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BOR - Papers in Press, published online ahead of print February 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025668
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BIOLOGY OF REPRODUCTION 70, 1731–1737 (2004)
DOI: 10.1095/biolreprod.103.025668
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Regulation of Mouse Spermatogonial Stem Cell Self-Renewing Divisionby the Pituitary Gland1

Mito Kanatsu-Shinohara3, Takeshi Morimoto4, Shinya Toyokuni5, and Takashi Shinohara2,3

Horizontal Medical Research Organization,3 Department of Clinical Epidemiology,4 Department of Pathologyand Biology of Diseases,5 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis originates in spermatogonial stem cells, which have the unique mode of replication. It is considered that a single stem cell can produce two stem cells (self-renewing division), one stem and one differentiating (asymmetric division), or two differentiating cells (differentiating division). However, little is known regarding how each type of division is regulated. In this investigation, we focused on the analysis of self- renewing division and examined the effect of the pituitary gland using two models of stem cell self-renewing division. In the first experiment using newborn mice, the administration of GnRH- analogue, which represses the release of gonadotropin, reduced the number of stem cells during postnatal testicular development, suggesting that the pituitary gland enhances stem cell self- renewing division. In the second experiment, however, the number of stem cells increased dramatically in hypophysectomized adult recipients after spermatogonial transplantation. Thus, the pituitary gland affects the self-renewing division of stem cells, but these contradictory results suggest that its role may be different depending on the stage of the testicular development.

gonadotropin-releasing hormone, pituitary, Sertoli cells, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis depends on a small population of cells that are called spermatogonial stem cells. There are 20 000– 30 000 stem cells in the mouse testis [1, 2], and these cells produce sperm throughout the life span of the animal [1, 3, 4]. An important characteristic of stem cells is their ability to self-renew. It is generally believed that stem cells have three modes of replication and that a single stem cell can produce 1) two stem cells (self-renewing division), 2) one stem cell and one differentiating cell (asymmetric division), or 3) two differentiating cells (differentiating division) [1]. This unique mode of replication is the hallmark of stem cells, and the manner in which it is regulated has biological significance. However, as stem cells can be identified only by functional assays that detect self-renewal activity, little progress has been made in the elucidation of the regulation of spermatogonial stem cell replication.

Although the numbers of stem cells are regulated to remain constant in the steady state, they can increase dramatically under several conditions. For example, the number of stem cells increases dramatically during the early postnatal period. Based on transplantation assays, we reported previously that the number of spermatogonial stem cells during the first week after birth increases from 2.5% to 16.2% of the total stem cell population in the adult testis [5]. On the other hand, active proliferation of stem cells is observed in the adult testis after transplantation into the seminiferous tubules of infertile recipients that lack endogenous spermatogenesis. Using the serial transplantation assay and the enhanced green fluorescent protein (EGFP) transgenic marker, we and others have found recently that stem cells increase in number in the developing colony [6, 7]. Because stem cells are considered to undergo self-renewing division to increase their number under these conditions, these models provide unique opportunities to examine the regulatory factors that are involved in self-renewing division of stem cells.

Spermatogenesis is unique among stem cell systems in that hormonal regulation plays a major regulatory role. Several lines of evidence support the association of spermatogenesis with the pituitary gland. In a classic experiment, removal of the pituitary gland (hypophysectomy) resulted in the cessation of spermatogenesis [8]. In mutant mice with gonadotropin-deficiency, germ cell differentiation is not observed beyond the spermatocyte stage [9]. Although gonadotropin is obviously necessary for the completion of meiosis and spermiogenesis, the effect on spermatogonia is unclear, as hypophysectomy results in a modest to profound reduction (20–50%) in the number of spermatogonia [10, 11]. However, Meistrich and Kangasniemi reported in 1997 that treatment with GnRH antagonist dramatically stimulated the recovery of rat spermatogenesis after irradiation [12]. As undifferentiated spermatogonia cannot initiate differentiation in a damaged environment, this result suggests that the pituitary gland affects the differentiation of undifferentiated spermatogonia or stem cells. This result was unexpected, as administration of the GnRH analogue usually inhibits spermatogenesis under physiological conditions.

Although these studies suggested a possible association between stem cells and the pituitary gland, they focused on the regeneration of spermatogenesis or development of mature spermatozoa, and the effect on stem cell division was not clear. The regeneration of spermatogenesis can be explained by enhanced differentiating division of stem cells to produce mature germ cells and does not necessarily indicate the self-renewing division of stem cells. In addition, it is difficult to distinguish the type of division by morphological assay, because stem cell is a functional definition. The changes in stem cell number must be determined only by quantifying stem cells with a functional assay, thereby enabling the analysis of stem cell replication.

The research described here was carried out to determine the effect of the pituitary gland on changes in the number of spermatogonial stem cells using spermatogonial transplantation [13]. In this technique, each colony is believed to originate from a single stem cell, which allows quantification of donor stem cell number [14]. The numbers of stem cells were determined in two models of stem cell self- renewing division (defined as the increase in the number of stem cells), namely in the immature testis and the transplanted mature testis. In both models, stem cells were allowed to proliferate in the absence of pituitary gland influence, which was brought about either by GnRH agonist injection or hypophysectomy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

In the first set of experiments, donor cells were collected from the testes of transgenic mice of the lineage C57BL/6J-TgN(MTnlacZ)204Bri (designated as MT-lacZ), which were purchased from Jackson Laboratory (Bar Harbor, ME) [15]. The MT-lacZ transgene is thought to be expressed initially in primary spermatocytes [15]. The donor mice were injected with leuprorelin acetate (0.19 mg per mouse; donated by Takeda Pharmaceutical Co., Osaka, Japan) at 1–3 days (i.v. injection) [16] and at 2 wk (i.p. injection) after birth. Cells for transplantation were collected from 5-wk- old mice. In the second set of experiments, donor cells were isolated from the testes of transgenic mice of the lineage C57BL/6 Tg14(act- EGFP)OsbY01 (designated as Green), which were provided by Dr. M. Okabe (Osaka University, Osaka, Japan) [17]. The spermatogonia and spermatocytes of these mice express the EGFP gene, the level of expression of which decreases gradually after meiosis [17]. Cells for transplantation were obtained from the testes of 8- to 12-wk-old animals. In both experiments, single-cell suspensions from the testes were prepared by two- step enzymatic digestion using collagenase and trypsin [18]. Briefly, the tunica albuginea was manually removed from the testes. The exposed seminiferous tubules were then dissociated with collagenase (1 mg/ml, type IV; Sigma, St. Louis, MO) at 37°C for 15 min. After being rinsed two times in Hanks balanced salt solution, the tissue was digested with 0.25% trypsin and 1 mM EDTA (both from Invitrogen, Carlsbad, CA) at 37°C for 10 min. The cells were suspended in Dulbecco modified Eagle medium/10% fetal calf serum (DMEM/FCS), supplemented as described previously [18].

The donor cells were transplanted into the testes of WBB6F1-W/Wv mice (designated W; purchased from Japan SLC, Shizuoka, Japan) at 6 to 10 wk of age. The W mice are histocompatible with the donor cells and are congenitally infertile, because they lack all the stages of differentiating germ cells due to mutations in the c-kit receptor tyrosine kinase [19, 20]. In some of the experiments in the second set using hypophysectomized and sham-operated control W mice, the operation was performed when the animals were 5 wk of age. Cells were transplanted at least 2 wk after the operation. The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols.

Flow Cytometry

Flow cytometric analyses were performed using a standard protocol [21]. Briefly, aliquots of 106 testis cells were suspended in 0.1 ml of PBS that contained 1% fetal bovine serum and were incubated with the primary antibodies. Testis cells were incubated with 5 µg/ml rat anti-TDA IgG (EE2) [22], which was provided by Dr. Y. Nishimune (Osaka University, Osaka, Japan). The primary antibody was detected using 5 µg/ml of allophycocyanin (APC)-conjugated goat anti-rat-IgG (Cedarlane Laboratories, ON, Canada). The control cells were not treated with the primary antibodies. The cells were kept in the dark on ice until analysis on a Becton Dickinson FACSCalibur (BD Biosciences, Franklin Lakes, NJ). At least 10 000 events were acquired for each sample.

Transplantation Procedure

For the testicular injections, aliquots of approximately 3 µl of the donor cell suspension were introduced into the seminiferous tubules of the testes of control W mice, while aliquots of 1.5 µl were introduced into the testes of hypophysectomized W mice, as the latter testes are smaller. In the first set of experiments, approximately 1.5 x 105 cells were injected into each testis of untreated W mice. In the second set of experiments, approximately 1.5 x 105 cells were injected into the hypophysectomized W recipients; whereas 3 x 105 cells were injected into the sham-operated control W recipients. For the serial transplantation, the cells from whole testes were collected from these primary recipients after analysis of colony numbers, and were dissociated in Eppendorf tubes. The cells were suspended in a volume of approximately 10–15 µl of DMEM/FCS and transplanted into 3–4 testes of secondary recipients. The number of cells injected per testis ranged from 0.2 x 105 to 7.6 x 105. The number of colonies was expressed per donor mouse testis.

Transplantation was by efferent duct injection [18], whereby 75–85% of the tubules of each recipient testis were filled.

Analysis of Recipient Testes

In the first set of experiments using the MT-lacZ mice, recipient mouse testes were recovered 3 mo after donor cell transplantation and analyzed by staining for the lacZ gene product, ß-galactosidase, with the substrate 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal) (Wako Pure Chemical Industries, Osaka, Japan) [14]. In the second set of experiments using Green mice [17], donor cell colonization was evaluated by observation of fluorescence under ultraviolet light [17]. These methods allowed the specific identification of donor germ cells, as the endogenous host testis cells did not stain with X-gal and had no endogenous fluorescence. A cluster of germ cells was defined as a colony when it occupied more than 50% of the basal surface of the tubule and was at least 0.1 mm in length [14]. The total number of colonies was counted under a stereomicroscope by dissecting the seminiferous tubules with fine forceps. Statistical analysis was conducted using the Student t-test. A P value less than 0.05 was considered to be statistically significant.

Histology

The testes were fixed in 10% neutral-buffered formalin (Wako Pure Chemical Industries) and processed for paraffin sectioning. All of the histological sections were stained with hematoxylin-eosin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pituitary Gland Enhances Stem Cell Proliferation During Postnatal Testicular Development

In the first set of experiments, we examined the effect of the pituitary gland on stem cells in the immature postnatal testis. For this purpose, neonatal MT-lacZ mice were injected with leuprorelin, which reduces the levels of LH and FSH [23]. The injected mice were returned to their mothers and the mice received a second injection 2 wk later. Five weeks after birth, the leuprorelin-treated mice weighed 16.0 ± 0.1 g (mean ± SEM, n = 5), whereas untreated control mice of the same litter weighed 19.0 ± 0.6 g (mean ± SEM, n = 3), which indicated that leuprorelin had reduced growth hormone release. The effect of leuprorelin was more dramatic in the testes; the testes of the leuprorelin-treated and untreated mice weighed 6.0 ± 0.9 mg (mean ± SEM, n = 10) and 60.5 ± 4.6 mg (mean ± SEM, n = 6), respectively, and this difference was statistically significant (Fig. 1A). Histological analysis revealed that germ cell differentiation was limited in the leuprorelin-treated testis (Fig. 1B). In the tubules that contained the highest number of differentiated germ cells, the most advanced germ cells were spermatocytes, whereas numerous elongated spermatids and spermatozoa were observed in the untreated control testes (Fig. 1B).



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  		FIG. 1. Leuprorelin injection into newborn MT-lacZ mice. A) Macroscopic appearance of MT-lacZ donor testes. (Left) A testis from a control litter MT-lacZ mouse. (Right) A testis from an MT-lacZ mouse that was treated with leuprorelin. Note the large difference in size, which reflects the suppression of spermatogenesis by leuprorelin treatment. B) Histological section of donor testes. (Top) Histological section from a control litter MT-lacZ mouse testis. (Bottom) Histological section from the testis of an MT-lacZ mouse testis that was treated with leuprorelin. Note the suppression of spermatogenesis in the leuprorelin-treated mouse testis. C) Characterization by flow cytometry of testis cells from leuprorelin-treated MT-lacZ mice. (Left) Control MT-lacZ mouse testis cells stained with the anti-EE2 antibody. (Right) Leuprorelin-treated MT-lacZ mouse testis stained with the anti-EE2 antibody. The shaded area shows the range of fluorescence in the control. Note the increased percentages of EE2-positive cells in the leuprorelin-treated mouse testis cell population. Bars = 1 mm (A) and 50 µm (B); stain, hematoxylin and eosin (B)

The effect of leuprorelin injection on spermatogenic cells was examined by flow cytometry. The testes from both types of animal were dissociated by enzymatic digestion and stained with EE2, which is a marker for spermatogonia and early meiotic cells [22]. The yield of testis cells was significantly lower from the leuprorelin-treated donor mice than from the untreated controls (2.6 ± 0.3 x 106 vs. 30.1 ± 2.1 x 106 cells/testis; mean ± SEM, n = 3) and the percentage of EE2-positive cells was significantly higher in the testis cell population from the leuprorelin-injected animals than in that from the untreated controls (24.1% ± 1.1% vs. 13.8% ± 2.5%; mean ± SEM, n = 4; Fig. 1C), which suggests that leuprorelin-treated testes are relatively enriched for early-stage germ cells, such as stem cells.

To investigate the effect of the pituitary gland on stem cells, we performed spermatogonial transplantation experiments [13]. Testis cells from both types of animals were microinjected into the testes of infertile W mice that have normal pituitary function. The W recipients lack all stages of endogenous germ cells and are capable of generating spermatogenesis from transplanted donor cells [5]. The recipient testes were recovered 3 mo after transplantation, and the testes were stained for lacZ activity (Fig. 2A). The average number of colonies per 105 injected cells was 24.0 ± 3.3 (mean ± SEM, n = 19) and 5.7 ± 0.7 (mean ± SEM, n = 22) for the leuprorelin-treated and untreated animals, respectively (Fig. 2B). Histological analysis of the recipient testes showed complete spermatogenesis from both types of donor cell (data not shown). Thus, the concentrations of stem cells were higher in the leuprorelin- treated testes than in the untreated control testes, and the stem cells in the leuprorelin-treated testis were able to complete differentiation after transplantation into surrogate, untreated, infertile recipients. As the recovery of cells from leuprorelin-treated animals was reduced to approximately 9% of that of the untreated controls, the total number of stem cells in leuprorelin-treated testis (stem cell concentration x total cell recovery) was 36% of the number in untreated testis (620 ± 80 vs. 1700 ± 120 stem cells per testis; mean ± SEM) (Fig. 2C), and this difference was statistically significant. These results indicate that the number of stem cells is reduced in the absence of effect from the pituitary gland during early postnatal testicular development.



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  		FIG. 2. Spermatogonial transplantation of leuprorelin-treated donor testis cells. A) Macroscopic appearance of recipient testes after spermatogonial transplantation of donor testis cells. (Left) A testis from a W recipient mouse 3 mo after transplantation with control MT-lacZ mouse testis cells. (Right) A testis from a W recipient mouse 3 mo after transplantation with leuprorelin-treated MT-lacZ testis cells. The same number of cells (1.5 x 105) was injected into each recipient testis. The blue stretches in the tubules represent colonies of spermatogenesis from lacZ-marked donor cells. B) Enhanced colonization of recipient testes by leuprorelin- treated donor MT-lacZ donor testis cells. The levels of colonization in the three experiments are indicated by the numbers of individual blue colonies. The values (mean ± SEM) for the control MT-lacZ testis cells and leuprorelin-treated MT-lacZ testis cells were 5.7 ± 0.7 (n = 22) and 24.0 ± 3.3 (n = 19) per 1 x 105 injected cells, respectively. C) The total number of stem cells in the testis. The values (mean ± SEM) for the control MT-lacZ testis cells and leuprorelin-treated MT-lacZ testis cells were 1700 ± 120 (n = 22) and 620 ± 80 (n = 19) cells per testis, respectively. Bar = 1 mm (A); stain, X-gal (A)

Enhanced Self-Renewing Division of Donor Spermatogonial Stem Cells in Hypophysectomized Mature Recipient Animals

In the second set of experiments, we used serial transplantation into mature animals to investigate the effect of the pituitary gland on the changes in the number of spermatogonial stem cells. For this set of experiments, we used Green mice [17]. As the spermatogenic colonies from these mice can be visualized under ultraviolet (UV) light, they can be counted and collected for transplantation into another animal for the assessment of stem cell numbers [6, 7].

Donor Green mouse testis cells were microinjected into the testes of hypophysectomized and sham-operated W recipients (primary recipient). Two months after transplantation, the number of donor-derived colonies was counted under UV light (Fig. 3A and Table 1). The numbers of colonies per 105 cells in the hypophysectomized and sham- operated control animals were 20.4 ± 2.4 (mean ± SEM, n = 19) and 5.8 ± 0.6 (mean ± SEM, n = 21), respectively, and the difference in colony numbers between the two types of recipient testis was significant. Although donor cell colonization was more extensive in the hypophysectomized recipients, histological analysis revealed limited meiotic differentiation of donor germ cells (Fig. 3B). In contrast, the donor germ cells in the sham-operated recipients were able to differentiate into elongated spermatids (Fig. 3B).



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  		FIG. 3. Spermatogonial transplantation in hypophysectomized mice. A) Macroscopic appearance of W recipient testes 2 mo after transplantation of GFP-marked testis cells. GFP-positive cells were injected into primary recipient testes. Cells from the primary recipient testis were subsequently transplanted into three to four secondary recipient testes 2 mo after the original transplantation. The green stretches in the tubules represent colonies of spermatogenesis from GFP-marked donor cells. (Left, Top) A sham-operated primary recipient testis that received GFP-marked testis cells (3 x 105 cells injected). (Left, Bottom) A hypophysectomized primary recipient testis that received GFP testis cells (1.5 x 105 cells injected). Note the smaller size of the hypophysectomized recipient testis. (Right, Top) A secondary recipient testis that received cells from a sham-operated primary testis. (Right, Bottom) A secondary recipient testis that received cells from a hypophysectomized primary testis. Note the increase in the number of colonies from the hypophysectomized primary testis despite the lower number of GFP testis cells originally injected. B) Histological section of primary recipient testes. (Top) Histological section of a sham- operated primary recipient testis. Note the normal-appearing spermatogenesis. Elongated spermatids are observed. (Bottom) Histological section of a hypophysectomized primary recipient testis. Note the incomplete spermatogenesis. Spermatogenesis was interrupted at meiosis. Bars = 1 mm (A) and 50 µm (B); stain, hematoxylin and eosin (B)


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  		TABLE 1. Serial transplantation experiments.a

To examine the number of stem cells in the primary recipients, whole testes from the primary recipients were dissociated and the testis cells were collected. The total cell recovery from the testes of hypophysectomized recipients was significantly lower than that from the testes of sham- operated recipients (2.1 ± 0.3 x 105 vs. 9.6 ± 0.5 x 105, mean ± SEM; n = 19 and 21 testes for hypophysectomized and sham-operated control, respectively), which reflects the size difference between the two types of recipient. The cells were then transplanted into untreated W recipients (secondary recipients). The cells from one testis were transplanted into three or four recipient testes. Two months after transplantation, the number of colonies in the secondary recipients was counted to evaluate the stem cell number in the primary recipient testis. The average numbers of colonies per 105 cells in the secondary recipients were 17.1 ± 2.9 (mean ± SEM, n = 19) and 2.6 ± 0.8 (mean ± SEM, n = 21) for hypophysectomized and sham-operated control, respectively. Assuming that 1) each colony is derived from a single stem cell [14], 2) 10% of the injected cells colonize [14], 3) the absence of the pituitary effect in the donor does not influence the seeding efficiency of stem cells, and 4) 100% of the cells were recovered by enzymatic digestion, the self-renewing activity of stem cells was evaluated by comparing the stem cell numbers in the primary recipient testes between the two time points (from the time of transplantation to the retransplantation into the secondary recipients). The ratio of stem cell number between the two time points was 7.6 ± 1.1 (mean ± SEM, n = 19) and 5.0 ± 1.5 (mean ± SEM, n = 21) for the hypophysectomized and sham-operated donors, respectively. Although no significant difference was noted between the two groups, the increment of stem cell numbers (the net increase in the number of stem cells during the 2-mo period in the primary recipient testis) was significantly greater in the hypophysectomized animals than in the sham-operated animals (150 ± 30 vs. 20 ± 10; mean ± SEM; n = 19 and 21 testes for hypophysectomized and sham-operated control, respectively). Regardless of the host type, complete spermatogenesis was observed from both donor cell types in the secondary recipients (data not shown), which indicates that the transplanted stem cells were functionally normal. Taken together, these data indicate that the self-renewing division of stem cells occurs more actively in hypophysectomized animals after spermatogonial transplantation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The involvement of the pituitary gland in spermatogonial proliferation has been addressed in numerous studies [1, 3, 4, 10, 11, 23–25]. As most of these studies focused on stem cell differentiation, the effects on stem cell self- renewing division were not determined due to the difficulty in distinguishing the type of division by morphological assay. To address this issue, we used two models of stem cell self-renewing division, namely the initiation of spermatogenesis in immature testes and the development of colonies in mature testes after spermatogonial transplantation. In both models, increases in the number of stem cells were previously demonstrated by spermatogonial transplantation [5–7], which is a functional assay to detect stem cells [13]. Using this technique, we examined the effect of the pituitary gland on the self-renewing division (as defined by the increase in the number of stem cells) of spermatogonial stem cells.

In the first experiment, we examined the effect of the pituitary gland on immature testes. Gonocytes are the only germ cells in the neonatal testis, and these cells divide and migrate toward the basement membrane to become spermatogonia during the first week after birth [26]. Although stem cell activity is detectable from the fetal or neonatal stage [27, 28], the number of stem cells increases dramatically during this period [5, 29]. In addition, the stem cell niche [30], which affects the stem cell division, also undergoes dynamic changes [5, 29]. The signals causing these changes have not been identified, but one possible candidate is a hormonal effect from the pituitary gland. The pituitary gland is known to affect testicular development from the fetal stage [26]; the division of Sertoli cells in decapitated embryos is reduced significantly [26], and transition from proliferating Leydig cell progenitor to differentiated adult Leydig cell is also regulated hormonally [31]. Previous reports have demonstrated the deleterious effect of neonatal suppression of gonadotropins. When neonatal rats were injected daily with testosterone at a dose that suppressed pituitary gonadotropins, spermatogenesis initiated at the expected time, but was interrupted at meiosis, which suggests that the pituitary gland influences meiosis during this period [32]. However, although a reduction in the number of spermatogonia was reported [33], it was not clear whether the treatment affected the stem cells.

In the present experiment, we treated neonatal animals with leuprorelin, which is the GnRH analogue that suppresses gonadotropin. Although the concentration of stem cells in the donor cell populations increased possibly due to the absence of mature germ cells, the total number of stem cells per testis was reduced by the treatment. At present, it is not clear how the GnRH analogue suppresses the increase in the number of stem cells, but one possible explanation is that administration of GnRH analogue inhibits the secretion of FSH, which is a major regulator of Sertoli cell proliferation [26, 34], thereby resulting in a lower number of Sertoli cells. Stem cells are considered to reside in a special microenvironment, or niche [30], which is distributed nonrandomly in the seminiferous tubule [35]. By definition, if space with the necessary microenvironment were limited, the number of stem cells also would be limited [30]. Therefore, limited proliferation of Sertoli cells is likely accompanied by a corresponding decrease in the number of niches, leading to fewer stem cells. Furthermore, GnRH treatment suppresses the glial cell line-derived neurotrophic factor (GDNF) expression in the Sertoli cells [36]. As GDNF is a positive regulator of stem cell self-renewal [37], the absence of the FSH signal from the pituitary gland results in lower GDNF levels in the testes [36], which may lead to fewer stem cells. Thus, the lowered number of Sertoli cells and reduced expression of GDNF may explain the reduced number of stem cells seen in the first experiments.

The purpose of the second experiment was to examine the effect of the pituitary gland in mature animals. Although stem cell numbers are kept constant in the steady- state conditions [1, 2], transplanted stem cells can be triggered to proliferate actively in the recipient testis in the absence of competing germ cells [6, 7]. In this sense, generation of spermatogenic colonies by transplantation into infertile recipients is one of the models to trigger self-renewing division in mature animals. Our result agrees with previous studies that have shown the beneficial effect of GnRH analogue on spermatogonial transplantation. Ogawa et al. originally showed that colonization of donor-derived stem cells was enhanced in GnRH analogue-treated mouse recipients [38]. A similarly beneficial effect was subsequently confirmed in rats [39]. A more recent study has shown that the numbers of type A and type B spermatogonia per fixed number of Sertoli cells increase in recipients that have undergone GnRH analogue treatment [23], probably due to increased proliferation and reduced apoptotic cell death [40]. Thus, both the numbers of colonies and the density of germ cells in the spermatogenic colonies increase in GnRH analogue-treated recipients. However, the effect of GnRH analogue on stem cell proliferation was not addressed in these studies. As stem cells cannot be distinguished morphologically from other committed spermatogonia, the number of stem cells must be determined by the retransplantation of recipient testis cells.

In the present experiments, we used serial transplantation to evaluate the self-renewing activity of stem cells in hypophysectomized animals. The finding of enhanced colonization in the primary recipient mice (hypophysectomized W) agrees with previous studies that used GnRH analogue- treated recipients [23, 38, 39]. Although this result strongly suggests that the pituitary gland affects the seeding efficiency of stem cells, it does not demonstrate higher self- renewing activities of stem cells in the recipient testis. Therefore, to evaluate the number of stem cells in the primary recipients, we transplanted the testis cells from the primary recipients into the untreated secondary recipients. Using this serial transplantation assay, we showed that self- renewing division of stem cells is stimulated in the absence of the pituitary gland. Thus, the present study extends the previous findings and demonstrates that stem cell proliferation is negatively regulated by the pituitary gland after spermatogonial transplantation.

However, this result appears to conflict with that of the first experiment, which showed that self-renewing division of stem cells is limited in the absence of a signal from the pituitary gland. A possible explanation for this discrepancy is that the difference in the testicular microenvironment affected the stem cell proliferation. While Sertoli cells proliferate in the immature testis [26, 34], they do not divide in the mature testis. Because the pituitary gland affects the proliferation of the Sertoli cells in the early postnatal stage [26, 34], self-renewing division of stem cells may have been influenced by the difference in the recipient environment. Another possible reason for the discrepancy is the presence of competing germ cells. The stem cells that were tested in the first set of experiments were from testes that contained germ cells in all the seminiferous tubules, whereas those that were used in the second set of experiments were transplanted into infertile animals that lacked endogenous germ cells. Interactions between different populations of germ cells have been suggested in several studies [41–43], and proliferation of undifferentiated spermatogonia can be influenced by other differentiated spermatogonia via the production of a tissue-specific inhibitor of cell proliferation, or chalone [44]. Therefore, the presence of other germ cells could influence stem cell behavior.

While these factors can partly explain the discrepancy between the two experiments, results of a recent study suggest a different scenario. It was shown that the number of stem cells increases dramatically in 3- to 4-day-old mice, whereas germ cells from the Day 0–3 mouse pups can colonize but do not establish spermatogenic colonies [45]. Based on these results, it was proposed that different signals control the migration to basement membrane, resumption of mitosis, and differentiation into stem cells [45]. Because FSH is shown to regulate Sertoli and spermatogonial proliferation in immature testis [34], it is possible that the pituitary gland is involved in any of these processes that regulate stem cell maturation. If that is the case, the increase in the number of stem cells in the newborn testis may not result from self-renewing division alone; it may reflect the combined effect of stem cell self-renewing division and the stem cell maturation process. Further studies are clearly required to elucidate the mechanism by which stem cells increase in the prepubertal testis. Such studies will increase our knowledge about the developmental biology of stem cells and resolve the discrepancy of our experiments.

In conclusion, we used a functional stem cell assay to demonstrate the involvement of the pituitary gland in stem cell proliferation. The mechanism by which the division of stem cells is controlled is one of the central issues in stem cell biology, and spermatogonial stem cells probably represent the best model, as methods to manipulate stem cells [46–50] and their environments are now well established [51–53]. Uncovering the regulatory mechanism for spermatogonial stem cell proliferation also has a practical application in correcting male infertility. Further studies on the effect of hormones on stem cells will not only lead us to a better understanding of stem cell biology but will also have important implications for medicine.


   ACKNOWLEDGMENTS
 
We thank Ms. S. Hashino for technical assistance.


   FOOTNOTES
 
1 M.K.-S. was supported by a grant from the Japan Society for the Promotion of Science. Financial support for this research was provided by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Back

2 Correspondence: Takashi Shinohara, Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 753 9306;takashi{at}mfour.med.kyoto-u.ac.jp Back

Received: 21 November 2003.

First decision: 18 December 2003.

Accepted: 2 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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