The Partial Female to Male Sex Reversal in Wnt-4-Deficient Females Involves Induced Expression of Testosterone Biosynthetic Genes and Testos
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《内分泌学杂志》
Biocenter Oulu (M.H., R.N., F.N., P.I., J.V., H.P., S.V.) and Departments of Biochemistry (M.H., H.P., S.V.), Physiology (J.L.), and Medical Biochemistry and Molecular Biology (R.P., F.N., P.I., S.V.), University of Oulu, FIN-90014 Oulu, Finland
Abstract
Wnt-4 signaling has been implicated in female development, because its absence leads to partial female to male sex reversal in the mouse. Instead of Mullerian ducts, Wnt-4-deficient females have Wolffian ducts, suggesting a role for androgens in maintaining this single-sex duct type in females. We demonstrate here that testosterone is produced by the ovary of Wnt-4-deficient female embryos and is also detected in the embryonic plasma. Consistent with this, the expression of several genes encoding enzymes in the pathway leading to the synthesis of testosterone in the mouse is induced in the Wnt-4-deficient ovary, including Cyp11a, Cyp17, Hsd3b1, Hsd17b1, and Hsd17b3. Inhibition of androgen action with an antiandrogen, flutamide, during gestation leads to complete degeneration of the Wolffian ducts in 80% of the mutant females and degeneration of the cortical layer that resembles the tunica albuginea in the masculinized ovary. However, androgen action is not involved in the sexually dimorphic organization of endothelial cells in the Wnt-4 deficient ovary, because flutamide did not change the organization of the coelomic vessel. These data imply that Wnt-4 signaling normally acts to suppress testosterone biosynthesis in the female, and that testosterone is the putative mediator of the masculinization phenotype in Wnt-4-deficient females.
Introduction
ALTHOUGH INHERITANCE of an X- or Y-chromosome from the male establishes the genetic sex of the mammalian embryo at fertilization, the genes that determine the primary sex and subsequently lead to the establishment of secondary sexual characteristics are not activated until the gonad has developed, on embryonic d 10.5 and later, in the mouse. Initially the gonad is bipotential, but as a result of activation of the sex-determining gene Sry, the male sexual pathway becomes induced (1), whereas Wnt-4, a gene encoding a factor from the Wnt family of signaling molecules, is important for female development (2). After commitment of the male pathway, androgens secreted by differentiated Leydig cells are needed for the formation of the male secondary sexual characteristics beginning during embryogenesis, whereas the ovary becomes hormonally active at puberty (3, 4, 5). Thereafter, estrogen receptor (ER) and ER are critical in maintaining female sexual characteristics, because in their deficiency the ovary undergoes partial sex reversal to male and displays Sertoli cells (for a review, see Ref.6).
The paired primordia of the sex organs are composed of the indifferent gonads and the two sex ducts on either side of the developing embryo, the Wolffian and Mullerian ducts. In males, testosterone is required for subsequent development of the Wolffian duct, and this generates the epididymides, vasa deferentia, and seminal vesicles, whereas the Mullerian duct typical of females regresses in males due to the action of a Sertoli cell product, anti-Mullerian hormone (AMH) (for a review, see Ref.7). Morphogenesis of the male external genitalia is, in turn, thought to be controlled by dihydrotestosterone (DHT) (3, 4) that is produced from testosterone by the enzymes 5-reductase types 1 and 2. The type 2 enzyme is important in humans, because mutations in it lead to pseudohermaphroditism, characterized by a defective prostate and external genitalia (for a review, see Ref.8), but in the mouse, testosterone is the only androgen required for differentiation of the male urogenital tract (9).
In females, the lack of testosterone production leads to regression of the Wolffian ducts, whereas the Mullerian ducts persist in the presence of Wnt-4 signaling and in the absence of AMH (2). The Mullerian ducts develop into the oviduct, uterus, and upper part of the vagina of the adult female (3, 4).
The Wnt-4 gene is widely expressed during fetal life and is functional in organs such as the kidney (10), gonad (2, 11, 12, 13), Mullerian duct (2), adrenal gland (14), mammary gland (15), and pituitary gland (16), suggesting a pleiotropic function for this factor. Studies of Wnt-4 function in the mouse have revealed that it is an essential element in female sex organogenesis, supporting the idea that the specification of female sex depends on cell signaling and is not a passive default developmental pathway (2).
Based on studies of Wnt-4-deficient female embryos, it has been suggested that Wnt-4 regulates femaleness in three distinct phases (2). First, Wnt-4 is expressed in the indifferent gonad and is perhaps required normally for suppression of the expression of the genes encoding enzymes in the testosterone biosynthetic pathway. Second, Wnt-4 signaling is necessary for development of the secondary female characteristics, i.e. the Mullerian ducts, because these ducts are not able to form in either sex in its absence. Third, it is important for maintaining the oocytes.
Sex reversal in Wnt-4-deficient females is characterized by the presence of a Wolffian duct. The masculinization of females is also manifested by the presence of round, encapsulated ovaries that have a closely associated fat body, all of which are characteristic of males, suggesting that masculinization takes place due to synthesis of the androgen testosterone (2).
In this study we report evidence that testosterone is indeed produced by the ovaries of Wnt-4-deficient newborn mice. It can be measured in the gonad and plasma and may be the driving force for the masculinization phenotype in females, because blocking of androgen action with an antiandrogen, flutamide, leads to degeneration of the Wolffian duct and the cortical layer of the ovary in Wnt-4-deficient females. These findings are in line with an earlier report showing that testosterone is sufficient to induce male reproductive structures in female fetuses (17). Consistent with the presence of testosterone in Wnt-4-deficient females, several genes encoding enzymes that are involved in the synthesis of testosterone, such as Hsd17b1 and Hsd17b3, are expressed by the Wnt-4-deficient ovary.
Materials and Methods
Animals and their treatment with the antiandrogen flutamide
Crosses between the mouse strains 129SV and CD-1, both heterozygous for the targeted Wnt-4 allele, were used throughout to obtain embryos and newborn mice for the analysis. The targeted allele of Wnt-4 was genotyped as reported previously (10). For generation of Wnt-4-deficient embryos, heterozygous Wnt-4-deficient female and male mice were mated, and the day when the vaginal plug was detected in the mated female was designated embryonic d 0.5 (E0.5). The Wnt-4-deficient newborn mice were recognized by their hypoplastic kidneys, which is a typically penetrant phenotype, whereas other animals were genotyped (10).
For the generation of flutamide-treated embryos or newborn mice, 23 pregnant Wnt-4 heterozygote females were treated with sc injections of flutamide (F9397, Sigma-Aldrich Corp., St. Louis, MO; 100 mg/kg·d daily) from E10.5 until they were killed, and nine mice were treated with the vehicle, turnip oil. Flutamide functions by competing with androgens for binding to the androgen receptor and is therefore a useful tool for blocking androgen action during embryogenesis (18). All of the animal experimentation described in this study was conducted according to accepted standards of humane animal care, and adequate permits were obtained from the local ethical committee.
Analyses of hormones
The blood of decapitated newborn mice from Wnt-4 heterozygous intercourses was collected into heparinized glass tubes. Samples were centrifuged for 10 min at 3000 rpm and stored at –20 C. The plasma samples were pooled, six samples per tube, to provide sufficient volume. Gonadal tissues or embryonic adrenal glands were collected, frozen quickly, and stored at –20 C. The tissues were sonicated for 15 sec in 100 μl distilled water and washed twice with 300 μl of a solution containing 9 vol diethyl ether and 1 vol ethyl acetate. After centrifugation, the water phase was taken for protein assays, and the organic phases were combined, evaporated, and assayed for the presence of steroids. Protein concentrations in the gonadal samples were measured using a protein assay kit from Bio-Rad Laboratories (Hercules, CA).
The evaporated organic phase was reconstituted with 500 μl RIA buffer, and double aliquots of 25–100 μl were subjected to RIA. Concentrations of testosterone and estradiol were measured in plasma and gonad, and testosterone was determined in adrenal tissue samples using RIA kits from Orion DG (Oulu, Finland). Concentrations of dehydroepiandrosterone (DHEA) and androstenedione were measured in gonad tissue samples only, with RIA kits from DRG Instruments GmbH (Marburg, Germany). Plasma DHT was measured with an RIA kit (Intertech Sarl, Dudelange, Luxemburg). Due to the small plasma volumes available (20–40 μl), 150 μl 0.9% NaCl were added to each sample, and the amounts of the solutions used in the extractions were halved. According to the manufacturer’s information, the cross-reactivity of DHT antiserum with testosterone is 0.19%, and that with estradiol, progesterone, or DHEA is less than 0.005%. The coefficient of variation is less than 5%, and the interassay variation is between 8.7% and 18.6%. The detection limits of the hormones analyzed were 0.5 pmol/ml for testosterone, 0.7 fmol/ml for DHEA, 1 fmol/ml for androstenedione, 0.8 pmol/ml for DHT, and 50 fmol/ml for estradiol. Values below the limit of detection were recorded as zero.
Examination of the anogenital distance
The pups derived from heterozygous Wnt-4 matings were killed by decapitation on the day of delivery and quickly analyzed in Dulbecco’s PBS (Invitrogen Life Technologies, Inc., Gaithersburg, MD) on ice. The anogenital distance was measured before the sex or genotype was determined in each newborn mouse using a dissecting microscope and an attached micrometer lens, after which the urogenital systems were dissected for analysis. The males and females were distinguished according to the morphology of the gonad.
Histology, whole mount, and section in situ hybridization
The testes and ovaries from isolated urogenital systems were prepared and fixed in 4% paraformaldehyde overnight at 4 C, dehydrated in a graded ethanol series, incubated in xylene, embedded in paraffin, sectioned at 5 μm, and used for histological staining or in situ hybridization (see below). Hematoxylin-eosin staining was performed according to routine procedures (19).
For whole mount in situ hybridization, the prepared testes and ovaries were fixed in 4% paraformaldehyde overnight, dehydrated in a graded methanol series, and stored in 100% methanol at –20 C until used. The whole mount and section in situ hybridizations were performed according to standard procedures (20). The probes used were Hsd3b1 and amh (Robin Lovell-Badge, National Institute for Medical Research, London, UK), Cyp17 (expressed sequence tag AA822113, National Center for Biotechnology Information), and Hsd17b1 (21). After linearization of the plasmids containing the respective cDNA sequences, digoxigenin-labeled riboprobes were produced (digoxigenin RNA labeling kit, Roche, Indianapolis, IN).
Fixed or stained samples of the freshly dissected gonads were photographed with a DP50 digital camera (Olympus, New Hyde Park, NY) mounted on an MZ FLIII microscope (Leica Microsystems, Deerfield, IL). The images were processed with Viewfinder 3.0.1 software (Pixera Corp., Los Gatos, CA) and the Adobe Photoshop and Corel Draw programs (Adobe Systems, San Jose, CA).
Changes in the organization of endothelial cells in developing gonads
To study the effects of flutamide on the development of the gonadal vasculature, the endothelial cell-specific Tie1LacZ reporter gene (22) was crossed into a heterozygous Wnt-4+/– background by breeding Tie1LacZ and Wnt-4+/– mice together. Embryos were collected from flutamide- or vehicle-treated pregnant females from matings of Tie1LacZ+ and Wnt-4+/– mice. The endothelial cells of the isolated gonads were visualized by X-gal staining according to Kispert et al. (23).
Isolation of the RNA preparation and microarray analysis
Gonads were prepared from Wnt-4-deficient and wild-type embryos on E12.5 and E14.5 and from newborn mice. The separated gonads were frozen immediately in liquid nitrogen and stored at –70 C until used for the extraction of RNA. Total RNA was purified using the RNeasy Kit (Qiagen, Chatsworth, CA). RNA (5 μg) was used as a template for synthesizing cDNA and for making biotinylated cRNA, performed according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA). The biotinylated cRNA was hybridized to the GeneChip Mouse Expression Set 430_2.0 Array, which represents approximately 45,000 mouse transcripts. The arrays were scanned with GeneChip Scanner 3000, and the resulting expression data were analyzed with the Affymetrix GeneChip Operating System. The intensities of the signals for all probe sets were scaled to a target value of 500.
Results
Testosterone is detected in the plasma and gonad of Wnt-4-deficient females
Our previous report (2) indicated that the lack of Wnt-4 signaling in females leads to further development of the male sex ducts, the Wolffian ducts, instead of the Mullerian ducts, which typically develop in normal females. This suggested a role for androgens, namely testosterone, in the maintenance of this type of sex duct in Wnt-4-deficient females.
Measurements of testosterone concentrations in newborn mice obtained from Wnt-4+/– intercrosses revealed the presence of testosterone in blood samples from wild-type males and Wnt-4-deficient males and females, but not from wild-type females, as expected (Fig. 1A).
We also ascertained whether testosterone synthesis could be detected in the gonads of Wnt-4-deficient embryos. Consistent with the results in plasma, testosterone production was identified in wild-type and Wnt-4-deficient testes and also in the Wnt-4-deficient ovary, whereas no synthesis was detected in the wild-type ovary (Fig. 1B). No testosterone production was detected in the adrenal glands of wild-type or Wnt-4-deficient males or females (data not shown). We conclude that testosterone is synthesized by the Wnt-4-deficient ovary, making it a candidate mediator for the partial male to female sex reversal phenotype in Wnt-4-deficient females.
Synthesis of precursors of testosterone, DHT, and estradiol in ovaries of Wnt-4-deficient female mice
Because testosterone is produced via precursors such as androstenedione and dehydroepiandrosterone (DHEA), and at least some of the genes that encode these critical enzymes in their biosynthesis are also expressed in the Wnt-4-deficient ovary (2), we were interested in measuring changes in the amounts of these precursors in the gonads. The concentration of androstenedione was below the limit of detection, but that of DHEA was elevated in Wnt-4-deficient females compared with wild-type females (P < 0.01), whereas no significant differences were observed between wild-type and Wnt-4-deficient males (P = 0.091; Fig. 1C). No notable differences were observed in the amount of plasma DHT between the mutant females and males and their wild-type littermates; the average values were 0.83 nmol/liter for wild-type females, 2.36 nmol/liter for males, 0.77 nmol/liter for Wnt-4-deficient females, and 1.35 nmol/liter for males.
In addition to measuring the precursors that lead to the synthesis of testosterone and DHT, we addressed possible changes in the concentrations of plasma or gonadal estradiol in Wnt-4 deficiency, but found them to be below the level of detection in all samples analyzed regardless of the genotype.
Changes in expression of the genes encoding enzymes in the biosynthetic pathway of hormones in Wnt-4-deficient ovaries
To reveal the genetic changes that lead to the synthesis of ovarian testosterone in Wnt-4-deficient females, we used Affymetrix oligonucleotide gene chips containing the major genes involved in the synthesis of hormones. The gonads were separated out from E12.5 and E14.5 embryos and newborn mice, total RNA was purified and subjected to the gene chips, and changes in the genes involved in hormone synthesis were measured. The results, summarized in Table 1, indicate that Cyp11a, Cyp11b2, Cyp17, Cyp19, Cyp21a, Hsd3b1, Hsd3b6, Hsd17b1, and Hsd17b3 gene expression is induced in the ovaries of Wnt-4-deficient females compared with wild-type ovaries. In situ hybridization analysis revealed that, consistent with the gene chip studies, the Cyp17 and Hsd171 genes are ectopically expressed in Wnt-4-deficient ovaries and are also expressed in a sexually dimorphic manner (Fig. 2, A–D and I–L). Thus, Cyp17 and Hsd17b1 are expressed in wild-type and Wnt-4-deficient testes, but not in ovaries of wild-type newborn mice.
We also analyzed changes in the expression of the ER and ER genes, because a deficiency in these has been reported to lead to sex reversal in the ER-deficient ovary at the onset of puberty (6). ER was down-regulated at all stages analyzed in Wnt-4-deficient ovaries compared with the wild type, whereas ER was somewhat increased, especially at the later stages of ovarian development (Table 1).
Blocking androgen action leads to degeneration of the male Wolffian ducts in Wnt-4-deficient females
The antiandrogen flutamide was used to determine whether testosterone is responsible for maintaining the Wolffian duct in Wnt-4-deficient females. The effect of flutamide on growth of the wild-type testis was used first to test the effectiveness of the concentration of flutamide used. Although daily injections of placebo did not affect the overall size of the testis (Fig. 3A), the administration of flutamide (100 mg/kg) led to a considerable reduction in its size (Fig. 3B) and a change in the ratio between the area of the seminiferous tubules and that of the interstitial tissue in the testis compared with the placebo controls, as demonstrated by hematoxylin-eosin staining (Fig. 3, C and D). The expression of the AMH (amh) and Hsd17b3 genes demark the seminiferous tubules in flutamide-treated testes (Fig. 3, E and F). This result is consistent with earlier findings (18) and suggests that in vivo administration of flutamide at the concentration used is sufficient to inhibit the action of androgens.
The treatment of pregnant females with flutamide generated a phenotype, compared with the placebo controls, in Wnt-4-deficient female embryos with no gonad-associated sex ducts, as seen in 16 of the 20 cases analyzed (Figs. 4). In these flutamide-treated newborn Wnt-4-deficient mice, the rudimentary ovaries were connected to the rest of the urogenital system by thin, poorly developed ligaments (Fig. 4, C and F). In the remaining four flutamide-treated embryos, the Wolffian ducts had degenerated unilaterally (data not shown). The coiled epididymis-like organization that was detectable in the ovaries of placebo-treated Wnt-4-deficient females (Fig. 4, B and E) had degenerated on account of flutamide exposure, as shown in Fig. 4, C and F. Similarly, the overall organization of the ovary in Wnt-4-deficient newborn mice had altered from the circular pattern seen in placebo controls to a more elongated one (compare the ovaries in Fig. 4, E and F). Flutamide did not have any effect on the expression of the Cyp17 and Hsd17b1 genes in either sex or genotype, as judged by in situ hybridization analysis (Fig. 2, E–H and M–P).
Because the development of external genitalia is regulated by testosterone (9) and DHT in males, we were also interested in assaying potential changes in the anogenital distance, which is characteristically longer in males than in females. The anogenital distances of newborn Wnt-4-deficient and wild-type males that had received daily injections of flutamide were significantly reduced compared with the controls, but not in Wnt-4-deficient females compared with wild-type females (Table 2).
Reduced androgen action leads to degeneration of the cortex of the ovaries in Wnt-4-deficient female embryos
The developing testis is covered by the tunica albuginea; no similar structure is apparent in the developing ovary (Fig. 5, A and B, arrow, compare with Fig. 5, C and D) (24). The ovary of a Wnt-4-deficient embryo is also surrounded by a thick membrane that structurally resembles the tunica albuginea and is thus clearly different from the corresponding wild-type structure (Fig. 5, E and F, arrow). No changes were detected in the cortical region of the wild-type testis or ovary after treatment with flutamide (Fig. 5, G and H, and Fig. 5, I and J), but the corresponding cortical structure in the ovaries of the Wnt-4-deficient females was either reduced or completely absent (Fig. 5, K and L, arrows).
Sexually dimorphic organization of endothelial cells depends on Wnt-4 signaling
Because Jeays-Ward et al. (25) showed that the coelomic vessels in Wnt-4-deficient females are organized as in males, we tested whether this specific pattern of endothelial cell assembly involves androgens by analyzing possible changes in endothelial organization in flutamide-treated embryonic gonads. Administration of flutamide did not change the sex-specific organization of endothelial cells in either sex or genotype (Fig. 6).
Discussion
The analysis of Wnt-4-deficient females revealed partial masculinization, because they had Wolffian ducts with structures resembling an epididymis and a fat body, and the ovary expressed some genes encoding enzymes involved in the testosterone synthesis pathway (2). Our results show that testosterone is indeed synthesized by the gonads of Wnt-4-deficient females and can also be detected in the plasma of Wnt-4-deficient newborn mice. It is likely that testosterone is also a functional mediator in the maintenance and further development of Wolffian ducts in Wnt-4-deficient females, because reduction of the action of androgens leads in most cases to degeneration of the single-sex duct type, the Wolffian duct. These results indicate that testosterone is the likely androgen synthesized in the Wnt-4-deficient ovary and that this is responsible for the maintenance and further development of the Wolffian duct in the mutant female. The findings are consistent with an earlier report showing that testosterone is sufficient to induce male reproductive structures in female fetuses (17).
The microarray gene chip studies revealed that several genes involved in the synthesis of hormones are induced in Wnt-4-deficient females. The enzymes involved in the stepwise synthesis of adrenal and gonadal hormones and those induced due to Wnt-4 deficiency are summarized in Fig. 7. Most of the pathway leading to the production of testosterone and also that of estradiol is induced in Wnt-4-deficient females. Consistent with the small elevation in the amount of DHEA in the gonads of Wnt-4-deficient mice, Cyp11a and Cyp17 gene expression, leading to the production of the respective enzymes and DHEA, was also induced in Wnt-4-deficient newborn females. The concentration of androstenedione in the gonad was below the limit of detection. This may also reflect the possibility that androstenedione is converted rapidly to testosterone by the enzymes Hsd17b1 and Hsd17b3, the expression of both of which is induced in Wnt-4-deficient newborn females compared with wild-type females. Because Hsd17b1 and Hsd17b3 are both capable of catalyzing the production of testosterone (for a review, see Ref.26), their induced expression is likely to explain the production of testosterone in the Wnt-4-deficient ovary.
The activity of the Cyp19 enzyme and that of Hsd17b1 and Hsd17b7 in females normally lead to the production of estrone and estradiol, respectively (26). All of the corresponding genes encoding these enzymes were expressed by the Wnt-4-deficient ovary. Expression of the Hsd17b1 gene was sexually dimorphic in newborn mouse ovaries and was thus detected in Wnt-4-deficient ovaries, but not in wild-type ovaries. Hsd17b1 gene expression was induced as much as 32-fold. Regardless of this, we could not detect the presence of estradiol in either the plasma or ovaries of Wnt-4-deficient newborn mice, suggesting that the enzymes concerned are not active at this stage. These findings are consistent with the fact that the biosynthesis of estrogen normally starts at a postnatal stage, in contrast to that of testosterone, which is already activated during fetal life. Because Hsd17b1 can also generate testosterone from androstenedione (26), its expression in the Wnt-4-deficient ovary may serve such a function. Nevertheless, we conclude that the Wnt-4-deficient ovaries still express a female character to some extent despite being masculinized.
It should be noted that we also observed significant down-regulation of the expression of the ER gene by a factor of 8.5 in Wnt-4-deficient newborn ovaries relative to wild type, whereas ER gene expression was slightly up-regulated at that time. This may be of significance for the partial sex reversal phenotype, because both ER and Wnt-4-deficient ovaries are characterized by the postnatal appearance of seminiferous tubule-like structures also expressing Sertoli cells markers such as AMH (2, 24). Hence, the reduced expression of ER may be a factor contributing to the sex reversal process in the Wnt-4-deficient ovary.
Wnt-4-deficient embryos produce testosterone, but this does not appear to be converted significantly to DHT, because we did not observe any changes in concentration in association with Wnt-4 deficiency. Moreover, the anogenital distance was not changed in response to flutamide, even though it was reduced by this antiandrogen in wild-type and Wnt-4-deficient males. Recent studies have demonstrated that 5-reductase-1 and -2 compound knockout mice are fertile, suggesting that testosterone is the major androgen responsible for the differentiation of the male urogenital tract (9). The amount of testosterone produced by the Wnt-4-deficient ovary is nevertheless likely to be too low to program the development of external genitalia in the male direction in Wnt-4-deficient females.
We previously reported that Wnt-4 is also functional in the development of the adrenal gland, so that a deficiency in signaling leads to reduced production of aldosterone (14). The presence of expression of the Cyp21 gene, which is normally expressed in the adrenal gland and gonad, suggested that some adrenal gland-derived cells may also enter the gonad during specification of the gonadal and adrenal gland primordia (14). This is consistent with observations by Jeays-Ward et al. (25). The gene chip analysis revealed that several of the genes that encode enzymes in the synthetic pathway for the mineralocorticoid aldosterone were expressed in the Wnt-4-deficient ovary, namely, Hsd3b1, Cyp21a, and Cyp11b2, but not Cyp11b1. We speculate that the lack of Wnt-4 signaling leads to a defect in cell sorting when the adrenal gonadal primordia are developing, and some adrenal gland cells may end up in the gonad. It is unlikely that these cells would contribute to the production of testosterone in the Wnt-4-deficient ovary, because the mouse adrenal gland does not produce testosterone (26), and consistent with this, we did not detect testosterone production in the adrenal gland of any genotype. We cannot exclude the possibility, however, that the original fate of the adrenal gland progenitors may undergo a change due to the lack of Wnt-4 signaling during their transition from the adrenal gland primordia to the gonad.
Jeays-Ward et al. (25) proposed that Wnt-4 signaling prevents formation of the male-specific coelomic vessel in the developing ovary. Because in this study we provided evidence that testosterone is produced by the Wnt-4-deficient ovary, an alternative option to consider is that testosterone would play a role in the organization of the male type of coelomic vessel in the developing testis and Wnt-4-deficient ovary. However, because administration of flutamide did not lead to a change in the organization of this vessel, this excludes the role of androgen action in the process.
In summary, we have shown that testosterone is detected in the ovaries and plasma of Wnt-4-deficient newborn mice and that androgens are involved in the generation of the partial female to male sex conversion due to deficiency in Wnt-4 signaling. Wnt-4 appears to play a critical role in controlling the expression of several genes involved in hormone synthesis, and the molecular mechanism of this control warrants additional investigations.
Acknowledgments
We thank Ms. Hannele Hrkman, Ms. Johanna Kekolahti-Liias, Ms. Jaana Kujala, and Ms. Mervi Matero for their excellent technical assistance. We are also grateful to Robin Lovell-Badge for the in situ hybridization probes.
Current address of H.P.: Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London, United Kingdom W12 0NN.
Footnotes
This work was supported by the Academy of Finland (Grants 107406 and 206038), the Sigrid Juselius Foundation, and the European Union (Grant LSHG-CT-2004-005085; to S.V.).
1 M.H. and R.P. contributed equally to the work.
Abbreviations: AMH, Anti-Mullerian hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E, embryonic day; ER, estrogen receptor.
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Abstract
Wnt-4 signaling has been implicated in female development, because its absence leads to partial female to male sex reversal in the mouse. Instead of Mullerian ducts, Wnt-4-deficient females have Wolffian ducts, suggesting a role for androgens in maintaining this single-sex duct type in females. We demonstrate here that testosterone is produced by the ovary of Wnt-4-deficient female embryos and is also detected in the embryonic plasma. Consistent with this, the expression of several genes encoding enzymes in the pathway leading to the synthesis of testosterone in the mouse is induced in the Wnt-4-deficient ovary, including Cyp11a, Cyp17, Hsd3b1, Hsd17b1, and Hsd17b3. Inhibition of androgen action with an antiandrogen, flutamide, during gestation leads to complete degeneration of the Wolffian ducts in 80% of the mutant females and degeneration of the cortical layer that resembles the tunica albuginea in the masculinized ovary. However, androgen action is not involved in the sexually dimorphic organization of endothelial cells in the Wnt-4 deficient ovary, because flutamide did not change the organization of the coelomic vessel. These data imply that Wnt-4 signaling normally acts to suppress testosterone biosynthesis in the female, and that testosterone is the putative mediator of the masculinization phenotype in Wnt-4-deficient females.
Introduction
ALTHOUGH INHERITANCE of an X- or Y-chromosome from the male establishes the genetic sex of the mammalian embryo at fertilization, the genes that determine the primary sex and subsequently lead to the establishment of secondary sexual characteristics are not activated until the gonad has developed, on embryonic d 10.5 and later, in the mouse. Initially the gonad is bipotential, but as a result of activation of the sex-determining gene Sry, the male sexual pathway becomes induced (1), whereas Wnt-4, a gene encoding a factor from the Wnt family of signaling molecules, is important for female development (2). After commitment of the male pathway, androgens secreted by differentiated Leydig cells are needed for the formation of the male secondary sexual characteristics beginning during embryogenesis, whereas the ovary becomes hormonally active at puberty (3, 4, 5). Thereafter, estrogen receptor (ER) and ER are critical in maintaining female sexual characteristics, because in their deficiency the ovary undergoes partial sex reversal to male and displays Sertoli cells (for a review, see Ref.6).
The paired primordia of the sex organs are composed of the indifferent gonads and the two sex ducts on either side of the developing embryo, the Wolffian and Mullerian ducts. In males, testosterone is required for subsequent development of the Wolffian duct, and this generates the epididymides, vasa deferentia, and seminal vesicles, whereas the Mullerian duct typical of females regresses in males due to the action of a Sertoli cell product, anti-Mullerian hormone (AMH) (for a review, see Ref.7). Morphogenesis of the male external genitalia is, in turn, thought to be controlled by dihydrotestosterone (DHT) (3, 4) that is produced from testosterone by the enzymes 5-reductase types 1 and 2. The type 2 enzyme is important in humans, because mutations in it lead to pseudohermaphroditism, characterized by a defective prostate and external genitalia (for a review, see Ref.8), but in the mouse, testosterone is the only androgen required for differentiation of the male urogenital tract (9).
In females, the lack of testosterone production leads to regression of the Wolffian ducts, whereas the Mullerian ducts persist in the presence of Wnt-4 signaling and in the absence of AMH (2). The Mullerian ducts develop into the oviduct, uterus, and upper part of the vagina of the adult female (3, 4).
The Wnt-4 gene is widely expressed during fetal life and is functional in organs such as the kidney (10), gonad (2, 11, 12, 13), Mullerian duct (2), adrenal gland (14), mammary gland (15), and pituitary gland (16), suggesting a pleiotropic function for this factor. Studies of Wnt-4 function in the mouse have revealed that it is an essential element in female sex organogenesis, supporting the idea that the specification of female sex depends on cell signaling and is not a passive default developmental pathway (2).
Based on studies of Wnt-4-deficient female embryos, it has been suggested that Wnt-4 regulates femaleness in three distinct phases (2). First, Wnt-4 is expressed in the indifferent gonad and is perhaps required normally for suppression of the expression of the genes encoding enzymes in the testosterone biosynthetic pathway. Second, Wnt-4 signaling is necessary for development of the secondary female characteristics, i.e. the Mullerian ducts, because these ducts are not able to form in either sex in its absence. Third, it is important for maintaining the oocytes.
Sex reversal in Wnt-4-deficient females is characterized by the presence of a Wolffian duct. The masculinization of females is also manifested by the presence of round, encapsulated ovaries that have a closely associated fat body, all of which are characteristic of males, suggesting that masculinization takes place due to synthesis of the androgen testosterone (2).
In this study we report evidence that testosterone is indeed produced by the ovaries of Wnt-4-deficient newborn mice. It can be measured in the gonad and plasma and may be the driving force for the masculinization phenotype in females, because blocking of androgen action with an antiandrogen, flutamide, leads to degeneration of the Wolffian duct and the cortical layer of the ovary in Wnt-4-deficient females. These findings are in line with an earlier report showing that testosterone is sufficient to induce male reproductive structures in female fetuses (17). Consistent with the presence of testosterone in Wnt-4-deficient females, several genes encoding enzymes that are involved in the synthesis of testosterone, such as Hsd17b1 and Hsd17b3, are expressed by the Wnt-4-deficient ovary.
Materials and Methods
Animals and their treatment with the antiandrogen flutamide
Crosses between the mouse strains 129SV and CD-1, both heterozygous for the targeted Wnt-4 allele, were used throughout to obtain embryos and newborn mice for the analysis. The targeted allele of Wnt-4 was genotyped as reported previously (10). For generation of Wnt-4-deficient embryos, heterozygous Wnt-4-deficient female and male mice were mated, and the day when the vaginal plug was detected in the mated female was designated embryonic d 0.5 (E0.5). The Wnt-4-deficient newborn mice were recognized by their hypoplastic kidneys, which is a typically penetrant phenotype, whereas other animals were genotyped (10).
For the generation of flutamide-treated embryos or newborn mice, 23 pregnant Wnt-4 heterozygote females were treated with sc injections of flutamide (F9397, Sigma-Aldrich Corp., St. Louis, MO; 100 mg/kg·d daily) from E10.5 until they were killed, and nine mice were treated with the vehicle, turnip oil. Flutamide functions by competing with androgens for binding to the androgen receptor and is therefore a useful tool for blocking androgen action during embryogenesis (18). All of the animal experimentation described in this study was conducted according to accepted standards of humane animal care, and adequate permits were obtained from the local ethical committee.
Analyses of hormones
The blood of decapitated newborn mice from Wnt-4 heterozygous intercourses was collected into heparinized glass tubes. Samples were centrifuged for 10 min at 3000 rpm and stored at –20 C. The plasma samples were pooled, six samples per tube, to provide sufficient volume. Gonadal tissues or embryonic adrenal glands were collected, frozen quickly, and stored at –20 C. The tissues were sonicated for 15 sec in 100 μl distilled water and washed twice with 300 μl of a solution containing 9 vol diethyl ether and 1 vol ethyl acetate. After centrifugation, the water phase was taken for protein assays, and the organic phases were combined, evaporated, and assayed for the presence of steroids. Protein concentrations in the gonadal samples were measured using a protein assay kit from Bio-Rad Laboratories (Hercules, CA).
The evaporated organic phase was reconstituted with 500 μl RIA buffer, and double aliquots of 25–100 μl were subjected to RIA. Concentrations of testosterone and estradiol were measured in plasma and gonad, and testosterone was determined in adrenal tissue samples using RIA kits from Orion DG (Oulu, Finland). Concentrations of dehydroepiandrosterone (DHEA) and androstenedione were measured in gonad tissue samples only, with RIA kits from DRG Instruments GmbH (Marburg, Germany). Plasma DHT was measured with an RIA kit (Intertech Sarl, Dudelange, Luxemburg). Due to the small plasma volumes available (20–40 μl), 150 μl 0.9% NaCl were added to each sample, and the amounts of the solutions used in the extractions were halved. According to the manufacturer’s information, the cross-reactivity of DHT antiserum with testosterone is 0.19%, and that with estradiol, progesterone, or DHEA is less than 0.005%. The coefficient of variation is less than 5%, and the interassay variation is between 8.7% and 18.6%. The detection limits of the hormones analyzed were 0.5 pmol/ml for testosterone, 0.7 fmol/ml for DHEA, 1 fmol/ml for androstenedione, 0.8 pmol/ml for DHT, and 50 fmol/ml for estradiol. Values below the limit of detection were recorded as zero.
Examination of the anogenital distance
The pups derived from heterozygous Wnt-4 matings were killed by decapitation on the day of delivery and quickly analyzed in Dulbecco’s PBS (Invitrogen Life Technologies, Inc., Gaithersburg, MD) on ice. The anogenital distance was measured before the sex or genotype was determined in each newborn mouse using a dissecting microscope and an attached micrometer lens, after which the urogenital systems were dissected for analysis. The males and females were distinguished according to the morphology of the gonad.
Histology, whole mount, and section in situ hybridization
The testes and ovaries from isolated urogenital systems were prepared and fixed in 4% paraformaldehyde overnight at 4 C, dehydrated in a graded ethanol series, incubated in xylene, embedded in paraffin, sectioned at 5 μm, and used for histological staining or in situ hybridization (see below). Hematoxylin-eosin staining was performed according to routine procedures (19).
For whole mount in situ hybridization, the prepared testes and ovaries were fixed in 4% paraformaldehyde overnight, dehydrated in a graded methanol series, and stored in 100% methanol at –20 C until used. The whole mount and section in situ hybridizations were performed according to standard procedures (20). The probes used were Hsd3b1 and amh (Robin Lovell-Badge, National Institute for Medical Research, London, UK), Cyp17 (expressed sequence tag AA822113, National Center for Biotechnology Information), and Hsd17b1 (21). After linearization of the plasmids containing the respective cDNA sequences, digoxigenin-labeled riboprobes were produced (digoxigenin RNA labeling kit, Roche, Indianapolis, IN).
Fixed or stained samples of the freshly dissected gonads were photographed with a DP50 digital camera (Olympus, New Hyde Park, NY) mounted on an MZ FLIII microscope (Leica Microsystems, Deerfield, IL). The images were processed with Viewfinder 3.0.1 software (Pixera Corp., Los Gatos, CA) and the Adobe Photoshop and Corel Draw programs (Adobe Systems, San Jose, CA).
Changes in the organization of endothelial cells in developing gonads
To study the effects of flutamide on the development of the gonadal vasculature, the endothelial cell-specific Tie1LacZ reporter gene (22) was crossed into a heterozygous Wnt-4+/– background by breeding Tie1LacZ and Wnt-4+/– mice together. Embryos were collected from flutamide- or vehicle-treated pregnant females from matings of Tie1LacZ+ and Wnt-4+/– mice. The endothelial cells of the isolated gonads were visualized by X-gal staining according to Kispert et al. (23).
Isolation of the RNA preparation and microarray analysis
Gonads were prepared from Wnt-4-deficient and wild-type embryos on E12.5 and E14.5 and from newborn mice. The separated gonads were frozen immediately in liquid nitrogen and stored at –70 C until used for the extraction of RNA. Total RNA was purified using the RNeasy Kit (Qiagen, Chatsworth, CA). RNA (5 μg) was used as a template for synthesizing cDNA and for making biotinylated cRNA, performed according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA). The biotinylated cRNA was hybridized to the GeneChip Mouse Expression Set 430_2.0 Array, which represents approximately 45,000 mouse transcripts. The arrays were scanned with GeneChip Scanner 3000, and the resulting expression data were analyzed with the Affymetrix GeneChip Operating System. The intensities of the signals for all probe sets were scaled to a target value of 500.
Results
Testosterone is detected in the plasma and gonad of Wnt-4-deficient females
Our previous report (2) indicated that the lack of Wnt-4 signaling in females leads to further development of the male sex ducts, the Wolffian ducts, instead of the Mullerian ducts, which typically develop in normal females. This suggested a role for androgens, namely testosterone, in the maintenance of this type of sex duct in Wnt-4-deficient females.
Measurements of testosterone concentrations in newborn mice obtained from Wnt-4+/– intercrosses revealed the presence of testosterone in blood samples from wild-type males and Wnt-4-deficient males and females, but not from wild-type females, as expected (Fig. 1A).
We also ascertained whether testosterone synthesis could be detected in the gonads of Wnt-4-deficient embryos. Consistent with the results in plasma, testosterone production was identified in wild-type and Wnt-4-deficient testes and also in the Wnt-4-deficient ovary, whereas no synthesis was detected in the wild-type ovary (Fig. 1B). No testosterone production was detected in the adrenal glands of wild-type or Wnt-4-deficient males or females (data not shown). We conclude that testosterone is synthesized by the Wnt-4-deficient ovary, making it a candidate mediator for the partial male to female sex reversal phenotype in Wnt-4-deficient females.
Synthesis of precursors of testosterone, DHT, and estradiol in ovaries of Wnt-4-deficient female mice
Because testosterone is produced via precursors such as androstenedione and dehydroepiandrosterone (DHEA), and at least some of the genes that encode these critical enzymes in their biosynthesis are also expressed in the Wnt-4-deficient ovary (2), we were interested in measuring changes in the amounts of these precursors in the gonads. The concentration of androstenedione was below the limit of detection, but that of DHEA was elevated in Wnt-4-deficient females compared with wild-type females (P < 0.01), whereas no significant differences were observed between wild-type and Wnt-4-deficient males (P = 0.091; Fig. 1C). No notable differences were observed in the amount of plasma DHT between the mutant females and males and their wild-type littermates; the average values were 0.83 nmol/liter for wild-type females, 2.36 nmol/liter for males, 0.77 nmol/liter for Wnt-4-deficient females, and 1.35 nmol/liter for males.
In addition to measuring the precursors that lead to the synthesis of testosterone and DHT, we addressed possible changes in the concentrations of plasma or gonadal estradiol in Wnt-4 deficiency, but found them to be below the level of detection in all samples analyzed regardless of the genotype.
Changes in expression of the genes encoding enzymes in the biosynthetic pathway of hormones in Wnt-4-deficient ovaries
To reveal the genetic changes that lead to the synthesis of ovarian testosterone in Wnt-4-deficient females, we used Affymetrix oligonucleotide gene chips containing the major genes involved in the synthesis of hormones. The gonads were separated out from E12.5 and E14.5 embryos and newborn mice, total RNA was purified and subjected to the gene chips, and changes in the genes involved in hormone synthesis were measured. The results, summarized in Table 1, indicate that Cyp11a, Cyp11b2, Cyp17, Cyp19, Cyp21a, Hsd3b1, Hsd3b6, Hsd17b1, and Hsd17b3 gene expression is induced in the ovaries of Wnt-4-deficient females compared with wild-type ovaries. In situ hybridization analysis revealed that, consistent with the gene chip studies, the Cyp17 and Hsd171 genes are ectopically expressed in Wnt-4-deficient ovaries and are also expressed in a sexually dimorphic manner (Fig. 2, A–D and I–L). Thus, Cyp17 and Hsd17b1 are expressed in wild-type and Wnt-4-deficient testes, but not in ovaries of wild-type newborn mice.
We also analyzed changes in the expression of the ER and ER genes, because a deficiency in these has been reported to lead to sex reversal in the ER-deficient ovary at the onset of puberty (6). ER was down-regulated at all stages analyzed in Wnt-4-deficient ovaries compared with the wild type, whereas ER was somewhat increased, especially at the later stages of ovarian development (Table 1).
Blocking androgen action leads to degeneration of the male Wolffian ducts in Wnt-4-deficient females
The antiandrogen flutamide was used to determine whether testosterone is responsible for maintaining the Wolffian duct in Wnt-4-deficient females. The effect of flutamide on growth of the wild-type testis was used first to test the effectiveness of the concentration of flutamide used. Although daily injections of placebo did not affect the overall size of the testis (Fig. 3A), the administration of flutamide (100 mg/kg) led to a considerable reduction in its size (Fig. 3B) and a change in the ratio between the area of the seminiferous tubules and that of the interstitial tissue in the testis compared with the placebo controls, as demonstrated by hematoxylin-eosin staining (Fig. 3, C and D). The expression of the AMH (amh) and Hsd17b3 genes demark the seminiferous tubules in flutamide-treated testes (Fig. 3, E and F). This result is consistent with earlier findings (18) and suggests that in vivo administration of flutamide at the concentration used is sufficient to inhibit the action of androgens.
The treatment of pregnant females with flutamide generated a phenotype, compared with the placebo controls, in Wnt-4-deficient female embryos with no gonad-associated sex ducts, as seen in 16 of the 20 cases analyzed (Figs. 4). In these flutamide-treated newborn Wnt-4-deficient mice, the rudimentary ovaries were connected to the rest of the urogenital system by thin, poorly developed ligaments (Fig. 4, C and F). In the remaining four flutamide-treated embryos, the Wolffian ducts had degenerated unilaterally (data not shown). The coiled epididymis-like organization that was detectable in the ovaries of placebo-treated Wnt-4-deficient females (Fig. 4, B and E) had degenerated on account of flutamide exposure, as shown in Fig. 4, C and F. Similarly, the overall organization of the ovary in Wnt-4-deficient newborn mice had altered from the circular pattern seen in placebo controls to a more elongated one (compare the ovaries in Fig. 4, E and F). Flutamide did not have any effect on the expression of the Cyp17 and Hsd17b1 genes in either sex or genotype, as judged by in situ hybridization analysis (Fig. 2, E–H and M–P).
Because the development of external genitalia is regulated by testosterone (9) and DHT in males, we were also interested in assaying potential changes in the anogenital distance, which is characteristically longer in males than in females. The anogenital distances of newborn Wnt-4-deficient and wild-type males that had received daily injections of flutamide were significantly reduced compared with the controls, but not in Wnt-4-deficient females compared with wild-type females (Table 2).
Reduced androgen action leads to degeneration of the cortex of the ovaries in Wnt-4-deficient female embryos
The developing testis is covered by the tunica albuginea; no similar structure is apparent in the developing ovary (Fig. 5, A and B, arrow, compare with Fig. 5, C and D) (24). The ovary of a Wnt-4-deficient embryo is also surrounded by a thick membrane that structurally resembles the tunica albuginea and is thus clearly different from the corresponding wild-type structure (Fig. 5, E and F, arrow). No changes were detected in the cortical region of the wild-type testis or ovary after treatment with flutamide (Fig. 5, G and H, and Fig. 5, I and J), but the corresponding cortical structure in the ovaries of the Wnt-4-deficient females was either reduced or completely absent (Fig. 5, K and L, arrows).
Sexually dimorphic organization of endothelial cells depends on Wnt-4 signaling
Because Jeays-Ward et al. (25) showed that the coelomic vessels in Wnt-4-deficient females are organized as in males, we tested whether this specific pattern of endothelial cell assembly involves androgens by analyzing possible changes in endothelial organization in flutamide-treated embryonic gonads. Administration of flutamide did not change the sex-specific organization of endothelial cells in either sex or genotype (Fig. 6).
Discussion
The analysis of Wnt-4-deficient females revealed partial masculinization, because they had Wolffian ducts with structures resembling an epididymis and a fat body, and the ovary expressed some genes encoding enzymes involved in the testosterone synthesis pathway (2). Our results show that testosterone is indeed synthesized by the gonads of Wnt-4-deficient females and can also be detected in the plasma of Wnt-4-deficient newborn mice. It is likely that testosterone is also a functional mediator in the maintenance and further development of Wolffian ducts in Wnt-4-deficient females, because reduction of the action of androgens leads in most cases to degeneration of the single-sex duct type, the Wolffian duct. These results indicate that testosterone is the likely androgen synthesized in the Wnt-4-deficient ovary and that this is responsible for the maintenance and further development of the Wolffian duct in the mutant female. The findings are consistent with an earlier report showing that testosterone is sufficient to induce male reproductive structures in female fetuses (17).
The microarray gene chip studies revealed that several genes involved in the synthesis of hormones are induced in Wnt-4-deficient females. The enzymes involved in the stepwise synthesis of adrenal and gonadal hormones and those induced due to Wnt-4 deficiency are summarized in Fig. 7. Most of the pathway leading to the production of testosterone and also that of estradiol is induced in Wnt-4-deficient females. Consistent with the small elevation in the amount of DHEA in the gonads of Wnt-4-deficient mice, Cyp11a and Cyp17 gene expression, leading to the production of the respective enzymes and DHEA, was also induced in Wnt-4-deficient newborn females. The concentration of androstenedione in the gonad was below the limit of detection. This may also reflect the possibility that androstenedione is converted rapidly to testosterone by the enzymes Hsd17b1 and Hsd17b3, the expression of both of which is induced in Wnt-4-deficient newborn females compared with wild-type females. Because Hsd17b1 and Hsd17b3 are both capable of catalyzing the production of testosterone (for a review, see Ref.26), their induced expression is likely to explain the production of testosterone in the Wnt-4-deficient ovary.
The activity of the Cyp19 enzyme and that of Hsd17b1 and Hsd17b7 in females normally lead to the production of estrone and estradiol, respectively (26). All of the corresponding genes encoding these enzymes were expressed by the Wnt-4-deficient ovary. Expression of the Hsd17b1 gene was sexually dimorphic in newborn mouse ovaries and was thus detected in Wnt-4-deficient ovaries, but not in wild-type ovaries. Hsd17b1 gene expression was induced as much as 32-fold. Regardless of this, we could not detect the presence of estradiol in either the plasma or ovaries of Wnt-4-deficient newborn mice, suggesting that the enzymes concerned are not active at this stage. These findings are consistent with the fact that the biosynthesis of estrogen normally starts at a postnatal stage, in contrast to that of testosterone, which is already activated during fetal life. Because Hsd17b1 can also generate testosterone from androstenedione (26), its expression in the Wnt-4-deficient ovary may serve such a function. Nevertheless, we conclude that the Wnt-4-deficient ovaries still express a female character to some extent despite being masculinized.
It should be noted that we also observed significant down-regulation of the expression of the ER gene by a factor of 8.5 in Wnt-4-deficient newborn ovaries relative to wild type, whereas ER gene expression was slightly up-regulated at that time. This may be of significance for the partial sex reversal phenotype, because both ER and Wnt-4-deficient ovaries are characterized by the postnatal appearance of seminiferous tubule-like structures also expressing Sertoli cells markers such as AMH (2, 24). Hence, the reduced expression of ER may be a factor contributing to the sex reversal process in the Wnt-4-deficient ovary.
Wnt-4-deficient embryos produce testosterone, but this does not appear to be converted significantly to DHT, because we did not observe any changes in concentration in association with Wnt-4 deficiency. Moreover, the anogenital distance was not changed in response to flutamide, even though it was reduced by this antiandrogen in wild-type and Wnt-4-deficient males. Recent studies have demonstrated that 5-reductase-1 and -2 compound knockout mice are fertile, suggesting that testosterone is the major androgen responsible for the differentiation of the male urogenital tract (9). The amount of testosterone produced by the Wnt-4-deficient ovary is nevertheless likely to be too low to program the development of external genitalia in the male direction in Wnt-4-deficient females.
We previously reported that Wnt-4 is also functional in the development of the adrenal gland, so that a deficiency in signaling leads to reduced production of aldosterone (14). The presence of expression of the Cyp21 gene, which is normally expressed in the adrenal gland and gonad, suggested that some adrenal gland-derived cells may also enter the gonad during specification of the gonadal and adrenal gland primordia (14). This is consistent with observations by Jeays-Ward et al. (25). The gene chip analysis revealed that several of the genes that encode enzymes in the synthetic pathway for the mineralocorticoid aldosterone were expressed in the Wnt-4-deficient ovary, namely, Hsd3b1, Cyp21a, and Cyp11b2, but not Cyp11b1. We speculate that the lack of Wnt-4 signaling leads to a defect in cell sorting when the adrenal gonadal primordia are developing, and some adrenal gland cells may end up in the gonad. It is unlikely that these cells would contribute to the production of testosterone in the Wnt-4-deficient ovary, because the mouse adrenal gland does not produce testosterone (26), and consistent with this, we did not detect testosterone production in the adrenal gland of any genotype. We cannot exclude the possibility, however, that the original fate of the adrenal gland progenitors may undergo a change due to the lack of Wnt-4 signaling during their transition from the adrenal gland primordia to the gonad.
Jeays-Ward et al. (25) proposed that Wnt-4 signaling prevents formation of the male-specific coelomic vessel in the developing ovary. Because in this study we provided evidence that testosterone is produced by the Wnt-4-deficient ovary, an alternative option to consider is that testosterone would play a role in the organization of the male type of coelomic vessel in the developing testis and Wnt-4-deficient ovary. However, because administration of flutamide did not lead to a change in the organization of this vessel, this excludes the role of androgen action in the process.
In summary, we have shown that testosterone is detected in the ovaries and plasma of Wnt-4-deficient newborn mice and that androgens are involved in the generation of the partial female to male sex conversion due to deficiency in Wnt-4 signaling. Wnt-4 appears to play a critical role in controlling the expression of several genes involved in hormone synthesis, and the molecular mechanism of this control warrants additional investigations.
Acknowledgments
We thank Ms. Hannele Hrkman, Ms. Johanna Kekolahti-Liias, Ms. Jaana Kujala, and Ms. Mervi Matero for their excellent technical assistance. We are also grateful to Robin Lovell-Badge for the in situ hybridization probes.
Current address of H.P.: Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London, United Kingdom W12 0NN.
Footnotes
This work was supported by the Academy of Finland (Grants 107406 and 206038), the Sigrid Juselius Foundation, and the European Union (Grant LSHG-CT-2004-005085; to S.V.).
1 M.H. and R.P. contributed equally to the work.
Abbreviations: AMH, Anti-Mullerian hormone; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E, embryonic day; ER, estrogen receptor.
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