Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print July 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.019323
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
69/6/1815    most recent
biolreprod.103.019323v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohta, H.
Right arrow Articles by Nishimune, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohta, H.
Right arrow Articles by Nishimune, Y.
Agricola
Right arrow Articles by Ohta, H.
Right arrow Articles by Nishimune, Y.
BIOLOGY OF REPRODUCTION 69, 1815–1821 (2003)
DOI: 10.1095/biolreprod.103.019323
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Proliferation and Differentiation of Spermatogonial Stem Cells in the W/Wv Mutant Mouse Testis1

Hiroshi Ohta3,4, Akira Tohda5, and Yoshitake Nishimune2,4

Department of Science for Laboratory Animal Experimentation,4 Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan Department of Urology,5 Osaka University Medical School, Osaka, 565-0871, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.

gamete biology, Sertoli cells, spermatogenesis, testis, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many mutations have been described for the dominant-white spotting (W) locus on mouse chromosome 5, which encodes the c-kit receptor. These mutations are known to induce pleiotropic effects, such as sterility, macrocytic anemia, and depletion of melanocytes and mast cells [1–3]. Most of the W mutant alleles, including W, Wv, Wj, and W44, cause severe impairment of fertility because of the almost total absence of germ cells in the gonads of homozygous or double-heterozygous mice [2, 4, 5]. The germ cell defect is apparent initially at Day 9 of gestation, both because of a reduction in the proliferative capacity of the primordial germ cells (PGCs) and because of retardation in their migration from the yolk sac splanchnopleure to the genital ridge [6, 7]. Moreover, several alleles that show homozygous lethality in utero, such as W35, W38, W40, W42, and W43, cause deleterious effects on postnatal germ cell differentiation, even in heterozygotes [5]. The phenotypes of the various W mutants are very similar to those of the Steel (Sl) mutants, which encode a ligand for the c-kit receptor, stem cell factor (SCF) [8]. Although phenotypic analyses of W or Sl mutant testes have been examined during early development [7] and in juvenile mice [4], to our knowledge no studies have considered the adult testicular germ cells of homozygous W mutants, because the spermatogonia of W mutants are believed to be degenerate in the adult testes (resulting from the loss of c-kit/SCF signaling).

We recently observed a small number of undifferentiated type A spermatogonia both in W and Sl mutants [9, 10]. The spermatogonia of the Sl mutant mice showed the capacity to differentiate after transplantation into the seminiferous tubules of W/Wv mutant mice [9, 11], whereas transplanted wild-type spermatogonia were able to proliferate, but not to differentiate, in the seminiferous tubules of Sl/Sld mutant mice that lacked the normal ligand [9]. These results indicate that the SCF is required not only for the migration and proliferation of PGCs but also for the differentiation of spermatogonia. The first step of spermatogonial cell differentiation, in which the cells respond to the SCF signal stimulation, is unknown. The elucidation of the detailed mechanisms of proliferation and differentiation of spermatogonial stem cells in the W mutant testis should answer this question. Furthermore, it is important to know the physiological background of germ cell differentiation in homozygous W mutants, because W/Wv mice are widely used in the germ cell-deficient testis model, which is utilized in many areas of research, including transplantation [9, 11] and molecular analysis [12–14]. In the present study, we used immunohistochemistry and germ cell transplantation techniques to perform a detailed analysis of the spermatogonia found in the W/Wv mutant testis during development.


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

Male WBB6F1 W/Wv or WBB6F1 Sl/Sld mice were purchased from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan) at 2 mo of age. To introduce the enhanced green fluorescent protein (eGFP) gene into W/Wv mice, the eGFP transgenic mice (C57BL/6TgN(acro/act-EGFP)OsbN01 [15]) were mated to W or Wv heterozygous males or females. The W or Wv heterozygous offspring of these matings were selected for the eGFP transgene and mated to each other to obtain W/Wv mice. The presence of the eGFP transgene in the homozygous mutant mice was examined by exposing individuals to excitation light. All animal experiments conformed to the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation (Research Institute for Microbial Diseases, Osaka University).

Immunohistochemistry

To obtain an overview of the spermatogonial contents of the seminiferous tubules of W/Wv mouse testes, a whole-mount immunohistochemical technique was performed using the TRA98 monoclonal antibody, which recognizes all the male germ cell nuclei [16]. Each testis was decapsulated, and the seminiferous tubules were unraveled with a forceps in Dulbecco modified Eagle medium that contained collagenase type I (1 mg/ml) and hyaluronidase (1 mg/ml). The tubules were fixed in Bouin solution for 30 min at room temperature, washed with PBS, and then incubated with the TRA98 antibody overnight at 4°C. Positive reactions were visualized by incubation with the fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G secondary antibody (ICN Pharmaceuticals, Inc., Cappel Research Products, Costa Mesa, CA).

To determine spermatogonial cell differentiation, serial cross sections were stained with either TRA98 or the anti-c-kit monoclonal antibody ACK2 [17]. In this case, freshly dissected testes were covered with OCT compound (Tissue-Tec; Sakura, Tokyo, Japan) and sections (thickness, 10 µm) were cut in a cryostat. The serial cross sections were fixed with 4% paraformaldehyde (PFA) for 30 min (for TRA98 staining) or with cold acetone for 10 min (for ACK2 staining). After fixation, the slides were incubated for 1 h at room temperature with TRA98 for germ cell-specific staining of nuclei or at 4°C overnight with 10 µg of purified ACK2 monoclonal antibody in 1 ml of PBS for the c-kit receptor. The TRA98 was visualized with diaminobenzidine, and the ACK2 antibody was detected by the avidin-biotin-peroxidase complex method with diaminobenzidine using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's recommendations.

Assessment of Germ Cell Differentiation in W/Wv Mutant Testis

Ten testes were examined at each time point. Two serial cross sections from a single testis were stained immunohistochemically with the TRA98 or ACK2 monoclonal antibodies as germ cell-specific or differentiated germ cell-specific markers, respectively. The seminiferous tubular cross sections that contained spermatogonia or differentiated (ACK2-positive) spermatogonia were counted by examining sections from 10 testes.

Transplantation of Spermatogonial Stem Cells

Donor cells were collected from the testes of eGFP-transgenic W/Wv mutant mice at 7–10 days of age using a previously described two-step digestion procedure [15, 18, 19] and subsequently transplanted into the testis via the efferent ductules [20]. To examine the proliferation of the transplanted green germ cells, the recipient testes were removed by a midabdominal incision under anesthesia, exposed to the excitation light of a fluorescent stereomicroscope, and photographed with a Leica DC200 camera (Leica Microscopy System Ltd., Tokyo, Japan). The abdominal wall was sutured carefully after the testes were returned to the scrotum. Thereafter, photographs of the testes were taken at appropriate intervals using the same method. In this way, the chronological changes in proliferation of the donor germ cells in the recipient testis could be monitored [9, 15]. For the detailed observations of colonization, the seminiferous tubules were isolated in PBS from the decapsulated transplanted testis, collected on glass slides, mounted with PBS, and observed under the fluorescent microscope.

Histological Analysis of the Testis after Green Germ Cell Transplantation

Recipient mice were killed by cervical dislocation at the appropriate time after transplantation. The testes were fixed in 4% PFA for 12 h and embedded in glycol methacrylate (Technovit 8100; Kulzer, Wehrheim, Germany) after dehydration in cold acetone for 1 h. Sections (thickness, 5 µm) were then prepared. The sections were observed and photographed under a fluorescent microscope, then stained with hematoxylin for further detailed observation under the light microscope.

GnRH-Antagonist Treatment

The GnRH-antagonist Nal-Glu ([Ac-D2-Nal1, D4Cl-Phe2, D3-Pal3, Arg5, D-Glu6 (AA), D-Ala10] GnRH) was supplied by the Contraception and Reproductive Health Branch, Center for Population Research, NICHD. The compound was suspended in distilled water at a concentration of 8 mg/ml and used to fill an osmotic pump (ALZET Model 2004; ALZA Corporation, Palo Alto, CA) designed to ensure continuous delivery over a period of at least 4 wk. The pump was placed under the skin on the back of the mouse, and Nal-Glu was delivered at a dose of 2500 µg kg-1 day-1, which is the dose that was previously reported to suppress the effects of gonadotropins and intratesticular testosterone [21–24]. Seven W/Wv mice were treated with Nal-Glu starting at 10 wk of age and were analyzed after 4 wk of treatment. In total, 14 testes were examined by immunohistological staining with the TRA98 and ACK2 monoclonal antibodies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of Spermatogonia in the W/Wv Mutant Mouse Testis

For detailed characterization of the spermatogonia in the W/Wv testes, cross sections and whole mounts of the seminiferous tubules from mice at 2, 4, or 10 wk of age were stained immunohistochemically with the germ cell-specific monoclonal antibody TRA98 [16]. Although a small number of spermatogonia were observed in the testicular cross sections of the W/Wv mutants at all ages examined, the numbers varied from testis to testis (Fig. 1, A and B, and Table 1). Even in the case of testes that contained germ cells, the spermatogonia were found as colonies that were localized in a very limited number of seminiferous tubules. In other words, some of the seminiferous tubules contained spermatogonia, but others did not (Fig. 1, C and D). However, in other cases, spermatogonia were completely absent (Fig. 1B and Table 1). Similar observations were made in the W/Wv testes of 22-mo-old mice (data not shown). These results indicate that a certain type of spermatogonia exists in the testes of W/Wv mutant mice, regardless of age. Furthermore, the probability of finding germ cells appears to be lower in prepubertal testes than in adult testes (Table 1).



View larger version (137K):
[in this window]
[in a new window]
  		FIG. 1. Characterization of spermatogonia in W/Wv mutant mice. A and B) Immunohistochemical staining with the germ cell-specific monoclonal antibody TRA98 of testicular cross sections of W/Wv mouse testes at 2 wk of age. Testicular cross sections that contain germ cells (A) or that lack germ cells (B) are shown. C and D) Whole-mount seminiferous tubules that were isolated from W/Wv mouse testes at 2 wk of age and that were immunostained with TRA98. Seminiferous tubules that contained spermatogonia were visualized with the fluorescein isothiocyanate-conjugated secondary antibody. In C, the arrowhead and arrows indicate the seminiferous tubules that contain spermatogonia and that lack spermatogonia, respectively. In D, the former situation is shown at a higher magnification. Bar = 200 µm (A and B), 100 µm (C), and 25 µm (D)


View this table:
[in this window]
[in a new window]
  		TABLE 1. Histological analysis of spermatogonia in W/ W v mutant mouse testis during development.a

To confirm that the W/Wv spermatogonia were capable of proliferation, a transplantation assay using eGFP-labeled W/Wv spermatogonia was performed. Eight weeks after transplantation, the donor spermatogonia had settled in the recipient seminiferous tubules and formed colonies (Fig. 2, A and B), whereas spermatogenesis derived from the donor eGFP-labeled W/Wv spermatogonia was not observed in the recipient nonlabeled W/Wv testis (Fig. 2, C and D). Chronological analysis of the recipient testis revealed that the transplanted spermatogonia could proliferate in the seminiferous tubules, as evidenced by the elongation of fluorescence-positive seminiferous tubules (Fig. 2, E and F). Immunohistochemical analysis revealed that c-kit-positive spermatogonia were not present in the transplanted testes (data not shown), which indicates that spermatogenesis is arrested at the point of undifferentiated type A spermatogonia.



View larger version (113K):
[in this window]
[in a new window]
  		FIG. 2. Transplantation of testicular cells from an eGFP-labeled W/Wv mouse testis into the seminiferous tubules of a nonlabeled W/Wv mouse testis. A) An isolated seminiferous tubule that contains colonized green spermatogonia 8 wk after transplantation as viewed under the fluorescent stereomicroscope. B) A higher-magnification view of A). C and D) Fluorescence micrograph (C) and light micrograph (D) of the same viewing field of the testicular cross section after hematoxylin staining. E and F) Chronological observation of a whole testis at the same position under a fluorescent stereomicroscope after transplantation of the eGFP-labeled W/Wv testicular cells. Eight weeks after transplantation (E), the donor green germ cells colonize the recipient seminiferous tubules. Twelve weeks after transplantation (F), the fluorescence-positive seminiferous tubules are elongated. The arrowheads and the arrow indicate the termini of a colony at 8 and 12 wk posttransplantation, respectively. Bar = 100 µm (A), 50 µm (B–D), and 250 µm (E and F)

The eGFP-labeled W/Wv spermatogonia were transplanted into the seminiferous tubules of W/Wv or Sl/Sld mice and monitored by fluorescence for the number and length of colonies to further confirm the settlement and proliferation of the spermatogonial stem cells. The SCF stimulation is known to be normal in the W/Wv mutant testis, whereas it is defective in the Sl/Sld mutant testis [9, 11]. If germ cell settlement and proliferation are affected by some unknown effect of SCF or via the mutant Wv receptor even though the tyrosine kinase is defective [8], we should observe some differences in the transplantation experiment. Both the number and average length of the colonies that formed in W/Wv testes were similar to those that formed in the Sl/Sld mouse, which lacks SCF function (Fig. 3). These observations demonstrate directly that c-kit/SCF signal transduction affects neither the settlement nor the proliferation of undifferentiated type A spermatogonia.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 3. Transplantation of eGFP-labeled W/Wv spermatogonia into the seminiferous tubules of W/Wv and Sl/Sld mutant mouse testes. A) Six weeks after transplantation, spermatogonial colonies that are derived from the donor cells are observed in the recipient Sl/Sld mouse testes. B) The transplanted eGFP-labeled W/Wv spermatogonia form chains or mesh structures in the recipient Sl/Sld mouse testis. C) Proliferative activity of the W/Wv spermatogonia in the W/Wv or Sl/Sld mouse testes. Donor spermatogonia from the eGFP-labeled W/Wv testes were transplanted into six testes each of W/Wv and Sl/Sld mutant mice. Six weeks after transplantation, 47 and 52 spermatogonial colonies were identified in the W/Wv and Sl/Sld mutant testes, respectively, and the lengths of the colonies were measured. The average length ± SD (error bars) is shown. No statistically significant difference was found between the groups (P = 0.41). Bar = 200 µm (A) and 50 µm (B).

Differentiation Capacity of W/Wv Mutant Spermatogonia

To examine the differentiation of W/Wv mutant spermatogonia, we performed immunohistochemical analysis using the anti-c-kit monoclonal antibody ACK2. The c-kit receptor is expressed on differentiated germ cells at the A1 spermatogonia to pachytene spermatocyte stages and also on Leydig cells; however, it is not expressed on undifferentiated type A spermatogonia that contain spermatogonial stem cells [9, 25, 26]. Likewise, the functionless c-kit protein in the W/Wv mutant is also expressed in many tissues in which the wild-type c-kit is supposedly expressed during normal differentiation, although c-kit signal transduction is impaired because of the W/Wv mutations in the receptor tyrosine kinase—that is, the deletion (W) and point mutation (Wv) [8]. Using ACK2 monoclonal antibody, which recognizes the extracellular domain of the murine c-kit receptor [17], we observed that Leydig cells in the W/Wv mutant were able to express c-kit protein strongly (Fig. 4, B, D, and F), like normal Leydig cells in wild-type mice. At 2 or 4 wk of age, some of the spermatogonia were observed to be c-kit positive, which indicates that spermatogonial cell differentiation occurred even in W/Wv mutant testes (Fig. 4, A and B, and Table 1). However, at 10 wk of age, all the spermatogonia were c-kit-negative undifferentiated type A spermatogonia (Fig. 4, C and D, and Table 1). These results indicate that spermatogonial cell differentiation occurs in the juvenile, but not in the adult, W/Wv mutant testis.



View larger version (129K):
[in this window]
[in a new window]
  		FIG. 4. Spermatogonial cell differentiation in the W/Wv mouse testis at various growth stages. Immunohistochemical staining with the TRA98 (A) or anti-c-kit ACK2 (B) monoclonal antibodies of two consecutive testicular cross sections from the W/Wv mouse at 2 wk of age is shown. The asterisks in A and B show the same seminiferous tubules, which contain differentiated spermatogonia that are positive for the c-kit receptor. Immunohistochemical staining with TRA98 (C) or ACK2 (D) of two consecutive testicular cross sections from the W/Wv mouse at 10 wk of age is also shown, as is induction of spermatogonial cell differentiation in the adult W/Wv mutant mouse by the administration of the GnRH antagonist (E and F). Administration of the GnRH antagonist was started at 10 wk of age, and the testes were analyzed 4 wk after the start of the treatment. Note the immunohistochemical staining with TRA98 (E) or ACK2 (F) of two consecutive testicular cross sections from the GnRH antagonist-treated W/Wv mouse. In B, D, and F, the interstitial tissue was also stained with ACK2, because Leydig cells in the testis are known to express the c-kit receptor. Bar = 50 µm (A, B, E, and F) and 100 µm (C and D)

While studying the mechanism of germ cell differentiation during the development of the juvenile W/Wv mutant testis, we realized that this phenomenon reflected the phenotype of the jsd/jsd (juvenile spermatogonial depletion) mutant, in which germ cell differentiation is observed only in juvenile testes [27]. In this case, the intratesticular androgen level is low, and differentiation can be resumed, even in the adult testis, if the testosterone level is decreased [22, 23]. The administration of Nal-Glu, a GnRH antagonist known to reduce intratesticular testosterone level, to W/Wv mice at 10 wk of age was able to promote differentiation of germ cells expressing c-kit protein after 4 wk of treatment (Fig. 4, E and F, and Table 2). These results indicate that spermatogonial cell differentiation in W/Wv mutant testis is induced by reducing the level of intratesticular testosterone, at least until the appearance of the c-kit-receptor protein, although the function of the c-kit-receptor tyrosine kinase is defective because of mutation. These results also indicate that the initial differentiation process does not require c-kit signal stimulation.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Induction of spermatogonial cell differentiation in W/ W v mutant mice by the administration of the GnRH antagonist Nal-Glu.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Deletions or mutations in the W and Sl genes, which encode the tyrosine kinase receptor (c-kit) and its ligand (SCF), respectively, produce sterility in mice because of the loss of germ cells [8]. Studies of these mutants have clearly established the importance of c-kit/SCF signal transduction in the normal development of PGCs, and analyses of W and Sl embryos have shown that the c-kit/SCF system plays an important role in the migration and/or proliferation of PGCs [7, 28, 29]. Consistent with this notion, in situ hybridization studies have shown that c-kit is expressed in PGCs before and after migration into the genital ridges during embryogenesis [30]. However, to the best of our knowledge, no reports have analyzed spermatogonia in the adult W mutant testis, probably because it was thought that spermatogonia were defective, very few in number, or degenerated in the adult testis. Nevertheless, the W mutant mice, such as the W/Wv mutants, are widely used as sources of germ cell-deficient testes. For example, W/Wv mutant testes have been used to prepare a subtracted cDNA library of genes that are expressed specifically in undifferentiated spermatogonia [12–14] or as recipient testes for the assessment of donor spermatogonia [31, 32]. The results of our present study indicate the existence of undifferentiated spermatogonia in the testes of the W/Wv mutant mouse even in adult mice. Previously, we transplanted jsd/jsd mutant spermatogonia into the seminiferous tubules of the W/Wv mutant mouse for the assessment of mutant spermatogonia [32]. At that time, we used the W/Wv mutant mouse after busulfan treatment to eliminate the endogenous spermatogonia from the W/Wv mutant testis. After the administration of busulfan, almost all the endogenous spermatogonia were eliminated from the W/Wv mutant testes, which could then be used as germ cell-deficient recipient testes [32].

Previously, we observed proliferating cell nuclear antigen-positive undifferentiated spermatogonia in the W/Wv mutant testis [10]. In the present study, we investigated in more detail the number of spermatogonia in W/Wv mutant mice (Table 1), and we characterized the proliferation and differentiation of W/Wv spermatogonia directly by transplantation into recipient testes (Figs. 2 and 3). The number of germ cells in the W/Wv mutant testes varied from testis to testis. This variability was probably caused by differences in the migration and proliferation of PGCs in W/Wv mutant testes [7]. By and large, the possibility of finding germ cell-positive tubules increases with age (Table 1), which offers circumstantial evidence for the proliferation of W/Wv mutant germ cells in the seminiferous tubules.

Although differentiated germ cells were not found in the adult W/Wv mouse testes, spermatogonial differentiation was observed in the juvenile testes (Fig. 4). This observation was quite surprising, because we had believed that c-kit signal stimulation was a prerequisite for the initial stage of testicular germ cell differentiation and because c-kit receptor-positive differentiated germ cells had not been observed previously in W/Wv mutant testes. Specific expression of c-kit-receptor protein on the cell surface of spermatogonia in the juvenile, but not in the adult, mouse may be caused by differences in the microenvironments of these two testes. Indeed, testicular germ cell differentiation is known to occur in juvenile Sl17H/Sl17H and jsd/jsd mutant mice, in which it is referred to as the "first wave of spermatogenesis" after birth, but it is subsequently inhibited in the adult testes [27, 33]. A similar pattern of differentiation was probably observed in the juvenile W/Wv mutant mice (Fig. 4). Recent experiments in which the intratesticular testosterone level was decreased by the administration of a GnRH antagonist demonstrated the induction of spermatogenesis in testes that had been damaged by radiation or drugs [34] and also in jsd/jsd mutant mice [22, 23]. In the present study, spermatogonial cell differentiation, which resembled that seen in the juvenile testis, was observed in GnRH antagonist-treated adult W/Wv mouse testes, which suggests that lowering the intratesticular testosterone level induces germ cell differentiation even in adult W/Wv mice. Therefore, it appears that some of the factors involved in promoting spermatogenesis are produced in the testis by reducing the testosterone concentration or are themselves repressed by testosterone.

Several factors, such as transforming growth factor {alpha} and insulin-like growth factor-1, are present consistently during prepubertal testicular development but decrease with sexual maturity [35–38]. These two factors are known to induce the differentiation of type A spermatogonia in vitro [39]. Thus, the testicular microenvironment changes during maturation and ends up being quite different in young and adult animals. Germ cell differentiation might occur only in favorable conditions as juveniles and in various defective conditions as W/Wv (Fig. 2 and Table 1), Sl17H/Sl17H [33], and jsd/jsd [27] mutant mouse testes. In addition, production of these factors may be regulated by the local level of testosterone. The observation of germ cell differentiation in the W/Wv mouse testes was limited to the c-kit-receptor expression in the present study. Although we do not have any efficient markers for early stages of germ cell differentiation at present, it would be important and interesting to find other novel differentiation markers. In any case, once we understand these mechanisms, we may be able to find new ways to promote spermatogenesis in adult testis as in the juvenile testis.


   FOOTNOTES
 
1 H.O. is the recipient of a Research Fellowship of the Japan Society for the Promotion of Science (2000–2003). Nal-Glu was synthesized at the Salk Institute (under contract NO1-HD-0-2906 with the NIH) and kindly provided by the Contraception and Reproductive Health Branch, Center for Population Research, NICHD. Back

2 Correspondence: Yoshitake Nishimune, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka, Japan 565-0871. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp Back

3 Current address: Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan Back

Received: 13 May 2003.

First decision: 2 June 2003.

Accepted: 16 July 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kitamura Y, Go S, Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 1978 52:447-452[Abstract/Free Full Text]
  2. Russell ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genet 1979 20:357-459[Medline]
  3. Green MC. Catalog of mutant genes and polymorphic loci. In: Lyon MF, Searle AG (eds.), Genetic Variants and Strains of the Laboratory Mouse. Oxford: Oxford University Press; 1990:383–386
  4. Coulombre JL, Russell ES. Analysis of the pleiotropism at the W locus in the mouse. The effects of W and Wv substitutions upon post natal development of germ cells. J Exp Zool 1954 126:277-296[CrossRef]
  5. Geissler EN, McFarland EC, Russell ES. Analysis of pleiotropism at the dominant white-spotting (W) locus of the house mouse: a description of ten new W alleles. Genetics 1981 97:337-361[Abstract/Free Full Text]
  6. Mintz B. Embryological development of primordial germ cells in the mouse: Influence of a new mutation, Wj. J Embryol Exp Morph 1957 5:396-403
  7. Mintz B, Russell ES. Gene-induced embryological modifications of primordial germ cells in the mouse. J Exp Zool 1957 134:207-237[CrossRef][Medline]
  8. Besmer P, Manova K, Duttlinger R, Huang EJ, Packer A, Gyssler C, Bachvarova RF. The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis. In: Signals, polarity and adhesion in development. Development 1993 (suppl) 125-137
  9. Ohta H, Yomogida K, Dohmae K, Nishimune Y. Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 2000 127:2125-2131[Abstract]
  10. Tadokoro Y, Yomogida K, Ohta H, Tohda A, Nishimune Y. Homeostatic regulation of germinal stem cell proliferation by the GDNF/FSH pathway. Mech Dev 2002 113:29-39[CrossRef][Medline]
  11. Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med 2000 6:29-34[CrossRef][Medline]
  12. Tanaka H, Yoshimura Y, Nishina Y, Nozaki M, Nojima H, Nishimune Y. Isolation and characterization of cDNA clones specifically expressed in testicular germ cells. FEBS Lett 1994 355:4-10[CrossRef][Medline]
  13. Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked genes expressed in spermatogonia. Nat Genet 2001 27:422-426[CrossRef][Medline]
  14. Tanaka K, Tamura H, Tanaka H, Katoh M, Futamata Y, Seki N, Nishimune Y, Hara T. Spermatogonia-dependent expression of testicular genes in mice. Dev Biol 2002 246:466-479[CrossRef][Medline]
  15. Ohta H, Yomogida K, Yamada S, Okabe M, Nishimune Y. Real-time observation of transplanted ‘green germ cells’: proliferation and differentiation of stem cells. Dev Growth Differ 2000 42:105-112[CrossRef][Medline]
  16. Tanaka H, Pereira LA, Nozaki M, Tsuchida J, Sawada K, Mori H, Nishimune Y. A germ cell-specific nuclear antigen recognized by a monoclonal antibody raised against mouse testicular germ cells. Int J Androl 1997 20:361-366[CrossRef][Medline]
  17. Nishikawa S, Kusakabe M, Yoshinaga K, Ogawa M, Hayashi S, Kunisada T, Era T, Sakakura T, Nishikawa S-I. In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development. EMBO J 1991 10:2111-2118[Medline]
  18. Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 1994 91:11298-11302[Abstract/Free Full Text]
  19. Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 1994 91:11303-11307[Abstract/Free Full Text]
  20. Ogawa T, Arechaga JM, Avarbock MR, Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 1997 41:111-122[Medline]
  21. Kangasniemi M, Dodge K, Pemberton AE, Huhtaniemi I, Meistrich ML. Suppression of mouse spermatogenesis by a gonadotropin-releasing hormone antagonist and antiandrogen: failure to protect against radiation-induced gonadal damage. Endocrinology 1996 137:949-955[Abstract]
  22. Matsumiya K, Meistrich ML, Shetty G, Dohmae K, Tohda A, Okuyama A, Nishimune Y. Stimulation of spermatogonial differentiation in juvenile spermatogonial depletion (jsd) mutant mice by gonadotropin-releasing hormone antagonist treatment. Endocrinology 1999 140:4912-4915[Abstract/Free Full Text]
  23. Tohda A, Matsumiya K, Tadokoro Y, Yomogida K, Miyagawa Y, Dohmae K, Okuyama A, Nishimune Y. Testosterone suppresses spermatogenesis in juvenile spermatogonial depletion (jsd) mice. Biol Reprod 2001 65:532-537[Abstract/Free Full Text]
  24. Tohda A, Okuno T, Matsumiya K, Okabe M, Kishikawa H, Dohmae K, Okuyama A, Nishimune Y. Restoration of spermatogenesis and fertility in azoospermic mutant mice by suppression and reelevation of testosterone followed by intracytoplasmic sperm injection. Biol Reprod 2002 66:85-90[Abstract/Free Full Text]
  25. Yoshinaga K, Nishikawa S, Ogawa M, Hayashi S, Kunisada T, Fujimoto T, Nishikawa S-I. Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 1991 113:689-699[Abstract]
  26. Vincent S, Segretain D, Nishikawa S, Nishikawa S-I, Sage J, Cuzin F, Rassoulzadegan M. Stage-specific expression of the Kit receptor and its ligand (KL) during male gametogenesis in the mouse: a Kit-KL interaction critical for meiosis. Development 1998 125:4585-4593[Abstract]
  27. Beamer WG, Cunliffe-Beamer TL, Shultz KL, Langley SH, Roderick TH. Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice. Biol Reprod 1988 38:899-908[Abstract]
  28. Bennett D. Developmental analysis of a mutation with pleiotropic effects in the mouse. J Morphol 1956 98:199-233[CrossRef]
  29. McCoshen JA, McCallion DJ. A study of the primordial germ cells during their migratory phase in Steel mutant mice. Experientia 1975 31:589-590[CrossRef][Medline]
  30. Orr-Urtreger A, Avivi A, Zimmer Y, Givol D, Yarden Y, Lonai P. Developmental expression of c-kit, a proto-oncogene encoded by the W locus. Development 1990 109:911-923[Abstract/Free Full Text]
  31. Johnston DS, Russell LD, Friel PJ, Griswold MD. Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation. Endocrinology 2001 142:2405-2408[Abstract/Free Full Text]
  32. Ohta H, Yomogida K, Tadokoro Y, Tohda A, Dohmae K, Nishimune Y. Defect in germ cells, not in supporting cells, is the cause of male infertility in the jsd mutant mouse: proliferation of spermatogonial stem cells without differentiation. Int J Androl 2001 24:15-23[CrossRef][Medline]
  33. Brannan CI, Bedell MA, Resnick JL, Eppig JJ, Handel MA, Williams DE, Lyman SD, Donovan PJ, Jenkins NA, Copeland NG. Developmental abnormalities in Steel17H mice result from a splicing defect in the steel factor cytoplasmic tail. Genes Dev 1992 6:1832-1842[Abstract/Free Full Text]
  34. Meistrich ML. Hormonal stimulation of the recovery of spermatogenesis following chemo- or radiotherapy. APMIS 1998 106:37-46[Medline]
  35. Mullaney BP, Skinner MK. Growth factors as mediators of testicular cell-cell interactions. Baillieres Clin Endocrinol Metab 1991 5:771-790[Medline]
  36. Mullaney BP, Skinner MK. Transforming growth factor-{alpha} and epidermal growth factor receptor gene expression and action during pubertal development of the seminiferous tubule. Mol Endocrinol 1992 6:2103-2113[Abstract]
  37. Schlatt S, Meinhardt A, Nieschlag E. Paracrine regulation of cellular interactions in the testis: factors in search of a function. Eur J Endocrinol 1997 137:107-117[Abstract]
  38. Shoba L, An MR, Frank SJ, Lowe WL Jr. Developmental regulation of insulin-like growth factor-I and growth hormone receptor gene expression. Mol Cell Endocrinol 1999 152:125-136[CrossRef][Medline]
  39. Tajima Y, Watanabe D, Koshimizu U, Matsuzawa T, Nishimune Y. Insulin-like growth factor-I and transforming growth factor-{alpha} stimulate differentiation of type A spermatogonia in organ culture of adult mouse cryptorchid testes. Int J Androl 1995 18:8-12[Medline]



This article has been cited by other articles:


Home page
 Stem CellsHome page
J. Hao, M. Yamamoto, T. E. Richardson, K. M. Chapman, B. S. Denard, R. E. Hammer, G. Q. Zhao, and F. K. Hamra
Sohlh2 Knockout Mice Are Male-Sterile Because of Degeneration of Differentiating Type A Spermatogonia
Stem Cells, June 1, 2008; 26(6): 1587 - 1597.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Kanatsu-Shinohara, K. Inoue, H. Miki, N. Ogonuki, M. Takehashi, T. Morimoto, A. Ogura, and T. Shinohara
Clonal Origin of Germ Cell Colonies after Spermatogonial Transplantation in Mice
Biol Reprod, July 1, 2006; 75(1): 68 - 74.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Kanatsu-Shinohara, M. Ikawa, M. Takehashi, N. Ogonuki, H. Miki, K. Inoue, Y. Kazuki, J. Lee, S. Toyokuni, M. Oshimura, et al.
From the Cover: Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells
PNAS, May 23, 2006; 103(21): 8018 - 8023.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
Z. Zhang, S. Shao, and M. L. Meistrich
Irradiated Mouse Testes Efficiently Support Spermatogenesis Derived From Donor Germ Cells of Mice and Rats
J Androl, May 1, 2006; 27(3): 365 - 375.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
R. M. David
Proposed Mode of Action for In Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract
Toxicol Pathol, April 1, 2006; 34(3): 209 - 219.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
S. M. Prabhu, M. L Meistrich, E. A McLaughlin, S. D Roman, S. Warne, S. Mendis, C. Itman, and K. L. Loveland
Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis.
Reproduction, March 1, 2006; 131(3): 489 - 499.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Kanatsu-Shinohara, K. Inoue, J. Lee, H. Miki, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara
Anchorage-Independent Growth of Mouse Male Germline Stem Cells In Vitro
Biol Reprod, March 1, 2006; 74(3): 522 - 529.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
M. Kanatsu-Shinohara, N. Ogonuki, T. Iwano, J. Lee, Y. Kazuki, K. Inoue, H. Miki, M. Takehashi, S. Toyokuni, Y. Shinkai, et al.
Genetic and epigenetic properties of mouse male germline stem cells during long-term culture
Development, September 15, 2005; 132(18): 4155 - 4163.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
M. Kanatsu-Shinohara, H. Miki, K. Inoue, N. Ogonuki, S. Toyokuni, A. Ogura, and T. Shinohara
Germline niche transplantation restores fertility in infertile mice
Hum. Reprod., September 1, 2005; 20(9): 2376 - 2382.
[Abstract] [Full Text] [PDF]

Home page
 GeneticsHome page
H.-B. Ruan, N. Zhang, and X. Gao
Identification of a Novel Point Mutation of Mouse Proto-Oncogene c-kit Through N-Ethyl-N-nitrosourea Mutagenesis
Genetics, February 1, 2005; 169(2): 819 - 831.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
S. Chuma, M. Kanatsu-Shinohara, K. Inoue, N. Ogonuki, H. Miki, S. Toyokuni, M. Hosokawa, N. Nakatsuji, A. Ogura, and T. Shinohara
Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis
Development, January 1, 2005; 132(1): 117 - 122.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
A. Nakata, T. Ito, M. Nagata, S. Hori, and K. Sekimizu
GRIP1{tau}, a novel PDZ domain-containing transcriptional activator, cooperates with the testis-specific transcription elongation factor SII-T1
Genes Cells, November 1, 2004; 9(11): 1125 - 1135.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
69/6/1815    most recent
biolreprod.103.019323v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohta, H.
Right arrow Articles by Nishimune, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohta, H.
Right arrow Articles by Nishimune, Y.
Agricola
Right arrow Articles by Ohta, H.
Right arrow Articles by Nishimune, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS