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Experimental Biology and Medicine 232:503-514 (2007)
© 2007 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Gene Expression Change in the Müllerian Duct of the Mouse Fetus Exposed to Diethylstilbestrol In Utero

Atsuko Suzuki*,{dagger},{ddagger},§, Hiroshi Urushitani{dagger},{ddagger}, Tomomi Sato||, Tomohiro Kobayashi, Hajime Watanabe{dagger},{ddagger},#, Yasuhiko Ohta{ddagger},§ and Taisen Iguchi{dagger},{ddagger},#,1

* United Graduate School of Veterinary Science, Yamaguchi University, Yoshida 753-8515, Japan; {dagger} Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama, Myodaiji, Okazaki 444-8787, Japan; {ddagger} Core Research for Evolutional Science and Technology, Japan Science and Technology, Kawaguchi 332-0012, Japan; § Laboratory of Experimental Animals, Department of Veterinary Medicine, Faculty of Agriculture, Tottori University, Koyama 680-8553, Japan; || Graduate School of Integrated Science, Yokohama City University, Seto, Kanazawa-ku, Yokohama 236-0027, Japan; Department of Pharmacology, GlaxoSmithKline K.K., Wadai 43, Tsukuba, Ibaraki, 300-4247, Japan; and # Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Higashiyama, Myodaiji, Okazaki 444-8787, Japan

1To whom requests for reprints should be addressed at Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5–1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. E-mail: taisen{at}nibb.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In utero exposure to diethylstilbestrol (DES) induces various abnormalities in the Müllerian duct of the mouse. In order to understand the underlying molecular mechanisms associated with DES-induced abnormalities of the Müllerian duct, gene expression was examined on Gestation Day (GD) 19 in mouse fetuses exposed to DES (67 µg/kg body weight) from GDs 10 to 18. Microarray analysis revealed that 387, 387, and 225 genes were upregulated and 177, 172, and 75 genes were downregulated by DES in the oviduct, uterus, and vagina, respectively. DES exposure in utero commonly upregulated 72 genes and downregulated 15 genes in these three organs. The present study demonstrated that organ-specific gene expression patterns in the mouse Müllerian duct were altered by in utero DES exposure. DES-induced changes in expression of genes such as Dkk2, Nkd2, and sFRP1 as well as changes in genes of the Hox, Wnt, and Eph families in the female mouse fetal reproductive tract could be the basis for various abnormalities in reproductive tracts following exposure to this estrogenic drug.

Key Words: microarray • gene expression • diethylstilbestrol • oviduct • uterus • vagina • mouse


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prenatal diethylstilbestrol (DES) exposure induces persistent malformations of male and female reproductive organs in mice. Female mice exposed perinatally to DES showed noncoiled oviducts, uterine metaplasia, disorganization of the uterine circular muscles, and ovary-independent vaginal epithelial stratification and cornification (1–6).

DNA microarray has been successfully used to analyze estrogen-responsive genes in the mouse uterus and vagina and to begin to identify those genes possibly related to persistent vaginal proliferation induced by neonatal DES exposure (7–12). Therefore, we studied global gene expression, including signal transduction and organogenesis genes in the Müllerian duct, after DES exposure in utero using microarray at Gestation Day (GD) 19, and we selected several genes for further study. We focused on the expression of ephrin, Eph family, Wnt, Wnt-antagonists, and Hoxa genes, since they showed altered expression by in utero DES exposure in the present study.

Hox genes, expressed in spinal cord, limb, and reproductive tracts, determine anterior to posterior body axis patterning. Hox genes are expressed in the Müllerian duct along its axis, with genes a-9, a-10, a-11, and a-13 exhibiting anterior to posterior expression pattern at GD 15.5 (13). With the positional expression of Hox genes along the anterior to posterior axis, the Müllerian duct differentiates into three distinct reproductive organs: oviduct, uterus, and upper vagina. Lack of positional Hox gene expression results in reproductive abnormalities due to the loss of organ specificity (14, 15). Lack of Hoxa-13 expression, for example, resulted in the loss of the caudal Müllerian duct (16). DES repressed the expression of Hoxa-10 and a-11 in the mouse uterus at GD 17, which is associated with reduced reproductive performance, including embryo implantation, in adult offspring (17, 18). The lack of Hox gene expression leads to disturbed patterning in the body axis, primarily in limbs, spinal cord, hindbrain, and reproductive tracts (14, 15, 19–22).

Hoxa-13 knockout mice showed the downregulation of the Eph receptor A7 and inhibition of mesenchymal cell adhesion and apoptosis in limbs (23), suggesting possible functions in reproductive tracts. Eph receptors and ephrin ligands regulate cell–cell communication, cellular movement, and mitogenic responses during development via the ERK/MAPK cell signaling pathway (24–26).

Epithelial–mesenchymal differentiation in the Müllerian duct is regulated by Wnt signaling correlated with expression of Hox genes. In female reproductive organs, Wnt-4, –5a, and –7a are expressed during development (27). Lack of Wnt-7a expression induced uterine metaplasia, an abnormality similar to that reported in mice exposed to DES in utero (28). Wnt-7a maintains the expressions of Hoxa-10 and a-11; thus, lack of Wnt-7a is considered to disrupt segmentation of the reproductive organs. Moreover, Wnt-4 is essential for the early development of female reproductive tracts (29).

Wnt signaling regulates vaginal growth and differentiation through epithelial–mesenchymal interactions. Perinatal DES exposure reduced expression of Wnt-7a, which resulted in uterine metaplasia (30), suggesting the presence of suppression factors for Wnt signaling. Secreted frizzled related protein (sFRP) was downregulated by 17ß-estradiol treatment in the adult mouse uterus and was shown to compete with Wnt and frizzled (Fz) receptors (31, 32). Dicknock (Dkk; Refs. 33–38) and Naked cuticle (Nkd; Ref. 39) have been reported as Wnt antagonists in various developing organs.

In order to understand the molecular mechanisms underlying the reproductive tract abnormalities reported in female mice exposed prenatally to DES, we analyzed expression changes in Eph family, Wnt, Wnt-antagonists, and Hoxa genes following exposure to this pharmaceutical estrogen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals.
Mice of the ICR/Jcl strain were kept under a 12:12-hr light:dark cycle at 23°C–25°C, were given a commercial diet (CE-2; CLEA, Tokyo, Japan) and tap water ad libitum. All experiments and animal husbandry protocols were approved by the animal care committee of the National Institutes of Natural Sciences. The day on which a vaginal plug was found was considered GD 0. DES (Sigma Chemical Co., St. Louis, MO) was dissolved in sesame oil. Pregnant mice were given daily subcutaneous injections of DES (67 µg/kg maternal body weight) or the oil vehicle alone from GDs 10 to 18 as described previously (40). These experiments were repeated three times.

DNA Microarray Analysis.
Total RNA was extracted from the oviduct, uterus, and vagina (7–12 pups/3 litters) at GD 19 using TRIzol (Invitrogen, Tokyo, Japan) and purified with the RNeasy mini kit (Qiagen, Tokyo, Japan). Total RNA quality was examined with a Bioanalyzer 2100 (Agilent Japan, Tokyo, Japan). Purified RNA was processed according to the manufacturer’s protocol to prepare the labeled cRNAs, which were hybridized to the mouse expression array 430A (Affymetrix Japan, Tokyo, Japan). Hybridization, washing, and scanning were performed according to the manufacturer’s protocol as described (7). Microarray analysis was performed in triplicate using three different samples.

Data Analysis.
Scanned data were analyzed with GeneChip Suit Analysis Software version 5.0 (Affymetrix Japan) to obtain the average intensity of each cell corresponding to each oligonucleotide probe. The averaged fluorescence intensity (2500) of each probe was further analyzed by dChip, a model-based expression analysis program (41), and expression levels were estimated. The PM-only model was used for the analysis, and the estimated values were transferred to the GeneSpring software program (Silicon Genetics, Redwood City, CA) and analyzed. To calculate changes in expression, genes for which average expression levels were more than 1000 fluorescence intensity units under at least one experimental condition were selected, and the average expression values of the treated samples was divided by those from control samples. For the clustering analysis, genes activated more than 2-fold by DES were selected, and similarities between experiments and expression levels were measured by standard correlation using the GeneSpring program as described (7–10). Gene expression changes were estimated by assigning the value of the control uterus as one. These selected genes were listed on http://www.nibb.ac.jp/bioenv1/suzuki/suzukidata004.html. These raw data were loaded into the National Center for Biotechnology Information’s Gene Expression Omnibus as the dataset GSE1886 (GEO: http://www.ncbi.nlm.nih.gov/geo/). Categories in DES-regulated genes were determined from the GEO database. Putative target genes were validated by quantitative real-time polymerase chain reaction (Q-PCR).

Quantitative Real-Time PCR.
Total RNA was purified as described above. Complementary DNA was synthesized from purified total RNA with Superscript II RT(-) (Invitrogen) and random primers at 42°C for 60 mins. PCR reactions were performed in the PE Prism 5700 Sequence Detection System (PE Biosystems, Tokyo, Japan) with SYBR-Green PCR core reagents (Applied Biosystems Japan, Tokyo, Japan) in the presence of the appropriate primers according to the manufacturer’s instructions. The primers were chosen to amplify short PCR products of less than 100 base pairs, and their sequences are listed in Table 1Go.


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  		Table 1. Sequences for Primers Using Quantitative RT-PCR
 
Each PCR amplification was performed in triplicate using the following conditions: 2 mins at 50°C and 10 mins at 95°C, followed by a total of 40 two-temperature cycles (15 secs at 95°C and 1 min at 60°C). Model 7000 software (Applied Biosystems, Foster City, CA) was used to construct amplification plots from extension-phase fluorescent emission data collected during PCR amplification. Threshold (C{tau}) values were calculated by determining the point at which fluorescence exceeds a threshold limit.

Gene expression levels were normalized to the expression levels of ribosomal protein L8 mRNA (U67771), and changes in concentration were calculated. Gel electrophoresis and melting curve analyses were performed to confirm correct amplicon size and the absence of nonspecific bands. Quantification of mRNAs was repeated three times with independent mice, and average levels of change were calculated. Statistical analyses of the Q-PCR data were conducted by ANOVA. Q-PCR data were expressed as the relative expression of each gene to that of the control uterus in order to compare differences in gene expression among the three organs derived from the Müllerian duct.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA Microarray Analysis.
We examined gene expression in the oviduct, uterus, and vagina at GD 19 in DES-exposed mice and oil controls. The correlation coefficients of microarray chips averaged 0.980 (minimum: 0.967; maximum: 0.996). As described above, genes showing at least a 2-fold expression change in DES-exposed mice have been listed at http://www.nibb.ac.jp/bioenv1/suzuki/suzukidata004.html.

To examine the gene expression changes by DES in the three organs of Müllerian duct origin, we analyzed the clustering pattern of DES-regulated genes. Clustering analysis in controls revealed that there was organ specificity in gene expression. Gene expression profiles in the uterus were more similar to those of the oviduct than the vagina. However, clustering analysis in DES-exposed mice revealed that DES-regulated genes in the three organs showed less organ specificity compared with controls (Fig. 1Go).


Figure 1
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  		Figure 1. Clustering analysis of DES-responsive genes in the mouse oviduct, uterus, and vagina at GD 19. Each colored bar indicates the expression level of one gene: red, induction; green, repression; yellow, average expression in six groups; gray, not detected. Only genes showing more than a 2-fold change in expression following in utero DES exposure are shown here.

 
DES exposure upregulated 387, 387, and 225 genes and downregulated 177, 172, and 75 genes in the oviduct, uterus, and vagina, respectively (Fig. 2Go). We observed 72 genes that were upregulated and 15 downregulated in common in the three organs. The number of genes showing altered expression by DES was largest in the oviduct and smallest in the vagina (Fig. 2Go).


Figure 2
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  		Figure 2. Venn diagrams of the number of upregulated and downregulated genes in the GD 19 oviduct, uterus, and vagina exposed to DES at GDs 10–18. (a) Number of upregulated genes by DES in the Müllerian duct. The number of DES-upregulated genes was the smallest in the vagina. (b) Number of downregulated genes by DES in the Müllerian duct.

 
We focused on genes related to signal transduction and organogenesis in DES-exposed Müllerian ducts (Table 2Go). Expressions of RAB 20 and E74-like factor 3 were upregulated in all DES-exposed organs. In contrast, expressions of prostaglandin E receptor 3, tumor necrosis factor receptor superfamily member 19, Eph receptor A7, and naked cuticle 2 (Nkd2) were downregulated in all DES-exposed organs (Table 2Go).


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  		Table 2. DES-Regulated Genes Related to Signal Transduction and Organogenesis in the Müllerian Duct at GD 19a
 
Several organ-specific genes were encountered in DES-exposed mice (Table 2Go). Forkhead box J1 (foxj1), expressed in ciliated cells in the oviduct (41), was one of the oviduct-specific genes upregulated in DES-exposed oviducts, whereas expression of homeobox, msh-like 1 (Msx1) and fibroblast growth factor 9 (Fgf9) was upregulated and that of insulin-like growth factor-I (IGF-I) was downregulated in the DES-exposed uterus only. In DES-exposed oviduct and uterus, Dickkopf (Dkk) homologs 2 and 3 were upregulated, whereas ephrin B2, growth differential factor 10, and secreted frizzled-related sequence protein 1 (sFRP1) were downregulated (Table 2Go).

Hoxa-11 and Hoxd-10 were repressed in DES-exposed oviducts. Moreover, expression of Hoxd-9 was down-regulated in DES-exposed oviduct and uterus (Table 2Go). Wnt-4 gene was induced only in DES-exposed vagina. Wnt-6, Wnt-7a, and Wnt-11 genes were commonly down-regulated in DES-exposed uterus.

Four genes of the Eph family, ephrin B2 and Eph receptor A3, A4, and A7, and three Wnt antagonists showed altered expressions in DES-exposed female reproductive tracts (Table 2Go). Thus, we further studied genes of the Hoxa, Wnt, and Eph families and Wnt antagonists by Q-PCR.

The DES-regulated common genes in the three organs derived from Müllerian duct at GD 19 are listed in Table 3Go.


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  		Table 3. DES-Regulated Common Genes in the Three Organs Derived from Müllerian Duct at GD 19a
 
Expression of Hox and Wnt Genes in Female Reproductive Tracts by Q-PCR.
At GD19, Q-PCR revealed that DES downregulated Hoxa-10 mRNA in the oviduct (Fig. 3Go), whereas in the uterus Hoxa-9 mRNA was upregulated but Hoxa-10 was downregulated. DES did not alter the expression of Hoxa-11 in any of the organs studied, nor did we observe a change in the expression of Hoxa-13 in the vagina. Upregulation of Hoxa-13 mRNA expression was observed in the oviduct and uterus following DES exposure. Expression of Wnt-7a mRNA was repressed by DES. Expression of Wnt-5a mRNA was elevated by DES in all organs. The expression of Wnt-4 was repressed in the oviduct. In the DES-treated uterus, the expression of Wnt-4 showed a tendency toward repression, but it did not reach statistical significance (P = 0.06 vs. organ-matched control). Although upregulation of Wnt-4 mRNA expression was observed in the DES-treated vagina, it also did not reach statistical significance (P = 0.06 vs. organ-matched control; Fig. 4Go).


Figure 3
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  		Figure 3. Quantification of Hoxa-9, a-10, a-11, and a-13 mRNA expressions in the oviduct, uterus, and vagina at GD 19 using Q-PCR. Results were normalized by ribosomal L8 expression. Ratios were calculated relative to expression levels in the control uterus. Ov, oviduct; Ut, uterus; Vg, vagina. #P < 0.05 vs. control uterus; *P < 0.05 vs. organ-matched control groups.

 

Figure 4
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  		Figure 4. Quantification by Q-PCR of Wnt-4, –5a, and –7a mRNA expressions in the oviduct, uterus, and vagina at GD 19 following exposure to DES at GDs 10–18. Results were normalized by ribosomal L8 expression. Ratios were calculated relative to expression levels in the control uterus. Ov, oviduct; Ut, uterus; Vg, vagina. #P < 0.05 vs. control uterus; *P < 0.05 vs. organ-matched control groups.

 
Gene Expression of Eph Family and Wnt Antagonists in the Müllerian Duct.
After DES exposure, ephrin B2 mRNA was downregulated in the oviduct and uterus, whereas Eph receptor A3 expression was upregulated in the oviduct and vagina. Eph receptor A4 and A7 mRNA were downregulated by DES in all organs studied (Fig. 5Go).


Figure 5
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  		Figure 5. Quantification by Q-PCR of ephrin B2 and Eph receptors A3, A4, and A7 mRNA expressions in the oviduct, uterus, and vagina at GD 19 following exposure to DES at GDs 10–18. Results were normalized by ribosomal L8 expression. Ratios were calculated relative to expression levels in the control uterus. Ov, oviduct; Ut, uterus; Vg, vagina. #P < 0.05 vs. control uterus; *P < 0.05 vs. organ-matched control groups.

 
In DES-exposed mice, expression of Dkk2 mRNA was upregulated in the oviduct and uterus but downregulated in the vagina (Fig. 6Go). Expression of Nkd2 mRNA was downregulated by DES in all organs studied, whereas expression of sFRP1 mRNA was downregulated in the oviduct and uterus only (Fig. 6Go).


Figure 6
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  		Figure 6. Quantification by Q-PCR of DKK2, Nkd2, and sFRP1 mRNA expression in the oviduct, uterus, and vagina at GD 19 following exposure to DES at GDs 10–18. Results were normalized by ribosomal L8 expression. Ratios were calculated relative to expression levels in the control uterus. #P < 0.05 vs. control uterus; *P < 0.05 vs. organ-matched control groups.

 
Some of the microarray (Table 2Go) and Q-PCR (Figs. 5Go and 6Go) data were not consistent for Eph receptor A3, Eph receptor A4, Nkd2, and sFRP1. Therefore, we relied on the Q-PCR data for this discussion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In utero exposure to DES has been repeatedly observed to induce various reproductive abnormalities in mice and humans (40, 43, 44). DES-induced malformations of the reproductive organs have been hypothesized to be caused by a disruption in the expression of Hoxa genes along the anterior to posterior axis of the developing Müllerian duct (17). Furthermore, Wnt signaling regulates and maintains Hoxa gene expression in the Müllerian duct (45). DES-induced repression of Wnt-7a gene has been linked to developmental effects on the mouse reproductive tract (17, 46, 47).

Previous studies reported downregulation of Hoxa-10 and a-11 at GD 17 in the DES-exposed uterus and downregulation of Hoxa-9 in the DES-exposed oviduct (17, 18). In the present study, we confirmed the decrease in Hoxa-10, but we did not observe a change in uterine Hoxa-11 expression following DES exposure. Four antisense cDNAs for Hoxa-11 have been described in a cDNA library from the mouse embryonic limb (48). It may be that changes in uterine Hoxa-11 mRNA were not detected by our Q-PCR because of the presence of an antisense strand DNA.

In the present study, DES did not downregulate expression of Hoxa-13 mRNA in the vagina at GD 19, yet the same DES treatment in utero has been reported to induce ovary-independent vaginal stratification and cornification in mice (40). Thus, the ovary-independent vaginal changes may not be related to changes in Hoxa-13 expression. Interestingly, cluster analysis performed here revealed that the pattern of gene expression in the vagina, either in the control or DES-treated animals, differed significantly from those of the oviduct and uterus. Hoxa-10 expression is required for oviductal formation and uterine growth (14, 15). The molecular mechanisms of growth and differentiation in the caudal Müllerian duct are apparently different from the other regions of the Müllerian duct–derived reproductive system.

Dkk2 acts as an antagonist of Wnt signaling to induce endocytosis of the Wnt-Fz receptor complex and is activated by ß-catenin (49). In the present study, DES downregulated Dkk2 expression in the vagina but upregulated it in the oviduct and uterus. Wnt signaling regulates vaginal growth and differentiation by epithelial–mesenchymal interaction. The importance of epithelial–stromal interaction has been previously reported as an important factor mediating the developmental effects of estrogens, including DES, in the vaginal epithelium of the neonatal mouse, which is mediated through the action of stromal estrogen receptors (50, 51). Loss of Wnt-7a expression caused vaginal adenosis and concretions (30), whereas loss of Wnt-5a expression caused the absence of the vagina (52). During the perinatal period, the developing vagina expressed Wnt-5a and –7a but not Wnt-4. Expression of Wnt-7a in the vagina disappeared by 10 days of age, and adult vagina expressed Wnt-4 and –5a genes in the epithelium only (27). In the normal neonatal vagina, Wnt-7a regulates the reduction of Wnt-4 expression (27). However, in DES-exposed vagina, the reduction of Wnt-7a expression may cause a reduction of Dkk2 expression. Thus, vaginal epithelial cells in the DES-exposed fetus differentiate into squamous cells like those seen in an adult, followed by repression of Dkk2.

DES repressed expression of Nkd2 and sFRP1 in the oviduct and uterus, and Nkd2 in the vagina. This is the first report showing expression of Wnt antagonists and their estrogen regulation in organs derived from the Müllerian duct. Further studies are needed to clarify the role of Wnt antagonists during development of the Müllerian duct.

Eph receptor–ephrin signaling is a trigger regulating developmental patterning (26). Eph family genes are downstream genes of Hox genes (23, 53). Hoxa-9 directly regulates the transcription of Eph receptor B4 in endothelial cells, followed by increased cell migration and tube formation (53). In embryo limb, misexpression of Hoxa-13 caused downregulation of the Eph receptor A7, resulting in an inhibition of apoptosis (23). In the present study, DES-induced downregulation of ephrin B2 mRNA, as well as Hoxa genes, was found in the oviduct and uterus. Moreover, DES exposure downregulated Eph receptor A4 and A7 expression in all three organs. The Eph family of proteins may regulate pattern development in the Müllrerian duct by inducing changes in cytoskeleton dynamics, mitogenesis, and integrin signaling, as reported in other organ systems (25).

In Figure 7Go we summarized the expression change of Eph families, Wnt, Wnt antagonist, and Hox genes induced by DES in utero in the three organs derived from the mouse Müllerian duct. Further studies are needed to understand the functional relationship among these genes in the developing mouse reproductive tract and their relationship to reproductive tract abnormalities induced by DES.


Figure 7
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  		Figure 7. Summary of mRNA expression of Eph family, Wnt, Wnt antagonists, and Hoxa genes in the DES-exposed Müllerian duct. Eph, Ephrin receptor.

 
Some of the microarray data and Q-PCR data were not consistent for Hoxa-11 in the oviduct, Eph receptor A3 in the vagina, Eph receptor A4 in all organs, Nkd2 in the vagina, and sFRP1 in the oviduct. Therefore, we relied on the Q-PCR data for discussion. Recently, a new microarray method has been proposed that uses a "per cell" normalization method for mRNA measurement (54), which will provide a more consistent correlation between data sets derived from microarray and Q-PCR analyses.

In conclusion, microarray analysis revealed the presence of organ-specific changes in gene expression profiles in the oviduct, uterus, and vagina following DES exposure, thus providing for future study candidate genes that could be related to the reproductive abnormalities reported following embryonic or neonatal exposure to this estrogenic pharmaceutical agent. About 400 genes were upregulated and 200 genes were downregulated in the oviduct and uterus by DES exposure in utero. The vagina showed changes in fewer than half the number of DES-regulated genes than those found in the oviduct and uterus. Downregulation of ephrin B2, Eph receptors A4 and A7, and Nkd2, accompanied with changes in Hox and Wnt gene expression, could lead to abnormalities of segment-related positional identity in the upper part of the Müllrerian duct following DES exposure. In addition, the downregulation of Nkd2 and Dkk2 in DES-exposed vagina needs to be studied in more detail to determine whether these factors contribute to the persistent vaginal epithelial stratification seen in DES-exposed females.


   Acknowledgments
 
We are grateful to Dr. Raphael Guzman, Cancer Research Laboratory and Department of Molecular Cell Biology, University of California at Berkeley, and Professor Louis Guillette, Department of Zoology, University of Florida, for their discussion and critical readings of the manuscript.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Health Sciences Research Grant from the Ministry of Health, Labour and Welfare, Japan.

Received for publication July 18, 2006. Accepted for publication October 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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S L Rodriguez-Zas, Y Ko, H A Adams, and B R Southey
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