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Poult Sci 2006. 85:1641-1647
© 2006 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Effects of In Ovo Exposure to Imazalil and Atrazine on Sexual Differentiation in Chick Gonads

S. Matsushita*, J. Yamashita{dagger}, T. Iwasawa*, T. Tomita{dagger} and M. Ikeda{dagger},{ddagger},1

* Shizuoka Swine and Poultry Experiment Station, Kikugawa, Shizuoka, 439-0037 Japan; {dagger} Institute for Environmental Sciences, University of Shizuoka, Shizuoka, 422-8526 Japan; and {ddagger} Department of Environmental and Disaster Research, Fuji Tokoha University, Shizuoka 417-0801 Japan

1 Corresponding author: ikedam{at}u-shizuoka-ken.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the effects of atrazine and imazalil, 2 commonly used pesticides, on sexual differentiation in chickens. Atrazine and imazalil were injected into fertile eggs on d 0. At hatching, sex genotype and phenotype were determined. Gonads were stereomicroscopically and histologically observed. In ovo exposure of atrazine (0.01 to 3 mg/egg) did not influence hatchability, whereas imazalil exposure (2 mg/egg) inhibited hatch-ability. The sex genotype matched the sex phenotype in controls, atrazine, and imazalil-exposed groups. In control females, the right gonad was regressed at hatching. Regression of the right gonad, however, was inhibited following atrazine and imazalil exposure. In atrazine-exposed female chicks, the left gonads had normal ovary structures, and the remaining right gonads had ovary medulla-like structures. In imazalil-exposed females, some left gonads had an ovary medulla-like structure without the cortex as well as tubules, and the right gonad had testis-like structures. There was no change in male gonads at hatching following atrazine and imazalil exposure. Aromatase activity of the left gonad from female chicks was not changed by any concentration of atrazine exposure. These results suggest that atrazine and imazalil inhibit regression of the right gonad in female chicks, although it is not clear whether the remaining right gonad has aromatase activity. In ovo exposure to atrazine influences sexual differentiation of the ovary by different mechanisms from imazalil, possibly by the induction of aromatase in the right gonad, whereas it is confirmed that imazalil inhibits in vitro aromatase activity in the chick ovary. The results indicated that in ovo exposure to imazalil inhibits sexual differentiation of the ovary by inhibiting aromatase activity.

Key Words: atrazine • imazalil • aromatase • chick • sexual differentiation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In contrast to mammals, the heterogametic sex (sex chromosome ZW) in avian species is a genetic female, whereas the homogametic sex (sex chromosome ZZ) is a genetic male. The W chromosome positively controls early aromatase synthesis and, consequently, estrogen production (Shimada, 1998, 2002). Estrogens and their receptors are crucial for female sexual differentiation. Chicken embryonic gonads are bipotential at an early stage. During development of the female, the left gonad differentiates to a single ovary or oviduct, and the right gonad regresses, developing a permanent female phenotype. This sexual differentiation occurs as a result of aromatase expression in the left gonad at d 6.5 and the production of estrogen from testosterone (Yoshida et al., 1996; Shimada, 1998). In the male genotype, both gonads develop into 2 testes (Shimada, 1998). The time- and sex-dependent expression of enzymes involved in steroid production, which determines the ratio of androgens:estrogens produced by the gonads, has been extensively investigated during the last 5 to 6 yr (Clinton, 1998; Nishikimi et al., 2000). The findings indicate that the lack of estrogen synthesis in the male is due to the extremely low levels of P450 aromatase expression (Nakabayashi et al., 1998; Yoshida et al., 1996; Shimada, 1998). In females, extensive expression of the aromatase gene (around d 5 to 6 of incubation), leading to estrogen synthesis (Abinawanto et al., 1998) and specific expression of the estrogen receptor mRNA in the left gonad (Nakabayashi et al., 1998), results in the development of a functional left ovary. Experimental sex reversal has been performed using antiestrogens, androgens, aromatase inhibitors, and synthetic steroids (Elbrecht and Smith, 1992; Abinawanto et al., 1998; Shimada, 1998). Differences between male and female gonadal differentiation and development depend on the absence of aromatase and estrogen, whereas an estrogen receptor is present in the gonads of males before sexual differentiation (Smith et al., 1997).

Differences between left and right ovarian development, however, depend on the specific expression of the estrogen receptor in the left gonad (Andrew et al., 1997; Nakabayashi et al., 1998; Bruggeman et al., 2002).

Atrazine is the most commonly used herbicide in the world. There are several reports on the adverse effects of atrazine exposure, such as atrazine-induced hermaphroditism in African clawed frogs and demasculinization of the larynx in male frogs (Hayes et al., 2002). Atrazine exposure decreases plasma testosterone concentrations in male frogs, and increases plasma estradiol concentrations in rats (Storker et al., 2000). Atrazine also increases aromatase activity in the human adrenocortical carcinoma cell line H295R by inducing aromatase mRNA (Sanderson et al., 2000, 2002).

A chlorine-containing pesticide, imazalil, is structurally similar to various imidazole-containing chemicals used clinically, such as the potent aromatase inhibitor fadrozole and numerous antifungal chemicals. These chemicals reversibly (although not necessarily competitively) inhibit aromatase activity in human placental microsomes (Mason et al., 1987; Ayub and Levell, 1988). Imazalil and difenoconazole inhibit aromatase activity in human adrenocortical carcinoma cell line H295R (Sanderson et al., 2002).

In this study, we studied gonad abnormality in chicks at hatching to clarify the toxic effects of in ovo exposure to an aromatase-activating chemical, atrazine, and an aromatase-inhibiting chemical, imazalil, on the sexual differentiation of the chick gonad.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
We purchased [1ß-3H]-Androsto-4-ene-3,17-dione ([3H]-androstendione) from New England Nuclear (Boston, MA). Additionally, 2-Chloro-4-ethylamino-6-isopropylamine-1,3,5-triazine (atrazine) and 1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl]-1-imidazole (imazalil) were obtained from Kanto Kagaku Chemical Co. Inc. (Tokyo, Japan) and Dr. Ehrenstorfer GmbH (Augsburg, Germany), respectively.

In Ovo Exposure and Bird Treatments
Rhode Island Red and Plymouth Rock chickens were obtained from the Hoshino Poultry Breeding Farm (Shizuoka, Japan) and maintained at the Shizuoka Swine and Poultry Experimental Station under natural sunlight (approximately 11L:13D). Rhode Island Red males were crossed with Plymouth Rock females to obtain fertile eggs for autosexing chicks. Atrazine (0.01, 0.05, 0.1, 1, and 3 mg) and imazalil (0.01, 0.1, 0.3, 1, 2, and 3 mg) were dissolved in 50 µL of propylene glycol and injected into the egg white on d 0 using a 1-mL syringe with a 23-gauge needle. In the control, 50 µL of propylene glycol was injected into the egg. The eggs were incubated at 37.6°C in a RH of 53% in a Showa Furanki incubator, model AH3 (Showa Furanki, Saitama, Japan). The eggs were automatically turned once per hour. After 10 d, the eggs were checked by candling, and the eggs without development were discarded. At hatching, sex genotype was determined by the color of the head feathers, legs, and mandible (Elbrecht and Smith, 1992). These genotypes were further confirmed by detection of a W chromosome-linked (female-specific) Xho1 repeat sequence using PCR amplification from liver genomic DNA (Kodama et al., 1987). The chicks were anesthetized with diethyl ether, and blood was collected from the heart. The gonads were stereomicroscopically observed to determine the sex phenotype, and photographs were taken to measure gonad surface area. The gonads were dissected under a stereomicroscope. The surface area of the gonad was traced using Photoshop (Adobe Systems Inc., San Jose, CA) and calculated using the public domain NIH image program (developed at the US National Institutes of Health and available on online at http://rsb.info.nih.gov/nih-image/).

Histologic Analysis
Some gonads observed abnormal with a stereomicroscope were fixed in a 10% neutral formalin buffer solution for 2 wk. The gonads were embedded in paraffin, and 5-µm thick paraffin sections were prepared. Each section was stained with hematoxylin and eosin for histologic analysis.

Assay for Aromatase Activity
Other chick ovaries (3 to 5 each) from control and atrazine-exposed females at hatching were homogenized and provided in a 10 mM potassium phosphate buffer (pH 7.4) containing 100 mM KCl, 1 mM EDTA, 10 mM dithiothreitol, and a protease inhibitor cocktail and centrifuged for 10 min at 1,900 x g. Aromatase activity in the supernatant was measured in terms of 3H2O release from [3H]-androstendione, as reported previously (Ikeda et al., 2002). Additionally, the supernatant in the control was incubated with imazalil or saline for 10 min at 37° C, and then aromatase activity was measured, as described above.

Assay for 17ß-estradiol Concentration
Serum (200 µL) was provided to 10 times the volume of diethyl ether and was centrifuged for 10 min at 1,900 x g. The organic layer was collected and evaporated under N2. The extract was dissolved in EIA buffer (Cayman Chemical Co., Ann Arbor, MI). Estradiol concentration was measured using an estradiol enzyme immunoassay kit (Cayman Chemical Co.) according to the manufacturers instruction.

Statistical Analysis
Data were analyzed using Student’s t-test, 1-way ANOVA, or {chi}2 test with StatView J5.0 (SAS Institute Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of In Ovo Exposure to Atrazine on Hatchability, Sex Ratio, and Gonads
In ovo exposure to atrazine (0.01 to 3 mg/egg) did not influence hatchability (Table 1Go). The genotype sex matched the phenotype sex in both control and atrazine-exposed groups. In control females, the left gonad had differentiated to an ovary, and the right gonad was involuted at hatching. Approximately 20% of atrazine-exposed females had a right gonad at hatching (Table 1Go). All concentrations of atrazine resulted in some instances of right gonad retention. The remaining right gonad was approximately 15% of the size of the left gonad; there was no atrazine dose-dependent effect on gonad size (Figure 1Go). The size of the left gonad in females tended to increase in an atrazine dose-dependent manner, but the effect was not statistically significant. There was no significant change in the size of the testes and also no atrazine exposure-induced change (Figure 1Go).


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  		Table 1. Effects of in ovo exposure to atrazine on hatchability, sex ratio, and gonads1
 

Figure 1
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  		Figure 1. Effect of in ovo exposure to atrazine on gonad size. Atrazine was injected into fertile eggs on d 0. At hatching, photographs of gonads were taken using a stereomicroscope, and the surface area of the gonad was measured. The columns and vertical bars indicate mean ± SD for 3 chicks. There was not significant difference between the control and exposure to atrazine in the left or right gonad size.

 
The left gonad from control females at hatching had a medulla containing some lacunae, and the cortex was filled with many primordial germ cells (Figure 2Go, panel A). Testes from control males at hatching had seminiferous tubules (Figure 2Go, panel B).


Figure 2
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  		Figure 2. Photomicrographs of control and atrazine-exposed gonads at hatching. Gonads were embedded in paraffin and 5-µm sections were prepared. Each section was stained with hematoxylin and eosin. Control ovary (A), control testis (B), female left and right gonads (C and D; 0.1 mg/egg) of atrazine-exposed chick; female left and right gonads (E and F; 3 mg/egg) of atrazine-exposed chick. m = medulla; c = cortex; l = lacunae.

 
The left gonad in females exposed to both 0.1 mg of atrazine/egg (Figure 2Go, panel C) and 3 mg of atrazine/egg (Figure 2Go, panel E) had normal ovary structure with a medulla and cortex. The remaining right gonads in females exposed to 0.1 mg of atrazine/egg (Figure 2Go, panel D) and 3 mg of atrazine/egg (Figure 2Go, panel F) had ovary medulla-like structures without a cortex.

Female left gonads from vehicle- and atrazine-exposed chicks at hatching were homogenized, and the aromatase activity was measured. Aromatase activity was 2.54 ± 0.14, 2.99 ± 0.33, 2.60 ± 0.52, and 2.99 ± 0.33 (Mean ± SEM) for control, 0.1, 1, and 3 mg/egg, respectively, and not changed by in ovo atrazine exposure. In control chicks at hatching, serum estradiol concentrations were significantly higher in females than in males (Figure 3Go). Serum estradiol concentrations were not significantly changed by in ovo atrazine exposure (Figure 3Go).


Figure 3
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  		Figure 3. Effect of in ovo exposure to atrazine on the serum estradiol concentration at hatching. Blood was obtained from vehicle- and atrazine-exposed chicks at hatching. Serum estradiol concentrations were analyzed using an enzyme immunoassay kit. The columns and vertical bars indicate mean ± SD for the number of chicks in parentheses. *P < 0.05 vs. control male by Student’s t-test.

 
Effects of In Ovo Imazalil Exposure on Hatchability, Sex Ratio, and Gonads
Aromatase activity in chick ovary homogenate was significantly inhibited by incubation with 10 µM of imazalil for 10 min (Figure 4Go). The inhibitory effect of imazalil on in vitro aromatase activity was confirmed.


Figure 4
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  		Figure 4. Inhibitory effect of imazalil on chick ovary aromatase activity in vitro. Chick ovaries at hatching were homogenized with phosphate buffer. Homogenate was centrifuged, and the supernatant was used for enzyme solution. Enzyme solution was incubated with or without 10 µM of imazalil for 10 min at 37° C, and then aromatase activity was measured. The columns and vertical bars indicate mean ± SD for 3 determinations. ***P < 0.001 vs. control by Student’s t-test.

 
Hatchability was not altered by low dose ( < 1 mg/egg) of in ovo imazalil exposure but was partially inhibited by 2 mg of imazalil/egg and inhibited by 3 mg of imazalil/egg (Table 2Go). The genotype sex matched the phenotype sex in both control and imazalil-exposed groups. The right gonad remained, following imazalil exposure at 0.01 to 1 mg/egg (Table 2Go). Imazalil exposure did not affect male gonads at hatching. In females, imazalil increased the size of the right gonad in a dose-dependent manner; however there was not a significant difference (Figure 5Go). Imazalil exposure did not affect the size of the left gonad in female or testes in males.


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  		Table 2. Effects of in ovo exposure to imazalil on hatchability, sex ratio, and gonads1
 

Figure 5
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  		Figure 5. Effect of in ovo exposure to imazalil on gonad size. Imazalil was injected into fertile eggs on d 0. At hatching, photographs of gonads were taken using a stereomicroscope, and the surface area of a gonad was measured. The columns and vertical bars indicate mean ± SD for 5 (0.01 and 0.1 mg of imazalil/egg) or 6 (0 and 1 mg of imazalil/egg) chicks. There was not significant difference between the control and exposure to imazalil in left or right gonad size.

 
In some imazalil- (1 mg/egg) exposed females, the left gonad had 2 portions, an ovary medulla-like structure and a testis-like structure (Figure 6Go, panels A and B). The cortex was not observed in the left gonad. The remaining right gonad had a testis-like structure and tubules similar to seminiferous tubules (Figure 6Go, panels C and D). In other imazalil- (1 mg/egg) exposed females, the left gonad had ovary structures comprised of cortex and medulla. The cortex contained degraded cells, however, and there were very few germ cells. Also, many cells in the medulla were degraded (Figure 6Go, panels E and F). The right gonad had ovary medulla-like structures without a cortex (Figure 6Go, panels G and H).


Figure 6
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  		Figure 6. Photomicrographs of female gonads from imazalil-exposed chicks. Gonads were embedded in paraffin, and 5-µm sections were prepared. Each section was stained with hematoxylin and eosin. Left (A and B) and right (C and D) gonads from 1 mg of imazalil/egg of imazalil-exposed chicks; Left (E and F) and right (G and H) gonads from 1 mg of imazalil/egg of imazalil-exposed chicks. m = medulla, c = cortex.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In female chicken embryonic gonads, the left gonad differentiates to a single ovary or oviduct and the right gonad regresses. This sexual ovarian or oviduct differentiation occurs as a result of aromatase expression in the left gonad at d 6.5, the production of estrogen from testosterone (Yoshida et al., 1996; Shimada, 1998), and the lack of estrogen receptors in the right gonad. In both female and male right gonads, however, the cortex is not noticeable by d 6 of incubation. The cortex in the right gonad is not developed because of the intrinsic absence of estrogen receptor gene expression. In contrast, the gene transcript for aromatase is present in the regressing right gonad (Nakabayashi et al., 1998). These findings indicate that aromatase is an important factor for regression of the medulla of the right gonad.

In atrazine-exposed female chicks, the left gonads had normal ovary structures; however, the right gonads remained and had ovary medulla-like structures. The cortex of the right gonad regressed. These results indicate that in ovo atrazine exposure influenced gonadal differentiation in females. Although the induction of aromatase activity by atrazine has been reported in H295R cells (Sanderson et al., 2000), and plasma estradiol concentrations in rats are increased by atrazine exposure (Storker et al., 2000), aromatase activity of the left gonad from female chicks was not changed by atrazine exposure in the present study. These results indicate that atrazine injected into the egg before incubation did not alter aromatase activity in the chicks hatched from these eggs. These results suggest that atrazine inhibited only the regression of the right gonad in female chicks, and the remaining right gonad might be due to the change in aromatase activity in the regressing right gonad. It is not clear whether an increase or a decrease in aromatase activity induces in right gonad regression. These effects appeared in even low-dose exposures that were reported in amphibians (Hayes et al., 2002) and a low-level atrazine mixture in rats (Boyd et al., 1990). Atrazine induces intratesticular testosterone in frogs and hermaphroditism in male frogs (Hayes et al., 2002). In the present study, stereomicroscopic observations indicated that atrazine exposure did not alter male gonad localization (left or right testis) at hatching. It has been reported that there is widespread contamination of atrazine in some agricultural areas such as groundwater, rainfall, and rivers. Low-dose endocrine-disrupting effects of atrazine might be a factor in the decline in the population of amphibians (Hayes et al., 2002) and may be associated with intersexual amphibians (Reeder et al., 1998). The results in the present study also indicate that endocrine-disrupting effects in wild bird in wild concentrations of atrazine need to be addressed.

Imazalil inhibits aromatase activity in a human placental cell line (Mason et al., 1987; Ayub and Levell, 1988) and a human adrenocortical carcinoma cell line H295R (Sanderson et al., 2000). Imazalil also inhibits aromatase activity in human placental microsomes at IC50=0.04 µM (Vinggaard et al., 2000). In the present study, aromatase activity in chick ovary homogenate was significantly inhibited to 30% of control by incubation with 10 µM imazalil for 10 min. Together, these findings suggest that in ovo exposure to imazalil might inhibit aromatase activity during development. Aromatase activity was not inhibited by imazalil, however, because there was no sex reversal (difference between phenotypic sex and genotypic sex) at hatching.

In imazalil-exposed female chicks, the left gonad had a portion containing tubules (like seminiferous tubules). The remaining right gonad had testis-like structures. These hermaphroditic histologic structures are the same as that reported in female embryonic gonads grafted with testis (Stoll et al., 1980). Another left gonad had a cortex that contained degraded cells and ovary-like medulla structures, and the remaining right gonad had a medulla structure without the cortex. These results suggest that imazalil partially inhibits female gonadal aromatase activity and partially inhibits the differentiation of female gonads. These adverse effects of imazalil were observed in a dose-dependent manner.

In conclusion, in ovo exposure to imazalil inhibits sexual differentiation of the ovary by inhibiting aromatase activity. In contrast, in ovo exposure to atrazine influences the sexual differentiation of the ovary by different mechanisms, possibly induction of aromatase in the right gonad.

Received for publication February 22, 2006. Accepted for publication May 4, 2006.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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