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. Author manuscript; available in PMC: 2014 Jun 27.
Published in final edited form as: Dev Biol. 2002 Jun 15;246(2):356–365. doi: 10.1006/dbio.2002.0663

Disruption of Testis Cords by Cyclopamine or Forskolin Reveals Independent Cellular Pathways in Testis Organogenesis

Humphrey Hung-Chang Yao 1, Blanche Capel 1
PMCID: PMC4073602  NIHMSID: NIHMS589684  PMID: 12051821

Abstract

Most studies to date indicate that the formation of testis cords is critical for proper Sertoli cell differentiation, inhibition of germ cell meiosis, and regulation of Leydig cell differentiation. However, the connections between these events are poorly understood. The objective of this study was to dissect the molecular and cellular relationships between these events in testis formation. We took advantage of the different effects of two hedgehog signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effect of Dhh−/− on formation of testis cords without influencing Sertoli cell differentiation. However, they exhibited different effects on other cellular events during testis development. Treatment with cyclopamine did not affect inhibition of germ cell meiosis and mesonephric cell migration but caused defects in Leydig cell differentiation. In contrast, forskolin treatment induced germ cell meiosis, inhibited mesonephric cell migration, and had no effect on Leydig cell differentiation. By carefully contrasting the different effects of these two hedgehog inhibitors, we demonstrate that although formation of testis cords and development of other cell types normally take place in a tightly regulated sequence, each of these events can occur independent of the others.

Keywords: Desert Hedgehog, Patched-1, Sertoli, Leydig, Germ cells, Meiosis, and PKA

INTRODUCTION

The embryonic gonad is unique in that it has the potential to develop into one of two organs, a testis or an ovary. In the presence of a single gene on the Y chromosome, Sry (Sex-determining Region of the Y chromosome), gonads develop into testes (Gubbay et al. 1990; Sinclair et al. 1990; Koopman et al. 1991). In the absence of the Sry gene or when the Sry gene is mutated, ovaries form (Lovell-Badge and Robertson 1990; Page et al. 1990; Hawkins et al. 1992). Intersex gonads with mixed testicular and ovarian structure (ovotestes) rarely develop. In cases where ovotestes arise, testicular and ovarian regions of the tissue are distinct and separate (Bradbury 1987; Eicher et al. 1995; Nagamine et al. 1998). Within testicular regions in the ovotestes, all characteristic features of testis development are coordinately present. Testis cords form with Sertoli cells and germ cells surrounded by peritubular myoid cells. In the interstitial space outside the testis cords, testis-specific vasculature develops and steroid-producing Leydig cells differentiate. Within ovarian regions in the ovotestis, no cord organization is present, germ cells enter meiosis, and sometimes follicle formation occurs (Roth et al. 1976; Ward et al. 1988). The vascular and steroid cell patterns are also ovarian. These observations indicate that formation of the testis or the ovary is strongly canalized.

Experiments using transgenics and gene mutations have revealed several genes at the top of the testis pathway that regulate formation of the testis. Transfer of Sry (Koopman et al. 1991; Eicher et al. 1995) or Sox9, an Sry-related HMG Box gene believed to be downstream of Sry (Schafer et al. 1995; Vidal et al. 2001), into XX embryos induced complete ovary to testis sex reversal. Mutations in either Sry (Hawkins et al. 1992; McElreavey et al. 1995) or Sox9 (Schafer et al. 1995) in XY individuals resulted in complete sex reversal of the gonad to form an ovary. Mutations in fibroblast growth factor 9 (Fgf9) in XY embryos either resulted in specification of limited testicular regions within the gonad or complete sex reversal and the formation of ovaries (Colvin et al. 2001). Results from mutations in these genes at the top of the testis pathway further strengthen the idea that gonad development is a canalized event. However, these mutations have given us a few tools to dissect the sequential steps of testis organogenesis and to understand the signaling relationships that lead to the differentiation of different cell types in the early testis.

In comparison to genes at the top of the testis pathway, a null mutation in the desert hedgehog (Dhh) gene revealed a more subtle role for Dhh in testis development. Dhh, which is expressed specifically in Sertoli cells, is the only mammalian hedgehog protein expressed in the XY gonad during testis organogenesis. Expression of the hedgehog receptor Patched-1 (Ptc-1) is under the positive control of Dhh and is expressed in a testis-specific manner in interstitial cells, including peritubular myoid cells, Leydig cells, and endothelial cells (Bitgood et al. 1996; Clark et al. 2000). On some genetic backgrounds, Dhh−/− mutants maintained normal testis structure, but showed defective spermatogenesis in adult life (Bitgood et al. 1996). However, on certain hybrid backgrounds, Dhh−/− mutants exhibited discrete defects in testis organization, including abnormal development of peritubular myoid cells, apolar Sertoli cells, absence of basal lamina, and anastomotic testis cords (Pierucci-Alves et al. 2001). Defects in adult Leydig cell differentiation were also reported (Clark et al. 2000).

Formation of testis cords is proposed to be the central cellular event downstream of Sry that regulates the development of germ cells and somatic cells in the male gonad. To understand the relationships between formation of testis cords and other cellular events downstream of Sry, we took advantage of the different effects of two hedgehog-signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effects of the Dhh null mutation on formation of testis cords. However, these two hedgehog inhibitors exhibited different effects on mesonephric cell migration, inhibition of germ cell meiosis, and Leydig cell differentiation. By contrasting the different effects of these inhibitors, we have uncoupled the formation of testis cords from differentiation of Sertoli and Leydig cells, and from the inhibition of germ cell entry into meiosis.

MATERIALS AND METHODS

Mouse Strains

CD1 random-bred mouse strains (Charles River) were used for organ culture, immunocytochemistry, and in situ hybridization. GFP transgenic mice (Stock TgN(GFPU)5Nagy, Jackson Labs #003115; (Hadjantonakis et al. 1998) were used for migration studies. The Ptc+/LacZ mice were generated as described by Goodrich and coworkers (Goodrich et al. 1997) and were kindly provided by Dr. Matthew Scott at Stanford University.

Organ Culture

Genital ridges (gonad + mesonephros) from 11.5 days post coitum (dpc) embryos (0.5 dpc = noon of the day when the vaginal plug was detected) were obtained for organ culture. To determine the sex of 11.5 dpc embryos, we used a staining method (Palmer and Burgoyne 1991) to detect the presence of XX specific Barr bodies in the amnion of individual embryos. Genital ridges were cultured on a 1.5% agar block for 48 hours in Dulbecco’s Minimal Eagle Medium (DMEM) supplemented with 10% fetal calf serum (Hyclone) and 50 ug/ml ampicillin at 37°C with 5% CO2/95% air. Cyclopamine (25 μM, TRC Biomedical Research Chemicals, New York, Ontario, Canada), forskolin (20 μM, Sigma), or Rp-cAMP triethylamine (1 μM, Sigma) was added to the culture medium to inhibit the hedgehog signaling pathway or the PKA pathway. These specific concentrations of cyclopamine and forskolin represent the minimal concentrations resulting in disruption of testis cord formation as determined by our preliminary studies. An equivalent volume of methanol (solvent for cyclopamine) or DMSO (solvent for forskolin) was added to other organ cultures as controls.

Immunocytochemistry

Samples were fixed overnight in 4% paraformaldehyde in PBS at 4°C. Samples were processed and cut into 10 μm frozen sections as described (Karl and Capel 1998). For detection of laminin and MIS, we used a rabbit polyclonal antibody against laminin-1 (1:200, kindly provided by Harold Erickson) and a rabbit polyclonal antibody against MIS (1:200). For double staining of germ cells and meiotic markers, we used a rat polyclonal antibody against mouse PECAM (1:1000, Pharmingen 01951D) and a rabbit polyclonal antibody against SYN1/COR1 (1:800; a kind gift of Peter Moens; (Dobson et al. 1994), or a rabbit polyclonal antibody against phosphorylated H2aX (Mahadevaiah et al. 2001). The secondary antibodies were FITC-conjugated goat anti-rabbit antibody and Cy3-conjugated goat anti-rat antibody (1:1000, Jackson Immunochemicals).

Whole Mount In Situ Hybridization

Samples were fixed overnight in 4% paraformaldehyde in PBS at 4°C and processed according to Henrique et al (Henrique et al. 1995). We used RNA probes for Dhh (Bitgood et al. 1996), SCC (Side Chain Cleavage enzyme), Sox9 (Kent et al. 1996), and Wnt-4 (Vainio et al. 1999).

Migration Assay

Gonads and mesonephroi from 11.5 dpc CD1 or GFP embryos were separated. A CD1 XY gonad and a GFP mesonephros were assembled on an agar block and cultured for 48 hours as described (Martineau et al. 1997a). Images were obtained using a Leica MZFLIII dissecting microscope with a GFP filter.

β-gal Stain

Samples were washed in PBS and fixed in 2% paraformaldehyde for 20 minutes at room temperature. Samples were then rinsed in washing solution (2 mM MgCl2, 0.02% Nonidet P-40 in PBS), incubated overnight at 37°C in β-gal stain (1 mg/mL X-gal, 200 mM K3Fe(CN)6, 200 mM K4Fe(CN)6), washed, and post-fixed in 4% paraformaldehyde.

RESULTS

Cyclopamine and Forskolin Phenocopy the Effects of a Null Mutation in Dhh on Testis Cord Formation

11.5 dpc XY genital ridges (gonad + mesonephros, Fig 1: G and M) were explanted to culture in the presence or absence of hedgehog signaling inhibitors cyclopamine or forskolin. These two hedgehog inhibitors have been shown to cause phenotypes similar to those with mutations in the hedgehog signaling pathway (Cooper et al. 1998; Incardona et al. 1998; Incardona et al. 2000; Taipale et al. 2000; Chen et al. 2001). Cyclopamine inhibits hedgehog signaling by inactivating Smoothened, the first downstream signaling molecule after binding of hedgehog protein to its receptor Ptc-1 (Taipale et al. 2000). Forskolin, on the other hand, activates Protein Kinase A (PKA) which blocks hedgehog signaling downstream of Smoothened (Li et al. 1995; Pan and Rubin 1995; Epstein et al. 1996; Hammerschmidt et al. 1996; Chen et al. 1998; Wang et al. 1999; Kiger and O’Shea 2001). In our culture system, treatment with either inhibitor disrupted deposition of laminin (a major component of the basal lamina surrounding testis cords) and formation of testis cords compared to the untreated controls (Fig. 1). Cyclopamine treatment caused scarce and disorganized laminin deposition and forskolin treatment almost completely abolished laminin deposition similar to cord disruption in Dhh−/− mice on mixed genetic backgrounds (Clark et al., 2000). To determine whether inhibition of hedgehog signaling by cyclopamine and forskolin results in down-regulation of the hedgehog receptor Ptc-1 as in Dhh−/− gonads (Bitgood et al., 1996), we cultured Ptc+/LacZ genital ridges in the presence or absence of cyclopamine or forskolin. Both cyclopamine and forskolin inhibited expression of Ptc-1 in all interstitial cells as seen in the Dhh −/− testis (Bitgood et al. 1996).

Fig. 1.

Fig. 1

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Effects of hedgehog inhibitors, cyclopamine and forskolin, on formation of testis cords and expression of Ptc-1. Explants of 11.5 dpc XY genital ridge with gonad (G) and mesonephros (M) were cultured in the absence (Control) or presence of cyclopamine or forskolin for ~48 h. Formation of testis cords is detected by immunocytochemistry for laminin (arrows). Genital ridges from PtcLacZ mice (Goodrich et al, 1997) were used for detection of Ptc-1 expression in explant cultures.

Sertoli Cells Differentiate in the Absence of Testis Cords

To determine whether Sertoli cell differentiation was affected in the absence of testis cords, we examined the expression of three markers for Sertoli cell differentiation in XY gonads treated with the two hedgehog signaling inhibitors. These three markers were Sox9 (da Silva et al. 1996; Kent et al. 1996), MIS (Müllerian Inhibiting Substance; (Tran and Josso 1982; Behringer et al. 1990), and Dhh (Bitgood et al. 1996) . When formation of testis cords was disrupted by cyclopamine or forskolin, the levels of Sox9 expression (Fig. 2, in situ hybridization), MIS expression (Fig. 2, immunocytochemistry), and Dhh expression (Fig. 2, in situ hybridization) remained similar to controls, indicating that Sertoli cell differentiation was normal.

Fig. 2.

Fig. 2

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Effects of hedgehog inhibitors, cyclopamine and forskolin, on differentiation of Sertoli cells. Explants of 11.5 dpc XY genital ridges were cultured in the absence (Control) or presence of cyclopamine or forskolin for ~48 h. Expression of Sox9 (in situ hybridization, purple stain), MIS (immunocytochemistry, white arrows) and Dhh (in situ hybridization, purple stain) were used as indicators for Sertoli cell differentiation.

Cyclopamine and Forskolin Exhibit Different Effects on Migration of Cells from the Mesonephros into the Gonad

Based on evidence that formation of testis cords depends on cell migration from the mesonephros (Buehr et al. 1993; Martineau et al. 1997b; Tilmann and Capel 1999), we investigated whether disruption of testis cords by hedgehog signaling inhibitors results from failure of cell migration from the mesonephros into the gonad. We assembled an 11.5 dpc CD1 XY gonad apposed to an 11.5 dpc mesonephros expressing GFP, and cultured the recombinant tissue for ~ 40 h with or without cyclopamine or forskolin. GFP expressing cells migrated from the mesonephros into the CD1 gonads in the absence or presence of cyclopamine (Fig. 3). However, forskolin treatment inhibited migration of GFP expressing cells from the mesonephros into the gonad (Fig. 3).

Fig. 3.

Fig. 3

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Effects of hedgehog inhibitors, cyclopamine and forskolin, on two separate cellular processes: cell migration from the mesonephros into the gonad and germ cell entry into meiosis. For migration experiments, a CD1 XY gonad and a GFP mesonephros were assembled, cultured for ~48 hours with or without hedgehog inhibitors, and assayed for migration of GFP-expressing cells (white arrows). For experiments on germ cell meiosis, 11.5 dpc XY genital ridges were cultured in the absence (Control) or presence of cyclopamine or forskolin for ~48 h and assayed for the presence of synaptonemal complex proteins (SYN/COR, green stain) in germ cells (PECAM, red stain).

Cyclopamine and Forskolin Exhibit Different Effects on Germ Cell Meiosis in 11.5 dpc XY Gonads

During sex determination, primordial germ cells exhibit a sexually dimorphic pattern of development. In the XX gonad, primordial germ cells enter prophase of meiosis I (Zamboni 1986); whereas, in the XY gonad, primordial germ cells cease proliferation once they are enclosed inside testis cords (McLaren 1985) and do not enter meiosis I until at least a week after birth. To investigate whether the formation of testis cords is responsible for inhibition of germ cell meiosis in the XY gonad, we disrupted formation of testis cords with hedgehog inhibitors and then examined the status of primordial germ cells. We used immunocytochemistry for a germ cell marker (PECAM) and a meiosis marker (SYN/COR) to identify meiotic germ cells. We found that when testis cord formation was disrupted by cyclopamine, primordial germ cells (Fig. 3, red staining for a germ cell marker, PECAM) followed normal XY specific development and did not enter meiosis. On the other hand, when testis cord formation was disrupted by the PKA activator forskolin, primordial germ cells entered meiosis as indicated by the presence of synaptonemal complex components detected by SYN/COR antibody (Fig. 3, green staining within germ cells in red).

Cyclopamine and Forskolin Exhibit Different Effects on Leydig Cell Differentiation

Lack of adult Leydig cells in the Dhh−/− testis (Clark et al. 2000) indicated that hedgehog signaling is responsible for adult Leydig cell differentiation. We examined the expression of SCC (Side-Chain Cleavage enzyme), a marker of fetal Leydig cell differentiation, when gonad development was disrupted by hedgehog inhibitors. Cyclopamine treatment disrupted cord formation and led to significant loss of SCC mRNA in Leydig cells (Fig. 4). Few cells expressing SCC were present in XY gonads treated with cyclopamine, whereas numerous SCC-expressing cells were found outside of testis cords in controls. On the other hand, forskolin treatment had no effects on SCC expression in Leydig cells despite its disruptive effects on cord formation. Expression of Wnt-4, a negative regulator of Leydig cell differentiation expressed in the XX gonad (Vainio et al. 1999), also was examined to determine whether cyclopamine and forskolin indirectly affect Leydig cell differentiation by regulating Wnt-4 expression. Wnt-4 expression was similar in XY controls, cyclopamine-, and forskolin-treated groups (Fig. 4).

Fig. 4.

Fig. 4

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Effects of hedgehog inhibitors, cyclopamine and forskolin, on differentiation of Leydig cells. Explants of 11.5 dpc XY genital ridge were cultured in the absence (Control) or presence of cyclopamine or forskolin for ~48 h. Expression of SCC (in situ hybridization, purple stain) was used as an indicator for Leydig cell differentiation. Expression of Wnt-4 (in situ hybridization, purple stain) was undetectable in both control and treated XY gonads.

Inhibition of PKA Does Not Affect Leydig Cell Differentiation and Germ Cell Meiosis

Forskolin treatment induced Leydig cell differentiation and germ cell entry into meiosis in gonad explant culture (Fig. 3 and 4) suggesting that the PKA pathway could play endogenous roles in gonad development. To test this possibility, we examined the effect of a PKA-specific inhibitor Rp-cAMP triethylamine on 11.5 dpc gonad explant cultures. Inhibition of PKA by this highly specific PKA inhibitor had no significant effects on the level of SCC expression in Leydig cells in XY gonads although in all treated samples the localization of Leydig cells seems disorganized (Fig. 5). We also found that inhibition of PKA had no effect on germ cell meiosis in XX gonads (Fig. 5, red= germ cells, green = γH2AX, a meiosis marker; Mahadevaiah et al. 2001).

Fig. 5.

Fig. 5

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Effects of the Protein Kinase A inhibitor, Rp-cAMP triethylamine, on differentiation of Leydig cells in XY gonads and on germ cell meiosis in XX gonads. Explants of 11.5 dpc XY or XX genital ridge were cultured in the absence (Control) or presence of Rp-cAMP triethylamine for ~48 h. Expression of SCC (in situ hybridization, purple stain) in Leydig cells and γH2aX proteins (immunocytochemistry, green stain) in germ cells (PECAM, red stain) were examined.

DISCUSSION

Organogenesis of the mammalian testis is controlled by a cascade of cellular events triggered by Sry. Identification of the components of the complex genetic circuitry has been promising, but painfully slow. To hasten this progress we have used two inhibitors of hedgehog signaling in organ culture assays to gain a better understanding of the interdependence of the cellular pathways downstream of Sry.

Formation of Testis Cords Requires Both Mesonephric Cell Migration and Dhh Signaling

Treatments with hedgehog inhibitors cyclopamine and forskolin phenocopied the effect of Dhh null mutation on testis cord formation. However, forskolin treatment caused more severe defects in cord organization. Forskolin treatment also inhibited mesonephric cell migration, whereas cyclopamine treatment had no effect on mesonephric cell migration (Table 1). The more severe effects on laminin deposition caused by forskolin treatment may result from the combination of inhibition of Dhh signaling and the loss of the migrating cell population. Results from cyclopamine treatment indicated that normal mesonephric cell migration was not sufficient to induce testis cord formation if hedgehog signaling was blocked, indicating that both mesonephric cell migration and Dhh signaling between Sertoli cells and myoid precursors are essential for proper formation of testis cords (Fig. 6. * & *).

Table 1.

Summary of the Effects of Hedgehog Inhibitors on Cellular Events Downstream of Sry

Inhibitor Ptc-1 Expression Sertoli Cell Differentiation Testis Cord Formation Mesonephric Cell Migration Germ Cell Meiosis Leydig Cell Differentiation
Fig. 1 Fig. 2 Fig. 1 Fig. 3 Fig. 3 Fig. 4
Cyclopamine X O X O X X
Forskolin (also a PKA activator) X O X X O O
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X = Inhibition

O = No effect

Fig. 6.

Fig. 6

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Proposed cellular pathways downstream of Sry. Formation of testis cords is accomplished through two independent pathways: * mesonephric cell migration and * Dhh signaling between Sertoli cells and myoid precursors for proper deposition of the basal lamina. * Leydig cell differentiation (expression of SCC) depends on Dhh signaling between Sertoli cells and fetal Leydig cell precursors. * Inhibition of germ cells entry into meiosis is mediated through unknown diffusible factor(s) from Sertoli cells or peritubular myoid and does not depend on Dhh signaling and formation of testis cords.

Despite disruption of testis cord formation by treatments with hedgehog inhibitors, differentiation of Sertoli cells was normal with respect to the expression of three markers (Sox9, MIS, and Dhh). Sertoli cells are considered to be the organization center for testis formation. Sry is expressed transiently (10.5–12.5 dpc) in mouse (Koopman et al. 1990; Hacker et al. 1995) by Sertoli cells precursors (Albrecht and Eicher, 2001) and serves as a genetic switch to induce the production of downstream molecules such as Sox9, MIS, and Dhh. It is not understood how the expression of these downstream molecules is maintained. Magre and Jost (Magre and Jost 1984) proposed that differentiation of Sertoli cells can be maintained in the absence of testis cords. Our results provide evidence for their hypothesis. Although Sertoli cells in the Dhh−/− XY gonads lost their polarity in relation to the basal lamina (Clark et al. 2000; Pierucci-Alves et al. 2001), their differentiation is maintained without a proper morphological arrangement in testis cords. Based on this evidence, we conclude that maintenance of Sertoli cell differentiation at least over the two day period of these experiments does not require formation of testis cords or epithelial polarity.

Leydig Cell Differentiation Requires Dhh Signaling but not Mesonephric Cell Migration after 11.5 dpc

The decreased Leydig cell population resulting from inhibition of Dhh signaling with the specific inhibitor cyclopamine strongly implicates Dhh as a positive regulator of Leydig cell differentiation (Fig. 6, *). While Wnt-4 seems to act as a negative regulator of Leydig cell differentiation in the XX gonad (Vainio et al. 1999), we found no changes in Wnt-4 expression when the Dhh signaling pathway was inhibited, indicating that Dhh does not act by inhibiting a negative regulator of Leydig cell differentiation. In the Dhh−/− gonad on some genetic backgrounds, Leydig cells were identified morphologically in EM micrographs (Clark et al. 2000). This suggests that Dhh signaling is not essential to establish Leydig cell progenitors, but is involved downstream in their differentiation. Detailed characterization of Leydig cell differentiation in the Dhh−/− XY gonads has provided further evidence for this hypothesis (Yao et al. 2002).

Some reports have suggested that Leydig precursors migrate from the mesonephros in recombinant organ cultures assembled at 11.5 dpc (Merchant-Larios and Moreno-Mendoza 1998). However, treatment with forskolin completely inhibited mesonephric cell migration yet ectopically activated Leydig cell differentiation (Table 1). In this case, the appearance of Leydig cells in the absence of mesonephric cell migration indicates that either Leydig cells derive from other sources such as the coelomic epithelium of the gonad (Karl and Capel 1998) or Leydig cell precursors migrate into the gonad before 11.5 dpc. Consistent with the finding in Wnt-4 −/− XX gonads that Leydig cells appeared in XX gonads in the absence of cords (Vainio et al. 1999), our results again demonstrate that Leydig cells can differentiate independently of the formation of testis cords.

Inhibition of Germ Cell Meiosis Does Not Require Formation of Testis Cords

McLaren and Southee (McLaren and Southee 1997) hypothesized that the inhibition of primordial germ cell meiosis in XY gonads is due to enclosure of germ cells in testis cords at 12.5 dpc. This hypothesis was based on the observation that in the XY embryo, primordial germ cells enter meiosis I when they develop in locations outside the XY gonads such as the adrenal gland (Upadhyay and Zamboni 1982; Zamboni and Upadhyay 1983), or when they are cultured in vivo (McLaren and Southee 1997). However, we found that primordial germ cells did not enter meiosis when testis cords were disrupted by the hedgehog inhibitor cyclopamine or in Dhh−/− XY gonads on a mixed genetic background (Clark et al., 2000). This result indicates: 1) enclosure of primordial germ cells in testis cords is not essential for inhibition of germ cell meiosis; 2) Inhibition of germ cell meiosis in the XY gonad probably depends on a diffusible factor; and 3) Dhh is not the diffusible meiosis inhibiting factor produced by the XY gonad (Fig. 6. *).

In contrast to cyclopamine treatment (Table 1), we found that treatment of XY gonads with forskolin led to the entry of germ cells into meiosis, consistent with previous findings (Byskov et al. 1993). This result suggested that in addition to blocking Dhh signaling for testis cord formation, activation of PKA could act ectopically to promote germ cell entry into meiosis. It is possible that PKA inhibits the production of the meiosis inhibiting factor(s) by the somatic cell populations in the gonad. One possibility is that the mesonephric cell migration blocked by forskolin treatment is critical to the production of this signal. Further experiments are needed to identify the cell types producing the meiosis inhibiting factor(s).

The PKA Pathway Is Unlikely to Be Involved in Early Gonad Development

Treatment with a PKA specific inhibitor had no effects on Leydig cell differentiation in XY gonads or on the entry of germ cells into meiosis in XX gonads, indicating that PKA probably does not normally play these roles in early gonad development. At birth and thereafter, Luteinizing hormone (LH) induces steroid production in Leydig cells by activating the PKA pathway (Hansson et al. 2000; Habert et al. 2001). Even though Leydig cell differentiation during gonad development does not require LH (El-Gehani et al. 1998), Leydig cells from fetal testes were capable of producing testosterone in response to LH (Habert et al. 2001). Our results demonstrate that although the signaling components of the PKA pathway are present in Leydig cells as early as 12.5–13.5 dpc, the early development of Leydig cells is not dependent on the PKA pathway.

It is impossible to unambiguously assign the roles of Dhh signaling using in vitro inhibitors which could have ectopic effects on gonad development. Nonetheless, the comparative use of these inhibitors provides us a useful tool to distinguish several morphological and cellular signaling pathways downstream of Sry. Results from these experiments illustrate that: (1) formation of testis cords requires both mesonephric cell migration and Dhh signaling; (2) molecular differentiation of Sertoli cells is not dependent on testis cord formation; (3) fetal Leydig cell differentiation depends on Dhh signaling among cells already present in the XY gonad by 11.5 dpc but is likely to be independent of cord formation; (4) inhibition of germ cell meiosis occurs even though germ cells are not enclosed in testis cords and therefore, probably depends on locally diffusible factors. Although these molecular and cellular events in testis organogenesis normally take place in a tightly regulated sequence, each can occur independent of the others.

Acknowledgments

We would like to dedicate this work to Dr. Lonnie Russell, whose lifetime contributions to the understanding of testis morphology have been seminal to the field. Within the lab, Kit Tilmann, Jennifer Brennan, Jennifer Schmahl, Andrea Ross, and Jordan Bachvarov have all contributed to useful discussions. We thank the following colleagues for their generous gifts of antibodies and probes: William Bonner, Harold Erikson, Peter Koopman, Robin Lovell-Badge, Peter Moens, Keith Parker, and Amanda Swain. This work was supported by a grant from the National Institutes of Health and a postdoctoral fellowship from the Lalor Foundation, Inc.

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