gene expression of sex-determining factors and...

General and Comparative Endocrinology 138 (2004) 148–156

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Gene expression of sex-determining factors and steroidogenic enzymes in the chicken embryo: inXuence of xenoestrogens�

Ryo Kamata,¤ Shinji Takahashi, and Masatoshi Morita

Endocrine Disrupter Research Laboratory, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

Received 15 January 2004; revised 29 March 2004; accepted 24 May 2004Available online 20 July 2004

Abstract

Many genes involved in gonadal development have been proposed for mammals. To elucidate if those genes play any critical rolein sexual diVerentiation of the avian gonad, we have examined expressions of the genes for proposed sex-determining factors (SF1,Sox9, DMRT1, Wpkci, and AMH), steroidogenic enzymes (P-450scc, 3�-HSD, P-450c17, 17�-HSD and aromatase) and the estrogenreceptor in the urogenital system during chicken embryogenesis (days 4–16 of incubation), using a semi-quantitative reverse tran-scription-polymerase chain reaction. Transcripts of the genes for sex-determining factors except Wpkci and AMH were detected inboth sexes but had no sexual dimorphism. Wpkci expression was female speciWc and constantly high throughout incubation. AMHwas expressed in both sexes from the earliest stages but was higher in males than in females after the onset of gonadal diVerentiation.Expressions of the genes for more downstream enzymes in a steroidogenic pathway, such as P-450c17, 17�-HSD and aromatase,were clearly higher in females than in males. In particular, 17�-HSD expression increased in the course of gonadal development infemales, whereas it was constantly low in males. Aromatase was highly expressed in females during gonadal diVerentiation but not inmales over the period. In addition, to elucidate the relationship between gene activation during embryogenesis and reproductiveabnormalities in wild birds, we examined expressions of these genes in embryos treated with various doses of diethylstilbestrol(DES), as a representative estrogenic compound. DES had no eVect on the expressions of all the genes in either sex during the periodsof gonadal diVerentiation (days 8, 12, and 16). Sexual dimorphism of the gene expression for steroidogenic enzymes appeared to beclosely related to gonadal development in the chicken embryo, especially in the female. However, all the genes examined here seemunlikely to respond to xenoestrogens. 2004 Elsevier Inc. All rights reserved.

1. Introduction

Sexual diVerentiation in higher vertebrates, such asmammals and birds, is considered to depend on tran-scripts of several genes on the sex chromosomes andrelated genes. The Y-linked male-determining gene inmammals, SRY/Sry, triggers male sex determination andinduces testicular diVerentiation from the immaturegonad (Koopman et al., 1991; Sinclair et al., 1990). Inaddition to Sry, Sry-related box gene 9 (Sox9) and the

� Sex-determining factors and steroidogenic enzymes in thechicken embryo.

¤ Corresponding author. Fax: +81-29-8502870.E-mail address: [email protected] (R. Kamata).

0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2004.05.011

autosomal gene encoding an orphan nuclear receptor,steroidogenic factor-1 (SF1), are considered to playimportant roles in testicular diVerentiation (Kent et al.,1996; Nachtigal et al., 1998). In birds, a critical gene forsex determination, such as Sry in mammals, has notbeen identiWed and the primary sex-determining signalis unknown. The constitution of the sex chromosomesin birds is ZW (female)/ZZ (male) diVerent than inmammals. Although several genes involved in sexualdiVerentiation in mammals have been detected in thegenital system of fowl embryos, the nature of the geneshas not been elucidated (Shimada, 2002). For example,Sox9 and SF1 are expressed in the developing gonadsof the avian embryo (Kent et al., 1996; Kudo andSutou, 1997). Moreover, the genes on the Z or W sex

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R. Kamata et al. / General and Comparative Endocrinology 138 (2004) 148–156 149

chromosome in birds, such as DMRT11 which encodesDoublesex and the Mab-3 related transcription factor1 and is located on the Z chromosomes and Wpkci,which encodes an altered form of the PKC inhibitor/interaction protein (PKCI) and is located on the non-heterochromatic end region of the W chromosome,have been proposed as candidates for key genes inavian sex determination (Hori et al., 2000; Raymondet al., 1999; Shan et al., 2000). However, the molecularfunctions of these genes in gonadal development arestill unclear.

Mortality and reproductive disorders in wild birdsexposed to environmental contaminants have beenreported for years. Indeed, many contaminants havebeen experimentally identiWed to possess reproductivetoxicity as a result of endocrine disrupting eVects (Fry,1995). In particular, compounds mimicking the actionof sex steroids are thought to impair diVerentiation ofthe reproductive and nervous systems during embryo-genesis and induce structural and functional abnormali-ties of the reproductive tract and brain in adult birds.Berg and colleagues have reported that embryonicexposure to synthetic estrogens such as diethylstilbes-trol (DES) and ethynylestradiol (EE2) induces morpho-logical abnormalities of the Müllerian ducts in bothsexes and of the testes in males and a failure in male-typical sexual behavior of Japanese quail (Berg et al.,1998, 1999, 2001; Halldin et al., 1999). Therefore, avianeggs were proposed as a test system for endocrine dis-rupting chemicals. Molecular mechanisms of the mal-formation of the reproductive systems by xenoestrogenshave not identiWed in detail. Because the onset of theseteratogeneses seems to be consistent with a criticalperiod of gonadal diVerentiation, transcripts of thegenes related to sex determination may participate inintra- and inter-cellular regulation of reproductivetissue diVerentiation.

The primary objective of the present study was toconWrm the expression proWles of sex-determining genesduring avian embryogenesis. Therefore, we examinedexpressions of the genes (SF1, Sox9, DMRT1, andWpkci) in the gonads of chicken embryos of both sexesusing a semi-quantitative reverse transcription-polymer-ase chain reaction (RT-PCR) method. To identify thegene expressions in detail throughout a critical period ofsexual diVerentiation, gonads were periodically harvested

1 Abbreviations used: AMH, Anti-Müllerian hormone; DMRT1,Doublesex and Mab-3 related transcription factor 1; ER, estrogenreceptor; 3�-HSD, 3�-hydroxysteroid dehydrogenase; 17�-HSD, 17�-hydroxysteroid dehydrogenase; P-450c17, 17�-hydroxylase/C17 ¡ 20 ly-ase; P-450scc, cholesterol side chain cleavage; PKC, protein kinase C;RT-PCR, reverse transcription-polymerase chain reaction; SF1, steroi-dogenic factor-1; Sox9, Sry-related box gene 9; SRY, sex-determiningregion on the human Y chromosome; Wpkci, gene for an altered formof PKC inhibitor/interaction protein and located on the nonhetero-chromatic end region of the W chromosome.

from early to late stages of incubation. The gene for theAnti-Müllerian hormone (AMH) was also examined. It isproposed that expression of AMH is regulated by SF1 inmammals and the resultant AMH acts to cause regres-sion of the Müllerian duct in the male embryo (Nachti-gal et al., 1998). Because endogenous estrogen can besynthesized at the time of gonadal diVerentiation andappears to be critical for ovarian diVerentiation in birds(Ottinger, 1989), expressions of the genes for steroido-genic enzymes, cholesterol side chain cleavage (P-450scc),3�-hydroxysteroid dehydrogenase (3�-HSD), steroid17�-hydroxylase/C17 ¡ 20 lyase (P-450c17), 17�-hydroxy-steroid dehydrogenase (17�-HSD), and aromatase (P-450arom), and the gene for estrogen receptor (ER) werealso examined to conWrm proWles of estrogen synthesis.

A second objective of this study was to evaluatewhether embryonic exposure to xenoestrogens aVectsexpressions of the genes related to avian sexual diVeren-tiation. The gene(s) that responds to treatment withxenoestrogens seems to be closely related to teratogene-sis of the genital systems in birds. Therefore, we exam-ined these gene expressions in developing embryostreated with DES, a typical xenoestrogen, and endocrinedisrupting chemical.

2. Materials and methods

2.1. Collection and sexing of tissue samples

Fertilized chicken (Gallus domesticus) eggs were incu-bated under humid conditions at 37.8 °C. According tothe method of Hamburger and Hamilton (1951), theembryo age was staged and measured in terms of thedays of incubation. Embryos were removed from eggs at4, 6, 8, 10, 12 and 16 days of incubation (stages 24, 29, 33,36, 38 and 42, respectively), and whole urogenital sys-tems and livers were excised. Genomic DNA wasextracted from each liver tissue using a Wizard GenomicDNA puriWcation kit (Promega, Madison, WI, USA),and then the sex of embryos was determined by PCRampliWcation of sex chromosomes-speciWc CHD1 genesfrom the genomic DNA template. PCRs were performedon an iCycler thermal cycler (Bio-Rad, Hercules, CA,USA) using sex chromosomes-speciWc primers 2550F(5�-GTTACTGATTCGTCTACGAGA-3�) and 2718R(5�-ATTGAAATGATCCAGTGCTTG-3�) (Fridolfs-son and Ellegren, 1999). PCR conditions were an initialdenaturing step at 94 °C for 2 min, followed by a “touch-down” scheme (Don et al., 1991) where the annealingtemperature was lowered 1 °C per cycle, starting from60 °C until a temperature of 50 °C was reached. Then 30additional cycles of 94 °C for 30 s, 50 °C for 30 s and72 °C for 30 s and a Wnal step of 5 min at 72 °C were con-ducted. Products of the reactions were analyzed by aga-rose gel electrophoresis (2% agarose, Takara Bio,

150 R. Kamata et al. / General and Comparative Endocrinology 138 (2004) 148–156

Kusatsu, Japan). Chromosomal sex was determined tobe ZZ (male) if the primers 2550F and 2718R give onefragment and ZW (female) if they give two fragments.

2.2. Semi-quantitative RT-PCR

Total RNA was extracted from urogenital systemswith a SV Total RNA Isolation System (Promega) todetermine the amount of transcript from genes and thequantity was determined by spectrophotometry at260 nm. An access RT-PCR system (Promega) was usedwith 100 ng total RNA for reverse transcription andampliWcation of each target mRNA, together with 1 �Meach of the speciWc primer combinations described inTable 1. The total volume of the RT-PCR samples was50 �l. The RT-PCR conditions were a reverse transcrip-tion step at 48 °C for 45 min, and then an initial denatur-ing step at 94 °C for 2 min, followed by 24–36 additionalcycles of 94 °C for 30 s, 56 or 64 °C for 40 s and 72 °C for1 min and a Wnal extension step of 5 min at 72 °C. Allprimers were designed so that they would anneal at 56 or64 °C and the appropriate cycle number for each genewas determined by preliminary examinations becausethese were clearly within the linear range of ampliWca-tion (Table 1). AmpliWcation products were subjected toelectrophoresis in 2% agarose gel beside molecularweight markers and stained with ethidium bromide. Thedensities of bands in the gels were analyzed using aChemiGenius 2 imaging system (Syngene, Cambridge,

UK) and the relative expression of each RT-PCR prod-uct quantiWed by normalization with the PCR productof �-actin using GeneTools software (Syngene). Since thelevel of expression of the �-actin gene was constant in allthe stages examined here and similar for the sexes(Fig. 1), �-actin was used as an internal control.

2.3. Xenoestrogen treatment

A small hole was punched with a needle at the bluntend of the shell of eggs at day 0. Eggs were injected intothe yolk with DES (Sigma, St. Louis, MO, USA) dis-solved in an emulsion of peanut oil, lecithin and water(Brunström and Darnerud, 1983; Brunström andÖrberg, 1982). The volume of all injections was 10 �l anddoses of test solution were adjusted to 0 (vehicle alone),1, 10, and 100 ng/egg. After injection, the shell was sealedwith paraYn wax. The eggs were incubated under theconditions described above and embryos were removedfrom eggs at 8, 12, and 16 days (stages 33, 38 and 42,respectively) of incubation, which were during and afterthe critical period of sexual diVerentiation. As men-tioned above, total RNA and genomic DNA wereextracted from whole urogenital systems for RT-PCR,and from livers for sexing. Semi-quantitative RT-PCRanalysis was carried out to determine expression pat-terns of each gene in response to a xenoestrogen underthe RT-PCR, electrophoresis, and gel analysis condi-tions described above.

Table 1Sequence of primers and conditions used for RT-PCR ampliWcation

Gene Accession No. F, forward primer; R, reverse primer Annealing temperature (°C)

Number ofPCR cycle

PCR product(bp)

Reference

SF1 AB002404 F: 5�-AGGATCTGGACGAGCTGTGT-3� 64 36 390R: 5�-ACAGCCCGTAGTCATTCTGCA-3�

Sox9 UI2533 F: 5�-CCCCAACGCCATCTTCAA-3� 56 36 381 Smith et al. (1999ab)R: 5�-CTGCTGATGCCGTAGGTA-3�

DMRT1 AF123456 F: 5�-CGCGTCTGCCCAAGTG-3� 64 32 367 Shan et al. (2000)R: 5�-GATGGAAGGGATGTCCTGAATGA-3�

Wpkci AB026677 F: 5�-GAGCAGAAGATTGTGGCGCA-3� 56 28 199R: 5�-GCGGTGCAAACATCTTAGCC-3�

AMH U61754 F: 5�-GTGGATGTGGCTCCCTACCC-3� 56 28 310 Neeper et al. (1996)R: 5�-GCAGCACCGAGGGCTCCTCC-3�

P-450scc D49803 F: 5�-TGCTCCCATGCTCTCCAGG-3� 64 32 382R: 5�-GCGGTAGTCACGGTATGCC-3�

P-450c17 M21406 F: 5�-GCTGCTGAAGAAGGGGAAG-3� 64 32 315R: 5�-CGTCGGTAGGAGGAGTTGA-3�

3�-HSD D43763 F: 5�-GGCAAGTTCCAGGGCAAGA-3� 64 32 408R: 5�-ACAGGTCACAAGCACGCCT-3�

17�-HSD AB004210 F: 5�-GGCCATGAGAGCAGTGTTT-3� 56 36 152 Nomura et al. (1998)R: 5�-AGTACACGGCGTTGAAGGG-3�

Aromatase D50335 F: 5�-TCCAGCAGGTTGAAAGGTAC-3� 56 36 111R: 5�-GCTTTGCTTTAGACAGAGGG-3�

ER� X03805 F: 5�-CAAGGCACTGAGCTGGAGA-3� 64 36 380R: 5�-CTGTAGAAGGCTGGAGGAG-3�

�-actin L08165 F: 5�-GCCAACAGAGAGAAGATGAC-3� 56 24 288R: 5�-CACAATTTCTCTCTCGGCTG-3�


2.4. Statistics

Statistical analyses were performed with StatView forWindows version 5.0 (SAS Institute, Cary, NC, USA).

One-way analysis of variance was used to examine diVer-ences between control groups and DES treatmentgroups for each measured variable, followed by post hocanalysis using Fisher’s PLSD test for signiWcance. All

Fig. 1. Time course of expression of the genes proposed as sex-determining factors in the urogenital system during chicken embryogenesis. The upperpanel for each gene shows an ethidium bromide gel of RT-PCR analysis from female (F) and male (M) embryos. In the lower panel, each mRNAdetected by semi-quantitative RT-PCR is presented as relative to �-actin (%). Only �-actin controls are shown as relative Xuorescence intensity (%) today 4 of incubation. Open circles and open triangles represent female and male values, respectively.


data are expressed as mean § SEM. Statistical signiW-cance is noted where P 0 0.05.

3. Results

3.1. Gene expressions of sex-determining factors

Transcripts of the genes for proposed sex-determiningfactors in the urogenital systems during chicken embryo-genesis were measured using a semi-quantitative RT-PCR method and compared between the sexes (Fig. 1).All the genes except Wpkci were expressed in all thestages examined in both male and female embryos. SF1and DMRT1 genes were weakly expressed on day 4 ofincubation in both sexes and the levels of the relativeexpressions to �-actin increased gradually over theperiod of the examination. Sox9 expression was consid-erably higher at the earliest stage and changed littlethroughout the experimental period in both sexes. AMHwas also expressed in both sexes from the earliest stageof the experimental period. The expression in males,however, increased from days 6 to 16 and was muchhigher than in females in days 8 to 16. As shown in Table2, a statistically signiWcant diVerence in the relative expres-sion of AMH to �-actin was observed between the sexeson day 12. For Wpkci, located on the W chromosome,expression was consistently high in female embryos over

the experimental period, whereas no expression wasobserved in males.

3.2. Gene expressions of steroidogenic enzymes and theestrogen receptor

Each of steroidogenic genes had a typical proWle ofexpression (Fig. 2). For the P-450scc and 3�-HSD genes,no or low expression was observed in the earlier stages(days 4 and 6) in both female and male embryos, but thelevel of expression of each of the genes increased in thecourse of incubation and was similar for the sexes. P-450c17 expression was also low in the earlier stages butincreased sharply over the experimental period in bothsexes. However, the onset of increase in the gene expres-sion in male embryos was later than in females with thelevel in males being lower than in females in days 8 to 12.17�-HSD expression in female embryos, like P-450c17,was also low in the earlier stages and increased over theexperimental period, whereas the expression in maleswas still low at the end of the stages examined. The rela-tive expression of P-450c17 and 17�-HSD was signiW-cantly diVerent between the sexes on day 12 (Table 2).Moreover, aromatase was expressed in female embryosbut not in males. The expression in females was Wrstobserved on day 8 and generally increased thereafter.

The ER� gene was expressed from the earliest stagethroughout the experimental period and the expres-

Table 2Relative expression in chicken embryo treated with DES (% of �-actin)

Each mRNA in the urogenital systems from chicken embryos detected by semi-quantitative RT-PCR is presented as a relative expression to �-actin(%). Asterisks represent signiWcant diVerences between female and male values: *P 0 0.05; **P 0 0.01. Each value is the mean § SE of six embryos.

Gene Sex DES treatment

Non-treated Vehicle 1 ng/egg 10 ng/egg 100 ng/egg

SF1 Female 45.3 § 9.70 39.9 § 4.63 41.5 § 5.19 39.8 § 6.74 36.5 § 4.03Male 37.7 § 6.69 38.2 § 8.99 41.0 § 9.48 36.4 § 9.54 37.8 § 10.1

Sox9 Female 79.3 § 5.91 79.7 § 4.94 81.3 § 5.28 82.7 § 3.71 77.3 § 4.88Male 83.3 § 5.70 72.8 § 5.39 76.7 § 4.80 80.6 § 4.73 75.6 § 5.42

DMRT1 Female 34.9 § 4.83 33.5 § 2.90 35.7 § 4.49 36.9 § 3.93 37.3 § 4.53Male 45.4 § 3.45 42.9 § 2.75 39.0 § 4.63 43.4 § 5.04 42.6 § 4.11

Wpkci Female 81.5 § 3.62 80.1 § 4.46 84.7 § 5.83 82.9 § 5.53 80.9 § 5.65Male ND ND ND ND ND

AMH Female 22.4 § 4.21** 20.8 § 3.58** 22.9 § 4.00** 21.0 § 2.94** 20.4 § 3.19**

Male 48.6 § 4.17 44.8 § 5.11 40.0 § 5.13 43.2 § 3.62 45.7 § 3.73

P-450scc Female 27.4 § 3.28 28.5 § 3.48 28.9 § 3.70 22.3 § 3.77 23.5 § 3.16Male 25.8 § 4.11 23.6 § 4.00 24.9 § 4.01 19.4 § 4.39 23.0 § 4.59

P-450c17 Female 62.1 § 3.27** 57.2 § 3.52** 58.1 § 4.30* 56.6 § 6.17** 58.1 § 3.32**

Male 38.6 § 5.74 38.8 § 3.71 41.5 § 7.28 34.4 § 4.45 30.7 § 3.293�-HSD Female 60.4 § 9.16 68.9 § 5.30 69.1 § 6.63 61.3 § 8.62 60.3 § 6.85

Male 67.4 § 10.5 61.7 § 9.83 62.3 § 8.07 52.8 § 12.5 59.9 § 12.217�-HSD Female 49.5 § 4.81** 47.4 § 4.89** 47.5 § 4.85** 44.6 § 4.25** 49.4 § 4.40**

Male 13.7 § 1.75 13.7 § 2.06 15.4 § 2.82 13.0 § 2.21 12.2 § 1.74Aromatase Female 88.8 § 4.85 88.4 § 4.57 90.2 § 7.01 89.7 § 6.58 87.6 § 4.94

Male ND ND ND ND NDER� Female 74.2 § 4.29 80.3 § 2.26 81.0 § 3.46 79.2 § 5.21 80.1 § 3.72

Male 82.4 § 2.96 82.1 § 3.10 81.5 § 4.78 75.7 § 5.01 75.6 § 6.67


Fig. 2. Time course of expression of the genes for steroidogenic enzymes and estrogen receptor � in the urogenital system during chicken embryogen-esis. The upper panel for each gene shows an ethidium bromide gel of RT-PCR analysis from female (F) and male (M) embryos. In the lower panel,each mRNA detected by semi-quantitative RT-PCR is presented as relative to �-actin (%). Open circles and open triangles represent female and malevalues, respectively.


sions in both sexes were similar and increased from day4 to 8.

3.3. In ovo treatment with diethylstilbestrol

To determine the inXuence of xenoestrogens on tran-scripts of the genes related to sexual diVerentiation,expression levels of the genes from chicken embryostreated with various dosages of DES were compared inboth female and male embryos. As shown in Table 2,although expressions of AMH, Wpkci, P-450c17, 17�-HSD and aromatase showed a physiological diVerencebetween the sexes, for all genes, expression levels on day12 of incubation were not signiWcantly diVerent betweenany dose groups of DES. In addition, there was no alter-ation in the expression levels of all genes on either days 8or 16 (data not shown).

4. Discussion

In the present study, the results of semi-quantitativeRT-PCR showed sexually dimorphic transcripts of thegenes for steroidogenic enzymes in the urogenital systemsduring chicken embryogenesis. Interestingly, the genes,such as P-450c17, 17�-HSD, and aromatase, for the moredownstream enzymes in the steroidogenic pathwayshowed diVerences between the sexes in their expressions.This result for the expression of the aromatase gene isconsistent with previous observations that the expressionsharply increases from about day 7 of incubation infemale embryos, but hardly or not at all in malesthroughout the incubation period (Nakabayashi et al.,1998; Nomura et al., 1999; Yoshida et al., 1996). Nomuraet al. (1999) showed that transcription of the P-450c17gene increased from day 6 to 9 of incubation in femaleembryos but not in males, whereas 17�-HSD showed nosigniWcant diVerence either in the course of incubation orbetween the sexes. Our data from proWles of P-450c17expression in early stages agreed with these observations.In addition, we reported here that P-450c17 expression inmale embryos increased sharply after day 10 and reacheda similar level in females at the end of the examinationperiod. Moreover, 17�-HSD expression showed sexualdimorphism after day 6 of incubation to the end of theincubation period in this study. Our observations on sex-ual dimorphism in steroidogenesis strongly support thetheory that the appearance of estradiol during earlyembryogenesis plays a key role in gonadal diVerentiationof the avian female embryo and later sexual develop-ment. In the synthesis of sex steroid hormones, 17�-HSDenzyme converts androstenedione to testosterone andthen aromatase Wnally converts testosterone to 17�-estra-diol, while P-450c17 exists upstream of the two enzymes.Therefore, the diVerence between the sexes in theamounts of 17�-estradiol in embryonic gonads appears

to be attributed to transcripts of the genes for steroido-genic enzymes, especially P-450c17, 17�-HSD and aro-matase. Furthermore, the beginning of the transcriptionof the estrogen receptor gene from the earliest stages alsoappears to play an important part in sexual dimorphism,even though the levels of the expression showed no diVer-ence between the sexes.

Compared with the steroidogenic enzymes, expres-sions of the genes proposed as sex-determining factorsexcept Wpkci and AMH did not show clear sexualdimorphism in the present study. In the mouse embryo,expression of the Sox9 gene increases from day 11.5 to17.5 postcoitum in male genital ridges but not in femalesusing an in situ hybridization method, although expres-sion of the gene was detected in female genital ridges byRT-PCR (Kent et al., 1996). Sox9 is also expressed ingenital ridges of male chicken embryos from day 6.5 to7.5 of incubation but not in females, and the expressionis greater in the right testis than in the left. By contrast,expression of the SF1 gene in chicken embryos isobserved in both sexes in the earlier stages (days 5.5 and6.5) and then increases only in developing ovaries afterthe onset of gonadal diVerentiation (day 7.5) (Smithet al., 1999a,b), although SF1 expression in mammals ishigher in developing testes than in ovaries (Ikeda et al.,1994). In the present study, neither the Sox9 nor SF1genes produced sexually dimorphic proWles of expres-sion, but expressions of the SF1 gene in both sexesshowed increases during the time course of incubation.Smith et al. (1999a) reported that in situ expression ofthe SF1 gene in the adrenal glands of the chickenembryo was strong and similar in both sexes. Therefore,the inconsistency in our results may be attributed to theuse of whole urogenital systems for examination;gonadal expressions of the gene may be masked by adre-nal expressions. On the other hand, no in situ expressionof the Sox9 gene was observed throughout the urogeni-tal systems except in the male gonads during earlyembryogenesis (Kent et al., 1996). Although the gene wasexpressed independently of sex and age in this study, wemay need to consider other ways in which Sox9 mighthave a primary sex-determining role.

In birds, AMH appears to be responsible for not onlyretrogression of the Müllerian ducts in the male embryobut that of the right duct in the female. Our observationthat expression of the AMH gene increases during theearly stages of incubation in male gonads but that thelevel of expression in female gonads is not as high as inmales is consistent with similar RT-PCR studies (Smithet al., 1999b). Although we found slight expressionsbefore the onset of Müllerian ducts retrogression (day 4)in both sexes, the coincidence of the increase in AMHtranscription and the retrogression of the Müllerianducts in males is appropriate to the current view.DMRT1 and Wpkci are proposed as participants in trig-gering sexual diVerentiation of the avian male and female


gonads, respectively, owing to proWles of the expressionsof the genes for these proteins. Because the DMRT1 geneis located on the Z chromosome, transcripts of the geneexist in both sexes. Our results agree with the previous insitu hybridization studies that DMRT1 is expressed inthe genital ridges of both sexes during early stages ofembryogenesis (Raymond et al., 1999; Shan et al., 2000),but we did not detect that the gene is expressed at higherlevels in the male embryo than in the female. The diVer-ence between the sexes in the amounts of expression ofthe DMRT1 gene may be less than implied by the resultsfor in situ hybridization. In comparison with DMRT1,expression of the Wpkci gene was detected only in thefemale embryo. It has been reported that the level ofexpression of the gene measured using in situ and North-ern blot hybridization is signiWcantly greater during theperiod before the onset of gonadal diVerentiation thansubsequently (Hori et al., 2000). However, expression ofthe gene in the present study was quite independent of thestages of incubation and showed no correlation withthe development of female gonads. Almost all studies ofthe genes involved in avian sex determination are basedon information about sex-determining factors in mam-mals, although avian sex-determining mechanisms arevery diVerent from mammals. The molecular functions ofthe genes have not yet been elucidated. Therefore, obser-vations on transcripts of the genes in birds may not pro-duce as satisfactory results as in mammals.

Exposure to exogenous steroids at an early stage ofembryogenesis has functional and morphological eVectson avian reproduction (Kamata and Morita, 2002). Fryand Toone (1981) Wrst reported that injection of someorganochlorine pesticides into eggs caused developmentof left ovotestis and both oviducts in male gull embryosand the right oviduct in females. Since these eVects have aclose resemblance to the results from synthetic estrogens,they appear to result from the estrogenic character ofthese compounds. In the present study, to elucidate tar-get gene(s) and mechanisms of teratogenesis, we adminis-tered DES to chicken eggs and investigated the eVect ofxenoestrogens on the action of the genes involved in sex-ual diVerentiation. However, the dosage of DES thatleads to malformation of the testes and oviducts (Berget al., 1998, 1999; Halldin et al., 1999) had no eVect ontranscription of all the genes examined here. Expressionof these genes seems unlikely to change either directly orindirectly in response to xenoestrogens in either sex dur-ing embryogenesis. In addition, these genes seem unlikelyto have any role in the malformation of testes and ovi-ducts. A gene that exhibits a dramatic response toxenoestrogens during avian embryogenesis has yet to berevealed. Although in oviparous animals measurement ofgene expression of the yolk lipophosphoglycoproteinvitellogenin in the liver is proposed as a bioassay methodfor estrogenic activity of environmental contaminants,the vitellogenin gene in the avian embryo has a poor

response to xenoestrogens (unpublished observation)and has no direct relation to sexual diVerentiation.Therefore, it is considered that new candidates are neces-sary for target genes and the mechanisms of teratogenesisand reproductive disorder in wildlife.

Acknowledgments

The authors thank Dr. John S. Edmonds for helpfulcomments on the manuscript. This work was supportedin part by Grants-in-Aid for ScientiWc Research from theMinistry of Education, Science, Sports, and Culture,Japan (Grant reference no. 14580577).

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