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Endocrinology, doi:10.1210/en.2006-0299
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Endocrinology Vol. 147, No. 8 3777-3788
Copyright © 2006 by The Endocrine Society

Sprouty2 Is Involved in Male Sex Organogenesis by Controlling Fibroblast Growth Factor 9-Induced Mesonephric Cell Migration to the Developing Testis

Lijun Chi, Petri Itäranta, Shaobing Zhang and Seppo Vainio

Department of Biochemistry (L.C., P.I., S.Z., S.V.), University of Oulu, FIN-90014 Oulu, Finland; and Department of Medical Biochemistry and Molecular Biology (L.C., P.I., S.V.), Biocenter Oulu, Laboratory of Developmental Biology, University of Oulu, FIN-90014, Oulu, Finland

Address all correspondence and requests for reprints to: Prof. Seppo Vainio, Biocenter Oulu, Laboratory of Developmental Biology, P.O. Box 5000, Aapistie 5A, FIN-90014 University of Oulu, Finland. E-mail: seppo.vainio{at}oulu.fi.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fibroblast growth factor 9 (FGF9) signal has a role in organogenesis of the mammalian testis by controlling migration of mesonephric cells to the XY gonad, but neither it nor the FGF receptors is expressed sex-specifically. Of the Sprouty genes encoding antagonists of receptor tyrosine kinases including FGFr, mSprouty2 expression was confined to the developing testis and mesonephros. Gain of SPROUTY2 function in the male genital ridge and mesonephros malformed the vas deferens and epididymis, and diminished the number of seminiferous tubules and interstitium associating with reduced mesonephric cell migration and Fgf9 expression in embryonic testis, whereas exogenous FGF9 signaling recovered mesonephric cell migration inhibited by SPROUTY2. These phenotypes associated also with the decreased expression of Sox9, Desert hedgehog, Hsd3ß, Platelet/endothelial cell adhesion molecule, and {alpha}-smooth muscle actin, which are markers of the Sertoli, Leydig, endothelial, and peritubular myoid cells of the developing testis. Based on these data, we propose that the Sprouty proteins are involved normally in mediating the sexually dimorphic signaling of FGF9 and controlling cell migration from the mesonephros during testis development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MALE AND FEMALE embryos possess initially a sexually indifferent gonad that becomes committed to ovary or testis development as a result of interactions between the mesenchymal cells derived from the mesonephros and the epithelial cells of the genital ridge (1, 2, 3). It is thought that the genital ridge epithelium generates the Sertoli cells, whereas many of the other somatic cell types, including at least endothelial cells and peritubular myoid cells, are derived from the mesonephros by migration into the XY gonad (4, 5, 6, 7). The extragonadal germ cells are encapsulated into the seminiferous tubules encircled by the peritubular myoid cells (8, 9).

One of the important steps in male sex organogenesis occurs when the differentiating Sertoli cells start to secrete anti-Müllerian hormone (amh), which induces regression of the female Müllerian duct, while the differentiating Leydig cells initiate the production of testosterone, which promotes the development of the other sex duct, the male Wolffian duct, into the epididymis, vas deferens, and seminal vesicles. Insulin-like 3, a factor produced by the Leydig cells, is also implicated in Wolffian duct development because its deficiency leads to bilateral cryptorchidism (10, 11).

In females, the absence of amh and testosterone and the presence of Wnt-4 and Wnt-7a signaling leads to development of the Müllerian duct into the oviduct, uterus, and upper part of the vagina, whereas the Wolffian duct degenerates in the absence of amh and testosterone (12, 13, 14, 15).

Male sex is thought to be determined as a result of the function of a single gene on the Y chromosome, the sex-determining region of the Y chromosome, or Sry (16), which is expressed transiently between embryonic day (E) 10.5 and E12.0 in the XY gonad (17, 18), whereas the female sexual differentiation pathway involves the action of at least the Wnt-4 and Dax1 genes (15, 19, 20, 21), also taking part in male sexual development (22, 23). The mechanism by which Sry induces male determination is still not well understood, and no direct targets for Sry have been identified so far. Sox9, which encodes a Sry-related HMG box DNA-binding protein, is a candidate downstream target gene of Sry and is expressed in the Sertoli cells (24, 25), and its overexpression induces female to male sex reversal (26). Heterozygous mutations in the Sox9 gene also lead to dwarfism and gonadal dysplasia in humans (27).

Fibroblast growth factor 9 (Fgf9), a member of the fibroblast growth factor gene family is a candidate downstream factor for Sry as well. It is expressed in the Wolffian duct, the mesonephric tubules, and the Sertoli cells of the developing testis, and deficient mice undergo sex reversal from male to female. FGF9 signaling stimulates mesenchymal cell proliferation and the migration of mesonephric cells into the testis (28), which is one critical cellular consequence of Sry activation, contributing to the formation of the interstitial compartment of the testis (29).

Desert hedgehog (Dhh), a member of the Hedgehog group of signals, is another secreted signal implicated in male sexual development, being expressed in the Sertoli cells. Dhh-deficient embryos have defects in the morphogenesis of the testis, so that the organization of the peritubular myoid cells is abnormal (30, 31). The kidneys are missing in embryos deficient in Lim1, which encodes like Sox9 a transcription factor (32). Lim1 also regulates Müllerian duct development (33).

Sprouty genes encode cytoplasmic membrane-associated proteins and function by inhibiting receptor tyrosine kinase signaling, i.e. the function of Fgf receptors, by blocking the action of Ras and the downstream pathway, such as the activation of ERKs (34, 35). Sprouties play a role in the morphogenesis of such organs as the lungs (36), kidneys (37, 38), limbs (39), and inner ear (40). Gain and loss of function studies have both given valuable information on the in vivo roles of the Sprouties, and indicated that they regulate epithelial branching in the developing lung and kidney (36, 37, 38). In addition, Sprouty2 controls cell proliferation and migration (38, 39, 41).

In this paper, we demonstrate that mSprouty2 expression is confined to the testis and mesonephric tubules at the early stages of organogenesis. A gain of SPROUTY2 function in the embryonic testis and mesonephric tubules disturbed the formation of the seminiferous tubules and epididymis and reduced the amount of testicular interstitial tissue associated with diminished expression of the Leydig cell marker Hsd3ß and Fgf9. The SPROUTY2-generated phenotype is probably caused by an inhibitory effect of the encoded protein on the migration of mesonephric cells into the testis promoted by FGF9. We propose that Sprouty2 is normally involved in organogenesis of the testis by regulating Fgf9-controlled mesonephric cell migration into the testis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation and genotyping of mouse strains
Generation of the mouse line in which the expression of SPROUTY2 and green fluorescent protein (GFP) is subject to the Pax2 promoter has been described earlier (38). Briefly, the full-length SPROUTY2 and GFP cDNAs were cloned at 3' of the 4.3-kb Pax2 promoter and transgenic mice were developed via pronuclear injections. To analyze the genetic status of the SPROUTY2 transgenic mice or embryos, DNA derived from ear or tail clips was digested with BamHI and the digests were run on agarose gels, blotted and probed with the 32P-labeled GFP probe (771 bp) for detection of the expressed transgene. PCR was also used for the genotyping (38). SPROUTY2/GFP-expressive embryos were generated by mating females and males with a wild-type C57BL/6J strain or with another SPROUTY2-expressive transgenic mouse. The CD-1 mouse line was a local colony, whereas the Rosa26 line, in which the LacZ gene is expressed under a constitutively active promoter, was obtained from Dr. Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA) (42, 43). After the initial analysis and comparison of the phenotypes in embryos obtained from crosses between the transgenic TG/WT, TG/TG, and wild-type (WT) embryos, the TG/WT embryos were used throughout the study. All animal experimentation described in this work has been conducted in accord with accepted standards of humane animal care and was approved by the local ethical committee.

Preparation of tissues, histology, and in situ hybridization
Embryonic reproductive systems at the ages indicated in the results section were dissected in ice-cold PBS, pH 7.4, fixed in Bouin’s solution, embedded in paraffin, and processed for histology by routine methods. Serial sections of 6 µm thickness were cut, dewaxed, stained with hematoxylin and eosin, and mounted for inspection. Testis sections (middle position) of newborn (NB) mice were analyzed at x10 magnification to count the number of seminiferous cords (44). The sections were photographed with a Leica CD 100 digital or Olympus DP 500 camera, and the photographs were processed with the Adobe Photoshop and Corel Draw programs.

Whole-mount and nonradioactive section in situ hybridizations were performed according to Zhang et al. (45) and Lin et al. (46). For better illustration of structures expressing the selected marker genes, the dissected organs were cultured on a nuclepore filter (pore size 0.1 µm; Whatman International Inc., Kent, UK) for few hours before fixation. Radioactive in situ hybridization was performed according to the protocols of Parr et al. (47) and Kispert et al. (48). 35S-UTP-labeled antisense probes were generated from linearized plasmids containing the respective cDNAs, which had been obtained as gifts. The Fgf9 cDNA was a gift from Dr. David Ornitz (Washington University School of Medicine, St. Louis, MO), Hsd3ß was obtained from Dr. Robin Lovel-Badge (MRC National Institute for Medical Research, London, UK), Sox9 was obtained from Dr. Peter Koopman (University of Queensland, Brisbane, Queensland, Australia), Dhh was obtained from Dr. Andy McMahon (Harvard University, Cambridge, MA), and Lim1 was obtained from Dr. Richard Behringer (University of Texas M. D. Anderson Cancer Center, Houston, TX). A minimum of three to five wild-type and transgenic gonads were analyzed for each gene and time point.

Organ culture and generation of the tissue combinants
Timed embryos were obtained by crossing the female and male SPROUTY2-expressive mice together or by crossing a SPROUTY2-expressive mouse with a CD-1 mouse and taking E0.5 to be attained at noon on the day after the appearance of the vaginal plug. The developmental stage of the dissected embryos was based on the embryonic days and on the count of tail somites. Embryos with 18–30 tail somites, corresponding to E11.5–12.5 (18), were used for dissection of the gonads and associated sex ducts for the culture and tissue combinants. The isolated organs were subjected to culture in DMEM supplemented with 10% fetal calf serum and antibiotics.

For generation of the tissue combinants, the embryonic urogenital system was first prepared at E12.0 and the mesonephros was then mechanically separated from the gonad and the metanephros. The combinants between the mesonephros and the testis were produced using organs of the same developmental stage.

To assay the influence of the gain of function of SPROUTY2 on testis-induced cell migration from the mesonephros, testes of wild-type or transgenic embryos expressing SPROUTY2 under the Pax2 promoter and mesonephros tissue from Rosa26 heterozygous embryos were combined in grooves on 2% agar blocks according to a previous method (5). FGF9 (25–50 ng/ml; PeproTech EC Ltd., Rocky Hill, NJ) was added to the culture medium (28) in some experiments, and the combinants were subcultured for 24–32 h, after which the tissues were fixed and stained with ß-Gal substrate for identification of the cells that expressed the LacZ gene (5).

The sex of the embryos was determined by PCR with primers corresponding to the Y chromosome-encoded Sry gene (49). The sequences of the primers were 5'-GGAAATGCGGTTACATGTTGACC-3' and 5'-CAGCTTGACCTGCAAAGGAAG-3', amplifying a 400-bp fragment in the males (data not shown). Sex typing was also performed by the more rapid method detailed in Ref. 50 , in which the amnions were prepared and stained directly to identify the XX chromosomes; the condensed X chromosome in the females appeared as a chromatin body.

Competitive RT-PCR assays
Competitive RT-PCR was performed as previously described (51, 52) to determine the effect of SPROUTY2 on gene expression in the embryonic testis, providing an accurate way to estimate the relative amounts of mRNA levels in the absence of contaminating DNA species. Total RNA was purified from E12.5 wild-type and transgenic testis using a RNeasy Plus Mini Kit (Qiagen, Valencia, CA). CDNA was synthesized using RevertAid first-strand cDNA synthesis kit (Fermentas Inc., Hanover, MD) according to the manufacturer’s instructions. Forward and reverse primer pairs used and the fragments amplified were the following: Fgf9, 5'-ATG GCT CCC TTA GGT GAA GTT GG-3' and 5-GCC TCC GCC TGA GAA TCC CCT TTA AAT G-3' (196 bp); Sox9, 5'-GTG GCA AGT ATT GGT CAA-3' and 5'-GAA CAG ACT CAC ATC TCT-3' (319 bp); Hsd3ß, 5'-CAG GAG CAG GAG GGT TTG T-3' and 5'-GTG GCC ATT CAG GAC GAT-3' (400 bp); and GAPDH, 5'-TGA TGA CAT CAA GAA GGT GGT GAA G-3' and 5'-TCC TTG GAG GCC ATG TAG GCC AT–3' (280 bp). PCR was initiated in the annealing temperatures appropriate to the selected primer pairs followed by 35 cycles of PCR consisting of 10 sec denaturation at 94 C, 30 sec annealing at 56 C, 58 C, or 60 C depending of the primers used, followed by 1 min extension at 72 C. The intensity of the amplified bands was evaluated by using the Quantity one program in relation to the reference (Bio-Rad, Hercules, CA).

Immunostaining of the explants as whole mounts
Testes that had been cultured for few hours were fixed in MeOH:DMSO (4:1) solution and processed for immunostaining as described (53). Rat antimouse Platelet/endothelial cell adhesion molecule [PECAM (CD-31)] monoclonal antibody (1:200; BD Biosciences, San Jose, CA) and monoclonal anti-{alpha}-smooth muscle actin ({alpha}-SMA; mouse IgG2a isotype; 1:200; Sigma-Aldrich, St. Louis, MO) were used as the primary antibodies. The PECAM antibody identifies endothelial cells and germ cells, whereas {alpha}-SMA is expressed in the peritubular myoid cells normally surrounding the developing seminiferous tubules. The secondary antibodies used were peroxidase-conjugated affinity-purified donkey antirabbit IgG (1:100; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and horseradish peroxidase-conjugated polyclonal rabbit antimouse Igs (1:100; DakoCytomation, Carpenteria, CA). 3,3'-Diaminiobenzidine (Vectstain ABC kit; Vector Laboratories, Burlingame, CA) was used as a substrate for the color reaction.

The basal lamina surrounding the Wolffian duct, mesonephric tubules, and seminiferous tubules was identified with a rabbit polyclonal antibody against laminin-5 (1:200; a gift from Dr. Sirpa Salo, University of Oulu, Oulu, Finland). The rabbit polyclonal phosphospecific anti-ERK1 and 2 antibody (1:100; Biosource International, Camarillo, CA) was used in some experiments. The secondary antibodies for these were Alexa 488 antirabbit IgG and Alexa 546 antirat IgG, respectively (Molecular Probes, Inc., Eugene, OR; Invitrogen Detection Technologies). Fluorescent images were collected with an Olympus Fluoroview FV1000 confocal microscope and a CellM Olympus video microscope. A minimum of five samples were analyzed for each developmental stage, antigen and genotype, and representative samples are shown.

Cell proliferation assay
Proliferating cells in the embryonic testes were detected with a cell proliferation kit (Amersham Biosciences, Buckinghamshire, UK), used according to the manufacturer’s instructions. Briefly, pregnant female mice were injected ip with a solution containing BrdU/FdU (bromo-deoxyuridine/fluoro-deoxyuridine; Amersham Biosciences). BrdU, an analog of thymidine that is incorporated into DNA during the S-phase of the cell cycle (54), was administered at a dose of 1 ml/100 g of body weight. The mice were killed 2 h postinjection, and the gonads with the associated mesonephros and sex ducts were separated from each embryo. The tissues were fixed overnight at 4 C with 4% paraformaldehyde diluted in PBS, dehydrated, embedded in paraffin, and serially sectioned to a thickness of 6 µm for detection of the cells that had incorporated BrdU. 3,3'-Diaminiobenzidine was also used as a substrate to visualize positive cells (Vector Laboratories). Five BrdU-labeled testes were analyzed to evaluate possible changes in cell proliferation due to SPROUTY2 expression.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sexually dimorphic expression of mSprouty2 during gonadogenesis
The mammalian gonad is initially sexually indifferent but becomes specified to an ovary or testis at around E11.5 of embryogenesis in the mouse. We analyzed whether the mSprouty2 gene is expressed during the early steps of sexual development. At E11.5, mSprouty2 gene expression was up-regulated in the testis and expression became sexually dimorphic, so that mSprouty2 transcripts were not detected in the developing ovary (Fig. 1Go, compare A and B, arrow). The metanephric kidney expressed mSprouty2 in the posterior region of the urogenital system in both sexes (Fig. 1Go, A and B, arrowheads). At E12.5, Sprouty2 expression was further up-regulated in interstitial cells and the seminiferous tubules of the testis and the associated mesonephric ducts and adjacent Wolffian duct in both sexes, whereas the ovary remained unstained (Fig. 1Go, C and D, arrows). Expression of the mSprouty1 and 4 genes was closely related to that of mSprouty2 in the gonad at these early developmental stages (data not shown). Hence, the Sprouty2 gene is expressed in the metanephric kidney in both sexes but expression in the gonad becomes sexually dimorphic, being confined to the testis, suggesting a role for Sprouty2 in male sexual development.


Figure 1
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  		FIG. 1. Expression of mSprouty2 in the gonad and targeted expression of SPROUTY2 and a reporter gene in the embryonic urogenital system with the Pax2 promoter. A–D, Whole-mount in situ hybridization demonstrating mSprouty2 expression in the XY and XX gonads. At E11.5 mSprouty2 is expressed in the presumptive testis (A, arrow), but no expression is detected in the presumptive ovary proper (B). Note that mSprouty2 is expressed in the metanephros in both sexes (A and B, arrowheads). C and D, At E12.5 mSprouty2 expression is confined to the testis (C, arrow) and the adjacent mesonephros, whereas no staining is detected in the ovary (D, arrow) or most of the mesonephros. No reporter-encoded GFP-derived fluorescence is seen in the normal male (XY) urogenital system or testis at E11.0–E14.5 (E–G), whereas Pax2 promoter-targeted GFP-derived fluorescence is seen in the mesonephric tubules and genital ridge at E11.0 (H) and testis at E12.0 (I). J, At E14.5, Pax2 promoter-driven GFP is detected in the interstitium and seminiferous tubules of the testis and in the mesonephric tubules. K, At E11.0, the SPROUTY2 transgene is expressed in the genital ridges (arrows), mesonephric tubules, and metanephros (arrowheads). L, SPROUTY2 is still expressed in the seminiferous tubules, interstitial cells of the testis, and Wolffian duct-derived epididymis at E17.5. A–K, Anterior at the top of each figure. Scale bar, 200 µm.

 
Expression of the SPROUTY2 and GFP genes in the testis, Wolffian duct, and mesonephric tubules with the Pax2 promoter
The developmental role of Sprouty2 was studied by a gain of function approach by cloning human SPROUTY2 and GFP cDNAs under the regulation of the Pax2 promoter (38).

Although no GFP-derived fluorescence was detected in the wild-type urogenital system at the initiation of gonadal development at E11.0 or E12.0 (Fig. 1Go, E and F), GFP and SPROUTY2 were expressed in the Wolffian duct (data not shown), genital ridge, and testis and the adjacent mesonephric tubules at these stages (Fig. 1Go, H, I, and K, arrows). At E14.5, the testis of a wild-type embryo did not show any fluorescence (Fig. 1GGo), but the transgenic testis maintained GFP expression in the mesonephric, interstitial cells, and seminiferous tubules (Fig. 1JGo). At E17.5, the seminiferous tubules, the interstitial cells of the testis, and the epididymis still expressed the SPROUTY2 transgene (Fig. 1LGo). No Pax2-directed transgene expression was detected in the ovary or Müllerian duct at any stage of development (data not shown). We conclude that the 4.3-kb Pax2 promoter directs SPROUTY2 and GFP expression to the Wolffian duct, embryonic testis, and adjacent mesonephric tubules, making it a useful tool for gain of function assays of SPROUTY2 in male sex organogenesis.

Developmental defects in the mesonephros derivatives and the testis due to gain of function of SPROUTY2 in the urogenital system
Even though SPROUTY2 expression did not lead to any notable changes in the sex ratio in the transgenic mice relative to the controls (data not shown), it did generate dysmorphologies in the male sex organs consistent with expression of the transgenes (Fig. 1Go, H–L).

Although the epididymis was coiled adjacent to the testis in the control samples (Fig. 2Go, A, F, and K), its coiling was reduced, and the vas deferens became cystic in many cases of a SPROUTY2-expressive transgenic urogenital system (Fig. 2Go, compare A, F, K with B, C, G, H, L, and M, arrows). The sex organs of the transgenic female embryos and NB mice were normal in appearance relative to the controls (Fig. 2Go, D, I, N, E, J, and O). The occurrence of the ductal phenotypes in the SPROUTY2-expressive individuals indicated that the defect in the ducts were more frequently observed if the transgene was inherited from both parents (Fig. 2UGo).


Figure 2
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  		FIG. 2. Gain of SPROUTY2 function leads to defects in the testis and mesonephric derivatives. A–E, Urogenital system of a NB mouse or 17.5-d-old embryo. A, Control male urogenital system. B and C, Gain of SPROUTY2 function leads to the development of cysts in the Wolffian duct derivatives and vas deferens and a reduction in the coiling of the epididymis (arrows). Development of the ovary is normal irrespective of SPROUTY2 expression (D), cf. control (E). The hydroureter and cystic kidneys are indicated with stars in B, C, and D. F–J, Higher magnifications of A–E, newborns. K–M, Histological micrographs of the testes shown in F–H. Arrows indicate the cystic or polycystic epididymis or vas deferens. N and O, The ovary is normal irrespective of SPROUTY2 expression compared with control (compare O with N). P–R, Higher magnifications of the testes shown in K–M (boxed areas). Note that the interstitium is looser in its organization in the transgenic testis compared with the wild type (compare Q and R with P). S and T, At E12.5, cell proliferation is not markedly changed due to SPROUTY2 expression compared with control. BrdU-labeled germ cells can be distinguished by their round big nucleus (arrows, insets of the testis). U, Percentage detection of Wolffian duct malformations in testes of transgenic embryos at given ages, including NB mice. The percentages depicted by the histograms indicate affected individuals as a proportion of the total number of embryos or NB mice collected: two of 56, seven of 24, two of 40, two of 12, six of 84, and four of 22. Numbers of seminiferous cords observed in wild-type and SPROUTY2-expressive testes of NB mice (***,P < 0.005) (V) and numbers of somatic and germ cells (GC) cells in the S-phase of their cycle in wild-type and transgenic testes at E12.5 (**, P < 0.01) (W). b, Bladder; t, testis; ep, epididymis; o, ovary; vd, vas deferens, ov, oviduct; u, uterus. WT, Wild type; TG, expression of SPROUTY2. Scale bar: A–O, 200 µm; P–R, 50 µm.

 
There was also a notable reduction in the appearance of the seminiferous cords in the testes of SPROUTY2-expressive embryos, and the associated interstitium was typically looser in its organization than in representative sections of the testis from control embryos (Fig. 2Go, compare L and M with K, and Q and R with P). Quantification of the seminiferous cords in the control and SPROUTY2-expressive testes of NB mice revealed a significant reduction in the latter (Fig. 2VGo), consistent with the idea that SPROUTY2 expression directly affects organogenesis in both the testis and the mesonephric derivatives.

The reduction in seminiferous tubule development may be a consequence of reduced proliferation in the coelomic epithelium, which may generate the encapsulated Sertoli cells of the seminiferous tubules (2, 29) and/or of the inhibition of mesonephric cell migration into the testis, which generates at least the pretubular myoid cells and endothelial cells (4). Possible changes in cell proliferation were analyzed at E12.5 by BrdU incorporation, marking the cells in the S-phase of their cycle.

Even though the overall localization of the cells in the S-phase of their mitosis was similar in the transgenic and wild-type testes (Fig. 2Go, S and T), the SPROUTY2-expressive testes had slightly less BrdU-labeled gonadal cells (Fig. 2WGo), although this did not apply to the germ cells, which can be distinguished from the rest of the gonadal cells due to their larger and round size (Fig. 2Go, S and T, arrows, insets, and W).

We assayed possible changes in the expression of markers of the seminiferous tubules at E12.5 and E17.5. The Sox9 and Dhh genes, which are both expressed in the differentiating Sertoli cells at E12.5 in normal development (Fig. 3Go, A and C, arrows), were present in the testis of the SPROUTY2 transgenic embryos, but expression was reduced in the testes processed simultaneously for the whole-mount in situ hybridization analysis (Fig. 3Go, compare B and D with A and C, arrows), consistent with the results of competitive RT-PCR (Fig. 4Go, G and H). We conclude that expression of the SPROUTY2 transgene leads to inhibition of seminiferous tubule formation associated with some reduction in Sox9 and Dhh expression.


Figure 3
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  		FIG. 3. Changes in the expression of certain genes implicated in urogenital system development due to gain of SPROUTY2 function. A–X, Whole-mount in situ hybridizations. A–T, E12.5; U–X, E12.0. A, Sox9 is normally expressed in Sertoli cells within the seminiferous cords (arrow). B, SPROUTY2 expression reduces Sox9 expression (arrow). The Sertoli cell marker Dhh is expressed in the seminiferous cords (C, arrow), and expression is reduced in the transgenic testis (D, arrow). Hsd3ß, a marker of Leydig cells, is expressed normally in the interstitium (E), and its expression is reduced in the transgenic testis (F). G and H, Egf expression is unchanged between the wild-type and transgenic testis. I–L, Pax2 is normally expressed in the Wolffian duct (arrows) and mesonephric tubules in both sexes, and its expression is locally reduced in the Wolffian duct of transgenic males (compare J with I, arrows). M–X, Expression of the Müllerian duct markers Lim1, Wnt7a, and Wnt4, that, in addition, are expressed in the developing ovary, does not differ between the wild-type and transgenic urogenital systems in either sex. wd, Wolffian duct. Scale bar, 200 µm.

 

Figure 4
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  		FIG. 4. Reduced expression of FGF9 and phospho ERK due to gain of function of SPROUTY2 in the embryonic urogenital system. A and B, Whole-mount in situ hybridizations; C–F, whole-mount immunostainings; and G and H, competitive RT-PCR. FGF9 is expressed in the Wolffian duct (arrowhead), mesonephric tubules and testis (arrow) of a normal embryo (A), but expression is reduced in the testis (arrow) and Wolffian duct (arrowhead) due to SPROUTY2 expression (B). C, Phospho ERK (pERK) is normally expressed in the embryonic testis (t), mesonephros (arrow), Wolffian duct (arrowheads), adrenal gland (a), and kidney (k). D, Expression is reduced in the testis, Wolffian duct (arrowheads), disorganized mesonephric tubules (arrows), and kidney due to SPROUTY2 expression. The same holds true at E13.5 in the testis, cf. wild type (compare F with E, arrows). Competitive RT-PCR for Fgf9, Sox9, and Hsd3ß, and Gapdh genes in wild-type or SPROUTY2 transgenic testis (G) and quantitation of the relative intensities of the amplified bands between wild-type and SPROUTY2 expressive testes at E12.5 (H). C and D, E12.5; E and F, E13.5. Scale bar, 200 µm.

 
Reduced expression of a Leydig cell marker gene Hsd3ß and a Wolffian duct marker Pax2, but not the Müllerian duct markers Lim1, Wnt-7a, or Wnt-4
The Hsd3ß (3ß-hydroxysteroid dehydrogenase) and Pax2 genes are implicated in male sexual development (1, 55) and could be involved in SPROUTY2-regulated dysmorphologies. Hsd3ß, a marker of differentiating Leydig cells (15), was expressed at a lower level in the transgenic than in the wild-type testis at E12.5 (Fig. 3Go compare F to E and Fig. 4Go, G and H). Hence, Sprouty may have an influence on the differentiation of the Leydig cell precursors, perhaps by affecting their migration from the mesonephros to the embryonic testis.

The Pax2 gene, which encodes a transcription factor of the Paired box family, is normally expressed in the Wolffian duct and mesonephric tubules in both sexes at E12.5 of development (Fig. 3Go, I, K and L; Refs. 56 and 57). Its expression was normal in the mesonephric tubules in the present case, but was locally reduced as compared with the wild-type controls in the Wolffian duct in the males at E12.5, due to SPROUTY2 expression (Fig. 3Go, compare J with L, arrows).

Lim1, a Lim homeodomain-containing protein, and Wnt-7a and Wnt-4 from the Wnt family of secreted signaling molecules are implicated in the control of Müllerian duct development in the female embryo (15, 58, 59). In view of their unchanged expression (Fig. 3Go, M–X), we conclude that Müllerian duct and early ovarian development is normal irrespective of the expression of the SPROUTY2 gene.

Changes in the expression of Fgf9 and phospho ERK in the embryonic urogenital system
FGF9 signaling is involved in organogenesis of the testis and controls the processes of mesonephric cell migration into the testis (28, 60). We observed reduced expression of the Fgf9 gene in the Wolffian duct and the XY gonad proper at E12.5 relative to the controls in samples processed simultaneously (Fig. 4Go, compare A with B, arrowheads and arrows), and in the competitive RT-PCR (Fig. 4Go, G and H). At still later developmental stages, Fgf9 was expressed at a lower level in the transgenic gonad and epididymis than in the testis of a wild-type embryo (data not shown). The changes in Fgf9 expression appeared to be fairly specific in the urogenital system at E12.5, since we did not record any differences between the wild-type and transgenic samples in the expression of certain other genes such as Egf, Fgf10, or Gdnf (Fig. 3Go, G and H, and data not shown).

Since Sprouty proteins inhibit receptor tyrosine kinase signaling and are involved in a negative feedback loop to fibroblast growth factors, for example (34, 61, 62), we also analyzed possible changes in the expression of phosphorylated ERK, which serves as an indicator of whether SPROUTY2 antagonizes the RAS/MAPK signaling pathway (63). Whole-mount immunostaining revealed that the normal embryonic testis and the associated sex ducts express phospho ERK at E12.5 and E13.5 (Fig. 4Go, C and E) in regions corresponding to those that express the Fgf9 gene, and that expression is reduced in the testis, mesonephric tubules, and Wolffian duct relative to controls due to SPROUTY2 expression (Fig. 4Go, compare D and F with C, E, and A, arrows, arrowheads; Ref. 63). The intensity of the phospho ERK immunostaining in the metanephric kidney was also reduced due to SPROUTY2 expression, consistent with the reported function of SPROUTY2 in the embryonic kidney (Fig. 4Go, compare D with C; Ref. 38). We conclude that FGF9 signaling is a candidate mediator of SPROUTY2 function in male sex organogenesis.

Changes in endothelial cells, smooth muscle cells, and laminin expression in the basal lamina of the embryonic testis in response to gain of function of SPROUTY2
Organogenesis of the testis involves development of the typical sexually dimorphic vascular pattern and coordinated migration of peritubular myoid cells from the mesonephros (25), which appears 12–24 h after the activation of Sry gene expression (5, 64). The Leydig cells may also have their origin in the mesonephros (4, 5, 65).

The organization of the testicular endothelial cells was analyzed with the PECAM antibody. The endothelial cell processes were thinner both within the testis and in its surface, representing the coelomic vessel in the transgenic relative to the wild-type testis (Fig. 5Go, A and B, arrow, color in green). No differences were detected in the germ cells, which also express the PECAM antigen in both the wild-type and transgenic testis (Fig. 5Go, C and D, arrows).


Figure 5
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  		FIG. 5. Changes in endothelial cells, peritubular myoid cells, and basement membrane due to gain of SPROUTY2 function. A, PECAM immunostaining highlights the coelomic vessel and thick endothelial cell projections (in green) in the normal testis. The red color indicates laminin-5 immunostaining. B, Coelomic vessel and endothelial cell projections are reduced (arrow) due to SPROUTY2 expression in the testis. Laminin-5 staining (in red) illustrates a thickened Wolffian duct (arrowheads). C and D, PECAM immunostaining also depicts the germ cells within the seminiferous tububules (arrows), which are not affected by transgene expression. {alpha}-SMA immunostaining marks the peritubular myoid cells around the seminiferous tubules in the normal testis (E, arrows) and is discontinuous and less intense in response to SPROUTY2 expression (F, arrows). Laminin-5 immunostaining visualizes the basement membrane around the Wolffian duct, and mesonephric and seminiferous tubules (G), whereas expression of SPROUTY2 has led to irregular organization of the Wolffian duct (arrowheads), and mesonephric (arrows) and seminiferous tubules (H). Scale bar, 200 µm.

 
Possible changes in the peritubular myoid cells of the SPROUTY2-expressive testis relative to the wild type were analyzed with the {alpha}-SMA antibody. Even though this was expressed in the transgenic testis, the staining was less continuous than in the wild type, especially in the cords of the seminiferous tubules (Fig. 5Go, E and F, arrows).

We used the laminin antibody to investigate possible changes in the fine organization of the seminiferous cord, Wolffian duct, and mesonephric tubules in response to SPROUTY2 expression. Structural changes relative to the controls did take place in the Wolffian duct and mesonephros. The seminiferous cords and Wolffian duct typically appeared to be irregularly organized, and the mesonephric tubules were already disorganized in their coiling in the transgenic samples at E12.5 (Fig. 5Go, H and G, arrows, arrowheads, see also B, arrowheads).

The studies with PECAM and {alpha}-SMA markers thus suggest that SPROUTY2 expression may inhibit testis development by playing a role in controlling the migration of mesonephric cells into the gonad, generating at least the peritubular myoid and endothelial smooth muscle cells of the testis, processes regulated by the gonadal signals (64).

Reduction in migration of the mesonephric cells to the developing testis
Because SPROUTY2 expression was targeted to the testis at the initiation of organogenesis (see Fig. 1Go, H, I, and K, arrows), we used the early testis in tissue combination experiments to test possible changes in gonad-induced mesonephric cell migration. The mesonephros was separated out from Rosa26 embryos (43) and combined with a gonad either from a wild-type embryo or from one expressing the SPROUTY2 transgene, both isolated at E12.0, to monitor changes in testis-induced mesonephric cell migration (Fig. 6Go, A and B; Ref. 65).


Figure 6
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  		FIG. 6. Reduced mesonephric cell migration into the testis due to SPROUTY2 gain of function is promoted by FGF9 signaling. A–E, Schematic layout of the experimental set-up used in A1–E3. Mesonephros samples from Rosa26 embryos (MN) were prepared and combined with the testis or ovary of a wild-type (WT) and testis of SPROUTY2-expressive embryo (TG) at E12.0 and subcultured without or with FGF9 protein. (A1 and A2) Combination of a wild-type testis with a Rosa26 mesonephros and subculture of conjugates for 24 or 32 h induces migration of mesonephric cells into the testis, as indicated by mesonephros-derived LacZ staining (arrows). A3, Sections reveal that mesonephros-derived cells migrate inside the testis as well (arrows). B1 and B2, Combination of a SPROUTY2-expressive testis with a Rosa26 mesonephros leads to a delay and reduction in mesonephric cell migration to the testis in culture. Note that only a few Rosa26 mesonephros-derived cells are detected inside the testis (arrow in B3). C1–C3, Wild-type testis conjugates with a Rosa26-derived mesonephros in the presence of FGF9 stimulate mesonephric cell migration into the testis up to the level of the celomic epithelium (arrows in C2 and C3). D1 and D2, Culture of a testis prepared from a SPROUTY2-expressive embryo with a Rosa26 mesonephros in the presence of FGF9 shows stimulated mesonephric cell migration to the testis (arrows) getting closer to the level promoted by the wild-type testis (compare D1–D3 and A1–A3 with B1–B3). D3, Note that administration of FGF9 promotes migration of mesonephric cells into the inner parts of the combined testis (arrows). E1 and E2, Wild-type ovary combined with a Rosa26 mesonephros in the presence of FGF9 stimulates some mesonephric cell migration to the ovary as well. E2, Wild-type ovary combined and subcultured for 32 h with a Rosa26 mesonephros does not stimulate cell migration. MN, Mesonephros. Dotted line in A3, B3, C3, and D3 demarcates the coelomic epithelium from the mesenchymal cells of the testis. Scale bars: A1–D2 and E1–E3, 200 µm; A3–D3, 100 µm.

 
When these tissue combinants were constructed using sex-typed tissues the wild-type testis was found to attract cells from the Rosa26 mesonephros (Fig. 6Go, A1 and A2, arrows), consistent with the data of Ref. 5 , but when a testis from a SPROUTY2-expressive transgenic embryo was combined with a mesonephros from a Rosa26 embryo, the process of mesonephric cell migration was delayed and reduced (Fig. 6Go, B1 and B2). This was more evident when the inner parts of the testis of a SPROUTY2-expressive embryo were compared with those of a similarly cultured wild-type testis (Fig. 6Go, B3 and A3, arrows).

Promotion of mesonephric cell migration into the SPROUTY2-expressive testis by FGF9 signaling
Because FGF9 has been implicated as a signal that controls mesonephric cell migration into the gonad (28), its expression was found to be reduced in this study in the SPROUTY2-expressive testes (Fig. 4Go, A, B, G, and H). We considered FGF9 as a candidate factor to be involved in the SPROUTY2 function of controlling testis development.

We introduced FGF9 recombinant protein (25–50 ng/ml) into the cocultures of wild-type testis and Rosa26 mesonephros and asked whether FGF9 could stimulate cell migration from the mesonephros induced normally by testis-derived signals in comparison to the untreated controls (Fig. 6CGo). Consistent with the earlier findings (28), FGF9 promoted mesonephric cell migration to the developing testis. It is noteworthy that migrated cells were detected also in the inner parts of the testis (Fig. 6Go, compare C1–C3 with A1–A3, arrows). We next tested whether Fgf9 signaling would be sufficient to promote even the SPROUTY2-reduced mesonephric cell migration to the developing testis in these experimental conjugates (Fig. 6DGo). Similarly to the wild-type samples, FGF9 signaling promoted mesonephric cell migration to the SPROUTY2-expressive testis over the controls, and cells had migrated again to the inner parts of the developing testis not seen as frequently when only SPROUTY2-expresssive testis was used in the combinants (Fig. 6Go, compare D1–D3 with B1–B3, arrows). FGF9 promoted some cells to migrate from mesonephros to the developing ovary (Fig. 6Go, E, E1, and E2), which does not normally induce such cell migration into it (Fig. 6EGo (3)].


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The sexually dimorphic expression of mSprouty2 suggests a role in male sex organogenesis
Mammalian sex organogenesis is regulated by epithelial and mesenchymal tissue interactions taking place as signaling between the cells of the developing mesonephros and the genital ridge epithelium. Our knowledge of the mechanisms of signal transmission and transduction that are involved in the control of gonadogenesis, its initiation, and subsequent morphogenesis is still limited, but we noted in this study that, of the mSprouty genes that encode cytoplasmic antagonists to the receptor tyrosine kinases (38, 61, 66, 67, 68), mSprouty2 expression was up-regulated upon specification of the testis at E11.5, whereas expression was not detected in the ovary at this stage. This sexually dimorphic up-regulation of mSprouty2 expression thus correlates with the primary male sexual commitment that involves Sry. At E12.5, mSprouty2 expression was maintained in the testis and the adjacent mesonephros, and mSprouties 1 and 4 were expressed at this time in the embryonic testis as well, suggesting some redundancy for these proteins in the developing testis. Given the expression of mSprouty2 in male sex organ primordia, we speculated that this gene may have a function in male sexual development.

Sprouty2 may function normally in testis development by controlling FGF9-induced mesonephric cell migration to the testis
The role of Sprouty2 in male sex organogenesis was addressed by a gain of function approach. The Pax2 promoter was used to target SPROUTY2 and GFP reporter gene expression in the embryonic urogenital system (38, 56, 57), directing these transgenes to the male genital ridge, testis, mesonephric tubules, and Wolffian duct, in part due to ectopic transgene activation in the early gonad. The gain of function of SPROUTY2 impaired morphogenesis in all these major constituents of the developing male sex organs. The Wolffian duct became cystic in a significant proportion of the transgenic embryos, the epididymis lost some of its coiling, the embryonic testis had less seminiferous tubules, and the interstitium was typically looser in its organization than in the wild-type controls. Hence, these gain of function data suggests that the corresponding mouse Sprouty2 takes part in the development of these structures.

Consistent with the changes in the embryonic testis, we noted that the expression of Sertoli, Leydig, peritubular myoid, and endothelial cell markers was already reduced at E12.5 in the developing testis. The reduction in the expression of Dhh in the Sertoli cells due to SPROUTY2 expression, for example, may explain the associated reduction in peritubular myoid cells, since Dhh encodes a Sertoli cell-derived signal and regulates differentiation of the peritubular myoid cells (30, 31). Like Dhh, Sox9 is expressed in the Sertoli cells, and consistent with the changes in Dhh; Sox9 levels were also lower due to SPROUTY2 expression. This reduction is in line with the finding that the seminiferous tubules were reduced in number in the transgenic testis. This reduction is likely to be secondary, since the Sertoli cells originate from the genital ridge epithelium (4, 5, 65, 69), where the transgenes were not expressed.

The endothelial cells, peritubular myoid cells, and perhaps also the hormone-producing Leydig cells may all have their origin in the mesonephros, so that their precursors migrate to the XY gonad at the initiation of gonad development in response to gonadal signals. Given the observed reduction in these major cellular components of the testis brought about by the gain of function of SPROUTY2, we speculate that the phenotypes involved in organogenesis of the testis are caused by an effect of the protein on the migration of the mesonephric cells to the testis. Inhibition of mesonephric cell migration would be expected to lead to a reduction in the amount of interstitium and also indirectly to affect the development of the seminiferous tubules.

The influence of SPROUTY2 on mesonephric cell migration was tested directly by combining the genetically marked wild-type mesonephros and with an embryonic testis that expressed the SPROUTY2 gene, following the method detailed in Ref. 5 , and embryonic organs from mice, as reported in Refs. 43 and 70 . The results showed that the SPROUTY2-expressive testes had a weaker capacity for inducing migration of the mesonephric cells than the wild-type testes. Given the role of Sprouty signaling in the regulation of Fgf signaling and FGF9 in the control of the migration of mesonephric cells into the embryonic testis (28), we speculate that the normal function of mouse Sprouty2 in testis development is to contribute to the cell migratory process regulated by Fgf9. A similar mode of regulation of Fgf receptors and ligands by Sprouties has been shown to apply in the developing inner ear, for example (40).

Several lines of evidence that have emerged in this study support the possibility that Sprouty signaling may involve Fgf9 in the control of testis development. First, mSprouty2 was expressed at the appropriate site and at the right time to be involved in the regulation of Fgf9 signaling for early testis development. Second, whole-mount in situ hybridization analysis demonstrated that SPROUTY2 expression in the embryonic urogenital system reduced Fgf9 gene expression in the Wolffian duct, mesonephros, and testis. Third, exogenous FGF9 in organ cultures composed of tissue combinants between a SPROUTY2-expressive testis and a Rosa26 embryo-derived mesonephros stimulated the SPROUTY2-inhibited migration of mesonephric cells into the embryonic testis. We interpret these data as supporting the conclusion that mouse Sprouty2 normally contributes to organogenesis of the testis by regulating mesonephric cell migration. This process in part involves Fgf9 signaling. At present we cannot exclude the possibility that SPROUTY2 may also influence testis development by controlling Fgf9 expression in the mesonephros as well.

Other Fgfs are also expressed in the gonad besides Fgf9. Fgf3 (Int-2) and Fgf4 are expressed at least in the adult testis (71, 72), whereas Fgf8 is transiently present in the embryonic testis (73), but it is not known whether these signals have a role in the embryonic urogenital system. A gain of Fgf3 function in vivo leads to defects in Wolffian duct development (74, 75) similarly to that reported in this study with SPROUTY2. Hence, changes in Fgf3 expression as well as that of Fgf9 may be involved in the generation of the Wolffian duct phenotype that resulted in this study also from the gain of SPROUTY2 function. Pax2 could be another factor contributing to the Wolffian duct phenotype generated by SPROUTY2, since Pax2 expression in this duct was locally reduced in the SPROUTY2 transgenic embryos relative to normal males, and as Pax2 is essential for the development of the urogenital system (55).

Even though FGF9 has a function in the male, neither it or any isoform of the mammalian FGF receptors (FGFr1, 3, and 4) is expressed in a sex-specific fashion, so that these could not explain the sexually dimorphic phenotype of Fgf9-deficient embryos. Given the fact that the sexually dimorphic expression of mSprouty2 is confined to the developing testis, Sprouty2 could contribute to the male-specific signaling of Fgf9. Hence, mSprouty2 could be involved in the sex-specific function of Fgf9 by controlling its receptors in the developing testis.

Recent studies with chick embryos have suggested a role for FGF receptors in gonad development (76), but we still know little about the function of FGF receptors in the mammalian gonad. Fgf9 regulates nuclear localization of Fgfr2 in the developing testis (60), but the role of Fgfr2 is open at the moment, as homozygous FGFr2 null mutants die before gonadogenesis is initiated (60, 77).

With regard to Sprouty-controlled signal transduction in developmental systems, these regulate Ca2+ and PKC signaling, whereas Spred molecules are responsible for MAPK activation (78). Sprouty can also inhibit FGF-induced ERK activation, and its expression is induced by signals such as FGF (38, 79). As ERK activity alters downstream of FGF receptor signaling (79), we tested whether a gain of SPROUTY2 function altered the expression of phosphorylated ERK in the embryonic testis. SPROUTY2 expression did indeed reduce the intensity of phospho ERK immunostaining in the embryonic testis and Wolffian duct, associating to dysmorphologies. Hence, ERK appears to be involved in mediating SPROUTY2 function in the embryonic testis.

Taken together, mSprouty2 expression becomes sexually dimorphic in association with the stages at which male sex is determined. Sprouty is likely to contribute to the regulation of testis development by controlling cell migration from the mesonephros into the XY gonad, which is an important step for organogenesis of the testis. Sprouty function involved, in part, Fgf9, since Fgf9 gene expression was down-regulated due to SPROUTY2 expression, and FGF9 signaling was sufficient to promote migration of mesonephric cells into the SPROUTY2-expressive testis. Sprouty2 signaling could contribute to normal testis development by controlling the male-specific function of Fgf signaling.


   Acknowledgments
 
We thank Hannele Härkman, Johanna Kekolahti-Liias, Jaana Kujala, and Mervi Matero for their excellent technical assistance; Gregory Dressler for the Pax2 promoter; and David Ornitz, Robin Lovell-Badge, Peter Koopman, Andy McMahon, and Richard Behringer for the in situ probes.


   Footnotes
 
This work was supported by the Academy of Finland (107406, 206038), the Sigrid Juselius Foundation, and the European Union (LSHG-CT-2004-005085).

Current address for S.Z.: Renal Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129.

Disclosure Statement: all authors have nothing to declare.

First Published Online May 4, 2006

Abbreviations: amh, Anti-Müllerian hormone; E, embryonic day; FGF, fibroblast growth factor; GFP, green fluorescent protein; NB, newborn; PECAM, platelet endothelial cell adhesion molecule; {alpha}-SMA, {alpha}-smooth muscle actin.

Received March 7, 2006.

Accepted for publication April 25, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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