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Research Article |
Divisions of Urology,4
Endocrinology and Oncology,5 Department of Surgery
Department of Pathology and Immunology,6 Washington University School of Medicine, St. Louis, Missouri 63110
ABSTRACT
Normal spermatogenesis is essential for reproduction and depends on proper spermatogonial stem cell (SSC) function. Genes and signaling pathways that regulate SSC function have not been well defined. We report that glial cell-line-derived neurotrophic factor (GDNF) signaling through the RET tyrosine kinase/GFRA1 receptor complex is required for spermatogonial self-renewal in mice. GFRA1 and RET expression was identified in a subset of gonocytes at birth, was restricted to SSCs during normal spermatogenesis, and RET expressing cells were abundant in a cryptorchid model of SSC self-renewal. We used the whole-testis transplantation technique to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice and found that each of these genes is required for postnatal spermatogenesis and not for embryological testesdevelopment. Each mutant testis shows severe SSC depletion by Postnatal Day 7 during the first wave of spermatogenesis. These defects were due to lack of SSC proliferation and an inability of SSCs to maintain an undifferentiated state. Our results demonstrate that GDNF-mediated RET signaling is critical for the fate of undifferentiated spermatogonia and that abnormalities in this pathway may contribute to male infertility and testicular germ cell tumors.
developmental biology, sperm, spermatogenesis, sperm maturation, testis
INTRODUCTION
Testicular germ cells are critical for maintaining spermatogenesis and reproduction. Spermatogenesis begins after birth and requires the activation of quiescent gonocytes into undifferentiated spermatogonia. These undifferentiated spermatogonia include spermatogonial stem cells (SSCs), a self-renewing population that provides a continuing source of haploid spermatozoa. The molecular pathways governing SSC self-renewal and differentiation are poorly characterized. Defining the mechanisms that regulate SSC function may help delineate the pathogenesis of male germ cell disorders, such as male infertility and germ cell tumors, and provide insights into their treatment.
The glial cell-line derived neurotrophic factor (GDNF) family ligands (GDNF, ARTN, NRTN, and PSPN) signal via their interaction with one of the GFRA1–4 coreceptors and the RET receptor tyrosine kinase (RTK) [1]. This interaction leads to RET activation of downstream signal transduction pathways, such as AKT1 and MAPK, that are important for cell survival, proliferation, and differentiation. In the testes, GDNF is secreted by Sertoli cells (somatic cells of the seminiferous tubules) and is an essential growth factor for the maintenance of SSCs in vitro [2–4]. The observations that GDNF haplo insufficiency results in a decreased number of germ cells and that testicular overexpression of GDNF results in germ cell proliferation and the development of seminomatous tumors, suggests that SSC fate is regulated by GDNF levels [3, 5, 6].
RET hypomorphic mice manifest abnormalities in germ cell maturation during the first wave of spermatogenesis, thus supporting the importance of GDNF-mediated RET activation in germ cell function [7]. However, a clear understanding of how RET signaling influences spermatogenesis has been hampered due to several factors. First, the temporal and cell-type-specific expression of GFRA1 and RET in germ cell derivatives (gonocytes, undifferentiated spermatogonia, SSC, differentiating spermatogonia, spermatocytes, spermatids, spermatozoa) is poorly defined [8]. Second, the neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice has prevented the study of spermatogenesis, which occurs postnatally.
In the present studies, we found that RET and GFRA1 are coexpressed and RET and GFRA1-expressing cells encompass a subpopulation of gonocytes at birth. With the progression of spermatogenesis, RET expression coincided with PLZF, a protein important for SSC function, and not with KIT, an RTK that is critical for spermatogonial differentiation [9–11]. We used testis transplantation technology to overcome the limitation imposed by the neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient animals on studies aimed at deciphering the role of these proteins in spermatogenesis. Using this strategy, we found that any disruption of GDNF-mediated RET signaling results in a failure of spermatogenesis due to deficits in SSC self-renewal.
MATERIALS AND METHODS
Animals
The Ret–/–, Gfra1–/–, and Gdnf –/– mutant mice have been previously described [12–14]. All surgical procedures were performed on mice under anesthesia. All animals were housed and maintained per protocols approved by the Institutional Animal Studies Committee. All investigations were conducted in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science).
Immunohistochemistry and Tissue Processing
Tissues were fixed in 4% paraformaldehyde or Bouin solution, paraffin embedded, serially sectioned at 6 µm, and processed as previously described [7]. Hematoxylin and eosin (hematoxylin-eosin) staining was performed for routine histological assessment. Immunohistochemical studies were performed on paraffin-embedded specimens as previously described [7]. Primary antibody incubations were for 12–18 h at 4°C, and washes were performed with PBS containing 0.1% Triton X-100. Secondary incubations were for 1–2 h at room temperature. Antibodies used to identify germ cells and Sertoli cells were anti-GCNA1 (1:200, rat polyclonal; a gift from George C. Enders, Ph.D., University of Kansas Medical Center, Kansas City, KS) and anti-GATA4 (1:200, goat polyclonal; Santa Cruz Biotechnology), respectively. The other primary antibodies used were anti-RET, anti-GFRA1 (1:100, goat polyclonal; Neuromics, Bloomington, MN), anti-KIT (1:100, goat monoclonal, M14; Santa Cruz Biotechnology), and anti-PLZF (1:50, rabbit polyclonal; Oncogene Research). Following primary antibody incubations, sections were treated with biotinylated secondary antibodies (1:250; Jackson ImmunoResearch) and the signals visualized with Cy3 (1:500) or Alexa488 (1:100) conjugated to streptavidin (SA), or with peroxidase-conjugated secondary antibodies (1:500) for visualization with 3,3'-diaminobenzidine (DAB).
For double-labeling experiments with RET where primary antibodies were from the same species (example, RET and KIT; RET and GFRA1), anti-RET antibody conjugated to biotin (1:200, goat polyclonal; Neuromics, Bloomington, MN) was used. In these experiments, the tissues were initially stained with anti-GFRA1 or anti-KIT and the respective secondary antibodies conjugated to Alexa488, blocked with goat serum (5% in PBS), and then treated with the biotinylated anti-RET antibody, followed by SA-Cy3 for visualization. Controls included omitting the primary or secondary antibodies, immunostaining with each antibody individually on adjacent sections, and/or staining tissues from the appropriate mutant mice.
In vivo 5-bromo-2-deoxyuridine (BrdU) labeling was performed by injecting BrdU intraperitoneally (200 mg/kg), and the animals were killed 2 h after injection. Tissues were prepared for immunohistochemistry and double-label RET and BrdU (1:200, mouse monoclonal; Roche) staining was used to detect proliferating SSCs [14]. Cell death was analyzed using TUNEL assay (Boehringer Mannheim) [15].
The fluorescent signals were visualized using a compound microscope equipped with epifluorescence (Nikon Eclipse 80i). Images were captured with a monochrome digital camera (Roper Scientific, CoolSnapES) and processed using Metamorph and Adobe Photoshop CS softwares.
Production of Cryptorchid Mice
The testes of 4- to 6-wk-old wild-type (WT) mice were moved to the peritoneum by ligating the epididymis and surrounding fat through a lateral oblique incision 2 mm caudal to the last rib [16]. The contralateral testis was sham operated by an incision on the skin and the peritoneum. Both testes were harvested 6 wk following the procedure and processed for subsequent studies.
Testes Transplantation Experiments
Testes from P0 mutant and WT mice were transplanted subcutaneously into the back/flank of castrated 4- to 8-wk-old male nude mice (Taconic #NCRNU-M, Germantown, NY) as previously described [17]. The grafted donor testes were harvested and processed for histological evaluation at the indicated time points.
Quantitative Cell Analyses
The numbers of RET+, GFRA1+, KIT+, PLZF+, or GCNA1+ cells per seminiferous tubule in WT, mutant, or the transplanted testes were determined from 50–100 random seminiferous tubules appearing in cross-section [7]. The percentages of RET+, GFRA1+, or KIT+ germ cells were determined from double-label immunofluoresence studies with the appropriate antibodies by counting 100 GCNA1+ cells in either WT or mutant testis. For determining percentage of RET-KIT, RET-PLZF, and KIT-PLZF, 100–200 immunopositive germ cells were evaluated at each time point examined (n 
3). To determine the percentage of proliferating SSCs (BrdU+RET+/RET+), 40 RET+ cells from each transplanted testes of WT mice (n = 3) and a total of 30 RET+ cells from testes of three different Gdnf –/– animals were evaluated for BrdU incorporation (note that fewer RET+ cells were evaluated in the mutant because of the severe reduction of SSCs in the mutant testes). Statistical analysis was performed using Student t-test (n 3) and differences were considered statistically significant at P < 0.05 (sigma plot). We used one-way ANOVA for multiple comparisons where indicated and the Bonferroni technique was used to maintain alpha levels at 0.05 (SPSS, Chicago, IL). The results are reported as the mean ± SEM.
RESULTS
RET and GFRA1 Are Coexpressed in Germ Cells During Early Spermatogenesis
GDNF signals through the RET/GFRA1 receptor complex to promote proper development of the kidney and peripheral nervous system. While GDNF is secreted by Sertoli cells, the testicular expression of the receptor components is not well defined [3]. To better understand how GDNF-mediated RET signaling might modulate spermatogenesis and to clarify cell type and temporal expression of RET and GFRA1, we used double-label immunofluorescence with RET or GFRA1 antibodies along with antibodies that recognize the germ cell marker GCNA1 on testicular sections from different aged mice. At birth, we found that RET is expressed in a subset of gonocytes, indicating the presence of both RET+ and RET– germ cell populations (Fig. 1A). As spermatogenesis progressed, RET expression was confined to a small number of germ cells located at the periphery of the seminiferous tubule in a position characteristic of undifferentiated spermatogonia and consistent with the location of RET+ cells in P14 testes [3].
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The quantification of RET+ germ cells during postnatal development showed that the number of RET+ cells in each seminiferous tubule remained relatively constant during the proliferative phase of spermatogenesis (P0–P10) (Fig. 1A, graph). However, the percentage of RET+ germ cells declined with increasing age, as RET+ cells were rare (less than 0.5%) in adult normal testes. We also observed that GFRA1 was expressed along with RET in germ cells at all time points examined (Fig. 1B and data not shown).
RET Is Expressed in SSCs
To gather further support that the RET+ germ cells represent SSCs, we performed colocalization studies in postnatal testis using antibodies against RET and PLZF, a well-characterized transcriptional repressor expressed in SSCs and crucial for SSC self-renewal [9, 10, 18]. Using double-label immunohistochemistry, we first detected PLZF expression at P3.5 when all PLZF+ cells also expressed RET (Fig. 2). With age, the majority of PLZF+ cells continued to express RET (92% at P7, 85% at P14).
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In contrast, at P7, when SSCs begin to differentiate, KIT expression, an RTK that is important for the function of differentiating spermatogonia [11], was observed in only a subset of RET+ or PLZF+ cells (Fig. 2, A and B).
We also examined an experimental model of cryptorchid testis (see Materials and Methods), where SSCs are enriched due to a block in spermatogonial differentiation but unimpaired SSC proliferation, to determine if there was an increase in RET+ germ cell population compared with the testes of untreated control mice [16]. Immunohistochemical analysis of cryptorchid testes revealed an increase in RET+ spermatogonia compared with sham-operated testis. In contrast, KIT+ cells were rare in the cryptorchid testes, consistent with the lack of differentiating spermatogonia in these testes (Fig. 3). From the above results and for consistency of terminology used by other investigators, we will refer to the undifferentiated spermatogonia that are PLZF+, RET+, or GFRA1+ as SSCs [3, 4, 9, 10].
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GDNF Signaling Through the RET/GFRA1 Complex Is Required for Spermatogenesis
To examine further the role of RET in SSC function and to overcome the limitation of neonatal lethality of Gdnf-, Gfra1-, and Ret-deficient mice on studies of spermatogenesis, we employed whole-testis transplantation. Spermatogenesis occurs in the transplanted neonatal testes in a manner that closely recapitulates the events of normal mammalian spermatogenesis [17]. Using this methodology, genes whose deficiency results in neonatal lethality can be tested for their role in spermatogenesis in a testis-autonomous manner. We typically achieve complete germ cell maturation in a subset of seminiferous tubules (20%), and maturation to spermatocytes (premeiotic products of spermatogonial differentiation) in nearly all seminiferous tubules (>95%) of the transplanted testes. We transplanted P0 testes from Gdnf-, Gfra1-, and Ret-deficient animals along with WT controls in the same recipient nude mouse, and examined these testes at various times after transplantation (1–8 wk). This analysis revealed that transplanted WT testes underwent spermatogenesis as evidenced by the presence of all cells derived from the germ cell lineage (spermatogonia, spermatocytes, spermatids, and spermatozoa). Conversely, we observed complete germ cell depletion in the transplanted testes from each of the mutant animals when examined at 8 wk after transplantation (Fig. 4). Germ cell loss was confirmed by the absence of GCNA1+ cells. In contrast, GATA4 immunohistochemistry demonstrated that Sertoli cells were unaffected despite the lack of GDNF or its receptor components (Fig. 4).
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To glean insights into when RET signaling is needed to support spermatogenesis, we examined key time points between birth and 4 wk of age for spermatogenic defects (in the murine spermatogenic cycle elongated spermatids appear by 1 mo) in testes transplanted from GDNF, GFR
1, and RET mutant mice. This period encompasses three main events: self-renewal and proliferation (P0–P10), differentiation and meiosis (P10–P21), and spermiogenesis (P21–P35) [19]. At birth, when gonocytes are the only germ cells in the seminiferous tubules, there was no difference in the number of GCNA1+ cells in WT vs. Gdnf –/–, Gfra1–/–, or Ret–/– testes (Fig. 5). The Posttransplant Day 7 (PT7) WT testes show a dramatic increase in germ cell number, reflecting expected self-renewal and proliferation. In contrast, there was a significant decrease in germ cells in PT7 testes in all three mutants compared with the WT. No differentiated germ cell products, such as spermatocytes, spermatids, and spermatozoa, were ever observed by hematoxylin-eosin-stained sections in testes from any of the three mutant mice, and by PT28, all germ cells were lost in Gdnf –/–, Gfra1–/–, and Ret–/– testes. To determine whether the absence of GDNF-mediated RET signaling alters either of the two distinct gonocyte populations (RET+ vs. RET–) at birth, we used anti-GFRA1 antibodies to identify these cells in Ret–/– animals and anti-RET antibodies to identify them in Gdnf –/– and Gfra1–/– mice. We found that these immunopositive germ cells per seminiferous tubules were comparable in WT and all mutant testes: RET+ cells in Gdnf –/– (2.6 ± 0.2) and Gfra1–/– (2.7 ± 0.2) testes; GFRA1+ cells (2.6 ± 0.1) in Ret–/– testes; or RET+ cells in WT (3.1 ± 0.2) testes (mean ± SEM, P > 0.05) (Fig. 6A).
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Failure of SSC Self-Renewal in the Absence of GDNF-Mediated RET Signaling
We used RET (in WT, Gdnf –/– and Gfra1–/– testes) or GFRA1 (in WT and Ret–/– testes) immunohistochemistry, as described in Figure 6A, to evaluate the SSC population in testes transplanted from each of these mutants as well as WT littermates. There was a marked reduction of the SSC population in testes from each mutant despite the presence of residual germ cells identified by GCNA1 immunohistochemistry (Fig. 6B). Specifically, the percentage of seminiferous tubules containing SSCs, the number of SSCs in each seminiferous tubule, and the percentage of germ cells that represent SSCs (i.e., percentage of GCNA1+ cells that express RET or GFRA1) were all decreased in the mutant mice (Figs. 6B and 7).
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The current view of stem cell self-renewal depicts a balance between stem cell proliferation and maintenance of their undifferentiated state (developmental potential) to ensure the preservation of the stem cell pool and the successful generation of differentiated products [20]. To examine this issue, we measured the proliferation index (measured by BrdU incorporation) of SSCs (RET+ cells) in transplanted testes from WT and Gdnf –/– mice at PT7 using double-label immunofluorescence with BrdU and RET antibodies. Because the SSC population is already severely decreased at PT7 due to Gdnf deficiency, we could only evaluate a limited number of RET+ cells for proliferation in the mutant testes. Nevertheless, we found a complete absence of proliferating SSCs (BrdU+RET+/RET+) in Gdnf –/– testes compared with the percentage of proliferating SSCs in WT testes at PT7 (Fig. 7A).
The inability of stem cells to maintain an undifferentiated state results in the elimination of the self-renewing population as the cells all differentiate into restricted progenitors [20]. If RET signaling plays an additional role of maintaining SSCs in the undifferentiated state, then production of restricted progenitors, such as KIT+ differentiating spermatogonia, should continue in the absence of RET signaling until the SSC population is depleted. This would be reflected as an increased ratio of differentiating spermatogonia to SSCs in the mutant vs. WT testes. To test this hypothesis, we counted the number of differentiating spermatogonia using KIT/GCNA1 double-label immunohistochemistry on transplanted WT, Gdnf –/–, and Ret–/– testes at PT7. We found that, while SSCs were only a minor component of the residual germ cells in these mutant testes, the number of KIT+ differentiating spermatogonia in WT and the mutant testes was similar at PT7 (Fig. 7B). Double-label immunofluorescence studies in WT and mutant transplanted testes with KIT and PLZF revealed that <3% of KIT+ cells express PLZF (Fig. 7C). This results in an increased differentiating spermatogonia:SSC ratio in both Gdnf –/– and Ret–/– testes, suggesting that, in addition to its regulation of SSC proliferation, GDNF-mediated RET signaling is also important in maintaining SSCs in an undifferentiated state. Despite the initial transition of SSCs into KIT+ germ cells in the absence of RET signaling, we did not observe additional downstream differentiated germ cell products, such as spermatocytes or spermatids in any of the mutant mice.
DISCUSSION
Previous work suggests that the GDNF regulates spermatogonial stem cell fate. In this study, we have examined the role of GDNF, GFRA1, and RET during the first wave of spermatogenesis. Our work confirms that RET tyrosine kinase and GFRA1 coreceptor are localized in germ cells. Interestingly, this temporal expression profile (i.e., high percentage of positive cells early in development, but rare positive cells in the mature organ) is often observed with proteins involved in stem cell function [9, 21]. These observations therefore suggest that the RET-expressing germ cells likely represent SSCs and that RET signaling is an important regulator of SSC function during spermatogenesis. Additionally, the early coexpression of RET and GFRA1 in germ cells strongly supports the idea that GDNF-mediated signaling through this receptor complex is physiologically important in spermatogenesis.
We next demonstrate that RET is expressed in SSCs through colocalization studies with known stem cell markers, as well as demonstrating increased RET expression in a cryptorchid testis model enriched for SSCs. These studies on colocalization of RET with the SSC marker, PLZF, and our recent observation that RET-expressing germ cells are absent in testes of animals lacking ERM (a Sertoli cell transcription factor necessary for SSC self-renewal; and ERM depletion results in SSC loss) [22] (data not shown) strongly indicate that the RET+ cells in the testes are SSCs, or a subset of SSCs. This claim is also consistent with recent reports demonstrating that RET is expressed by SSCs cultured in vitro and that GFRA1 and RET mRNA expression is closely related to spermatogonial stem cell activity in tissue culture and absent in differentiated germ cells [4, 23–25].
The subset of germ cells that expressed RET, PLZF, and KIT at P3.5 are most likely germ cell precursors that are in transition toward becoming either the functional SSCs (RET+PLZF+KIT–) or the differentiating spermatogonia (KIT+RET–PLZF–) (Figs. 2, A and B, and 8A). This hypothesis is further supported by previous observations that KIT is coexpressed with SSC markers in a few SSCs at P7, before its exclusive expression in differentiating spermatogonia, and suggests that RET and KIT activities are important in two distinct germ cell populations: RET in the SSC and KIT in the differentiating spermatogonia [26]. Although definitive proof that RET+ cells are the SSCs would require transplantation of purified RET+ germ cell population into testes of infertile recipient mice, as described for other testicular stem cell markers [4, 27], the above results (Figs. 1–5) strongly support that GDNF-mediated RET signaling is essential for SSC function.
Proper function of SSCs is essential for spermatogenesis, yet little is known concerning the factors that are critical for SSC activity. The first indication that signaling by GDNF may be important for SSC function in vivo came from the observation that GDNF heterozygous mice, although fertile, show reduced spermatogonial proliferation in adulthood and that GDNF overexpression in testes results in seminomatous tumors [3]. The consequences of total deficiencies of GDNF or of components of the RET/GFRA1 receptor on SSC function are unknown due to the neonatal lethality of mice lacking these molecules. Furthermore, GDNF has been reported to signal through RET-independent pathways, such as NCAM1. Because NCAM1 is expressed by SSCs, it is important to definitively determine if GDNF-mediated RET activation is the physiologically important pathway for SSC function and spermatogenesis [4, 28]. We previously demonstrated that mice with decreased RET activity show germ cell maturation arrest during the first wave of spermatogenesis [7]; however, it is unclear if spermatogenic defects resulting from diminished RET signaling are due to RET's role in SSC self-renewal and/or differentiation. Our in vivo results clearly establish, through whole-testis transplantation techniques to overcome the limitation of perinatal lethality of gene-deficient mouse models, that GDNF-mediated RET signaling in the testis is essential for spermatogenesis.
We further demonstrate failure of SSC self-renewal in the absence of GDNF-mediated RET signaling. After birth, the gonocytes normally migrate to the periphery of the seminiferous tubules, and a subset of these gonocytes, the SSCs, enters mitosis by P3–4 [29, 30]. One of the daughter cells possesses the self-renewal properties (proliferation and maintenance of undifferentiated state) of the parent SSC, and the other daughter cell differentiates into a restricted progenitor that produces mature germ cells after subsequent cycles of proliferation [20, 31]. Defects affecting SSC migration, survival, and/or self-renewal may lead to germ cell loss and failure to establish the spermatogonial lineage. Because RET signaling is known to promote mitogenesis in other systems and is important for neural crest stem cell survival and/or proliferation, the dramatic reduction in germ cells by PT7 in testes from Gdnf –/–, Gfra1–/–, and Ret–/– mice could be due to abnormal SSC self-renewal [15, 32, 33]. These results demonstrate that RET signaling is essential for SSC self-renewal during the first postnatal week.
Abnormal SSC self-renewal can result from deficits in proliferation or in maintenance of the undifferentiated state or both. Proliferation defects cause depletion of stem cells due to their inability to replenish the undifferentiated cell population. The dramatic reduction in the SSC pool in early spermatogenesis (by PT7), coupled with the histological absence of differentiated germ cell progeny (between PT7 and PT28) in testes from Gdnf –/–, Gfra1–/– , and Ret–/– mice, strongly suggest that RET signaling is necessary for SSC proliferation. The reduced SSC proliferation in Gdnf –/– testes, along with previously documented germ cell proliferation defects in GDNF heterozygote and RET hypomorphic mice [3, 7], as well as the requirement for GDNF to maintain SSCs in culture [4], demonstrate that SSC proliferation in vivo is regulated by GDNF-stimulated RET activation. Because we also observed increased differentiating spermatogonia in the testes of the mutant animals, the overall role of GDNF-mediated RET signaling is likely regulating both SSC proliferation and differentiation.
Perioperative inflammatory changes in the transplanted testes before PT7 prevented us from examining if the migration of gonocytes from the lumen to the basement membrane of the seminiferous tubules was abnormal in the mutant testes. However, the fact that we observed KIT+ differentiating spermatogonia in mutant transplanted testes at the periphery of the seminiferous tubules suggests that the decreased number of germ cells is unlikely to be caused by abnormalities in gonocyte migration. Similarly, the number of apoptotic cells (assessed by TUNEL staining) was similar in the transplanted mutant and WT testes, suggesting that RET, like TAF4b and ERM (recently characterized genes important for SSC function), does not play a significant role in SSC survival during early spermatogenesis [18, 22] (data not shown).
The present studies, along with data obtained from the examination of GDNF heterozygous mice as well as mice with deficient KIT signaling or decreased RET activity, lead us to propose the following model of RET's role in regulating spermatogenesis (Fig. 8) [3, 7, 11]. Among the two germ cell populations at birth (RET+KIT+PLZF– and RET–KIT–PLZF–), the functional SSC population is derived from the RET+KIT+ gonocytes that also express PLZF by P3–4 (coincides with the time of migration of gonocytes to the basement membrane) (Fig. 8A). As spermatogenesis progresses, RET and PLZF expression is limited to SSCs and both these proteins are essential for SSC self-renewal. In contrast, with increasing age, KIT expression is predominantly in the premeiotic differentiating spermatogonia, consistent with the known postnatal role of KIT in spermatogonial differentiation. The absence of GDNF, GFRA1, or RET results in SSC depletion and a Sertoli cell-only phenotype (Fig. 8B). This phenotype arises from defects in SSC self-renewal due to deficits in SSC proliferation and maintenance of the undifferentiated state in the absence of GDNF-mediated RET signaling. Thus, RET is the prime regulator of SSC self-renewal and functions before KIT during spermatogenesis.
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We also propose from our studies that spermatogenesis is highly sensitive to the level of RET activity in SSCs. Mild perturbations in RET signaling, such as those that occur in Gdnf heterozygous mice, allow the initial establishment of the male germ cell lineage, but abnormalities in SSC proliferation prevent repetitive spermatogenic cycles and result in successive germ cell depletion with increasing age [3]. Moderate decreases in RET activity, as seen in hypomorphic RET mice [7], lead to more severe spermatogenesis defects because the first round of spermatogenesis is incomplete. In the complete absence of RET signaling, the total lack of SSC proliferation results in rapid germ cell depletion.
The ability to successfully use the testis transplantation technology to examine testis-autonomous functions of proteins whose deficiency results in neonatal lethality has broad applications. In this instance, we found that GDNF-mediated RET signaling is critical for SSC function in early spermatogenesis. Further delineation of the RET signaling pathways that modulate SSC function may provide new insights into the pathogenesis of male germ cell diseases, such as infertility and cancer.
ACKNOWLEDGMENTS
We are grateful to Amanda Knoten for histological assistance and Rex A. Hess from University of Illinois, Urbana, IL, for valuable discussions. We thank George C. Enders, Ph.D., University of Kansas Medical Center, Kansas City, KS, for the generous gift of the GCNA1 antibody.
FOOTNOTES
1 Supported by NIH grants HD44766–02 (C.K.N.), HD047396–01 (S.J.), AG13730 and NS39358 (J.M.), and by a Faculty Scholarship of American College of Surgeons (C.K.N.). 
2 Correspondence: Cathy K. Naughton, 660 South Euclid Avenue, Box 8118, St. Louis, MO 63110. FAX: 314 576 8880; naughtonc{at}msnotes.wustl.edu
3 These authors contributed equally to this work.
Received: 12 September 2005.
First decision: 26 September 2005.
Accepted: 12 October 2005.
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Z. He, J. Jiang, M. Kokkinaki, N. Golestaneh, M.-C. Hofmann, and M. Dym Gdnf Upregulates c-Fos Transcription via the Ras/Erk1/2 Pathway to Promote Mouse Spermatogonial Stem Cell Proliferation Stem Cells, January 1, 2008; 26(1): 266 - 278. [Abstract] [Full Text] [PDF]
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Z. He, J. Jiang, M.-C. Hofmann, and M. Dym Gfra1 Silencing in Mouse Spermatogonial Stem Cells Results in Their Differentiation Via the Inactivation of RET Tyrosine Kinase Biol Reprod, October 1, 2007; 77(4): 723 - 733. [Abstract] [Full Text] [PDF]
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B. Zhang, S. Jain, H. Song, M. Fu, R. O. Heuckeroth, J. M. Erlich, P. Y. Jay, and J. Milbrandt Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome Development, September 1, 2007; 134(17): 3191 - 3201. [Abstract] [Full Text] [PDF]
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 J. M. Oatley, M. R. Avarbock, and R. L. Brinster Glial Cell Line-derived Neurotrophic Factor Regulation of Genes Essential for Self-renewal of Mouse Spermatogonial Stem Cells Is Dependent on Src Family Kinase Signaling J. Biol. Chem., August 31, 2007; 282(35): 25842 - 25851. [Abstract] [Full Text] [PDF]
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M. Tokuda, Y. Kadokawa, H. Kurahashi, and T. Marunouchi CDH1 Is a Specific Marker for Undifferentiated Spermatogonia in Mouse Testes Biol Reprod, January 1, 2007; 76(1): 130 - 141. [Abstract] [Full Text] [PDF]
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S. Jain, J. P. Golden, D. Wozniak, E. Pehek, E. M. Johnson Jr, and J. Milbrandt RET Is Dispensable for Maintenance of Midbrain Dopaminergic Neurons in Adult Mice J. Neurosci., October 25, 2006; 26(43): 11230 - 11238. [Abstract] [Full Text] [PDF]
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 C. Payne and R. E. Braun Glial cell line-derived neurotrophic factor maintains a POZ-itive influence on stem cells PNAS, June 27, 2006; 103(26): 9751 - 9752. [Full Text] [PDF]
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