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Environmental influences on fertility: can we learn lessons from studies of wildlife?

2008; Elsevier BV; Volume: 89; Issue: 2 Linguagem: Inglês

10.1016/j.fertnstert.2007.12.019

1556-5653

Louis J. Guillette, Thea M. Edwards,

Pharmaceutical and Antibiotic Environmental Impacts

Wildlife are good models for understanding the impacts of environmental contamination on fertility because [1] exposure doses are representative of actual contaminant mixtures and concentrations in the environment, [2] wildlife populations are more genetically diverse than laboratory animal populations, and [3] both individual and population level effects of fertility compromise can be observed. Wildlife are good models for understanding the impacts of environmental contamination on fertility because [1] exposure doses are representative of actual contaminant mixtures and concentrations in the environment, [2] wildlife populations are more genetically diverse than laboratory animal populations, and [3] both individual and population level effects of fertility compromise can be observed. Since the publication of Rachel Carson's legendary book "Silent Spring" in 1962 (1Carson R. Silent spring. Houghton Mifflin Co, Boston1962Google Scholar), wildlife have been seen as important sentinels of ecosystem health. The perception by many, however, is that the major endpoint worth examining is mortality, and thus, declining population numbers. A close reading of Carson's book and many scientific studies that have followed illustrates a very different series of endpoints. By no means is mortality excluded, but altered population size also can be attributed to alterations in fertility and fecundity of the individuals that comprise the population under study. In the early 1990s, a growing series of studies began to associate environmental contamination with altered reproductive performance in wild populations of fish, amphibians, reptiles, and birds (2Colborn T. vom Saal F.S. Soto A.M. Developmental effects of endocrine-disrupting chemicals in wildlife and humans.Environ Health Perspect. 1993; 101: 378-384Crossref PubMed Scopus (2969) Google Scholar). This literature documented the endocrine disruptive effects of a wide array of environmental pollutants, including pesticides, contaminants in sewage such as surfactants (e.g., octylphenol and nonylphenol) and pharmaceutical agents (e.g., ethynylestradiol from birth control pills), plasticizers (e.g., phthalates), flame retardants (PCBs, PBDEs), and industrial pollutants (e.g., heavy metals, dioxin, PAHs) [for reviews, see (3Tyler C.R. Jobling S. Sumpter J.P. Endocrine disruption in wildlife: a critical review of the evidence.Crit Rev Toxicol. 1998; 28: 319-361Crossref PubMed Scopus (914) Google Scholar, 4McLachlan J.A. Environmental signaling: what embryos and evolution teach us about endocrine disrupting chemicals.Endocr Rev. 2001; 22: 319-341Crossref PubMed Scopus (463) Google Scholar, 5Guillette Jr., L.J. Gunderson M.P. Alterations in the development of the reproductive and endocrine systems of wildlife exposed to endocrine disrupting contaminants.Reproduction. 2001; 122: 857-864Crossref PubMed Scopus (165) Google Scholar, 6Iguchi T. Watanabe H. Katsu Y. Developmental effects of estrogenic agents on mice, fish and frogs: a mini-review.Horm Behav. 2001; 40: 248-251Crossref PubMed Scopus (75) Google Scholar, 7Milnes M.R. Bermudez D.S. Bryan T.A. Edwards T.M. Gunderson M.P. Larkin I.V. et al.Contaminant-induced feminization and demasculinization of nonmammalian vertebrate males in aquatic environments.Environ Res. 2006; 100: 3-17Crossref PubMed Scopus (88) Google Scholar)]. Many of these compounds alter estrogen, androgen, or thyroid signaling, which are essential for normal embryonic development and reproductive activity in all vertebrates studied to date (4McLachlan J.A. Environmental signaling: what embryos and evolution teach us about endocrine disrupting chemicals.Endocr Rev. 2001; 22: 319-341Crossref PubMed Scopus (463) Google Scholar, 8Gray LE Jr, Wilson VS, Stoker T, Lambright C, Furr J, Noriega N, et al. Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals. Int J Androl 29:96–104.Google Scholar, 9Zoeller R.T. Dowling A.L.S. Herzig C.T.A. Iannacone E.A. Gauger K.J. Bansal R. Thyroid hormone, brain development, and the environment.Environ Health Perspect. 2002; 110: 355-361Crossref PubMed Scopus (211) Google Scholar). As more studies are published, it is becoming clear that multiple endocrine signaling pathways are targeted by these chemicals, as well as neural and immune signaling pathways (10Guillette Jr., L.J. Endocrine disrupting contaminants—beyond the dogma.Environ Health Perspect. 2006; 114: 9-12PubMed Google Scholar, 11Osteen K.G. Sierra-Rivera E. Does disruption of immune and endocrine systems by environmental toxins contribute to development of endometriosis?.Semin Reprod Endocrinol. 1997; 15: 301-308Crossref PubMed Scopus (40) Google Scholar, 12Fournier M. Brousseau P. Tryphonas H. Cyr D. Biomarkers of immunotoxicity: an evolutionary perspective.in: Guillette Jr., L.J. Crain D.A. Endocrine disrupting contaminants: an evolutionary perspective. Francis and Taylor Inc., Philadelphia, PA2000: 182-215Google Scholar). Moreover, the mechanisms of action of these various signal-disrupting compounds are not limited to receptor interactions; studies have shown these compounds can alter hepatic biotransformation and clearance of hormones as well as alterations in hormone synthesis and storage on plasma proteins (5Guillette Jr., L.J. Gunderson M.P. Alterations in the development of the reproductive and endocrine systems of wildlife exposed to endocrine disrupting contaminants.Reproduction. 2001; 122: 857-864Crossref PubMed Scopus (165) Google Scholar). Furthermore, disrupted regulation of gene expression (e.g., DNA methylation, RNA stability, protein degradation) has been observed in studies of chemically exposed humans and animals [reviewed by (13Edwards T.M. Myers J.P. Environmental exposures and gene regulation in disease etiology.Environ Health Perspect. 2007; 115: 1264-1270Crossref PubMed Scopus (203) Google Scholar)]. In some cases, altered DNA methylation patterns have been shown to be heritable (14Anway M.D. Cupp A.S. Uzumcu M. Skinner M.K. Epigenetic transgenerational actions of endocrine disruptors and mate fertility.Science. 2005; 308: 1466-1469Crossref PubMed Scopus (1887) Google Scholar, 15Crews D. Gore A.C. Hsu T.S. Dangleben N.L. Spinetta M. Schallert T. et al.Transgenerational epigenetic imprints on mate preference.Proc Nat Acad Sci USA. 2007; 104: 5942-5946Crossref PubMed Scopus (331) Google Scholar). Determination and differentiation of the gonad in vertebrates, other than mammals, can involve either specific genes (genetic sex determination—GSD) or environmental factors such as temperature (environmental sex determination—ESD) (16Edwards T.M. Moore B.C. Guillette Jr., L.J. Reproductive dysgenesis in wildlife—a comparative view.Int J Androl. 2005; 29: 109-121Crossref Scopus (105) Google Scholar). Those with ESD have been hypothesized to be more sensitive to disruption by environmental factors, because in many of the species studied, estrogens play a central role in the differentiation of the ovary. Exposure of reptilian embryos to endogenous (estradiol-17β), pharmaceutical (ethynylestradiol, diethylstilbestrol), or contaminant (DDT, DDE, bisphenol-A, trans-nonachlor) estrogens, during a critical window of development, induces sex reversal at male incubation temperatures leading to highly skewed female sex ratios (17Willingham E. Crews D. Sex reversal effects of environmentally relevant xenobiotic concentrations on the red-eared slider turtle, a species with temperature-dependent sex determination.Gen Comp Endocrinol. 1999; 113: 429-435Crossref PubMed Scopus (113) Google Scholar, 18Milnes M.R. Bryan T.A. Medina J.G. Gunderson M.P. Guillette Jr., L.J. Developmental alterations as a result of in ovo exposure to the pesticide metabolite p,p'-DDE in Alligator mississippiensis.Gen Comp Endocrinol. 2005; 144: 257-263Crossref PubMed Scopus (45) Google Scholar, 19Matter J.M. Crain D.A. Sills-McMurry C. Pickford D.B. Rainwater T.R. Reynolds K.D. et al.Effects of endocrine-disrupting contaminants in reptiles: alligators.in: Kendall R. Dickerson R. Giesy J. Suk W. Principles and processes for evaluating endocrine disruption in wildlife. SETAC Pr, Pensacola, FL1998: 267-289Google Scholar, 20Stoker C. Rey F. Rodriguez H. Ramos J.G. Sirosky P. Larriera A. et al.Sex reversal effects on Caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol A.Gen Comp Endocrinol. 2003; 133: 287-296Crossref PubMed Scopus (102) Google Scholar). Interestingly, embryonic exposure to concentrations of contaminants too low to cause sex reversal, alter steroidogenesis of the ovary or testis in neonates and juveniles (21Willingham E. Rhen T. Sakata J.T. Crew D. Embryonic treatment with xenobiotics disrupts steroid hormone profiles in hatchling red-eared slider turtles (Trachemys scripta elegans).Environ Health Perspect. 2000; 108: 329-332Crossref PubMed Scopus (43) Google Scholar). Altered steroidogenesis, and consequently altered gametogenesis, is a common feature observed after embryonic or neonatal exposure to contaminants [e.g., see (22Guillette Jr., L.J. Arnold S.F. McLachlan J.A. Ecoestrogens and embryos—is there a scientific basis for concern?.Anim Reprod Sci. 1996; 42: 13-24Abstract Full Text PDF Scopus (52) Google Scholar, 23Mylchreest E. Sar M. Wallace D.G. Foster P.M.D. Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate.Reprod Toxicol. 2002; 16: 19-28Crossref PubMed Scopus (260) Google Scholar, 24Sharpe R.M. Irvine D.S. How strong is the evidence of a link between environmental chemicals and adverse effects on human reproductive health.Br Med J. 2004; 328: 447-451Crossref PubMed Scopus (335) Google Scholar)]. In fish and amphibians with GSD, sex reversal does not occur following exposure to endocrine active compounds; rather, one observes aberrant gonadal morphology. For example, in male rats and fish, the presence of oocytes in the testis or altered Leydig or Sertoli cell morphology or abundance has been reported following contaminant exposure early in life (23Mylchreest E. Sar M. Wallace D.G. Foster P.M.D. Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate.Reprod Toxicol. 2002; 16: 19-28Crossref PubMed Scopus (260) Google Scholar, 25Jobling S. Beresford N. Nolan M. Rodgers-Gray T.P. Brighty G. Sumpter J. et al.Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents.Biol Reprod. 2002; 66: 272-281Crossref PubMed Scopus (347) Google Scholar). A number of studies have examined fertility and fecundity in wildlife, and some of the best examples come from fish and alligators. Below we briefly review these studies relative to implications for human health. Fertility in fish is best measured by egg fertilization rates and offspring survival. The most widely studied chemical from a fish fertility perspective is ethynylestradiol (EE2). EE2 is the synthetic estrogen found in birth control pills, and is, consequently, an increasingly important contaminant in treated sewage effluent. In a well-done series of studies, Jobling and colleagues (25Jobling S. Beresford N. Nolan M. Rodgers-Gray T.P. Brighty G. Sumpter J. et al.Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents.Biol Reprod. 2002; 66: 272-281Crossref PubMed Scopus (347) Google Scholar, 26Jobling S. Beresford N. Nolan M. Rodgers-Gray T.P. Brighty G. Sumpter J. et al.Wild intersex roach (Rutilus rutilus) have reduced fertility.Biol Reprod. 2002; 67: 515-524Crossref PubMed Scopus (338) Google Scholar) have described the high incidence of intersex (feminized male phenotype) among wild roach (Rutilus rutilus) from rivers that receive treated sewage effluent. Compared with normal males, intersex fish exhibit malformations of the reproductive ducts, including duct occlusion, concomitant with lower released milt volumes and decreased sperm density and motility. Furthermore, intersex fish are less likely to successfully fertilize eggs and produce viable offspring. Fertilization success was 75% lower among severely feminized fish compared with unaffected males (26Jobling S. Beresford N. Nolan M. Rodgers-Gray T.P. Brighty G. Sumpter J. et al.Wild intersex roach (Rutilus rutilus) have reduced fertility.Biol Reprod. 2002; 67: 515-524Crossref PubMed Scopus (338) Google Scholar). Intersex has also been observed in pearl dace (Margariscus margarita) taken from a lake that was experimentally treated with EE2 over a 3-year period (27Palace V.P. Wautier K.G. Evans R.E. Blanchfield P.J. Mills K.H. Chalanchuk S.M. Biochemical and histopathological effects in pearl dace (Margariscus margarita) chronically exposed to a synthetic estrogen in a whole lake experiment.Environ Toxicol Chem. 2006; 25: 1114-1125Crossref PubMed Scopus (55) Google Scholar). Mean concentrations of EE2 ranged from 4.5–8.1 ng/L during the 3 years. This range is considered environmentally relevant. In addition to intersex, Palace et al. (27Palace V.P. Wautier K.G. Evans R.E. Blanchfield P.J. Mills K.H. Chalanchuk S.M. Biochemical and histopathological effects in pearl dace (Margariscus margarita) chronically exposed to a synthetic estrogen in a whole lake experiment.Environ Toxicol Chem. 2006; 25: 1114-1125Crossref PubMed Scopus (55) Google Scholar) observed ovarian edema and poor testicular development among exposed fish, compared with fish captured from a reference lake. Young-of-the-year size classes were also less abundant in the EE2-treated lake, suggesting reduced reproductive success at the population level. Interestingly, lake trout (Salvelinus namaycush) taken from the EE2-treated lake did not exhibit significant reproductive abnormalities, indicating differential sensitivity to EE2 among species (28Werner J. Palace V.P. Wautier K.G. Mills K.H. Chalanchuk S.M. Kidd K.A. Reproductive fitness of lake trout (Salvelinus namaycush) exposed to environmentally relevant concentrations of the potent estrogen ethynylestradiol (EE2) in a whole lake exposure experiment.Sci. Mar 2006; 70: 59-66Google Scholar). Experimental studies with low, environmentally relevant concentrations of EE2 have yielded results similar to the field studies described above. Male fathead minnows (Pimephales promelas) with lifetime exposure to 0.32 and 0.96 ng/L EE2 exhibited reduced ability to fertilize eggs (29Parrott J.L. Blunt B.R. Life-cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and demasculinizes males.Environ Toxicol. 2005; 20: 131-141Crossref PubMed Scopus (247) Google Scholar). Similarly, sand gobies (Pomatoschistus minutus), exposed for 7 months to 6 ng/L EE2 or 0.3% or 0.03% v/v sewage effluent, exhibited impaired male maturation, decreased female fecundity, and low egg fertility. As a result, fertile egg production by the EE2-exposed population was reduced by 90% compared with controls (30Robinson C.D. Brown E. Craft J.A. Davies I.M. Moffat C.F. Pirie D. Effects of sewage effluent and ethynyl oestradiol upon molecular markers of oestrogenic exposure, maturation and reproductive success in the sand goby (Pomatoschistus minutus, Pallas).Aquat Toxicol. 2003; 62: 119-134Crossref PubMed Scopus (92) Google Scholar). In a third study, exposure of breeding zebrafish and their offspring to 5 ng/L EE2 resulted in a 56% decline in fecundity and no fertilization success among the F1 generation. The male infertility resulted from developmental abnormalities in the gonads (31Nash J.P. Kime D.E. Van der Ven L.T.M. Wester P.W. Brion F. Maack G. et al.Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish.Environ Health Perspect. 2004; 112: 1725-1733Crossref PubMed Scopus (496) Google Scholar). Other aquatic contaminants associated with reduced fertility in fish include tributyltin (antifouling agent used on boats), bisphenol A (plasticizer), tetrabromobisphenol A (TBBPA) (widely used flame retardant), and nitrate. Haubruge et al. (32Haubruge E. Petit F. Gage M.J.G. Reduced sperm counts in guppies (Poecilia reticulata) following exposure to low levels of tributyltin and bisphenol A.Proc R Soc Lond Ser B-Biol Sci. 2000; 267: 2333-2337Crossref PubMed Scopus (78) Google Scholar) observed that adult male guppies (Poecilia reticulata) exposed for 21 days to 11.2–22.3 ng/L tributyltin or 274–549 μg/L bisphenol A in experimental aquaria had significant declines (40%–75%) in total sperm counts compared with controls. Kuiper et al. (33Kuiper R.V. van den Brandhof E.J. Leonards P.E.G. van der Ven L.T.M. Wester P.W. Vos J.G. Toxicity of tetrabromobisphenol A (TBBPA) in zebrafish (Danio rerio) in a partial life-cycle test.Arch Toxicol. 2007; 81: 1-9Crossref PubMed Scopus (101) Google Scholar) observed significantly decreased egg production among female zebrafish (Danio rerio) exposed for 30 days to 0.047–1.5 μM TBBPA. This exposure level resulted in accumulated body concentrations of 3.9 μg/g lipid weight and up. This is higher than concentrations measured in wild fish (63–583 ng/g lipid weight). However, testing lower concentrations of TBBPA is limited by detection capabilities, leaving open the possibility that lower exposures can also reduce zebrafish fertility. Hatching of TBBPA-exposed larvae was decreased at doses as low as 0.023 μM, and posthatching mortality was 81% among juveniles exposed to 1.5 uM TBBPA. Females were more likely to survive than males. Finally, Edwards et al. (34Edwards T.M. Miller H.D. Guillette Jr., L.J. Water quality influences reproduction in female mosquitofish (Gambusia holbrooki) from eight Florida springs.Environ Health Perspect. 2006; 114: 69-75PubMed Google Scholar) observed that female mosquitofish (Gambusia holbrooki) caught from two springs in north-central Florida with nitrate concentrations of 4–5 mg/L NO3-N were significantly less likely to be pregnant during the reproductive season, and those that were pregnant had smaller offspring on average, compared with fish captured from six springs with lower nitrate concentrations (0.2–1.7 mg/L NO3-N). The US EPA drinking water standard for nitrate is 10 mg/L NO3-N. The American alligator is now common to many inland waters of the southeastern United States. Previous studies from our laboratory over the last decade and a half have shown that alligators living in central Florida (USA) lakes polluted with agricultural and storm water runoff exhibit a number of alterations of the reproductive and endocrine systems. Many of these modifications appear to be developmental defects that are detectable at hatching and persist throughout juvenile life stages. Lake Apopka is a large, eutrophic, and polluted lake northwest of Orlando, Florida. In 1980, it was the site of a spill of the pesticide dicofol, and has been the recipient of extensive agricultural pesticide and nutrient runoff during the last 40 years. In the 5 years following the pesticide spill, juvenile recruitment plummeted on Lake Apopka because of depressed clutch viability and juvenile mortality (35Woodward A.R. Jennings M.L. Percival H.F. Moore C.T. Low clutch viability of American alligators on Lake Apopka.Fl Sci. 1993; 56: 52-63Google Scholar). The juvenile population remained depressed until the early 1990s [see (35Woodward A.R. Jennings M.L. Percival H.F. Moore C.T. Low clutch viability of American alligators on Lake Apopka.Fl Sci. 1993; 56: 52-63Google Scholar, 36Rice K.G. Percival H.F. Woodward A.R. Abercrombie C.L. Wilkinson P.M. Clutch viability, population trends and nesting female demographics. In Effects of environmental contaminants on the demographics and reproduction of Lake Apopka's alligators and other taxa. Report No. #53. Florida Cooperative Fish and Wildlife Research Unit, Gainesville, FL1996Google Scholar)]. Recruitment increased the juvenile population during the 1990s, although pre-1980 population levels have not been observed. The rise in juvenile recruitment reflects the rise in clutch viability. Clutch viability, the number of eggs that hatch versus the number laid, remained near or below 20% from 1983–1991 at Lake Apopka (35Woodward A.R. Jennings M.L. Percival H.F. Moore C.T. Low clutch viability of American alligators on Lake Apopka.Fl Sci. 1993; 56: 52-63Google Scholar). Other lakes in Florida averaged approximately 50% (37Masson G.R. Environmental influences on reproductive potential, clutch viability and embryonic mortality of the American alligator in Florida.Ph.D. Dissertation. University of Florida, 1995Google Scholar). This is still significantly lower than the 85% clutch viability rate observed at Lake Woodruff National Wildlife Refuge (Guillette et al., unpublished data). The Lake Woodruff National Wildlife Refuge receives little to no agricultural runoff. Thus, alligators from Lake Apopka, as well as many other freshwater lake systems in Florida, exhibit reduced clutch viability when compared with reference sites in the same broad watershed. Although clutch viability rose to approximately 40% and juvenile recruitment increased during the 1990s on Lake Apopka, viability of clutches and neonates was still depressed compared with nonpolluted populations. In addition, a number of sublethal problems continued to be reported (38Guillette Jr., L.J. Woodward A.R. Crain D.A. Pickford D.B. Rooney A.A. Percival H.F. Plasma steroid concentrations and male phallus size in juvenile alligators from seven Florida (USA) lakes.Gen Comp Endocrinol. 1999; 116: 356-372Crossref PubMed Scopus (73) Google Scholar). Examinations of the reproductive and endocrine systems of hatchling and juvenile alligators from Lake Apopka have demonstrated alterations in plasma estrogen, androgen, and thyroid hormone concentrations as well as morphologic abnormalities of the testis and ovary (described below) (39Guillette Jr., L.J. Gross T.S. Masson G.R. Matter J.M. Percival H.F. Woodward A.R. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida.Environ Health Perspect. 1994; 102: 680-688Crossref PubMed Scopus (950) Google Scholar, 40Milnes M.R. Bermudez D.S. Bryan T.A. Gunderson M.P. Guillette Jr., L.J. Altered neonatal development and endocrine function in Alligator mississippienesis associated with a contaminated environment.Biol Reprod. 2005; 73: 1004-1010Crossref PubMed Scopus (37) Google Scholar, 41Rooney A.A. Crain D.A. Woodward A.R. Guillette Jr., L.J. Seasonal variation in plasma sex steroid concentrations in juvenile alligators.Gen Comp Endocrinol. 2004; 135: 25-34Crossref PubMed Scopus (25) Google Scholar). The alterations in plasma hormone concentrations have persisted and have been observed repeatedly [for review, see (7Milnes M.R. Bermudez D.S. Bryan T.A. Edwards T.M. Gunderson M.P. Larkin I.V. et al.Contaminant-induced feminization and demasculinization of nonmammalian vertebrate males in aquatic environments.Environ Res. 2006; 100: 3-17Crossref PubMed Scopus (88) Google Scholar)]. Alterations in plasma testosterone concentrations throughout early life would lead to altered anatomic structures dependent on this hormone, and we have reported that neonatal and juvenile male alligators had reduced phallus size (42Guillette Jr., L.J. Pickford D.B. Crain D.A. Rooney A.A. Percival H.F. Reduction in penis size and plasma testosterone concentrations in juvenile alligators living in a contaminated environment.Gen Comp Endocrinol. 1996; 101: 32-42Crossref PubMed Scopus (331) Google Scholar, 43Guillette Jr., L.J. Woodward A.R. Crain D.A. Pickford D.B. Rooney A.A. Percival H.F. Plasma steroid concentrations and male phallus size in juvenile alligators from seven Florida lakes.Gen Comp Endocrinol. 1999; 116: 356-372Crossref PubMed Scopus (104) Google Scholar). Recent work on a group of boys from mother's exhibiting normal pregnancies reported that a reduction (feminization) of anogenital distance in these boys was related to elevated maternal prenatal urinary concentrations of four phthalate metabolites (monoethyl phthalate, mono-n-butyl phthalate, monobenzyl phthalate, and monoisobutyl phthalate) (44Swan S.H. Main K.M. Liu F. Stewart S.L. Kruse R.L. Calafat A.M. et al.Decrease in anogenital distance among male infants with prenatal phthalate exposure.Environ Health Perspect. 2005; 113: 1056-1061Crossref PubMed Scopus (1259) Google Scholar). Swan et al. (42Guillette Jr., L.J. Pickford D.B. Crain D.A. Rooney A.A. Percival H.F. Reduction in penis size and plasma testosterone concentrations in juvenile alligators living in a contaminated environment.Gen Comp Endocrinol. 1996; 101: 32-42Crossref PubMed Scopus (331) Google Scholar) also reported that reduced anogenital distance was correlated with reduced penis volume and elevated incidence of cryptorchidism. Previous studies of phthalate- or pesticide-exposed rodents support the above observations of a syndrome of incomplete virilization in males. It is now apparent that numerous environmental contaminants have the potential to act as antiandrogenic compounds, altering androgen signaling during development in various vertebrate species, including humans (8Gray LE Jr, Wilson VS, Stoker T, Lambright C, Furr J, Noriega N, et al. Adverse effects of environmental antiandrogens and androgens on reproductive development in mammals. Int J Androl 29:96–104.Google Scholar). In addition to the genital abnormalities described above, we have also observed at least one major organizational defect of the ovary that could explain the continued reduction in clutch viability. Exposure of the developing ovary to endocrine active compounds, especially those with estrogenic activity, alters folliculogenesis leading to a pathologic condition termed the multioocytic follicle [see (45Guillette L.J.J. Moore B.C. Contaminants, fertility and multioocytic follicles: a lesson from wildlife?.Sem Reprod Med. 2006; 24: 134-141Crossref PubMed Scopus (56) Google Scholar)]. We observed such a condition in alligator neonates obtained from eggs collected from Lake Apopka (39Guillette Jr., L.J. Gross T.S. Masson G.R. Matter J.M. Percival H.F. Woodward A.R. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida.Environ Health Perspect. 1994; 102: 680-688Crossref PubMed Scopus (950) Google Scholar). The same ovarian pathology was first reported in neonatal mice exposed to diethylstilbestrol developmentally (46Iguchi T. Takasugi N. Polyovular follicles in the ovary of immature mice exposed prematally to diethylstilbestrol.Anat Embryol. 1986; 175: 53-55Crossref PubMed Scopus (61) Google Scholar, 47Iguchi T. Takasugi N. Bern H.A. Mills K.T. Frequent occurrence of polyovular follicles in ovaries of mice exposed neonatally to diethylstilbestrol.Teratology. 1986; 34: 29-35Crossref PubMed Scopus (85) Google Scholar, 48Iguchi T. Fukazawa Y. Uesugi Y. Takasugi N. Polyovular follicles in mouse ovaries exposed neonatally to diethylstilbestrol in vivo and in vitro.Biol Reprod. 1990; 43: 478-484Crossref PubMed Scopus (127) Google Scholar). Experimental studies exposing neonatal mice to phytoestrogen genistein, have described similarly altered folliculogenesis (49Jefferson W.N. Padilla-Banks E. Newbold R.R. Adverse effects on female development and reproduction in CD-1 mice following neonatal exposure to the phytoestrogen genistein at environmentally relevant doses.Biol Reprod. 2005; 73: 798-806Crossref PubMed Scopus (139) Google Scholar, 50Jefferson W. Newbold R. Padilla-Banks E. Pepling M. Neonatal genistein treatment alters ovarian differentiation in the mouse: inhibition of oocyte nest breakdown and increased oocyte survival.Biol Reprod. 2006; 74: 161-168Crossref PubMed Scopus (159) Google Scholar), as have studies in which the inhibin α subunit was overexpressed in the developing ovary (51McMullen M.L. Cho B.N. Yates C.J. Mayo K.E. Gonadal pathologies in transgenic mice expressing the rat inhibin alpha-subunit.Endocrinology. 2001; 142: 5005-5014Crossref PubMed Scopus (81) Google Scholar). Multioocytic follicles are associated with infertility and early embryonic loss in diethylstilbestrol-treated mice (52Iguchi T. Cellular effects of early exposure to sex hormones and antihormones.Int Rev Cytol. 1992; 139: 1-57Crossref PubMed Scopus (148) Google Scholar), and we have hypothesized that this condition could be the underlying basis for reduced clutch viability in contaminant-exposed alligator populations. Current research is testing this hypothesis. What induces the endocrine alterations and altered fertility described above? We have hypothesized that embryonic exposure to contaminants acting as endocrine disruptors, causes permanent organizational changes in the developing alligator, both male and female (53Guillette Jr., L.J. Crain D.A. Rooney A.A. Pickford D.B. Organization versus activation: the role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife.Environ Health Perspect. 1995; 103: 157-164Crossref PubMed Scopus (285) Google Scholar). If this hypothesis is to have a factual basis, embryos and subsequent neonatal and juvenile alligators must be exposed to contaminants that disrupt endocrine signaling at physiologically relevant concentrations. This prerequisite appears to be fulfilled, as we have reported in a number of studies that many of the contaminants found in the embryonic or juvenile environment exhibit competitive binding to receptors and/or disrupt hormone synthesis or hepatic clearance at concentrations found in the environment (54Guillette Jr., L.J. Brock J.W. Rooney A.A. Woodward A.R. Serum concentrations of various environmental contaminants and their relationship to sex steroid concentrations in juvenile American alligators.Arch Environ Contam Toxicol. 1999; 36: 447-455Crossref PubMed Scopus (159) Google Scholar, 55Vonier P.M. Crain D.A. McLachlan J.A. Guillette Jr., L.J. Arnold S.F. Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator.Environ Health Perspect. 1996; 104: 1318-1322Crossref PubMed Scopus (214) Google Scholar, 56Guillette Jr., L.J. Vonier P.M. McLachlan J.A. Affinity of the alligator estrogen receptor for serum pesticide contaminants.Toxicology. 2002; 181–182: 151-154Crossref PubMed Scopus (24) Google Scholar, 57Gunderson M.P. LeBlanc G.A. Guillette Jr., L.J. Alterations in sexually dimorphic biotransformation of testosterone in juvenile American alligators (Alligator mississippiensis) from contaminated lakes.Environ Health Perspect. 2001; 109: 1257-1264Crossref PubMed Scopus (41) Google Scholar). In conclusion, as studies of the underlying mechanisms of infertility expand over the next decade, it is essential that environmental factors be considered as major players in this phenomenon. A growing literature has established a basis for concern, as various ubiquitous contaminants have been shown to depress, but not necessarily preclude, fertility in a wide array of vertebrate species. The concentrations of chemicals needed to induce such effects are well within the exposure levels experienced by some human populations. Moreover, exposure during embryonic and neonatal periods appears to be critical, as our understanding of the development of the reproductive system clearly demonstrates that potential adult fertility is affected by contaminant exposure events occurring early in life.