Mouse Genetics and Husbandry

The mice used in the present study were kept under standard animal housing conditions. The Institutional Animal Care and Use Committee at Massachusetts General Hospital approved all protocols involving animal experimentation. All mice used in the present study were maintained on C57BL/6;129/SvEv mixed genetic background. Amhr2^{tm3(cre)Bhr/+} mice [20] were mated with Ctnnb1^{tm1Mmt/tm1Mmt}, which encodes phosphorylation sites required for its degradation by the adenomatosis polyposis complex [21], to generate Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice. The genetic crosses used to generate Amhr2-cre/+;YFP/+ has been described previously [16, 22]. Genotyping was performed on DNA from tail biopsies using standard PCR protocols. The age of embryos was determined by the day on which a vaginal plug was detected (considered to be Embryonic Day 0.5). The Embryonic Day 14, 16, and 18 as well as the 1-day-old and 8-wk-old testes were collected from Amhr2^{tm3(cre)Bhr/+};YFP/+ mice and were viewed directly using YFP (yellow fluorescent protein) fluorescence. The testes collected from Amhr^{2tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control mice were photographed by using a Nikon SMZ1500 microscope with an attached Spot camera (Diagnostic Instruments).

Histology Analysis, Immunofluorescence, Immunohistochemistry, and TUNEL staining

Murine testes used in the present study were immersion-fixed in Bouin fluid (for histological analysis) or 4% paraformaldehyde for 10–12 h at 4°C and then transferred to 70% ethanol until processing. The fixed tissues were dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin wax. Embedded tissue samples were sectioned (thickness, 5 μm) and then mounted on slides. Hematoxylin-and-eosin and periodic acid-Schiff staining were performed on Bouin-fixed sections using standard histological techniques.

Serial sections of tissues were deparaffinized with xylene and rehydrated with a graded series of ethanol (absolute, 95%, 80% and 50% ethanol, respectively, and distilled water), followed by two washes of 5 min each in PBS containing 0.05% Tween 20 (PBS-T). Tissue sections were then incubated for 10 min in 3% (v/v) hydrogen peroxide in methanol to block endogenous peroxidase activity before immunohistochemistry (IHC). After a wash with PBS-T, antigen retrieval was performed by boiling the tissue sections in 0.01 M citrate buffer (pH 6) for 20 min. Sections were then washed for 5 min in PBS-T and blocked at room temperature for 1 h using 2% normal donkey or goat serum, 2% bovine serum albumin, and 0.1% Triton-X in PBS. Tissues sections were then incubated in humidified chamber overnight at 4°C with primary antibody. Sections were subsequently washed with PBS-T and incubated at room temperature for 1 h with secondary antibody. After a wash with PBS-T, sections for IHC were incubated with ready-to-use streptavidin peroxidase (Lab Vision) for 10 min at room temperature. Afterward, diaminobenzidine (Vector Laboratories) was used to develop color. Sections were counterstained with hematoxylin (IHC) and 4′,6′-diamidino-2-phenylindole (DAPI; immunofluorescence). Both immunofluorescence and IHC images were photographed using a microscope (Nikon Eclipse TE 2000-S) equipped with a Spot digital camera. The primary and secondary antibodies used were as follows: anti-β-catenin (1:250; BD Transduction Laboratories), anti-mouse DDX4 (1:250; Abcam), anti-KIT (1:200; Abcam), anti-GFP (1:200; Abcam), anti-GDNF (1:200; Santa Cruz Biotechnology), anti-AMH (1:100; Santa Cruz Biotechnology), anti-3-hydroxysteroid dehydrogenase (HSD3B1; 1:200; Santa Cruz Biotechnology), vimentin (1:100; Santa Cruz Biotechnology), anti-SOX9 (Chemicon) anti-PCNA (ready to use; Zymed), anti-cyclin D1 (ready to use; Neomarkers), anti-germ cell nuclear antigen 1 (GCNA1; 1:100; Dr. George Enders [23]), AlexaFluor second antibodies (1:500; Invitrogen), and biotinylated donkey anti-mouse or anti-rabbit antibody F(ab)₂ (1:1000; Jackson ImmunoResearch Laboratories). TUNEL staining was performed according to instructions provided with the cell death detection kit (Roche).

Counting SOX9-, TUNEL-, and Cyclin D1-Positive Cells

The testicular sections were stained for SOX9, TUNEL, and cyclin D1 as described above. The counting method used in the present study has been described previously and used by Costoya et al. [24]. Briefly, after serial sectioning of mutant and control testes, sections were randomly selected to represent different depths of the whole testes. Sections selected for counting were at least 25 μm apart. SOX9-, TUNEL-, and cyclin D1-positive cells were scored from 40 round tubules of Postnatal Day (PND) 7 and PND 17 testes (n = 3 mice for each genotype). Twenty round tubules from three different independent microscopic fields were counted for PND 35 and adult testes (n = 3 mice for each genotype).

Transfection and Reporter Assays

The luciferase reporter assay was performed as described in our previous studies [25, 26]. Briefly, HEK293 cells (American Type Culture Collection [ATCC]) were plated at a density of 50 000 cells/well in a 24-well plate. The day after plating, cells were transfected with AMH reporter construct [27], GDNF reporter construct [28], and TOPflash construct [29] using Lipofectamine 2000 (Invitrogen). The following day, cells in duplicate wells were treated with indicated concentrations of LiCl and NaCl for 24 h. Results were obtained using the luciferase reporter assay according to the manufacturer's instructions (Promega). Activity was read in a Wallac Victor 3 luminometer (PerkinElmer), and results are shown as an average of three independent experiments. Promoter sequence analyses were performed with MacVector software (MacVector, Inc.).

Statistical Analysis

The unpaired t-test was used to test for differences between groups, and P < 0.05 was considered to be statistically significant. The statistical analyses were performed using Prism software (GraphPad Software, Inc.).

β-Catenin Expression During Postnatal Testicular Development

To determine where β-catenin might play a role in regulating testicular functions, we first analyzed the expression of β-catenin in testis at various ages by IHC (Fig. 1). We examined testes isolated from mice at PND 7, PND 17, and PND 35 and at adulthood because of the known morphological and functional changes that occur in the juvenile (PND 7 and PND 17) testes as well as those near (PND 35) and at sexual maturity [30]. In PND 7 and PND 17 testes, β-catenin was highly expressed across the width of seminiferous epithelium, specifically in the cytoplasm and membranes of Sertoli cells and also at Sertoli-germ cells junctions (Fig. 1, A and B). In PND 35 and adult testis, β-catenin was highly expressed at basal compartment of seminiferous tubule (Fig. 1, C and D). We also observed weak staining for β-catenin in the nuclei of Sertoli cells at all ages (Fig. 1, A–D, insets), suggesting that active β-catenin signaling is physiologically significant in Sertoli cells. Consistent with previous results [31], very weak or no β-catenin was detected in the interstitial cells of testis (Fig. 1).

FIG. 1.

Localization of β-catenin in testes of both Amhr2^{tm3(cre)Bhr/+};Ctnnb1^flox/+ and control postnatal mice. β-Catenin is detected by immunohistochemistry with an anti-β-catenin antibody (brown), and nuclei are counterstained with hematoxylin (blue). In control testes (A–D), β-catenin is expressed mainly in plasma membrane and cytoplasm of Sertoli cells, but weaker staining is also observed in nuclei of some Sertoli cells (see arrowheads and insets without hematoxylin). In Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testis (F–I), strong expression of β-catenin is detected in both nuclei (arrowheads) and the cytoplasm of Sertoli cells. E and J) Colocalization of vimentin (red) and β-catenin (green) in control and mutant adult testes. Arrowheads represent nuclear accumulation of β-catenin in vimentin-positive cells. The arrow points to vimentin-negative cells without nuclear accumulation of β-catenin (arrow). Bar = 50 μm.

Activation of β-Catenin in Sertoli Cells Leads to Abnormal Development of Testis

Because we observed β-catenin expression in Sertoli cells at all stages of postnatal development, we investigated the effect of conditionally expressing an activated allele of β-catenin on Sertoli cells functions and Sertoli-germ cell interactions. Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice were examined for the effect of dominant stable form of β-catenin, whereas Ctnnb^1tm1Mmt/+ littermates were used as controls. We detected strong accumulation of β-catenin in the nuclei of a few Sertoli cells in each tubule of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes at PND 7 (Fig. 1F), compared to the weak staining for β-catenin detected in nuclei of Sertoli cells in control testis at any age (Fig. 1, A–D). Morphologically, the early PND 7 tubules of the Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes appeared to be relatively normal when compared to control littermate tubules. As the Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice aged, more nuclei in each tubule showed strong nuclear β-catenin expression (Fig. 1, G and H), and in adult (age, 12 wk) mice, nearly all Sertoli cell nuclei displayed this pattern (Fig. 1I). To show the activation of β-catenin is limited to the Sertoli cells in seminiferous tubules, we performed experiments to colocalize vimentin, a Sertoli cell marker, with β-catenin (Fig. 1, E and J). We observed that cells with the nuclear accumulation of β-catenin were also positive for vimentin, whereas cells negative for vimentin showed no nuclear accumulation of β-catenin.

Consistent with the findings of Boyer et al. [19], we observed that Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice had much smaller testes than control littermates and that the weight of adult testes in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice was relatively less than the weight in younger mice when compared to control animals, suggesting a progressive decrease in testicular tissue in the mutant animals (Supplemental Fig. S1; all Supplemental Data are available online at  www.biolreprod.org). Although mature testes of the Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice were smaller, early postnatal testes did not appear to be much different histologically from control testes of littermates (Fig. 1, A and B), despite the fact that Amhr2-Cre is expressed in embryonic gonads [16]. This suggested three plausible explanations: that Amhr2-Cre expression was not faithfully recapitulating endogenous Amhr2 expression, that β-catenin is not normally expressed prenatally in Sertoli cells, or that nuclear β-catenin does not affect early postnatal Sertoli cell functions. Therefore, we investigated the expression of Amhr2-Cre at embryonic and in postnatal testes. We generated Amhr2^{tm3(cre)Bhr/+};YFP mice as described in our previous study [16]. In these mice, we were able to detect Amhr2-Cre-driven YFP reporter expression in Sertoli cells of Embryonic Day 14, 16, and 18 as well as 1-day-old and 8-wk-old testes, confirming the expression of Amhr2-Cre in both embryonic and postnatal Sertoli cells (Fig. 2, A–E). Interestingly, in both embryonic and PND 1 testes, Amhr2-Cre-driven YFP expression was limited to the few Sertoli cells (Fig. 2, A–D). However, all Sertoli cells and some interstitial cells were YFP positive in the adult testis (Fig. 2E). To confirm that Amhr2-Cre-driven YFP expression in Leydig cells, we performed colocalization of YFP and HSD3B1 and showed that the YFP-positive cells were also positive for HSD3B1 (Fig. 2F). Our previous study also has shown that the Amhr2 mRNA is expressed in both embryonic and adult testis [32]. Therefore, although Amhr2-Cre expression was detected in prenatal Sertoli cells, we did not observe histologically any defect in embryonic and early postnatal testicular development in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice.

FIG. 2.

Expression analysis of a floxed YFP reporter driven by Amhr2-Cre in embryonic and postnatal testes. A–D) In Embryonic Day 14, 16, and 18 as well as Postnatal Day 1 testes, few Sertoli cells were YFP (green) positive (arrowhead), whereas the remaining cells were negative (arrow). YFP expression was also present in interstitial cells and the tunica albuginea. In 8-wk-old testis (E) almost all Sertoli cells were YFP positive (arrowhead). YFP positivity was also observed in interstitial cells (asterisk). Inset picture represent the negative controls used for the primary antibodies. A–E) Germ cells were stained with GCNA (red). F) Colocalization of HSD3B1 (red), a Leydig cell marker, and YFP (green). Three animals were examined for each stage of testicular development. Bar = 50 μm.

Expression of a Stable Form of β-Catenin in Sertoli Cells Disrupts the Germ Cell Microenvironment and Leads to Infertility

As Boyer et al. [19] also showed, no detectable difference was observed histologically between Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control testes at PND 7 (Supplemental Fig. S2A, a–d). In contrast, in PND 17 mutant testes, we found that lumen formation was inhibited in the vast majority of tubules, which persisted in all Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice through adulthood (Supplemental Fig. S2A, e–q). At PND 35, round and elongated spermatids are observed in control testes (Supplemental Fig. S2A, j and l) but were absent in mutant testes, except in some tubules where very few round spermatids were observed (Supplemental Fig. S2A, k and m). The presence of round spermatids in the some tubules of mutant testis is consistent with nuclear accumulation of β-catenin in Sertoli of PND 35 mutant testes, where some Sertoli cells still do not show the nuclear accumulation of β-catenin and are able to support germ cell development. In adult Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice, most of the germ cells are lost, with the exception of a layer of germ cells with Sertoli cells remaining at the base of the seminiferous tubules. We also observed a complete absence of sperm in the tails of the epididymides, further indicating defective spermatogenesis (Supplemental Fig. S2B, a and b). Additionally, multinucleated giant cells were observed in the lumen of many seminiferous tubules of the mutant mice, which likely result from abnormal and incomplete differentiation of germ cells (Supplemental Fig. S2Bc). The identity of these multinucleated cells as germ cells was also confirmed with DDX4 immunostaining (Supplemental Fig. S2Bd).

To determine the identity and the differentiation status of the germ cells that remained in the seminiferous tubules of the mutant mice, we analyzed the expression of known germ cell markers, GCNA1, DDX4 (also known as mouse vasa homolog [mvh]), and the receptor for stem cell factor, KIT, by immunofluorescence. The combination of multiple germ cell markers has been used successfully to identify and isolate different stages of testicular germ cells [34]. For example, in wild-type testes, strong GCNA1 expression is detected in spermatogonia, whereas low levels of expression are detected in spermatocytes and round spermatids [23]. In early postnatal stages of testicular development, gonocytes express mvh; in older but still sexually immature mice, mvh is expressed in germ cell stages from spermatogonia to round spermatids. In adults, the strongest mvh expression is detected at early spermatocyte stages, but lower levels are detected in spermatogonia located at the basal membrane [35, 36]. KIT is a differentiation marker for spermatogonia [37, 38]. In early prepubertal testes, KIT expression is detected on the surface of SSCs. During later stages of testicular development, c-kit is expressed mainly in spermatogonia committed to differentiation [38]. Using an approach involving a combination of these markers, we attempted to identify the status of germ cell development in the Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice.

In PND 7 testes, the GCNA1-, DDX4-, and KIT-positive cells normally were arranged at the periphery of seminiferous tubules of both Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control mice (Fig. 3, A–F), which is consistent with the normal histology of mutant testes we had observed at this age. In PND 17 and PND 35 testes, GCNA1- and DDX4-positive cell normally were arranged at the periphery of seminiferous tubules of the control mice (Fig. 3, G, I, M, and O). However, in PND 17 and PND 35 mutant testes, GCNA1- and DDX4-positive cells also were present in the luminal area of tubules (Fig. 3, H, J, N, and P). In control mice, KIT staining was bright and positive (Fig. 3, K and Q); however, KIT expression was very weak or almost absent in the seminiferous tubules of the mutant mice (Fig. 3, L and R). In adult control testes, strongly GCNA1-positive and weakly DDX4-positive cells were present at the circumference of seminiferous tubules, and weakly GCNA1-positive and strongly DDX4-positive cells were present across the width of seminiferous epithelium (Fig. 3, S and U). Similarly, KIT-positive cells also were present in and around rim of seminiferous tubules (Fig. 3W), consistent with previous observations [39–41]. In adult mutant testes, only strongly GCNA1-positive, weaker DDX4-positive, and KIT-negative cells were observed (Fig. 3, T, V, and X).

FIG. 3.

Stabilization of β-catenin in Sertoli cells causes disruption in the arrangement of germ cells in spermatogenic tubule. Antibodies against GCNA1 (red), DDX4 (green), and KIT (red) were used to detect germ cells in testicular sections by immunofluorescence; nuclei were detected with DAPI (blue). In Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice testes at Postnatal Day 7, the arrangement of GCNA1-, DDX4-, and KIT-positive cells is similar to that in control littermates (A–F). In testes at Postnatal Days 17 and 35, GCNA1- and DDX4-positive cells are randomly arranged and also observed in the luminal area of mutant tubules compared to controls (G–J and M–P). KIT expression in mutant testes at Postnatal Days 17 and 35 is much weaker than in the controls, particularly in the cells present in the luminal area of mutant testes (K, L, Q, and R). In adult Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes, strongly GCNA1-positive, weakly DDX4-positive, and KIT-negative cells are observed in all the seminiferous tubule compared to controls in which GCNA1-, DDX4-, and KIT-positive cells are present across the width of seminiferous epithelium (S–X). Bar = 50 μm.

Strong GCNA1 and weak DDX4 expression, coupled with absence of KIT expression, reflects the undifferentiated status of spermatogonia. The cluster of germ cells observed in the center of seminiferous tubules of PND 17 and PND 35 Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes are GCNA1 and DDX4 positive and very weakly KIT positive, suggesting defective differentiation of spermatogonia (Fig. 3, H, J, L, N, P, and R). The germ cells present in seminiferous tubules of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ adult testes are strongly GCNA1 and weakly DDX4 positive (Fig. 3V) as well as KIT negative, confirming their status as undifferentiated spermatogonial cells (Fig. 3, T, V, and X). This phenotype in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes is similar to the phenotype of GDNF-overexpressing transgenic mouse testes [4]. In 3-wk-old GDNF transgenic mice testes, spermatogonia accumulate in clusters in the center of tubules, and by 10 wk, most of the germ cells are lost except for a layer of spermatogonia at the periphery of seminiferous tubules [4]. Therefore, we hypothesized that conditional expression of activated β-catenin in Sertoli cells might be either upstream or downstream of GDNF or indirectly affecting the GDNF signaling pathway.

Conditional Activation of β-Catenin in Sertoli Cells Maintains High Expression of GDNF and AMH

The striking similarity in phenotype of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and GDNF transgenic mice testes led us to investigate expression of GDNF in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes. GDNF is thought to be a master regulator of SSC function; when Sertoli cells express GDNF, SSCs proliferate and remain undifferentiated [4]. In control mice, GDNF expression is down-regulated after PND 7 and remains low throughout adulthood (Fig. 4, A–G). In contrast, Sertoli cells of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testis retained high levels of GDNF expression from PND 7 through adulthood (Fig. 4, B–H).

FIG. 4.

Sertoli cells of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice testes express higher levels of GDNF than littermate control mice. Anti-GDNF was used to detect GDNF by immunofluorescence in testicular sections (green). Nuclei were detected with DAPI (blue). In controls, GDNF protein is detected in Sertoli cells of Postnatal Day 7 (A) and Postnatal Day 17 (C) mice testes but not in Postnatal Day 35 (E) and adult (G) mice testes. In Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice testes, Sertoli cells express GDNF at all the ages examined (B, D, F, and H). Bar = 50 μm.

To further determine the effect of stabilization of β-catenin on the differentiation of Sertoli cells, we analyzed the expression of anti-Müllerian hormone (AMH, also known as Müllerian-inhibiting substance), a classic marker for immature Sertoli cells, in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control mice. Normally, AMH expression in Sertoli cells is down-regulated shortly after birth to a very low basal level in adult testes [42]. Similar to GDNF expression in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testis, AMH expression also is maintained at high levels in Sertoli cells through adulthood in comparison to controls, in which AMH expression is very low or undetectable after PND 17 (Fig. 5, A–H).

FIG. 5.

Sertoli cells with activated β-catenin maintain high levels of AMH expression, an immature Sertoli cell marker. AMH protein was detected in testicular sections by immunofluorescence with an anti-AMH (red), and nuclei were detected with DAPI (blue). In control mice testes, AMH expression level is high at Postnatal Day 7 (A), and then a progressive decrease occurs up to adulthood (C, E, and G). In contrast, much higher levels of AMH expression were detected in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice testes compared to controls at all ages except Postnatal Day 7, when expression level is similar to that in control (B, D, F, and H). Bar = 50 μm.

Persistent WNT/β-Catenin Signaling in Sertoli Cells Maintains Their Proliferation in Adult Testis

Since we observed expression of AMH and GDNF in Sertoli cells of adult mutant testis, we hypothesized that Sertoli cells in mutant testes are relatively immature and proliferating. To assess the number of Sertoli cells between control and mutant testis, we analyzed the expression of SOX9, a Sertoli cell marker. We observed more SOX9-positive cells at all ages in mutant testes compared to control except at PND 1, when no difference was observed (Fig. 6, A–J). To quantify the number of Sertoli cells in control and mutant testis, we performed a SOX9-positive cell count and observed a significantly higher number of Sertoli cells in mutant testes than control at all stages except PND 1 (Fig. 6K). Additionally, colocalization of SOX9 and PCNA indicated no proliferating Sertoli cells in control adult testis (Fig. 7, A–C). In contrast, proliferating Sertoli cells were observed in mutant testis (Fig. 7, D–F, arrowheads), suggesting that the increase in the number of Sertoli cells in mutant testis is because of continued proliferation of Sertoli cells. Collectively, these findings indicate that the Sertoli cells in mutant testis are maintained in an immature state.

FIG. 6.

Localization of SOX9 in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control testes. At Postnatal Day 1 (A and B), no difference was observed between mutant and control testes. At later stages of testicular development (C–J), more SOX9-positive Sertoli cells were observed in mutant testes (arrowheads). The graph (K) shows mean of number of SOX9-positive cells per tubule in testes of Amhr2^{tm3(cre)Bhr/+}; Ctnnb1^tm1Mmt/+ and control mice at various ages. Error bars represent the SEM, and an asterisk (*) above a given column indicates a significant difference from control (P < 0.05). Bar = 50 μm.

FIG. 7.

Activation of β-catenin leads to sustained proliferation of Sertoli cells. We performed coimmunostaining of PCNA (green), a marker of proliferating cells, and SOX9 (red), a Sertoli cell marker to observe the proliferative activity of Sertoli cells in adult testis. A) SOX9-positive cells (arrowhead) in adult control testis. B and C) PCNA-positive (green) in control testis and colocalization of SOX9- and PCNA-positive cells, respectively. All SOX9-positive (arrowhead) cells are negative for PCNA in controls. D and E) Localization of SOX9 and PCNA in adult mutant testis, respectively. F) Coimmunostaining of SOX9 and PCNA in adult mutant testis. Arrowheads represent cells positive for both SOX9 and PCNA. Bar = 50 μm.

Activation of β-Catenin in Sertoli Cells Leads to Decreased Proliferation and Increased Apoptosis of Germ Cells

To determine whether accumulation of undifferentiated spermatogonia in tubules of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testes is caused by a spermatogonial differentiation defect or by hyperproliferation of spermatogonia, we performed expression analysis of cyclin D1, a marker for mitotic spermatogonia [24]. Normally, mitotically active germ cells are located at or near the basal compartment, whereas meiotic germ cells are present in the more adluminal compartment of the seminiferous tubule. However, in the mutant mice, we observed mitotically active germ cells in the lumen (Fig. 8). To assay the number of cyclin D1-positive cells in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control testes, we scored cyclin D1-positive cells. The number of mitotic spermatogonia, from PND 17 to adulthood, in testis of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ was significantly decreased compared to that in littermate controls (Fig. 8), indicating that a differentiation defect, not hyperproliferation, of spermatogonia causes accumulation of germ cells in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testis.

FIG. 8.

Representative testicular sections from Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ (B, D, F, and H) and control mice (A, C, E, and G) stained with cyclin D1, a marker for mitotic spermatogonia. Anti-cyclin D1 was used to detect cyclin D1 by immunohistochemistry (brown); nuclei are counterstained with hematoxylin. In Postnatal Day 7 testes, cyclin D1-positive cells are similarly arranged in control and mutant testes (A and B). In mutant testes at Postnatal Days 17 and 35 and adulthood, cyclin D1-positive cells are also present in the center of the seminiferous tubules (arrowheads), whereas in controls, positive cells are only present at the periphery of tubules (C–H). Inset in G represent the control with no primary antibody. The graph (I) shows the mean number of cyclin D1-positive cells per tubule in testes of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control mice at various ages. At all ages, fewer cyclin D1-positive spermatogonia are observed in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice than in control mice except for Postnatal Day 7 testes, in which the number of cyclin D1-positive cells are similar for both groups. Three animals were analyzed for each time points and genotype. Error bars represent the SEM, and an asterisk (*) above a given column indicates a significant difference from control (P < 0.05). Bar = 50 μm.

A block in spermatogonial differentiation or accumulation of spermatogonia in seminiferous tubules has been shown to induce germ cell death in juvenile spermatogonial depletion (Utp14b) and Bax knockout mice [43, 44] as well as in Bcl2l1 or Bcl2 transgenic and GDNF-overexpressing mice testes [4, 43–45]. This led us to hypothesize that the germ cell loss we observed in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ adult testis was the result of increased apoptosis caused by accumulation of spermatogonia undergoing defective spermatogenesis. To test this hypothesis, we performed TUNEL analyses to assess the degree of cell death in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control testes. To estimate the total number of apoptotic cells, we used the counting method described above for estimating cyclin D1-positive cells. We observed a significantly higher average number of TUNEL-positive cells in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ than in control testis (Fig. 9) at all ages that we examined. Additionally, we did not observe any TUNEL-positive cells in the center of the mutant tubules (Fig. 9F, arrowhead), suggesting that the cyclin D1-positive cells observed in the center of the mutant tubules are not dead cells. Collectively, these findings support the hypothesis that defective differentiation of spermatogonia leads to their accumulation in tubules and increased apoptosis.

FIG. 9.

Increased apoptosis in testicular sections from Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice. Apoptotic cells (green) were observed by the TUNEL assay, which detects fragmented DNA. Control mice (A, C, E, and G) have fewer TUNEL-positive cells at all the ages compared to the mutant mice (B, D, F, and H). The graph (I) shows the mean number of apoptotic cells in testes of Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ and control mice. At all the ages, a significantly higher number of TUNEL-positive cells is present in mutant testes than in controls. Forty tubules per testis per animal for each genotype were scored for each of first two time points (Postnatal Days 7 and 17), whereas 20 or more tubules per testis per animal from three different microscopic fields were scored for Postnatal Day 35 and adult testis. Three animals were analyzed for each time point and genotype. The arrowhead in F represents the cells present in the center of the tubules in mutant testes that are TUNEL negative. Error bars represent the SEM, and an asterisk (*) above a given column indicates a significant difference from control (P < 0.05). Bar = 50 μm.

WNT/β-Catenin Directly Induces GDNF and AMH Expression

Because constitutive activation of β-catenin signaling leads to persistent high expression of GDNF and AMH in Sertoli cells, we examined whether β-catenin regulated both of these genes at the transcriptional level. Primary Sertoli cells are not easily transfected, and the TOPflash-optimized β-catenin luciferase reporter [29] does not respond to the GSK3B inhibitor, LiCl, in the TM4 Sertoli cell line (data not shown). Therefore, we used HEK293 cells, which are transfectable and have an intact WNT/β-catenin signaling system, to study transcriptional regulation of the Amh and Gdnf promoters. We observed a twofold increase in Amh promoter activity with 10 mM LiCl compared to control (10 mM NaCl treatment), but this was less than similarly treated cells transfected with the control TOPflash, β-catenin luciferase promoter (Fig. 10A). On the basis of these results and our previous observations described above, we hypothesized that continued AMH expression in Sertoli cells may be responsible, at least in part, for the mutant phenotype and that deletion of Amh or Amhr2 would be able to rescue the phenotype observed in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ mice testes. For this reason, we developed Amhr2^{tm3(cre)Bhr/Amhr2tm3(cre)Bhr};Ctnnb1^tm1Mmt/+ mice, in which activated β-catenin is expressed on an Amhr2 receptor-null background. We did not observe a difference in the testicular phenotype of 7-wk-old Amhr^{2tm3(cre)Bhr/Amhr2tm3(cre)Bhr};Ctnnb1^tm1Mmt/+ (Fig. 10B) and Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testis, indicating that continued expression of Amh in Sertoli cells in response to constitutive expression of activated β-catenin does not play an essential role in the Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+ testicular phenotype. We also observed that activation of β-catenin by treatment with 10 mM LiCl caused a fivefold increase in Gdnf promoter activity (Fig. 10C), which was greater than the induction observed with the optimized TOPflash reporter. Sequence analyses of the proximal Gdnf promoter showed a perfect match to the Tcf7 consensus binding site [46] (Fig. 10D). We also observed several core Tcf7/Lef1 consensus [47] sites in both the Gdnf and Amh promoters (data not shown). Collectively, these results suggest that GDNF expression is directly regulated by β-catenin.

FIG. 10.

WNT/β-catenin signaling directly regulates Amh and Gdnf promoter activity. A) HEK293 cells were transfected with either the rat Amh or the TOPflash luciferase reporters and treated as indicated. B) Hematoxylin and eosin-stained section of mouse testis shows that conditional ablation of AMH signaling does not rescue mutant phenotype observed in Amhr2^{tm3(cre)Bhr/+};Ctnnb1^tm1Mmt/+testes. C) HEK293 cells were transfected with either the mouse Gdnf or the TOPflash luciferase reporters and treated as indicated. D) Schematic presentation of GDNF proximal promoter showing the consensus TCF/LEF binding site. Luciferase results in A and B are representative of mean values of three independent experiments performed in duplicate. Error bars represent the SEM, and an asterisk (*) above a given column indicates a significant difference from control (P < 0.05). Bar = 50 μm.