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Endocrinology Vol. 148, No. 8 3704-3710
Copyright © 2007 by The Endocrine Society

Distinct Roles for Steroidogenic factor 1 and Desert hedgehog Pathways in Fetal and Adult Leydig Cell Development

Susan Y. Park, Minghan Tong and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Department of Medicine, Northwestern Memorial Hospital, Galter Building 3-150, 251 East Huron Street, Chicago, Illinois 60611. E-mail: ljameson{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Testicular Leydig cells produce testosterone and provide the hormonal environment required for male virilization and spermatogenesis. In utero, fetal Leydig cells (FLCs) are necessary for the development of the Wolffian duct and male external genitalia. Steroidogenic factor 1 (Sf1) is a transcriptional regulator of hormone biosynthesis genes, thus serving a central role in the Leydig cell. Desert hedgehog (Dhh), a Sertoli cell product, specifies the FLC lineage in the primordial gonad through a paracrine signaling mechanism. Postnatally, FLCs are replaced in the testis by morphologically distinct adult Leydig cells (ALCs). To study a putative interaction between Sf1 and Dhh, we crossed Sf1 heterozygous mutant mice with Dhh homozygous null mice to test the function of these two genes in vivo. All of the compound Sf1+/–; Dhh–/– mutants failed to masculinize and were externally female. However, embryonic gonads contained anastomotic testis cords with Sertoli cells and germ cells, indicating that sex reversal was not attributable to a fate switch of the early gonad. Instead, external feminization was attributable to the absence of differentiated FLCs in XY compound mutant mice. ALCs also failed to develop, suggesting either a dependence of ALCs on the prenatal establishment of Leydig cell precursors or that Sf1 and Dhh are both required for ALC maturation. In summary, this study provides genetic evidence that combinatorial expression of the paracrine factor Dhh and nuclear transcription factor Sf1 is required for Leydig cell development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROIDOGENIC FACTOR 1 (Sf1) is a nuclear receptor that regulates the expression of a large number of genes involved in adrenal and gonadal development (1). At mouse embryonic day 9.5, Sf1 is expressed in the primitive urogenital ridge (2) and is one of the earliest markers of coelomic epithelial cells, the precursors of the somatic lineages of the gonad. A homozygous mutation of Sf1 leads to apoptosis of adrenogenital progenitors at about 11.5 days post coitum (dpc), resulting in the complete absence of the adrenal gland and gonads (3). In the testis, Sf1 expression marks a sexually dimorphic proliferative expansion of pre-Sertoli and other precursors cells, including the Leydig cell lineage (4). Hence, Sf1 is indispensable for the survival and proliferation of progenitor somatic cells of the male gonad.

In the Sertoli cell, Sf1 functions within a regulatory complex that also contains Sox9 (Sry-related HMG box gene 9), Wt1 (Wilms’ tumor), and the GATA4 transcription factor to promote anti-Müllerian hormone (Amh) expression beginning at 11.5 dpc (5, 6, 7, 8). By 14.5 dpc, Sf1 immunostaining can be detected in the fetal Leydig cells (FLCs) (9), which reside in the interstitial compartment between the testis cords. In FLCs, Sf1 controls the expression of steroid biosynthetic enzyme genes, including StAR (steroidogenic acute regulatory protein), Cyp11a1 (cholesterol side-chain cleavage), 3ß-hydroxysteroid dehydrogenase, and Cyp17, which encode proteins that mediate the stepwise conversion of cholesterol to testosterone (10, 11, 12).

Haploinsufficiency of Sf1 is permissive for testis development, although there is a temporal delay in the expression of both Sertoli cell and FLC markers (13). For example, in Sf1 heterozygote male gonads, the Sertoli cell expression of Amh is low at 11.5 dpc but recovers by 12.5 dpc. Also, the FLC expression of Cyp11a1 and Cyp17 is attenuated at 13.5 dpc but recovers by 14.5 dpc, in parallel with the earlier restoration of Sertoli cell markers. Nonetheless, the testis and accessory sex organs ultimately develop normally, and the Sf1 heterozygous mice are fertile. Previous studies showed that the combination of Sf1 heterozygous mutation and the loss of Dax1 (DSS-AHC critical region on the X chromosome) which encodes an X-linked nuclear receptor, resulted in a more severe delay in Desert hedgehog (Dhh) expression than seen with either mutation alone (13). These results raise the possibility that the Leydig cell phenotype associated with Sf1 haploinsufficiency might be caused in part by a transiently reduced expression of Dhh.

Dhh is secreted by the Sertoli cell and acts in a paracrine manner to induce differentiation of FLCs and peritubular myoid (PTM) cells, which surround the testis cords (14, 15). Dhh binds to the Ptc1 (Patched 1) receptor on the Leydig cell surface, relieving repression of Smo (Smoothened), which mediates downstream signaling events (15). Ptc1 expression in the interstitial space is undetectable in Dhh null mutants (16), indicating that it is a downstream target of Dhh signaling in the testis. The testes of Dhh null mice have anastomotic cords as a result of diminished PTM cell number (14). In the absence of Sertoli cell and myoid cell contacts, the formation of basal lamina surrounding the seminiferous tubules is impaired (17). Because of its role in FLC development, loss of Dhh signaling is associated with severely diminished Cyp11a1 [P450 side-chain cleavage (P450 scc)] mRNA expression and reduced Sf1 immunostaining (15). The adult Dhh homozygous mutant males lack mature sperm and are infertile (16). On a mixed genetic background, the Dhh null phenotype varies from hypogonadal male mice to external feminization (17).

The goal of this study was to understand the role of Sf1 in relation to Dhh. We generated mice that are haploinsufficient for Sf1 and homozygous deficient for Dhh (Sf1+/–; Dhh–/–) to assess the contributions of these pathways to testis development and differentiation. If Sf1 is an upstream regulator of Dhh signaling in the Sertoli cell, one would expect a similar phenotype in Sf1+/–; Dhh–/– compound mutants and in Dhh–/– single gene knockout males or perhaps a more pronounced defect in double heterozygotes. Alternatively, given the diverse regulatory role of Sf1 in the FLC, there is the possibility of a distinct phenotype in Sf1+/–; Dhh–/– gonads. Examination of compound mutants revealed unique features not seen in mice with mutations in the Dhh or Sf1 pathways alone. In particular, the combined mutation precludes development of either FLCs or adult Leydig cells (ALCs), resulting in the absence of virilization and XY phenotypic sex reversal in all affected animals. There was not, however, a switch of Sertoli cell fate. These results provide genetic evidence that FLC and ALC development require combinatorial expression of Sf1 and Dhh.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Genetic crossbreeding and genotyping
Dhh null animals on a 129/B6/Swiss background were a gift from Andrew McMahon (Harvard University, Cambridge, MA) and Ann Clark (Curis Inc., Cambridge, MA). All Dhh colony animals were interbred, and 100% of Dhh homozygous XY animals were externally male without ambiguity. Sf1 heterozygous mice from The Jackson Laboratory (Bar Harbor, ME) were maintained on the DBA strain. A two-generation breeding scheme was used to yield Sf1+/–; Dhh–/– animals of interest. Genotyping of adult animals and embryos was performed on genomic DNA extracted from tail biopsy for Dhh, Sf1, and Sry (sex-determining region of the Y chromosome) by PCR. All genotypes were represented in predicted Mendelian ratios. Externally feminized animals that were Sry positive were found only in the Sf1+/–; Dhh–/– group. Mating of animals for timed pregnancies was set up in the evening and the following morning was designated 0.5 dpc.

Histology and immunohistochemistry
Adult tissues and embryos were dissected and fixed in 10% neutral buffered formalin. Processed tissue was embedded in paraffin, and 4 µm sections were stained with hematoxylin (Harris Hematoxylin; Surgipath, Richmond, IL) and eosin (Protocol Eosin; Fisher Scientific, Pittsburgh, PA). Immunohistochemistry for Amh (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), P450 scc (1:50; Santa Cruz Biotechnology) or estrogen sulfotransferase (EST) (1:200 gift from Dr. Wen-Chao Song, University of Pennsylvania, Philadelphia, PA) was performed on deparaffinized slides. Antigen retrieval used 20 mM sodium citrate (pH 6.0), followed by blocking in 10% donkey serum (Jackson ImmunoResearch, West Grove, PA) in antibody diluent (Zymed, South San Francisco, CA). The primary antibody was recognized using donkey antigoat Alexa 488 or goat antirabbit Alexa 488 secondary antibody (Invitrogen, Carlsbad, CA). Slides were counterstained and mounted with 4',6'-diamidino-2-phenylindole Hard Set (Vector Laboratories, Burlingame, CA) and viewed by confocal microscopy (UV Laser Scanning Microscope 510 Meta). Images were evaluated with LSM software version 3.2.

In situ hybridization
Embryonic gonads were dissected at 14.5 dpc. After fixation and tissue processing, antisense probe for P450 scc was hybridized overnight. Bound probe was detected by anti-digoxigenin antibody and the nitroblue-tetrazolium-chloride/5-bromo-4-chloro-indolyl-phosphate enzyme-substrate reaction (Roche, Indianapolis, IN) to yield a purple color product for mRNA transcripts. A minimum of four gonads were tested for each genotype and were obtained from different litters.

Fetal gonadal hormone measurements
Paired testes were dissected and homogenized in 250 µl PBS [0.01 M (pH 7.2)] at room temperature. The homogenized samples were transferred in a 10-ml glass tube, and 2 ml diethyl ether (Sigma, St. Louis, MO) was added, vortexed for 2 min, and allowed to stand at room temperature for 10 min. The upper phase was removed, and the lower aqueous phase was reextracted with 2 ml diethyl ether and kept at –80 C for 10 min to separate the ether and aqueous phases. The ether phases from two extractions were combined, allowed to evaporate using the Genevac EZ-2 plus evaporation system (Genevac, Ipswich, UK), and dissolved in 50 µl PBS. RIAs were performed by the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Male-to-female external sex reversal in mice with combined loss of Sf1 and Dhh
A two-generation breeding scheme was used to generate Sf1+/–; Dhh–/– animals to evaluate the consequences of Sf1 haploinsufficiency in the context of Dhh homozygous null animals. All genotypes were represented in predicted Mendelian ratios. Genetic males were confirmed by testing for the presence of the Sry locus on the Y chromosome. Sf1+/–; Dhh+/– double-heterozygous males (Fig. 1AGo) and Sf1+/– single heterozygotes (Fig. 1BGo) developed normally and had mature testes, epididymes, vas deferentia, and seminal vesicles. Dhh–/– homozygotes (Fig. 1CGo) had features similar to those reported previously (13, 17). The testes were small but the male the reproductive tract was developed, consistent with the capacity for testosterone-mediated virilization. No uterine structures were observed, confirming the action of Amh. In contrast, the XY Sf1+/–; Dhh–/– compound mutants consistently developed as phenotypic females (26 of 26). Examination of the internal genitalia from Sf1+/–; Dhh–/– animals revealed a severely underdeveloped gonad remnant (Fig. 1Go, D and E). The phenotypic sex of the gonad was not clearly discerned by visual inspection attributable to adipose tissue in the normal location of the gonad, caudal to the kidneys. Bilaterally, the gonad remnant was associated with a hypoplastic secondary duct structure. There was no evidence of virilized seminal vesicles. Nevertheless, the reproductive tract was distinguishable from development of the ovary, oviduct, and uterus, as seen in the phenotypically normal XX female (Fig. 1FGo).


Figure 1
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  		FIG. 1. Gross anatomy of internal genitalia of adult animals with compound Sf1+/–; Dhh–/– mutations. All animals were 12- to 16-wk old. A, Double-heterozygous males showed a complete male phenotype consisting of testis, epididymis, vas deferens, and seminal vesicle virilization. B, Sf1 heterozygotes showed no apparent defects in male differentiation. C, Dhh homozygous nulls were hypogonadal yet maintained male secondary reproductive structures. D, XY Sf1+/–; Dhh–/– animals were externally female in appearance but had Wolffian-like tissue and no evidence of retained Müllerian ducts. Higher magnification (E) revealed a primitive gonad (arrow) that had failed to increase in size beyond the embryonic stage. The XX Sf1+/–; Dhh–/– females developed normal ovary, oviduct, and uterus (F).

 
Lack of Wolffian derivative virilization in mice with combined loss of Sf1 and Dhh
Male secondary reproductive structures, including the epididymis, vas deferens, and seminal vesicle, develop from the embryonic Wolffian duct in response to testosterone. Virilization of external genitalia relies on the testosterone derivative dihydrotestosterone. Histology of the prepubertal (4-wk-old) reproductive tract was used to characterize the differentiation of the Wolffian duct derivatives. Double-heterozygous Sf1+/–; Dhh+/– males (Fig. 2AGo) formed distinct seminiferous tubules with differentiated Leydig cells residing in the interstitium (Fig. 2BGo). The epididymis and vas deferens exhibited androgenized features (Fig. 2Go, C and D, respectively). Single-mutant Dhh–/– homozygotes (Fig. 2EGo) lacked mature spermatids (Fig. 2F–HGo), as described in previous studies (16), and they were infertile. Sf1+/–; Dhh–/– combined mutant gonads (Fig. 2IGo) maintained testis cord-like structures that contained Sertoli cells and sparse spermatogonia (Fig. 2JGo). The rostral portion of the epididymis was recognizable; however, the epithelial lining of the vas deferens was severely undervirilized (Fig. 2Go, K and L). The presence of Amh action in Sf1+/–; Dhh–/– animals was supported by the absence of Müllerian derivatives, such as the oviduct and uterus, that were seen in an XX control female (Fig. 2Go, M–P).


Figure 2
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  		FIG. 2. Prepubertal reproductive organ histology in hormone-responsive tissues of compound mutant mice. Postnatal reproductive organs were obtained at 4 wk of age. Double-heterozygous (A–D) and Dhh–/– (E–H) males had virilized testis, epididymis, and vas deferens. Consistent with the originally reported Dhh null phenotype, spermatogenesis is severely affected (F). Sf1+/–; Dhh–/– gonad sections reveal impaired spermatogenesis (I and J). Examination of the interstitial space showed undifferentiated cells that were shaped like mesenchyme. The epididymis (K) and vas deferens (L) were found but were severely hypoplastic (note that D, H, and L are the same magnification). Histological staining demonstrated undervirilized cellular structures in the epithelium of the vas deferens, but XY Sf1+/–; Dhh–/– animals did not exhibit female differentiation as shown for the control XX female (M–P). Black scale bars, 50 µm; red scale bars, 100 µm. Gross anatomy (A, E, I, and M) taken at x10 magnification.

 
Embryonic gonads of Sf1+/–; Dhh–/– mice contain testis cords, interstitial cells, and primordial germs cells in mitotic arrest
Histological detail of gonads at 14.5 dpc showed that the embryonic gonads in Sf1+/–; Dhh+/– double heterozygotes (Fig. 3AGo) and Sf1+/– single-gene mutants (Fig. 3BGo) develop with identifiable testis cords containing Sertoli cells and germ cells, surrounded by PTM cells. In contrast, anastomotic testis cords were present in both the Dhh–/– single null and in Sf1+/–; Dhh–/– compound mutant gonads (Fig. 3Go, C and D, yellow brackets). Primordial germ cells entered mitotic arrest in all XY gonads as evident by prominent nucleoli (Fig. 3Go, A–D, arrows and insets) and the absence of condensed chromatin nuclei as found in the female (Fig. 3EGo, arrow and inset). The interstitial space contained cells consistent with the FLC lineage in all XY gonad tissue, including Sf1+/–; Dhh–/– mutants (Fig. 3Go, A–D). The presence of these cells is compatible with the initial lineage commitment of FLCs despite the combined absence of Dhh and Sf1 haploinsufficiency. These features indicate that the external sex reversal in Sf1+/–; Dhh–/– combined mutants was not caused by a change in fate of the bipotential gonad.


Figure 3
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  		FIG. 3. Characteristics of primordial germ cells in compound mutant mice. Sections from 14.5 dpc gonads were examined for testis cords, interstitial cell morphology, and sexual differentiation of primordial germ cells. Normal round testis cords are observed in cross-sections from double heterozygotes and Sf1+/– gonads (A and B). The presence of dysmorphic testis cords was apparent in both Dhh–/– and Sf1+/–; Dhh–/– gonads (yellow brackets, C and D). Cord formation is a sexually dimorphic event that is not seen in the female (E). Female-specific meiotic entry was apparent only in the nuclei of female germ cells (darkened condensed chromatin; arrow and inset in E). In contrast, the germ cells in all male mutant gonad tissue were arrested in mitosis (arrows and insets in A–D).

 
Amh production is preserved in Sf1+/–; Dhh–/– gonads
Sertoli-derived Amh is a direct target gene of Sf1 regulation. Amh was detectable in testis cords of all genetic male gonads (Fig. 4Go, A–D) and absent in females (Fig. 4EGo). These features are consistent with relatively normal Sertoli cell differentiation and function in Sf1+/–; Dhh–/– genetic males and are in agreement with the observation that Müllerian duct regression occurs in these mice.


Figure 4
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  		FIG. 4. Müllerian duct regression in compound mutant mice. Immunohistochemical staining for Amh peptide was conducted. The four male mutant gonads (A–D) showed Amh staining (pink) within testis cords, indicating preservation of male Sertoli cell fate. Amh was not detected in female control tissue (E). Scale bar, 50 µm.

 
Absence of P450 scc expression and gonadal testosterone production indicates failure of FLC cell steroidogenesis in Sf1+/–; Dhh–/– mice
Based on the external sex reversal and lack of virilized appearance of the internal reproductive structures of compound mutant mice, we hypothesized that Dhh and Sf1 act in a genetic cascade essential for FLC differentiation. Cytochrome P450 side-chain cleavage (also, Cyp11a1), a steroidogenic enzyme expressed in FLCs, was analyzed by in situ hybridization at 14.5 dpc. In Sf1+/–; Dhh+/– double-heterozygous (Fig. 5AGo) and Sf1+/– single-heterozygous (Fig. 5BGo) testes, P450 scc expression occurred in a characteristically branched pattern of interstitial steroidogenic cells that reside between the testis cords. The phenotype of the Sf1+/–; Dhh+/– double-heterozygote mutant is similar to that of the Sf1+/– heterozygote (Fig. 5Go, A and B), suggesting that Sf1 gene dosage effects are not significantly changed by a reduction in Dhh gene dosage. Consistent with previous reports (15), P450 scc expression was reduced in Dhh–/– single-mutant gonads, and the positive cells were located centrally in the gonad (Fig. 5CGo). In the Sf1+/–; Dhh–/– combined mutant gonads, P450 scc was undetectable at this stage of normally robust steroidogenesis, indicating impaired FLC differentiation (Fig. 5DGo). Female gonads served as a negative control for P450 scc expression (Fig. 5EGo). Measurement of gonadal testosterone at 18.5 dpc, when fetal testosterone peaks, confirmed that the FLCs of the compound mutant are not capable of testosterone production compared with levels secreted by control male gonads (Fig. 5FGo).


Figure 5
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  		FIG. 5. FLCs in compound mutant testes. Whole-mount in situ hybridization detected P450 scc transcripts on 14.5 dpc embryo gonads. Double-heterozygous and Sf1 heterozygous male gonads displayed interstitial recognition of the P450 scc detection riboprobe (A and B). Meanwhile, Dhh–/– gonads were characteristically deficient in P450 scc expression (C), except for limited positivity in the central region. Remarkably, Sf1+/–; Dhh–/– gonads were deficient in P450 scc transcription (D), consistent with failed FLC maturation. Female gonads (E) served as negative control and had no detectable P450 scc expression as expected. F, Fetal gonadal testosterone measurements (18.5 dpc) show that compound mutants do not produce testosterone. Scale bar, 500 µm.

 
ALCs fail to develop in Sf1+/–; Dhh–/– mice
One-week-old and adult testes were examined by immunohistochemistry for markers of FLCs and ALCs, respectively. At 1 wk of postnatal life, FLCs predominate in the testis interstitium before the maturation of ALCs. Immunodetection of the steroidogenic enzyme P450 scc highlighted well-differentiated, round Leydig cells in distinct clusters in both wild-type and Sf1+/– heterozygote gonads (Fig. 6Go, A and B). Dhh–/– gonads also displayed these FLCs at 1 wk, although the interstitial regions were less clearly demarcated because the testis cords are less developed (Fig. 6CGo). In Sf1+/–; Dhh–/– gonads, P450 scc staining was weak and limited to undifferentiated, striated interstitial cells (Fig. 6DGo). Thus, based on cellular morphology, we conclude that there is failed differentiation of steroidogenic Leydig cells from fetal life through early postnatal age in the Sf1+/–; Dhh–/– mutant mice.


Figure 6
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  		FIG. 6. FLCs and ALC populations fail to differentiate in compound mutant testes. Steroidogenic activity was ascertained by immunohistodetection of P450 scc in putative FLCs (postnatal wk 1) and EST in ALCs. EST is a differential marker expressed only in ALCs. Wild-type male (A), Sf1 single-heterozygous (B), and Dhh null (C) postnatal gonads contained distinct clusters of steroidogenic Leydig cells in the interstitium (green). Sf1+/–; Dhh–/– gonads (D) displayed sparse staining of P450 scc in cells located between primitive testis cord-like structures. However, these cells remained morphologically undifferentiated. ALCs were observed in wild-type, Sf1 heterozygous, and Dhh null gonads (F, G; green). In contrast, there was no evidence of a population of ALC in Sf1+/–; Dhh–/– gonads (H). Scale bar, 25 µm.

 
Given the severity of the internal reproductive phenotype, it seemed likely that ALC differentiation might be abnormal. EST is differentially expressed in ALCs but not in FLCs (18). At 12 wk, EST was robustly detected in both wild-type, Sf1 heterozygous, and Dhh–/– testis sections (Fig. 6Go, E–G). There was, however, no detection of EST in Sf1+/–; Dhh–/– gonads (Fig. 6HGo), consistent with the absence of ALCs in the gonads of combined mutant mice. These data suggest that adult steroidogenic cell differentiation may depend on the fetal testicular development, either as a source of progenitor cells or to maintain seminiferous tubule maturation and to promote a defined interstitium.

Serum testosterone levels in adult mice (12–16 wk of age) were consistent with defective ALC function in Sf1+/–; Dhh–/– gonads. Testosterone levels were 4.87 ng/ml in wild-type males, 1.20 ng/ml in Dhh–/– males, 0.053 ng/ml in combined Sf1+/–; Dhh–/– males, and 0.066 ng/ml in control females. Estradiol levels were similar among all groups tested but were lowest in the combined Sf1+/–; Dhh–/– mutant (data not shown). Thus, the gonad remnant in Sf1+/–; Dhh–/– mutant mice does not produce physiological levels of either male or female sex steroids.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that the loss of Dhh, when combined with Sf1 haploinsufficiency, leads to male-to-female external sex reversal and minimal virilization of the Wolffian derivatives, reflecting impaired development of the FLCs and ALCs. Testis determination, Sertoli cell differentiation, and Müllerian regression still occurred, but spermatogenesis was blocked, likely because of minimal testosterone production. These findings underscore an essential role for Sf1 and Dhh in the development of steroid-producing Leydig cells of the testis.

The goal of this study was to assess the relative roles of Sf1 and Dhh in Leydig cell development. In humans, SF1 haploinsufficiency is consistently associated with impaired Leydig cell function, resulting in XY gonadal dysgenesis, underandrogenization, and a female phenotype (19). In mice, heterozygous Sf1+/– mutants show delayed expression of steroidogenic enzyme genes, but they recover normal gene expression and Leydig cell function (13), suggesting compensatory pathways or less dependence on Sf1 gene dosage in contrast to humans. Mutation of the DHH gene in humans also causes gonadal dysgenesis (20, 21, 22). Previous analyses of homozygous Dhh–/– single-mutant knockouts reported the absence of ALCs in a subset of null animals, in part dependent on genetic background (17). In the current study, all Dhh homozygotes developed unambiguously as males, and ALCs were clearly distinguishable. Therefore, the absence of ALCs in all compound Sf1+/–; Dhh–/– mutant gonads illustrates distinct phenotypic features compared with Dhh–/– null mutants alone. Specifically, loss of one Sf1 allele (+/–) on the background of the homozygous Dhh–/– mutant results in complete and consistent loss of FLCs and ALCs and testosterone production. Although Sertoli cell development and Amh production were preserved in the Sf1+/–; Dhh–/– mutant, spermatogenesis was blocked, likely because of minimal androgen production. These findings are consistent with independent roles for Sf1 and Dhh in Leydig cell development. Of note, the compound heterozygous mutant (Sf1+/–; Dhh+/–) was minimally affected, suggesting that these pathways are not strongly interdependent, at least in terms of dosage sensitivity.

Although they share the property of sex hormone production, the lineage relationship, if any, between the FLC population and ALCs is unclear. The precursors to ALCs (before the onset of differentiation markers) are thought to arise from peritubular mesenchymal cells and perivascular smooth muscle cells that undergo transdifferentiation into steroidogenic cells (23). Additionally, spindle-shaped cells located in the peritubular region have been demonstrated to have stem cell properties (24). When these "stem Leydig cells" were reintroduced into adult host testes depleted of Leydig cells by ethane dimethane sulfonate treatment, they were capable of repopulating the ALC lineage (24). Because the precursors to FLCs have not been identified, it remains plausible that a precursor cell population gives rise to FLCs in fetal life and that the same population gives rise also ALCs postnatally. FLCs and ALCs are generally considered distinct in their origins, with FLCs arising during fetal development and ALCs arising as a distinct population after birth. Although FLC differentiation was severely affected in testes of compound mutants, we were surprised to find that the adult generation of Leydig cells, ALCs, were also significantly impaired in their maturation. These data then suggested a possible dependence of ALC differentiation on the FLC population. For example, FLCs might be a source of progenitor cells at the time of neonatal ALCs differentiation. Alternatively, the presence of steroidogenic FLCs is necessary to maintain seminiferous tubule maturation neonatally, which then provides a tissue environment capable of supporting ALC development postnatally. Either scenario could provide a possible explanation for the concomitant lack of functional FLCs and ALCs observed in compound mutants testes. However, it is also possible that the Sf1 and Dhh pathways are necessary for FLC and ALC differentiation or survival, independent of their cellular origin.

It is also notable that the interstitial space located between the seminiferous tubules was essentially obliterated in the Sf1+/–; Dhh–/– mutant. This might reflect the absence of Leydig cell development in this region. Alternatively, maintenance of the interstitial space may be necessary to generate the Leydig cell lineage, perhaps because it provides a niche for progenitor cells. The interstitial region of mutant gonads is occupied by striated cells and is relatively devoid of vascularization. Consequently, one might anticipate impaired transmission of endocrine and paracrine growth signals. For example, in addition to locally produced growth factors, the gonadotropins, FSH and LH may have limited access to Sertoli and Leydig cells, respectively. Thus, the ability of FSH and LH to induce testosterone and support spermatogenesis may be mitigated (25). This structural abnormality may therefore provide another reason why the testis remnant in Sf1+/–; Dhh–/– animals persists as a rudimentary tissue into adulthood and is limited in the onset of ALC expansion.

In summary, we sought to clarify the genetic interaction of Sf1 and Dhh in the process of FLC development. Interestingly, in genetic females, the same genotype has no consequence on female reproduction, because counterpart XX Sf1+/–; Dhh–/– animals are fertile. Our results confirm the sexually dimorphic attributes of the developmental genes Sf1 and Dhh. Control of FLC differentiation by overlapping pathways of Sf1 and Dhh ensures steroidogenesis in the male embryo and later affects ALC development. Genetic crosses to generate double mutants, such as this report of the compound phenotype of Sf1+/–; Dhh–/– animals, are valuable for defining the hierarchical nature of genes that govern testis development.


   Acknowledgments
 
We thank Ann M. Clark (Curis Inc., Cambridge, MA) and Andrew P. McMahon (Harvard University, Cambridge, MA) for Dhh null mice. The P450 scc riboprobe plasmid was provided by Blanche Capel (Duke University, Durham, NC). The antibody to estrogen sulfotransferase was a gift from Dr. Wen-Chao Song (University of Pennsylvania, Philadelphia, PA). Serum hormone assays were conducted at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core (National Institutes of Health Grant U54 HD28934). Jeanne O’Brien, Miranda Bernhardt, Donna Emge, and Liza Pfaff are gratefully acknowledged for statistical analysis and technical assistance. We are grateful to Jeff Weiss for valuable advice.


   Footnotes
 
This work was supported by National Institutes of Health Grant R01HD044801. S.Y.P. is a recipient of a Dolores Zohrab Liebmann Fellowship.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 10, 2007

Abbreviations: ALC, Adult Leydig cell; Amh, anti-Müllerian hormone; Dhh, Desert hedgehog; dpc, days post coitum; EST, estrogen sulfotransferase; FLC, fetal Leydig cell; P450 scc, P450 side-chain cleavage; PTM, peritubular myoid; Sf1, steroidogenic factor 1.

Received December 22, 2006.

Accepted for publication April 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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