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All of the control animals reared to sexual maturity (n = 40) were males, on the basis of external morphology, whereas only 90% of the atrazine-treated animals (36 of 40) appeared male at sexual maturity (on the basis of the presence of keratinized nuptial pads on the forearms and the absence of cloacal labia). The other 10% of atrazine-exposed animals (n = 4) lacked visible nuptial pads on the forearms and had protruding cloacal labia, typical of females (Fig. 1). Upon dissection of two of the apparent females and laparotomy in another two, we confirmed that animals with cloacal labia were indeed females from the present study, on the basis of the presence of ovaries (Fig. 1F). To date, two atrazine-induced females have been maintained, mated with control males (Fig. 1G), and produced viable eggs (Fig. 1H). The resulting larvae were all male when raised to metamorphosis and sampled (n = 100), confirming that atrazine-induced females were, in fact, chromosomal males. Furthermore, atrazine-induced females lacked the DM-W further confirming that these atrazine-induced females were indeed chromosomal males [F6]–These ZZ females expressed gonadal aromatase[F7], as did true ZW females (n = 4, from our stock colony), but ZZ males (n = 8, control or treated)— 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–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–Previous studies showed that atrazine demasculinizes (chemically castrates) and feminizes exposed amphibian larvae, resulting in hermaphrodites (8, 10) or males with testicular oocytes (7, 9) at metamorphosis. Since our initial publications (7, 9, 10), the effects of atrazine on amphibian development and the significance of these effects to amphibian declines have been a subject of debate (30, 35, 36). Although some investigators, including Carr et al. (6), reported statistically significant effects of atrazine on gonadal morphology in X. laevis (P < 0.0003 for multiple testes and P = 0.0042 for hermaphrodites), others, using different experimental conditions and different populations of the same species, suggested that atrazine had no effect (29).
Essential to this debate, however, is (i) the terminology used to describe gonadal abnormalities; (ii) the expertise and ability of other researchers to recognize abnormalities; (iii) the possibility of natural variation in sex differentiation processes between species and even between populations (or strains) within a species (37); and (iv) the long-term consequences and significance of the observed abnormalities to amphibian reproductive fitness. Here we describe complete and functional female development in genetic (ZZ) males exposed to atrazine, not the production of hermaphrodites or males with testicular oocytes. Thus, there is no confusion in the present study regarding proper terminology or proper identification. Furthermore, because we used an all genetic (ZZ) male colony and genotyped the atrazine-induced ZZ females, there is no question that atrazine completely sex-reversed genetic (ZZ) males, resulting in reproductively functional females. — The present study thoroughly examines the long-term effects of atrazine on reproductive function in amphibians.
Although a single published study attempted to examine long-term reproductive effects of atrazine in amphibians (38), the authors did not report examinations of morphology. Furthermore, their examination of fertility and breeding of atrazine-exposed males was conducted after animals were injected with reproductive hormones (human chorionic gonadotropin, hCG), effectively providing “hormone replacement therapy” and reversing the effects of atrazine. The present study represents a more thorough examination of the effects of atrazine on sex hormone production, testosterone-dependent development and morphology, male reproductive behavior, and fertility.
Transcription and subsequent translation of aromatase leads to estrogen production, which in turn directs differentiation of the ovary from the undifferentiated gonad. Just as exogenous estrogen results in the differentiation of ovaries in exposed genetic (ZZ) male X. laevis (45), induction of aromatase and subsequent estrogen production likely explain the complete feminization of genetic male– Furthermore, why only some males (10% in the present population) are completely feminized, whereas their siblings are merely demasculinized, remains to be explored–Furthermore, sex-reversed males (ZZ females) are only capable of producing genetic male (ZZ) offspring, so the sex ratio in exposed populations would be skewed both by the production of atrazine-induced ZZ females as well as by the fact that ZZ females can only produce ZZ (genetically male) offspring. In fact, mathematical models suggest that this very mechanism (the production of sex-reversed all male-producing animals) could drive populations to extinction.
Where is Atrazine used
Atrazine is used on crops such as sugarcane, corn, pineapples, sorghum, and macadamia nuts, and on evergreen tree farms and for evergreen forest regrowth. It has also been used to keep weeds from growing on both highway and railroad rights-of-way- Atrazine is used specifically to control broadleaf weeds (such as milkweed, thistle, or pigweed)