preterm parturi- classical ER (now termed ER␣) (Gigue`re et al., 1998); and (2) tion (Marselos and Tomatis, 1992). Despite a 1953 report of the unique ability of DES to suppress the transactivational the ineffectiveness of DES for this indication (Dieckmann activity of the orphan nuclear receptors ERR␣, ERR␤, and et al., 1953), clinical use increased in the years following ERR␥ (Tremblay et al., 2001). (Marselos and Tomatis, 1992). In 1971, following two re- Laboratory studies have also indicated that epigenetic ports of a causal link between vaginal clear-cell adenocar- mechanisms, independent of direct interaction with genetic cinoma, a rare cancer, and in utero DES exposure (Green- material but leading to permanent changes in gene expres- wald et al., 1971; Herbst et al., 1971), the U.S. FDA sion, may also contribute to the ultimate effects of DES proscribed the use of DES for pregnancy support (Herbst, (Liehr, 2000). Perinatal DES exposure in rodents causes 2000). To date, DES remains the only drug in which attenuated uterine responses to subsequent estrogen stimu- transplacental carcinogenesis in man has been proven lation during adulthood, characterized as decreased growth (Marselos and Tomatis, 1992). More prevalent than cancer and secretory activity and altered cellular differentiation are the multiple teratogenic effects attributed to in utero (Maier et al., 1985; Medlock et al., 1988, 1992). Similar DES DES exposure, including vaginal/cervical epithelial adeno- treatments in mice lead to constitutive up-regulation of sis and squamous metaplasia, transverse vaginal ridges, and lactoferrin expression in adult uteri (Nelson et al., 1994), structural malformations of the cervix and uterus (Herbst, seminal vesicle (Beckman et al., 1994), and the urethropros- 2000; Marselos and Tomatis, 1992; Mittendorf, 1995). In tatic complex (Salo et al., 1997). Moreover, Li et al. (1997) males, in utero DES exposure is associated with increased demonstrated distinct alterations in the methylation pat- risk of testicular cancer, epididymal cysts, cryptorchidism, tern of the lactoferrin gene promoter in uterine tissue of and testicular hypoplasia (Herbst, 2000; Marselos and mice neonatally exposed to DES, providing support for an Tomatis, 1992). epigenetic pathway by which DES permanently alters gene Over the past 30 years, rodent models that effectively expression. Similar changes in gene expression following assimilate the effects of developmental DES exposure in neonatal DES exposure are reported for epidermal growth humans have been thoroughly characterized, yet the under- factor (EGF), EGF-receptor, c-fos, c-jun, c-myc, bax, and lying mechanisms remain poorly understood (Marselos and bcl-2 in the reproductive tract of sexually mature mice and Tomatis, 1992, 1993). It is widely accepted that E2 or DES hamsters (Falck and Forsberg, 1996; Nelson et al., 1994; exposure during critical developmental periods elicits dis- Zheng and Hendry, 1997). Once again, the role of the ER in tinct and permanent alterations in the Mu¨llerian-derived mediating this effect of DES remains unknown. structures of the female reproductive tract (oviduct, uterus, In contrast to the hormonal and epigenetic mechanisms, cervix, upper vagina) (Cunha et al., 1991). Interestingly, null there is evidence of direct genotoxic actions of DES and its mouse models have illustrated that a lack of estrogen metabolites, presumably independent of ER action. These signaling has little effect on reproductive tract development mechanisms are more often applied to explain the carcino- (Couse et al., 1999; Couse and Korach, 1999; Fisher et al., genic properties of DES and include the induction of DNA 1998), whereas aberrant estrogen exposure during develop- adducts, microsatellite instability, sequence deletions or ment is detrimental (Cunha et al., 1991). Hence, the under- insertions, and single-strand breaks in both in vitro and in lying mechanisms of developmental DES exposure likely vivo systems (Liehr, 2000). Furthermore, DES and its me- involve its ability to bind the nuclear estrogen receptor (ER) tabolites are reported to elicit aneuploidy and cell transfor- and mimic the broad spectrum of E2 actions (Korach et al., mation in Syrian hamster embryo cells lacking measurable 1978, 1989). This aberrant stimulation of estrogen signal- ER levels, possibly via interfering with microtubule func- ing by DES during development may disrupt the proper tion (Tsutsui et al., 1983). Supporting evidence of DES- expression of estrogen-regulated genes and the ultimate induced genetic instability is provided by descriptions of differentiation/proliferation profile of cell populations. Re- aneuploid DNA in premalignant vaginal lesions in “DES ported alterations in the expression of Hoxa9, Hoxa10, daughters” (Welch et al., 1983) and DES-exposed mice Hoxa11 (Block et al., 2000; Ma et al., 1998), and Wnt7a (Hajek et al., 1993). (Miller et al., 1998a) in the developing murine female Therefore, existing evidence supports both ER-dependent reproductive tract following DES exposure support such a and -independent mechanisms as mediators of the develop- mechanism. Furthermore, null mouse models of the Hox mental and carcinogenic actions of DES. In this report, we (Dolle et al., 1991; Hsieh-Li et al., 1995; Satokata et al., describe our use of the estrogen receptor-␣ knockout mouse 1995) and Wnt (Miller et al., 1998a; Parr and McMahon,