Hayes TB , Khoury V , Narayan A , Nazir M , Park A , Brown T , Adame L , Chan E , Buchholz D , Stueve T , Gallipeau S .

Howard Hughes Medical Institute

	Fig. 1. Atrazine feminized exposed males. Cloaca (A–C) and gonads (D–F) for control male (A and D), atrazine-exposed male (B and E), and atrazine-exposed female (C and F) ZZ animals (genetic males). (G) Atrazine-induced female (genetic male, ZZ) copulating with an unexposed male sibling. (H) Same pair as in G, producing eggs. Eggs (H) were viable and produced larvae that survived to metamorphosis and adulthood. Yellow coloration (F) is the result of fixation in Bouin’s solution. Brackets (B and C) indicate protruding cloacal labia. (Scale bar in A applies to A–C; in D applies to D and E.)
	Fig. 2. Atrazine-induced females expressed aromatase in their gonads. (Top) DMRT-1 and DM-W genes from a representative control and an atrazine-exposed adult male (M) and female (F). Morphologic sex was assigned on the basis of the presence of testes (males) or ovaries (females). (Middle and Bottom) Cyp-19 aromatase expression from gonads of the same animals genotyped at Top, along with the control gene, rpL8.
	Fig. 3. Atrazine-demasculinized male morphology as shown in the nuptial glands and the larynx. (A and B) Forearms, showing nuptial pads from control (A) and atrazine-exposed males (B). Note the reduced nuptial pads in the atrazine-exposed male (B). Black arrowheads in A and B show boundaries of nuptial pads. (C and D) Representative largest breeding gland (selected from the midpoint of the nuptial pad) from control (C) and atrazine-exposed (D) males. The area of the largest section of the largest gland was determined for each sample. Control males had significantly larger glands (E). (F–H) Transverse cross-sections through the dissected larynges of a representative sexually mature control male (F), atrazine-exposed male (G), and control female (H) X. laevis. Atrazine-exposed males had a laryngeal morphology intermediate between unexposed males and females. The dilater larynges (DL) extended well beyond the thiohyral (TH) in control males, but very little (or not at all, as in the example shown) in atrazine-exposed males. This measure was quantifiable and significantly different between controls and atrazine-exposed animals, regardless of whether the absolute length of the muscle was measured (I) or the straight-line distance (J). Black arrowhead in F indicates the slip of the dilator larynges. Horizontal dashed lines in F and G indicate the midpoint of the thiohyral. ATR, atrazine-exposed; BG, breeding gland; CC, cricoid cartilage; CON, control; E, epidermis; EC, epithelial cells. *P < 0.05; n = 14 for breeding glands, n = 11 for larynges. (Scale bar in B applies to A and B; in D applies to C and D; in H applies to F–H.)
	Fig. 4. Control males out-competed atrazine-exposed males to copulate with females. Amplexus data from four mate choice trials for control (Con) and atrazine-treated (Atr) males (A). Eleven of 16 control males out-competed atrazine-exposed males for amplexus with females. Only two atrazine-exposed males in a single trial achieved amplexus. Male size did not affect breeding success (B). In all four trials, there was no difference (P > 0.05) in size between control (black symbols and bars) and atrazine-exposed males (red symbols and bars). Furthermore, in all trials smaller individuals from controls out-competed larger atrazine-exposed individuals. Filled circles show successful males, open circles show unsuccessful males, and horizontal bars show group means. (C) Testosterone levels for control and atrazine-treated males for all four trials. Filled symbols show successful (amplectant) males, and open symbols show unsuccessful males. Solid horizontal bars show mean testosterone levels for successful males, and open bars show the mean for unsuccessful males.
	Fig. 5. Atrazine decreased androgen-dependent sperm production, mating behavior, and fertility. (A and C) Largest testicular cross-sections for representative control (A) and atrazine-exposed males (C) from 2007. (B and D) Magnification of individual tubules for control (B) and atrazine-exposed (D) males. Arrowheads in B and D show outline of tubules. Control tubules are typically filled with mature spermatozoa bundles, whereas the majority of tubules in atrazine-exposed males lack mature sperm bundles and are nearly empty, with only secondary spermatocytes (SS) along the periphery of the tubule. (E) Fertility for control (Con) and atrazine-exposed (Atr) males. Pooled data from both 2007 and 2008 study are shown. *P < 0.005 (ANOVA). (F) Fertility plotted against sperm content (percentage of tubules with mature sperm bundles) for control males (black symbols) and atrazine-exposed males (red symbols) for the 2007 (circles) and the 2008 (squares) studies. Dashed lines indicate the lower limit for controls for fertility and sperm content. Sample size differs from the number of trials because no data are available from females that did not lay eggs. (Bar in A applies to A and C; in B applies to B and D.)
	Fig. 6. Other studies have shown that atrazine alters sex ratios. Data from Oka et al. (39) (A) and Suzawa and Ingraham (5) (B) showing a concentration-dependent decline in males due to atrazine exposure in African clawed frogs (A) and zebrafish (B). The dashed line shows the 50% mark in both cases.

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