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BOR - Papers in Press, published online ahead of print February 5, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013276
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BIOLOGY OF REPRODUCTION 68, 2304–2313 (2003)
DOI: 10.1095/biolreprod.102.013276
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Increment of Murine Spermatogonial Cell Number by Gonadotropin-Releasing Hormone Analogue Is Independent of Stem Cell Factor c-kit Signal1

Masako Ohmura3, Takehiko Ogawa2,4, Michio Ono3, Mari Dezawa3, Masahiko Hosaka4, Yoshinobu Kubota4, and Hajime Sawada3

Department of Anatomy3 Urology,4 Yokohama City University School of Medicine, Yokohama 236-0004, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.

sperm, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a complex but highly organized process that continuously produces a great number of spermatozoa throughout life. This process starts with cell division, both self-renewing and differentiating, of spermatogonial stem cells. Spermatogonia, which are daughter cells of spermatogonial stem cells, actively proliferate on the basement membrane of the seminiferous tubules. However, since approximately 50%–75% of spermatogonia are estimated to die out by apoptosis [1, 2], an endpoint increase in the number of spermatogonia ("net proliferation") is achieved as a result of overall balance between the spermatogonial divisions and their disappearance by apoptosis. The mechanism controlling this balance, thus maintaining the appropriate number of spermatogonia, however, is poorly understood.

In 1997, it was reported that high-dose GnRH-agonist treatment in rats after irradiation substantially improved the regeneration of spermatogenesis [3]. This result was unexpected because the predominant effect of GnRH-analogues had been believed to inhibit spermatogenesis, as GnRH-analogue administration reduces LH and FSH, thereby suppressing testosterone level [4, 5]. Subsequent studies, however, have confirmed that GnRH-analogues, both agonist and antagonist, can promote the regeneration process of spermatogenesis following testicular injuries by irradiation or cancer chemotherapy drugs [6–8]. Another study showed that GnRH-antagonist enhanced the proliferation and differentiation of type A spermatogonia, probably due to reduced levels of apoptotic cell death [9]. While all these studies were performed with rats as a model species, we have previously shown that a GnRH-agonist stimulates spermatogenic colony formation following spermatogonial transplantation in mice as well as in rats [10–12]. The GnRH-agonist administered to the recipient mice significantly increased the number and size of colonies, suggesting enhanced spermatogonial proliferation during regeneration of spermatogenesis following transplantation [10, 11].

The mechanism of actions of GnRH-analogues on spermatogonial proliferation/survival and subsequent regeneration of spermatogenesis is still elusive. It has been suggested that the level of testosterone in the testis plays a key role in the mechanism. GnRH-analogues promote the proliferation of spermatogonia, and this action is reversed by administration of testosterone in a dose dependent manner [13]. Furthermore, a GnRH-antagonist significantly suppresses the intratesticular testosterone level and restores spermatogenesis in juvenile spermatogonial depletion (jsd/jsd) mutant mice, which are infertile due to arrested spermatogonial differentiation from at Aal to A1 stages [14, 15]. This effect is enhanced when the GnRH-antagonist is administered together with androgen receptor antagonist [15, 16]. In the seminiferous epithelium of the testis, testosterone acts on Sertoli cells by binding to androgen receptor (AR) [17–19]. It is thus hypothesized that Sertoli cells play a central role to increase the net spermatogonial proliferation in response to the reduced levels of intratesticular testosterone following the administration of GnRH-analogues. Altered testosterone levels in the testis should affect gene expression activities of Sertoli cells, leading to regulation of factors that control spermatogonial proliferation, differentiation, and apoptosis. There are several growth factors produced by Sertoli cells that specifically influence the activity of spermatogonia, including stem cell factor (SCF) [20, 21], activin [22], insulin-like growth factor I (IGF-I) [23], and glial cell line-derived neurotrophic factor (GDNF) [24]. Among these growth factors, SCF are the best studied growth factor, and the signaling cascade involving the SCF receptor, c-kit, has been shown to be indispensable for spermatogenesis and spermatogonial proliferation/differentiation/survival [21, 25]. Expression of SCF was reported to be regulated by FSH [26–28]. On the other hand, testosterone has not so far been indicated as a regulator of SCF [27, 28]. Based on its important role in spermatogenesis, we focused in this study on the functions of SCF as a possible mediator of GnRH-analogues to induce spermatogonial proliferation/survival and promote regeneration of spermatogenesis. In order to examine the relationship between SCF and spermatogonial proliferation, we devised an experimental system by combining the technique of spermatogonial transplantation and laser confocal microscopic observation of whole-mount preparation of seminiferous tubules. By using the Green fluorescent protein (GFP) transgenic mouse as donor, we were able to monitor the number of spermatogonia over large areas of the recipient seminiferous tubules during the treatment with leuprorelin, a GnRH-analogue. In experiment 1, using wild-type mice as recipients, we evaluated the effect of leuprorelin treatment on germ cell number following spermatogonial transplantation. In experiment 2, we made similar analyses as in experiment 1, using the Sl/Sld mice as recipients of spermatogonial transplantation. Sl/Sld mice are infertile because the membrane-bound form of SCF is not expressed in Sertoli cells even though the secreted form is produced. The absence of functional SCF leads to defects in spermatogonial proliferation/differentiation/survival and a complete lack of spermatogenesis [25]. Therefore, this mutant mouse strain is an ideal model to investigate the role of SCF in spermatogenesis regeneration induced by a GnRH-analogue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Care

All animals were housed in a light-controlled facility with an illumination schedule of 14L:10D. The room was maintained at a constant temperature of 24 ± 1°C and 55 ± 1% humidity. All animal housing and surgical procedures were in accordance with the guidelines of the institutional animal care and use committee of the Animal Research Center, Yokohama City University School of Medicine.

Donor Cell Preparation

Donor cells for transplantation were obtained from transgenic mouse line C57Bl/6, pCNX-eGFP [29]. Every tissue and organ of this transgenic mouse, with exception of erythrocytes and hairs, shows green fluorescence under excitation light [30]. In order to enrich the spermatogonial stem cells, cryptorchid testes were produced in donor GFP mice [31]. Cryptorchid surgery was performed at 1 to 3 mo of age and used 2 to 5 mo later. Testis cells for transplantation were prepared using enzymatic digestion to produce a single cell suspension of testicular cells, as previously described [32]. The concentration of the cell suspension for transplantation was 2–9 x 107 cells/ml. Donor cells were transplanted into the testes of C57BL/6 or WBB6F1-Sl/Sld recipient mice in experiments 1 and 2, respectively.

Recipient Mice Preparation for Experiment 1

C57BL/6 mice, immunologically compatible with donor mice, were used as recipients. They were injected with busulfan at 50 mg/kg body weight intraperitoneally to destroy endogenous spermatogenesis [33]. Busulfan treatment was made at 7 to 8 wk of age and 7 wk before transplantation. Approximately 4–8 µl of donor cell suspension were injected into each testis of recipient mice as described previously [32]. Twelve mice received two injections of leuprorelin acetate (Takeda Pharmaceutical Co., Osaka, Japan), 0.19 mg/mouse, at 3 wk before and 1 wk after the cell transplantation (Fig. 1). The dose and schedule (Fig. 1) of leuprorelin were chosen based on previous reports [10, 11]. Eleven mice received two subcutaneous injections of solvent and served as sham-treated control. These recipient mice were sacrificed by CO2 asphyxiation at 3, 4, or 5 wk after the transplantation. The weights of body, testis, epididymis, and seminal vesicle were recorded at sacrifice.



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  		FIG. 1. Schematic representation of experimental design. In experiment 1, wild-type recipient mice were treated with busulfan (B) at 7 wk before spermatogonial transplantation (T). All mice received two injections of the leuprorelin or solvent (L) at 3 wk before and 1 wk after transplantation and were analyzed (A) at 3, 4, and 5 wk after transplantation. In experiment 2, Sl/Sld mutant mice were used as recipients. In protocol 1, all recipients received two injections of the leuprorelin or solvent (L) at 3 wk before and 1 wk after transplantation (T) and were analyzed (A) at 6 wk after transplantation. In protocol 2, all recipient mice received a single injection of leuprorelin or solvent (L) at 4 wk after transplantation and were analyzed (A) at 10 wk after transplantation (T)

Recipient Mice Preparation for Experiment 2

WBB6F1-Sl/Sld steel mutant mice, immunologically compatible with donor mice, were used as recipients at 9 to 12 wk of age. As shown in Figure 1, in protocol 1, four mice received two injections of the leuprorelin acetate, 0.19 mg/mouse, at 3 wk before and 1 wk after the cell transplantation. Three mice received two injections of solvent and served as sham-treated control with the same schedule. Mice were sacrificed at 6 wk after the transplantation by CO2 asphyxiation. In protocol 2, four mice received a single injection of the leuprorelin acetate, 0.19 mg/mouse, at 4 wk after the cell transplantation, and five mice received a single injection of solvent and served as sham-treated control (Fig. 1). Mice were sacrificed at 10 wk after the transplantation by CO2 asphyxiation. The weights of body, testis, epididymis, and seminal vesicle were recorded at sacrifice.

Evaluation of the Colonized Tubules

For detailed observation of colonized seminiferous tubules, the transplanted testis was decapsulated, and the seminiferous tubules were mechanically separated from the interstitium in PBS. GFP-positive tubules were cut out under a stereomicroscope (Olympus SZX12, Tokyo, Japan). The isolated segments of the tubules were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 6 h at 4°C. The tubular whole mounts were prepared according to a previous report [34]. Briefly, the segments were washed with PBS and dehydrated with 25%, 50%, and 75% ethanol containing 0.1% Tween 20/PBS (PBT) and 100% ethanol for 5 min, respectively, at 4°C. The samples were stored in 100% ethanol at -20°C for later observation. At the time of observation, the samples were rehydrated with 75%, 50%, and 25% ethanol containing PBT and 0.1% Tween 20/PBS for 5 min each at room temperature. After washing with PBS, the tubules were stained with TOTO-3 (1:500 in PBS: Molecular Probes) for 30 min for nuclear staining. Dual images of GFP and TOTO-3 were obtained with excitation wavelengths of 488 and 633 nm, respectively, using a confocal laser scanning system (Radiance2000; BioRad, NJ) with an Axioplan epifluorescence microscope (Carl Zeiss, Germany).

In order to observe the three-dimensional architecture of the spermatogonial networks on the basement membrane, laser confocal pictures were taken sequentially across the field every 2 µm parallel to the longitudinal axis of the seminiferous tubules with objective lens of 40x magnification and zoom 1.5x. The data from each point of the focal plane were reconstructed to a three-dimensional image. Based on the established histological criteria [35], germ cells of different developmental stages were distinguished in the laser confocal microscopic images.

For the evaluation of each colony, three kinds of measurements of cell numbers were made in five to eight colonies per mouse: all types of donor-derived GFP-positive germ cells (total germ cells), donor-derived spermatogonia, and recipient Sertoli cells. About 100 Sertoli cells were counted in each colony. The GFP germ cell numbers were presented as those in every 100 Sertoli cells [9, 36]. Previous studies showed that the number of Sertoli cells per tubules cross section was unaffected by GnRH-analogue [37].

Whole-Mount Immunohistochemistry for c-kit

In experiment 2, recipient tubules were analyzed for c-kit expression in donor germ cells by whole-mount immunohistochemistry because c-kit is the cell surface receptor of SCF and is a molecular marker of differentiating spermatogonia [20, 38]. Samples of tubules stored in ethanol were rehydrated as described previously. They were washed with PBS and incubated for blocking in 1.0% BSA (bovine serum albumin)/PBS for 30 min at room temperature. The tubules were reacted with c-kit antibody M14 (1:50 in 0.5% BSA/PBS; sc-1494, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at room temperature. After washing in PBS, they were reacted with Alexa546 labeled donkey anti-goat IgG (1:100 in 0.5% BSA/PBS Molecular Probes) for 2 h at room temperature. After washing with PBS, the tubules were stained with TOTO-3 (1:500 in PBS; Molecular Probes) for 30 min. Images of GFP, c-kit protein, and TOTO-3 were obtained with excitation wavelengths of 488, 546, and 633 nm, respectively, using a confocal laser scanning system (Radiance2000; BioRad, Hercules, CA) with an Axioplan epifluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Statistical Analysis

Results were presented as mean ± standard error of the mean (SEM). Data were analyzed by analysis of variance (ANOVA) followed by the Fisher least significant difference (LSD) test. The P-value of <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Leuprorelin Treatment on Body and Organ Weights

Table 1 summarizes the data of weights of bodies, testes, epididymides, and seminal vesicles. The weights of these organs were presented relative to the body weight. Although leuprorelin treatment had little effect on body weight, it markedly decreased the weights of testes, epididymides, and seminal vesicles at all time points in experiments 1 and 2. These results indicate that leuprorelin effectively reduced the function of the testes and suppressed serum testosterone levels.


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  		TABLE 1. Parameters shown are number of mice analyzed, weights of body, organ weight relative to body weight, and number of spermatogenic colonies analyzed in each experimental group. Weight and relative weight are presented as mean ± SEM. BW, body weight; TW, testis weight; EW, weight of epididymis; SVW, weight of seminal vesicle

Experiment 1. Effects of leuprorelin on colony formation of germ cells using wild-type recipient mice: Analysis of whole-mount sample Following transplantation, spermatogonial stem cells of donor GFP mice established spermatogenic colonies along seminiferous tubules. In these colonies, the donor germ cells start cell differentiation mostly at the center of the colony. At the periphery of the colony, on the other hand, undifferentiated spermatogonia actively keep dividing, which leads to expansion of the colony [39]. Therefore, we made detailed observations in the center of each colony in our whole-mount samples prepared for confocal laser microscopy. Laser confocal pictures were taken sequentially parallel to the longitudinal axis of the seminiferous tubules. Reconstruction of confocal pictures demonstrated a three-dimensional image of the colony in which donor GFP cells were clearly recognized (Fig. 2, B and D). By using TOTO-3 nuclear stain, we found that Sertoli cells were readily distinguishable from germ cells by their prominent nucleoli and characteristic nuclear shape (Fig. 2, A and C). We observed no donor-derived Sertoli cells colonizing recipient seminiferous tubules in this study as in other previous experiments of testis cell transplantation. Visualized by TOTO-3 nuclear stain, undifferentiated spermatogonia had relatively large oval nuclei with homogeneous matrix texture (Fig. 2, A and C). While type A differentiated spermatogonia showed small nucleoli located at the center with a significant amount of heterochromatin spots (Fig. 2C), type B spermatogonia had smaller nuclei with denser heterochromatin patches (Fig. 2C). In addition, we found that the level of GFP emission was strongest in undifferentiated spermatogonia (Fig. 2, B and D), followed by differentiated type A spermatogonia, and weakest in type B spermatogonia (Fig. 2D). Based on these differences in nuclear morphology and GFP emission levels, we identified type A undifferentiated and differentiated spermatogonia and type B spermatogonia in the subsequent studies.



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  		FIG. 2. Confocal observation of spermatogenic colony of GFP donor with TOTO-3 nuclear stain. A and B) Spermatogenic colony of donor germ cell in Sl/Sld recipients at 3 wk after transplantation. Undifferentiated type A spermatogonia (open arrowhead) have large oval nuclei that are faintly stained with TOTO-3 (blue color-coded) (A). Sertoli cells are distinguished by their characteristic "dumbbell shape" nucleoli (asterisk) (A and B). The donor GFP germ cells (green color-coded) are forming a network arrangement on the basement membrane (B). C and D) Five weeks after transplantation. In wild-type recipient mouse testis, differentiated donor GFP germ cells can be observed. Differentiating type A spermatogonia (closed arrowhead) have small nucleoli with scattered heterochromatin along the nuclear envelope. The nucleus of type B spermatogonia (arrow) is smaller than type A spermatogonia and has strongly stained heterochromatin of small patches. Extent of GFP emission is different among these spermatogonia; strongest in undifferentiated type A, followed by differentiating type A, then by type B spermatogonia (D). Scale bars are 25 µm in A–D. Two images of TOTO-3 (blue color-coded) and GFP (green color-coded) were merged in B and D.

Effects of Leuprorelin on the Number of Spermatogonia

The number of donor-derived spermatogenic colonies per testis was between 4 and 25. These colonies were clearly recognizable at 3 wk after transplantation. At this early stage of colony formation, the majority of donor spermatogonia were undifferentiated spermatogonia spreading on the basement membrane. Most of them were connected to each other with intercellular bridges forming a paired or aligned pattern (Fig. 3, A–D). The number of GFP germ cells per 100 Sertoli cells in the leuprorelin-treated group increased 1.51-fold compared to that of control mice, although the increment was not significant at this time point (Fig. 4, A and B). At 4 wk after transplantation, donor spermatogonia spreading on the basement membrane included type A, both undifferentiated and differentiating, and type B spermatogonia (Fig. 3, F and H). Spermatocytes were occasionally observed in some colonies. In confocal images, we observed more GFP germ cells in the leuprorelin-treated group than those in the control group (Fig. 3, E and F vs. G and H). A careful quantification analysis demonstrated a significant increase in total number of GFP germ cells per 100 Sertoli cells in the leuprorelin-treated group than that of control group mice (1.81-fold, P < 0.01, Fig. 4A). In addition, when only spermatogonia among donor-derived germ cells were counted per 100 Sertoli cells, 1.58-fold more spermatogonia were detected in the leuprorelin-treated group than in the control group (P < 0.05, Fig. 4B). At 5 wk, 20.0% and 13.6% of colonies in the control and leuprorelin-treated groups, respectively, contained spermatocytes or spermatids. Spermatocytes and spermatids were identified with their location in the intraluminal compartment above the basal membrane (Fig. 3, I–L). The number of total GFP germ cells per 100 Sertoli cells in the leuprorelin group increased 1.27-fold more than that of the control group, although with no significant difference (Fig. 4A). When only spermatogonia were counted, however, a significant increase was detected in the leuprorelin-treated group than in the control group (1.64-fold, P < 0.01, Fig. 4B).



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  		FIG. 3. Spermatogenic colony formations in seminiferous tubules of wild-type mouse. Donor GFP (green color-coded) germ cells forming spermatogenic colonies in seminiferous tubules of wild-type recipient mice at 3, 4, and 5 wk after transplantation. TOTO-3 nuclear stain (blue color-coded) was employed in B, D, F, H, J, and L. A, B, C, and D) At 3 wk after transplantation, GFP positive undifferentiated type A spermatogonia (open arrowhead) form chains of cells spreading on the basement membrane, control (A and B) and leuprorelin-treated (C and D). Asterisks indicate Sertoli cells. E, F, G, and H) At 4 wk after transplantation, the density of GFP positive germ cells in leuprorelin-treated group (G, H) was higher than that of control mice (E and F). These GFP positive germ cells included type A, both undifferentiated (open arrowhead) and differentiating (closed arrowhead), and type B spermatogonia (arrow). I, J, K, and L) At 5 wk after transplantation, some colonies contained spermatocytes and spermatids in intraluminal part of the tubules, both in the control (I and J) and in the leuprorelin-treated group (K and L). Scale bars are 100 µm in A, C, E, G, I, and K; 25 µm in B, D, F, H, J, and L



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  		FIG. 4. Leuprorelin increases the number of total germ cells and spermatogonia in wild-type recipient testes. A) The values show total numbers of GFP positive germ cells, including all differentiated stages of spermatogenic cells, per 100 Sertoli cells. B) Number of GFP positive spermatogonia per 100 Sertoli cells. Values: mean ± SEM. {dagger}, P < 0.05; {ddagger}, P < 0.01; *, P < 0.05; **, P < 0.01

In summary, the results of experiment 1 showed that the number of GFP germ cells per 100 Sertoli cells increased time dependently from 3 to 5 wk both in the control and the leuprorelin-treated group (Fig. 4A). The number of GFP spermatogonia per 100 Sertoli cells similarly increased from 3 to 4 wk. However, the increase from 4 to 5 wk is not significant, apparently because the density of spermatogonia on the seminiferous epithelium came close to saturation.

Experiment 2. Effects of leuprorelin on colony formation using Sl/Sld recipient mice In order to examine the involvement of SCF in the mechanism of spermatogonial proliferation and/or survival induced by a GnRH-analogue, we used Sl/Sld mouse as recipients. Since Sl/Sld mice lack spermatogenesis due to the absence of functional SCF, this strain of mutant mice served as an ideal model to investigate whether SCF mediates the actions of GnRH-analogue on spermatogonia.

Following transplantation, GFP donor germ cells colonized the Sl/Sld mouse testes and were clearly identified as an assembly of spermatogonia spreading over the basement membrane of the seminiferous tubules. Intercellular bridges connecting spermatogonia were observed due to the GFP positive cytoplasm, but some were present as isolated single spermatogonia, that is, spermatogonial stem cells (Fig. 5).



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  		FIG. 5. Spermatogenic colony formations in seminiferous tubules of Sl/Sld mouse. Both in protocol 1 (A–F) and in protocol 2 (G–L), GFP positive undifferentiated type A spermatogonia (open arrowhead) form chains of cells spreading on the basement membrane in control group (A, B, G, and H) at 6 and 10 wk after transplantation, respectively. In the leuprorelin-treated group (D, E, J, and K), the density of GFP positive germ cells is apparently higher than that of control group (A, B, G, and H) and occupying most of the seminiferous epithelium in some areas. In the cross section, there are no differentiated germ cells in recipient's seminiferous tubules (C, F, I, and L). Scale bars are 100 µm in A, D, G, and J; 20 µm in B, E, H, and K; 22 µm in C, F, I, and L. *, Sertoli cells. GFP emission (green color-coded). TOTO-3 nuclear stain (blue color-coded)

In protocol 1, the number of donor GFP germ cells per 100 Sertoli cells increased 1.66-fold in leuprorelin-treated mice compared with control mice (P < 0.01, Fig. 7), indicating that the undifferentiated spermatogonia were stimulated to increase in number by leuprorelin treatment in Sl/Sld testes. In protocol 2, the value also increased 1.84-fold in the leuprorelin-treated group compared with the control group (P < 0.01, Fig. 7). Taken together, these results demonstrate that the net spermatogonial proliferation was increased by leuprolein treatment of recipient mice in the absence of functional SCF, and the level of increase was similar to that observed in experiment 1 using wild-type recipient mice. In addition, the results also indicate that the effect of leuprorelin was independent of the treatment schedule during transplantation experiments and was not due to an increased colonization efficiency of transplanted germ cells because the number of donor germ cells increased at a similar rate in both protocols.



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  		FIG. 7. Leuprorelin increases the number of undifferentiated type A spermatogonia in Sl/Sld mutant mice. Data represent the number of GFP positive spermatogonia per 100 Sertoli cells. Values are the mean ± SEM. *, P < 0.01

As leuprorelin significantly increased the number of spermatogonia, we further examined if these cells included more differentiated spermatogonia or spermatocytes. Both in protocol 1 and 2, 6 and 10 wk after transplantation, respectively, we could find only type A spermatogonia but no further differentiated germ cells, although there were a few isolated GFP cells apart from the basement membrane of seminiferous tubules (Fig. 5, C, F, I, and L). The nuclear morphology of the observed type A spermatogonia indicated that they were all undifferentiated subtype (Fig. 5, B, E, H, and K). Immunohistochemical analysis showed that these cells were all negative for c-kit staining, confirming that these spermatogonia were undifferentiated subtypes (Fig. 6, C–F).



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  		FIG. 6. Whole-mount immunostaining with c-kit antibody. A and B) GFP (green color-coded) germ cells in wild-type recipient mouse, 4 wk after transplantation, are stained with c-kit antibody (red color-coded) and TOTO-3 nuclear stain (blue color-coded) as positive control. The c-kit is positive on differentiating type A spermatogonia (closed arrowhead) but not on undifferentiated type A spermatogonia (open arrowhead) (B). C–F) In the testes of Sl/Sld mice, in both protocol 1 and protocol 2 experiments, no c-kit stain is observed on germ cells either in the control or in the leuprorelin-treated group. Scale bars are 15 µm in A and B; 20 µm in C–F. *, Sertoli cells


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies have discovered a novel effect of GnRH-analogues on regeneration of spermatogenesis that is seemingly opposed to their well-known suppressive effect on normal, steady-state spermatogenesis. Earlier studies have shown that when administered to normal adult males, GnRH-analogues reduce the secretion of LH and FSH from the pituitary gland [4, 5]. The reduction of gonadotropin secretion results in lower testosterone levels in testes, leading to the inhibition of spermatogenesis [40], particularly in postmeiotic phase [4, 41, 42]. In contrast, recent studies have shown that GnRH-analogues stimulate regeneration of spermatogenesis after testicular damage caused by irradiation or chemical toxins [3, 6, 8]. A study using irradiated testis model has suggested that GnRH-antagonist reduces spermatogonial apoptosis, thus increasing the number of spermatogonia [9]. The results of these studies suggest that GnRH-analogues stimulate regeneration of spermatogenesis in damaged testes by acting on spermatogonia rather than meiotic germ cells as seen in intact testes. Furthermore, a similar stimulatory effect of GnRH-analogue was also demonstrated in spermatogonial transplantation studies. Following donor cell transplantation into the testes of chemically induced infertile recipients, treatments with a GnRH-agonist of recipient animals increased the number and size of spermatogenic colonies, suggesting the GnRH-agonist enhanced proliferation and/or survival of spermatogonia in this experimental system [10, 11]. To further extend these previous results, we carried out experiment 1 in the present study and demonstrated the positive effect of leuprorelin on donor spermatogonial proliferation in whole-mount preparations of the recipient seminiferous tubules. We chose 3, 4, and 5 wk after the transplantation for observation time points because donor spermatogonia can be clearly observed during these early periods by this method. The number of donor spermatogonia counted every 100 Sertoli cells in each colony was higher in the leuprorelin-treated group than in the control group at every time point, with a statistically significant difference at 4 and 5 wk. This finding together with previous transplantation experiments [10, 11] demonstrated that the GnRH-analogues enhance donor spermatogonial proliferation and/or survival, leading to increased cell number and colony size enlargement. Although similar findings showing GnRH-analogue's effect on spermatogonial proliferation on rats were reported previously [9], this is the first report, to our best knowledge, that demonstrated the same effect of GnRH-analogue on mouse spermatogonia in mouse testis.

The mechanism by which GnRH-analogues stimulate regeneration of spermatogenesis in damaged testes has not been well described. However, because both agonists and antagonists of GnRH act on the regeneration of spermatogenesis in a similar manner, the effect of GnRH-analogues is likely indirect and intervened by altered conditions in the testicular microenvironment. Recent studies indicate that lower testosterone levels in the testis caused by GnRH-analogue treatment are beneficial for the regeneration of spermatogenesis. For instance, the effect of GnRH-analogue administration on irradiated rats, which promoted the regeneration of spermatogenesis, was hampered by administration of supplemental exogenous testosterone. Furthermore, additional treatment with flutamide, an androgen receptor antagonist, together with GnRH-antagonist, gave further support on regeneration of spermatogenesis [9, 13]. These data support the finding that low intratesticular testosterone level is favorable for the proliferation of spermatogonia remaining in damaged testes. More physiological evidence on the relationship between low intratesticular testosterone levels and active spermatogonial proliferation can be seen during postnatal development of testes. The testosterone level in the testis is very low in immature mice around 10–20 days old when spermatogonia extensively proliferate as the testis grows [43]. Taken together, the results of previous studies suggest that spermatogonia better increase in number when testosterone level in the testis is lower.

Since testosterone does not directly act on germ cells [44], the positive effect of reduced intratesticular testosterone level on proliferation of spermatogonia and regeneration of spermatogenesis is thought to be mediated by Sertoli cells in the seminiferous epithelium. In this regard, Blanchard et al. have shown that GnRH-analogue treatment increases the expression level of mbSCF, a growth factor produced by Sertoli cells, in the testes damaged by 2,5-hexanedione [45]. SCF is one of the best-studied Sertoli cell-derived growth factors and is indispensable for spermatogenesis [46]. While there are two variant forms of SCF, membrane-bound (mbSCF) and secretory SCF (sSCF) [47], the mbSCF is more essential for the maintenance of normal spermatogenesis [46]. In vivo and in vitro studies have shown that SCF protects germ cells from apoptosis and acts as a survival factor of spermatogonia [48, 49]. Therefore, it can be hypothesized that SCF mediates the effects of GnRH-analogue on spermatogonia. However, our present study clearly shows that this is not the case. The results of experiment 2 showed that leuprorelin significantly increased the number of spermatogonia in the testis of Sl/Sld mouse, leading us to conclude that SCF is not required for spermatogonial proliferation induced by leuprorelin. This conclusion is based on three reasons: First, Sl/Sld mouse does not produce mbSCF; therefore, donor spermatogonia in the Sl/Sld recipient mouse testis received no signal of mbSCF. Second, the proliferated spermatogonia in the Sl/Sld mouse testis were undifferentiated spermatogonia that do not express c-kit, the receptor of SCF, as confirmed by immunohistochemical staining (Fig. 6). Third, we observed no donor-derived Sertoli cells colonizing seminiferous tubules of Sl/Sld mice; thus, the possibility is ruled out that normal Sertoli cells and functional SCF were supplied by the donor cell population as a consequence of transplantation. Therefore, the present study demonstrates that SCF-c-kit signals are not essential for the effects of GnRH-analogue on spermatogonial proliferation or survival. However, we do not rule out the possibility that provided with functional Sertoli cells as in experiment 1, SCF could support the action of GnRH-analogue by maintaining differentiating spermatogonia, from types A1 through B, which express c-kit.

The results thus far published, including those of this study, collectively suggest a possible process toward regeneration of spermatogenesis in response to administration of GnRH-analogues after severe testicular injuries suffered from irradiation or chemical toxins: 1) GnRH-analogues reduce secretion of gonadotropins, LH and FSH, in the pituitary gland. 2) Due to the reduction of LH, testosterone production by Leydig cells in the testis is greatly reduced. 3) The reduction of intratesticular testosterone level affects seminiferous epithelium microenvironment, particularly Sertoli cells and their activities. 4) Altered microenvironment subsequently promotes the net proliferation of spermatogonia, as shown in this study, and also probably their maintenance. 5) The increment of spermatogonial number up-regulates the regeneration process of spermatogenesis. In this study, we have shown that SCF is not directly involved in this process. It is important to study further and identify which factors mediate the positive action of GnRH-analogues on regeneration of spermatogenesis. Such studies would provide opportunities to develop efficient strategies for male fertility restoration following testicular injuries. In addition, identification of factors that propagate spermatogonia would facilitate development of male germ cell culture systems.

The experimental system we used in this study, combining spermatogonial transplantation with GFP donor mice and whole-mount observation of seminiferous tubules by laser confocal microscopy, is an excellent method for the observation of spermatogonia and quantitative analyses of their activity. In particular, this system allows clear identification of spermatogonial stem cells, As spermatogonia, in a whole-mount sample. The spermatogonial stem cells are the only germ cells in adults that possess the capacity of self-renewal proliferation. Due to this unique capacity, the spermatogonial stem cell can maintain lifelong continuity of spermatogenesis. Elucidation of the nature of spermatogonial proliferation has, therefore, important implications in the development of animal transgenesis, reproductive engineering, and reproductive medicine. Such studies would be greatly facilitated using the novel approach of combining spermatogonial transplantation and highly sensitive confocal microscopic analyses as demonstrated in this study.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Nagano for his critical reading of the manuscript. We also appreciate Takeda Pharmaceutical Co. for providing leuprorelin acetate (Leuplin Depot).


   FOOTNOTES
 
1 This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (13671663 to T.O. and 12370025 to H.S.), and a Grant-in-Aid from Yokohama Foundation for Advancement of Medical Science (to T.O.). Back

2 Correspondence: Takehiko Ogawa, Department of Urology, Yokohama City University School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan. Fax: 81 45 786 5775; ogawa{at}med.yokohama-cu.ac.jp Back

Received: 13 November 2002.

First decision: 8 December 2002.

Accepted: 24 January 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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