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Endocrinology, doi:10.1210/en.2006-1054
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Endocrinology Vol. 148, No. 2 719-725
Copyright © 2007 by The Endocrine Society

Congenital Hypothyroid Female Pax8-Deficient Mice Are Infertile Despite Thyroid Hormone Replacement Therapy

Jens Mittag, Elke Winterhager, Karl Bauer and Ruth Grümmer

Max-Planck-Institut für Experimentelle Endokrinologie (J.M., K.B.), Department of Neuroendocrinology, D-30625 Hannover, Germany; and Institut für Anatomie (E.W., R.G.), Universitätsklinikum Essen, University Duisburg-Essen, D-45122 Essen, Germany

Address all correspondence and requests for reprints to: PD Dr. Ruth Grümmer, University Hospital Essen, Institute of Anatomy, Hufelandstrasse 55, 45122 Essen, Germany. E-mail: ruth.gruemmer{at}uni-due.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Absence of the Pax8 gene results in congenital hypothyroidism in mice, and mutations of the Pax8 gene have been associated with thyroid hypoplasia in humans. As in humans, treatment of congenital hypothyroid Pax8 null mice with thyroxine normalizes the known deficits. However, we report here that thyroxine-substituted female Pax8–/– mice are infertile because they lack a functional uterus revealing only remnants of myometrial tissue. In addition, the vaginal opening is absent. Interestingly, oviduct, cervix, and upper parts of the vagina are not affected, although Pax8 expression has been described in the entire Müllerian duct before. Because the natural outflow of the oviduct is impaired, a hydrosalpinx develops frequently. Folliculogenesis, ovarian hormone production, and transcription of pituitary hormones are in a normal range. Thus, infertility in Pax8–/– mice seems to be due to a defect in development of the Müllerian duct rather than to hormonal imbalance, pointing to a direct morphogenic role for Pax8 in uterine development. Because we demonstrated Pax8 expression not only in the uterine epithelium of mice but also in the human endometrium, it remains to be elucidated whether adequate development of the uterus may also be affected in congenital hypothyroid female patients with mutations in the Pax8 gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PAIRED BOX (Pax) gene family plays an essential role in body patterning during development. It has been shown that congenital hypothyroidism (CH), a disorder caused by thyroid dysgenesis or agenesis affecting one in 3500 newborns (1, 2), is associated with mutations in the Pax8 gene in humans, which result in severe thyroid hypoplasia (3). In mice where the Pax8 gene has been deleted, the thyroid gland is completely devoid of all thyroid hormone-producing follicular cells (4), making these athyroid mice an ideal animal model for CH. Although during embryogenesis Pax8 is expressed not only in the thyroid gland but also in other tissues such as the metanephros, the midhindbrain boundary region (5, 6), as well as in the Müllerian duct (7), defects have not been observed yet in these tissues. This fact is most likely explained by a partial redundancy of the highly homologous Pax2 and Pax5 gene products (4, 8). In most cases of congenital hypothyroid patients, virtually all the symptoms of cretinism such as growth and mental retardation can be reversed by the timely institution of thyroid hormone replacement therapy (9). Therefore, substitution of Pax8–/– mice with thyroid hormone was initiated already at postnatal d 2. As expected, these animals developed nicely without any overt deficits, whereas untreated Pax8–/– animals were severely growth retarded and died around weaning time (10). T4-treated Pax8–/– female mice, however, did not become pregnant when caged together with fertile wild-type males. This observation was surprising because in rodents, infertility is not generally associated with hypothyroidism (11). Even the hyt/hyt mouse, which is severely hypothyroid due to a point mutation in the TSH receptor of the thyroid gland, responds to thyroid hormone therapy with improved fertility (12). Analysis of the reproductive system of T4-treated female Pax8–/– mice revealed that the female infertility is not caused by any disturbances in pituitary gland hormones or ovary dysfunction. However, these animals fail to develop a functional uterus as well as a vaginal opening. These developmental defects are most likely related directly to the lack of the Pax8 transcription factor and became obvious only after appropriate T4 substitution enabling the knockout mice to survive to adulthood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals and human tissue samples
Animal procedures were approved by the animal welfare committee of the Medizinische Hochschule Hannover. Pax8+/– and Pax8–/– mice (4) were kept at a constant temperature (22 C) and automatic light cycle control (12 h light, 12 h dark) and were provided with standard laboratory chow and tap water ad libitum. Pax8–/– females were injected daily with thyroxine (18 ng/g body weight sc; Sigma Chemie, Deisenhofen, Germany) from postnatal d 2 onwards which restores a euthyroid status in these animals (10). Wild-type controls were litter mates of the Pax8–/– mice. Genotyping of Pax8 mice was performed as described elsewhere (13). All experiments were carried out in accordance to German laws for animal protection and with permission of the state (approval no. AZ 509c-42502-99/156, Bezirksregierung Hannover, Germany).

Endometrial tissue was obtained from women undergoing endometrial biopsy or hysterectomy at the Department of Gynecology, University Hospital Essen (Germany). Institutional ethical approval was obtained, and all women provided written informed consent. The stage of the menstrual cycle was confirmed by serum hormone determination using competitive immunoassays for estrogen (06792063, Bayer Diagnostics, Leverkusen, Germany) and progesterone (Bayer Diagnostics 01586287) (progesterone concentrations > 1.0 ng/ml were allocated to the secretory phase, and samples below that level were allocated to the proliferative phase) and by histological staging according to Noyes et al. (14).

In situ hybridization (ISH)
After the animals were decapitated, tissues were removed rapidly, embedded in Tissue-Tek medium (Sakura Finetek, Torrance, CA), and frozen on dry ice. Sections (16 µm) were cut on a cryostat (Leica, Bentheim, Germany), thaw-mounted on silane-treated slides, and stored at –80 C until further processing. ISH histochemistry was carried out as described previously (15). Briefly, frozen sections were fixed in a 4% phosphate-buffered paraformaldehyde solution (pH 7.4) for 1 h at room temperature, rinsed with PBS, and treated with 0.4% phosphate buffered Triton X-100 solution for 10 min. After washing with PBS and water, tissue sections were incubated in 0.1 M triethanolamine (pH 8) containing 0.25% (vol/vol) acetic anhydride for 10 min. After acetylation, sections were rinsed several times with PBS, dehydrated by successive washing with increasing ethanol concentrations, and air dried.

Radioactive-labeled probes were generated from cDNA subclones in Bluescript SKII+ plasmids. In vitro transcription was carried out according to standard protocols with (35S)-UTP and (35S)-CTP as labeled nucleotides (nt) (16). The probe was prepared from a cDNA fragment corresponding to nt 887-1177 (accession no. NM_011040) of Pax8.

Radioactive cRNA probes were diluted in hybridization buffer [50% formamide, 10% dextran sulfate, 0.6 M NaCl, 10 mM Tris · HCl (pH 7.4), 1x Denhardt’s solution, 100 µg/ml sonicated salmon sperm DNA, 1 mM EDTA, and 10 mM dithiothreitol] to a final concentration of 5 x 104 cpm/ml. After application of the hybridization mix, sections were coverslipped and incubated in a humid chamber at 58 C for 16 h. After hybridization, coverslips were removed in 2x standard saline citrate [SSC; 0.3 M NaCl, 0.03 M sodium citrate (pH 7.0)]. The sections were then treated with ribonuclease A (20 µg/ml) and ribonuclease T1 (1 U/ml) at 37 C for 30 min. Successive washes followed at room temperature in 1x, 0.5x, and 0.2x SSC for 20 min each and in 0.2x SSC at 65 C for 1 h. The tissue was dehydrated and exposed to Biomax MR Film (Eastman Kodak, Rochester, NY; Sigma Chemie) for 48 h. For microscopic analysis, sections were dipped in NTB2 (Eastman Kodak; INTEGRA Biosciences GmbH, Fernwald, Germany) nuclear emulsion and stored at 4 C. After exposure for 14 d, autoradiograms were developed in D19 (Eastman Kodak; Sigma Chemie) for 4 min and fixed in Rapid Fix (Eastman Kodak; Sigma Chemie) for 4 min. If required, sections were counterstained with cresyl violet and then photographed under dark- or bright-field illuminations.

Digoxigenin-labeled probes were generated from cDNA subclones in pGEM-plasmids (Promega, Mannheim, Germany) with a DIG RNA Labeling Kit (Boehringer, Mannheim, Germany). In vitro transcription was carried out according to standard protocols. Probes were generated from cDNA fragments corresponding to nt 190–445 (accession no. M10902) of ß-TSH, nt 248–445 (accession no. U62779) of GH, nt 1566–1749 (accession no. J00769) of prolactin (PRL), nt 56–526 (accession no. J00759) of proopiomelanocortin (POMC), nt 1–880 (accession no. M36804) of ß-FSH, and nt 31–488 (accession no. NM_012858) of ß-LH.

The digoxigenin-labeled probes were diluted in hybridization buffer to a final concentration of 5 ng/µl. Hybridization and posthybridization were performed as described for radioactive ISH. Sections were then rinsed with P1 [(100 mM Tris, 150 mM NaCl (pH 7.5)] and incubated for 2 h in blocking solution provided by the manufacturer of the kit. After incubation overnight with antidigoxigenin antibody conjugated with alkaline phosphatase (1:1000 dilution, Boehringer), the tissue sections were washed with P1. Staining proceeded for 2–6 h in substrate solution containing nitroblue tetrazolium chloride (340 µg/ml; Biomol, Hamburg, Germany), X-Phosphate (5-bromo-4-chloro-3-indolyl phosphate, 175 µg/ml; Biomol), 100 mM Tris, 100 mM NaCl, and 50 mM MgCl2 (pH 9.0).

Histology and immunohistochemistry
Seven 7-wk-old and four 6-month-old mice were killed by cervical dislocation and examined macroscopically in comparison with four wild-type mice. Organs of the female reproductive tract (ovaries, oviduct, uterus, cervix, vagina) were dissected, and the tissues were fixed overnight in 4% phosphate-buffered paraformaldehyde in PBS at pH 7.2, dehydrated through an ethanol series, and embedded in paraffin. For histological examination, serial sections of 7 µm were prepared from all organs of wild-type and T4-treated Pax8–/– mice and stained with hematoxylin and eosin.

For immunostaining, paraffin sections were deparaffinized and rinsed in PBS containing 0.5% BSA to reduce nonspecific antibody binding. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. After washing in PBS, sections were incubated for 1 h at room temperature with rabbit-anti-{alpha}-smooth muscle actin (RB-9010, Lab Vision, Fremont, CA) and biotinylated goat antirabbit immunoglobulin (E0432, DakoCytomation Denmark A/S, Glostrup, Denmark) as secondary antibody. The chromogenic reaction was carried out by incubating the sections with the peroxidase substrate 3,3'-diaminobenzidine for 5 min; sections were rinsed in PBS, dehydrated in ascending ethanol concentrations, and coverslipped. To demonstrate specificity of the staining, consecutive sections were stained with the same protocol, except that the primary antibody was omitted. To confirm both orientation and localization, parallel sections were stained with hematoxylin-eosin and recorded with a Zeiss Axiophot photomicroscope (Carl Zeiss GmbH, Jena, Germany).

Hormone measurements
Trunk blood was obtained from 13 Pax8–/– and from 16 wild-type mice and collected in microtubes without anticoagulant. After coat formation at 4 C for 4 h, the samples were centrifuged, and the recovered serum was stored at –20 C until assayed. Serum hormone levels were measured using competitive immunoassays for estrogen (Bayer Diagnostics 06792063) and progesterone (Bayer Diagnostics 01586287).

Statistical analysis
The differences in hormone levels were analyzed with the Mann-Whitney test for the nonparametric independent two-group comparisons (SPSS version 10, SPSS Inc., Chicago, IL). Differences with P < 0.05 were regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T4-substituted Pax8–/– mice are infertile
As a consequence of their athyroidism, Pax8–/– mice die around weaning time. Because thyroid hormone replacement therapy generally restores the deficits observed in congenital hypothyroid Pax8–/– mice (10, 17), these animals were treated with T4 from postnatal d 2 onwards in the expectation that they survive to adulthood and become fertile. Although T4-treated female Pax8–/– animals developed similarly to wild-type litter mates (10, 18), they did not become pregnant when caged together with fertile male wild-type mice.

Expression of pituitary hormones in T4-substituted Pax8–/– mice
Untreated Pax8–/– mice exhibit a dramatically distorted cellular composition of the anterior pituitary with hypertrophy and hyperplasia of the thyrotropes, an almost complete absence of lactotropes, and a drastically reduced number of somatotropes (10). We therefore analyzed the mRNA expression of ß-TSH, PRL, GH, LH (ß-LH), ß-FSH, and POMC by ISH in T4-substituted Pax8–/– mice to assess whether an impaired expression of these hormones might be the cause for their infertility. In contrast to 3-wk-old untreated Pax8–/– mice, pituitaries of T4-treated female Pax8–/– mice at 3 months of age showed no obvious differences in the mRNA expression of the hormones analyzed or in the cellular composition of the gland compared with wild-type litter mates (Fig. 1Go). This was even true for the expression of ß-TSH, which is tightly controlled by the negative feedback of thyroid hormones, thereby clearly illustrating the euthyroid status of the T4-substituted animals.


Figure 1
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  		FIG. 1. In situ hybridization revealing mRNA expression of ß-TSH, PRL, GH, LH (ß-LH), ß-FSH, and POMC in anterior pituitaries of female wild-type and T4-treated Pax8–/– mice at 3 months of age as well as untreated Pax8–/– mice at 3 wk of age. Scale bar, 500 µm.

 
Histomorphology of ovaries and production of ovarian steroid hormones
All T4-substituted female Pax8–/– mice exhibited normal ovarian morphology at 6 months of age as shown by the presence of different stages of follicle development similar to wild-type controls (Fig. 2Go, A and B). Besides mature follicles, corpora rubra and corpora lutea could also be seen (data not shown) giving proof of ovulation. Serum hormone levels of 17ß-estrogen and progesterone did not show significant differences in these adult T4-substituted Pax8–/– mice when compared with wild-type controls (Fig. 2Go, C and D).


Figure 2
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  		FIG. 2. Histological and appearance of ovaries and serum hormone levels of wild-type and T4-substituted Pax8–/– mice at 6 month of age. No difference in development of ovarian follicles can be seen in T4-treated Pax8–/– mice (B) compared with wild-type mice (A). Likewise, there was no significant difference in serum estrogen (C) and progesterone (D) levels between these two populations (P < 0.05). Scale bar in A and B, 300 µm.

 
Macroscopic evaluation of the female genital tract
At external inspection, all T4-substituted Pax8–/– mice investigated at 7 wk as well as at 6 months of age exhibited a vagina closed by a lucent membrane (Fig. 3BGo). After laparotomy, ovarian gross morphology was inconspicuous; however, in the abdominal situs, the two uterine horns were missing (Fig. 3DGo). Instead, only a very thin ligament could be seen in all T4-substituted Pax8–/– mice investigated. An obvious manifestation of a dilated, fluid-filled fallopian tube (hydrosalpinx) had developed in nine of 11 (82%) of mice within 6 months after birth (Fig. 3Go, D and F).


Figure 3
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  		FIG. 3. Gross anatomy of the female genital tract of wild-type and T4-substituted Pax8–/– mice. In contrast to wild-type mice (A), adult 6-month-old T4-treated Pax8–/– mice lack a vaginal opening (B, arrow). Inspection of the abdominal situs (C and D) revealed a very thin structure at the topographic site of the uterus (Ut) in T4-treated Pax8–/– mice, and a prominent fluid-filled fallopian tube was observed in 82% of mice [hydrosalpinx (HS)] (D). This is even more obvious when comparing the isolated female reproductive tract of wild-type (E) and T4-treated Pax8–/– (F) mice. Scale bar, 3.5 mm. Ov, Ovary.

 
T4-substituted Pax8–/– mice lack a functional uterus
Histological examination confirmed the lack of a normal uterus in T4-substituted Pax8–/– mice. Surrounded by fat, we found only small remnants of ligament-like tissue (Fig. 4BGo), which considerably differed in size compared with wild-type mice (Fig. 4AGo). Histologically, this residual tissue revealed the morphology of smooth muscle cells that was confirmed by staining for {alpha}-smooth muscle actin (Fig. 4Go, C and D), pointing to a myometrial origin. The endometrial compartment and the formation of a lumen were missing.


Figure 4
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  		FIG. 4. Histomorphology of the female reproductive tract of T4-substituted Pax8–/– mice. In comparison with wild-type uteri (A), T4-substituted Pax8–/– mice at 6 months of age lack a uterus and reveal at the same magnification only small remnants of uterine tissue surrounded by fat tissue (B). These tissue residues of T4-treated Pax8–/– uteri showed a histological appearance of smooth muscle cells (Sm, C), which could be confirmed by staining of a parallel section for {alpha}-smooth muscle actin (D). Most regions of the fallopian tube of T4-substituted Pax8–/– mice appeared histologically normal (E). However, those segments where a hydrosalpinx had developed were dilated and revealed a flattened mucosal layer (F). The cervices were adequately developed showing the typical wall structure of this organ but also a dilated fluid-filled lumen (G). The vagina wall of the T4-treated Pax8–/– mice showed normal histomorphology with squamous epithelium and underlying stroma (H). Scale bar in A, B, and H, 400 µm; in C and D, 150 µm; in E and F, 300 µm; and in G, 125 µm).

 
In contrast to the uterus, the oviduct of T4-treated Pax8–/– mice appeared macroscopically normal and revealed no obvious histomorphological differences compared with wild-type mice (Fig. 4EGo), apart from those segments that were dilated by the development of a hydrosalpinx (Fig. 4FGo). Here, an extended fluid-filled lumen lined by a flattened mucosal layer was observed. Interestingly, the cervices were appropriately developed showing the typical wall morphology of this organ. Due to the lack of the natural outflow of secreted fluid via the vaginal opening, however, dilated fluid-filled lumina could be observed also in the cervix (Fig. 4GGo). The vagina itself had developed normally, showing an appropriate differentiation into a squamous epithelium with underlying submucosa and smooth muscle cell layers organized in a loose arrangement together with connective tissue (Fig. 4HGo).

Pax8 mRNA expression in the murine and human uterus
To investigate whether the observed phenotype might be related to the inactivation of the Pax8 gene, we analyzed by ISH the mRNA expression of Pax8 in the female reproductive system of 3-wk-old as well as sexually mature wild-type mice. Pax8 transcripts were detected in the entire uterine epithelium (Fig. 5Go, A, D, and E) as well as in the luminal epithelium of the oviduct (Fig. 5Go, B and C) and vagina but not in the ovaries of the immature mice. The same staining pattern was observed in the reproductive system of fertile 9-month-old wild-type females (not shown).


Figure 5
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  		FIG. 5. In situ hybridization analysis of Pax8 mRNA expression in the female reproductive tract of wild-type mice. Dark-field illumination reveals a clear staining of Pax8 mRNA in the epithelial lining of the oviduct (Ov) and the uterus (Ut) (overview in A, higher magnification of the ovary in B and of the uterus in D). C and E, Corresponding cresyl violet counterstaining to B and D, respectively. Scale bar in A, 3 mm; and in B and E, 1 mm. *, Ovary.

 
To further analyze whether Pax8 gene expression may also play a role in the human reproductive system, Pax8 mRNA expression was analyzed in human endometrial tissue samples from the secretory (Fig. 6Go, A and B) and the proliferative phase (Fig. 6Go, C and D) of the menstrual cycle. Corresponding to the situation in the mouse, strong signals of Pax8 transcripts were observed in both phases of the cycle in the luminal and glandular epithelium of the human endometrium.


Figure 6
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  		FIG. 6. In situ hybridization analysis of Pax8 mRNA expression in the human endometrium. Dark-field illumination illustrates Pax8 mRNA expression in endometrial tissue samples from the secretory (A) and proliferative phase (C) with the corresponding neighboring sections counterstained with cresyl violet (B and D). In both phases of the menstrual cycle, the glandular (GE) as well as the luminal (LE) epithelial cells revealed a clear staining for Pax8. Scale bar, 150 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Pax8 null mutant mouse completely lacks thyroid hormone-producing follicular cells (4, 10, 19, 20), which makes it a perfect animal model for CH. Apart from this thyroid defect, no further abnormalities have been described so far in other Pax8-expressing tissues (4). Because physiological disturbances due to hypothyroidism can be normalized by adequate substitution with T4 (10), it was very surprising that these mice were infertile.

It is well known that reproduction is influenced by thyroid hormones. Although hypothyroidism has been implicated in adult women with a broad variety of reproductive disturbances (11), fetal hypothyroidism does not affect female reproductive tract development in humans (21). In rats, fetal hypothyroidism has been associated with smaller ovaries and follicles, an underdeveloped uterus and vagina, as well as delayed vaginal opening (11). Because Pax8–/– animals are born by euthyroid Pax8+/– mothers, fetal thyroid hormone supply should not be seriously affected in these mice. Indeed, the ovaries of the T4-treated Pax8-deficient animals are normal; however, the uterus is absent, and vaginal opening does not occur at all. Taken together with the fact that the hormonal situation was also found to be normal after T4 treatment and the observation that TR{alpha}1–/–TRß–/– mice, being devoid of all functional TH receptors, are generally able to reproduce (22), the strong developmental impairment of the female reproductive tract in Pax8–/– mice might rather be directly related to the lack of the Pax8 gene during uterine development than to hormonal imbalance.

The murine Pax gene family consists of nine members (Pax1–9) that regulate fundamental events in body patterning during development (23). Pax8 is expressed during embryogenesis in various tissues such as the thyroid gland, inner ear, pro-, meso- and metanephros, the cloaca, and the midhindbrain boundary region (5, 6). Additionally, Pax8 expression was also found in the epithelium of the Müllerian duct (7), the origin of the female reproductive tract in mammals that differentiates into the oviducts, uterus, cervix, and the upper portion of the vagina along the anteroposterior axis. In rodents, the epithelial invaginations of the mesonephros forming the Müllerian duct occur around embryonic d 11.5 and extend posteriorly reaching the cloaca at approximately embryonic d 13.5 (24), where limited Müllerian duct fusion leads to the formation of a duplex uterus and dual cervix (for review, see Ref. 25). The different segments of the female reproductive tract exhibit distinct morphologies and cytoarchitecture (26), and the postnatal establishment of uterine histoarchitecture forming the different elements of the uterine wall, that is endometrium, myometrium, and perimetrium, is not completed until 2 wk after birth (27, 28, 29). As shown in the present study, Pax8 seems to play an important role in this postnatal differentiation of the Müllerian duct forming an adequate histoarchitecture of the uterus because Pax8 null mutant mice only show remnants of myometrium and a complete lack of endometrial structures. Interestingly, the appropriate development of the oviduct, cervix, and vagina is not affected.

Patterning events required for differentiation from the Müllerian derivatives occur in both the anteroposterior and radial axes (25). Obviously, anteroposterior patterning establishing histologically distinct segmental boundaries between the oviducts and the uterus as well as between the uterus and cervix is not disturbed in T4-treated Pax8–/– mice. However, these animals display defects in radial patterning establishing the tissue-specific uterine morphology.

In addition to the lack of a functional uterus, adult T4-substituted Pax8-deficient mice also lack a vaginal opening, although the vaginal development is normal. In wild-type mice, the opening of the vagina occurs at about 6–7 wk of age, around the onset of sexual maturity (30). It is known that developmental defects in the caudal sections of the urogenital tract can result in an imperforate vagina, which may lead to the accumulation of secretion products and subsequently to a marked distention of the vagina (31). The persistence of an imperforate vagina in the mouse is believed to be inherited in a recessive manner (32), and the role of Pax8 in this developmental step is not known yet.

Obviously, the lack of Pax8 expression can be compensated for by other factors during fetal development of the oviduct, cervix, and vagina but not in the postnatal development of the uterine histoarchitecture and during vaginal opening. Although Pax8 has been shown to be expressed in the entire Müllerian duct (4, 24), the absence of the Pax8 gene leads to a lack of the mucosal layer, the endometrium, only in the uterus, but not in the other tissues developing from this embryonal structure.

Redundantly acting transcription factors could be responsible for the normal development of these other parts of the reproductive tract. Coexpression of Pax2 with Pax8 has already been demonstrated in the developing Müllerian duct as well as in the kidney (4, 24), and an overlapping gene function has been shown for Pax2 and Pax8 during development of the mouse urogenital system (5, 33) as well as in otic development (33). This compensation obviously does not apply for endometrial development. Although embryonic development of the Müllerian duct does not seem to be disturbed in Pax8-deficient mice, the degeneration of the Müllerian duct during embryogenesis in Pax2 null mutants cannot be compensated for by Pax8, leading to a complete lack of the uterus and oviducts in these mice (34). However, one may speculate that Pax2 can partly compensate for Pax8 during the development of the oviduct, cervix, and vagina. Furthermore, in the developing uterus, Pax8 might be important for interactions with other transcription factors. However, as yet, only a small set of transcription factors and signaling molecules that are essential for female reproductive tract formation and differentiation have been identified in mice (for review, see Refs. 24 , 25 , 35). For example, LIM class homeodomain transcription factor-1 has been described to be expressed in the Müllerian duct epithelium (36) and thus could be a candidate transcription factor to interact with Pax8 because the myometrial part of the uterus is still recognizable. In addition, members of the Wnt family, Wnt5a and Wnt7a, have been shown to play a role in endometrial adenogenesis (37, 38) and should also be analyzed with regard to their possible interactions with Pax8.

Prenatal organogenesis as well as postnatal morphogenesis are obviously complex, multifactorial processes. According to our analysis, Pax8 seems to be an important player in the genetic pathway leading to female reproductive tract development. Similar to mouse embryonic development, Pax8 is also expressed in various structures of the human embryo such as the thyroid anlage, the otic vesicle, the midhindbrain boundary, and in the metanephric blastema and derivatives (39). Mutations of Pax8 found in human patients have been associated with the pathogenesis of thyroid dysgenesis and hypoplasia (3, 40, 41). These disorders do not lead to symptoms of cretinism nowadays because congenital hypothyroid patients are identified in early postnatal screenings and adequately treated with T4. To our knowledge, however, female patients with Pax8 mutations have not been analyzed with regard to fertility and the development of their uterus. Our observation that Pax8 is expressed not only in the uterine epithelium of mice but also in human tissue samples seems to indicate that the development of the endometrium might also be impaired in these women. This aspect warrants careful investigation.


   Acknowledgments
 
We thank Melanie Kraus, Petra Affeldt, and Georgia Rauter for excellent technical assistance and genotyping. We also thank Valerie Ashe for linguistic help and Ahmed Mansouri and Peter Gruss for kindly providing Pax8+/– mice.


   Footnotes
 
The authors have nothing to disclose.

First Published Online November 2, 2006

Abbreviations: CH, Congenital hypothyroidism; ISH, in situ hybridization; nt, nucleotide(s); POMC, proopiomelanocortin; PRL, prolactin; SSC, standard saline citrate.

Received August 2, 2006.

Accepted for publication October 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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