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BOR - Papers in Press, published online ahead of print December 26, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.024612
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BIOLOGY OF REPRODUCTION 70, 1286–1291 (2004)
DOI: 10.1095/biolreprod.103.024612
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Commitment of Fetal Male Germ Cells to Spermatogonial Stem Cells During Mouse Embryonic Development1

Hiroshi Ohta3,4, Teruhiko Wakayama4, and Yoshitake Nishimune2,3

Department of Science for Laboratory Animal Experimentation,3 Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan Laboratory for Genomic Reprogramming,4 Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.

developmental biology, gamete biology, sperm, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogonial stem cells originate from primordial germ cells (PGCs), which were first identified as alkaline phosphatase-positive cells within the extra-embryonic mesoderm at 7.2 days postcoitum (dpc) in the mouse [1– 3]. PGCs require several members of the bone morphogenetic protein (BMP) ligand family for development [4–7], after which they proliferate and migrate to the genital ridges, where they become enclosed in the seminiferous cords by 11 dpc. During this period, the PGCs are proliferating so rapidly that the initial founder population of about 40 PGCs gives rise to about 25 000 germ cells by the time proliferation ceases at 13.5 dpc [8]. By 13.5 dpc, the germ cells are also undertaking sexually dimorphic patterns of development. Male germ cells enter a mitotic arrest that will be maintained until after birth, whereas female germ cells directly enter meiotic prophase. During spermatogenesis after birth, spermatogonia proliferate and some undergo meiosis to give rise to haploid spermatids, which are then remodeled into spermatozoa. Spermatogonial stem cells are responsible for maintaining spermatogenesis throughout life in males by proliferation and differentiation; these characteristics rely on the ability to self-renew and to produce daughter cells that differentiate into sperm. Thus, male germ cells follow several complex steps during development and after birth; however, it is unclear when fetal male germ cells acquire the ability of spermatogonial stem cells to produce mature sperm in the seminiferous tubules.

A germ cell transplantation technique has been established, in which donor germ cells are transplanted into recipient seminiferous tubules, whereupon the transplanted germ cells initiate complete spermatogenesis [9, 10]. This technique allowed us to examine the ability of donor male germ cells to function as spermatogonial stem cells [11– 13] and will be applicable for the functional assessment of fetal male germ cells. In the present study, we transplanted fetal male germ cells into the seminiferous tubules of adult testis to examine the timing of the commitment of fetal male germ cells to differentiate into spermatogonial stem cells.


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

Two-month-old WBB6F1-W/Wv mice and pregnant female C57BL/6 mice were purchased from Shizuoka Laboratory Animal Center, Hamamatsu, Japan. W/Wv mutants, which lack endogenous germ cells, were used as recipients for transplantation. Green fluorescence protein (GFP) transgenic mice carrying pCX-EGFP alone [14] or double transgenic mice carrying both acrosin/eGFP (Acr3-EGFP) [15] and pCX-EGFP transgenes (C57BL/6TgN(acro/act-EGFP)OsbC3-N01-FJ002) were used as donors of testicular germ cells [16]. All mice used in the intracytoplasmic sperm injection experiment were also purchased from the Shizuoka Laboratory Animal Center. 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).

Donor Cell Preparation

To examine the spermatogonial stem cell activity of fetal germ cells, donor cells for transplantation were prepared from gonads at Embryonic Days 12.5, 14.5, 16.5, and 18.5. Male embryonic gonads can be easily distinguished from female by morphological differences after 12.5 dpc. Briefly, pregnant females were killed by cervical dislocation and the embryos were collected. After dissection from the embryos, the male gonads were separated from mesonephros with fine scissors and placed in PBS containing 0.05% trypsin and 0.1 mM EDTA and incubated for 15 min at 37°C with manual agitation at 5-min intervals to obtain a donor cell suspension. After the addition of a half volume of Dulbecco modified Eagle medium containing 10% fetal bovine serum, the cell suspension was washed twice with PBS and resuspended in injection medium [9]. The same procedure was performed to prepare donor cells from testes of infant (3 and 4 days postpartum ["p]) transgenic mice. The concentration of donor cells was adjusted to 1 x 108 cells/ml; approximately 90% of the cells were alive as judged by trypan-blue dye exclusion after the transplantation procedure.

Germ Cell and Gonadal Tissue Transplantation

The transplantation of germ cells into seminiferous tubules was performed via the efferent ductules as described previously [16, 17]. To determine the number of transplanted germ cells, each aliquot of donor cell suspension was stained with the germ-cell-specific monoclonal antibody TRA 98, which recognizes all male germ cell nuclei [18]. Fetal male germ cells from wild-type embryonic gonad (without green fluorescence) were transplanted into the seminiferous tubules of W/Wv mutant mice, which lack endogenous spermatogenesis. Each recipient testis was analyzed at 10 wk after transplantation.

To investigate the interaction of fetal germ cells with supporting cells, fetal male gonad tissue was transplanted into recipient testes. Briefly, fetal gonads were dissected from embryos and placed in PBS. The gonads were dissected from mesonephros and cut in half with fine scissors, and the tissue was transplanted under the tunica of W/Wv mice testes.

Histological Analysis

The testes were fixed with Bouin solution and embedded in paraffin; 5-µm serial cross sections at 50-µm intervals were prepared and stained with hematoxylin. The recipient testis was evaluated and classified into one of two categories: spermatogenesis positive if spermatogenesis was observed in the testicular cross sections, or spermatogenesis negative if it was not observed. Serial cross sections of more than half of each testis were observed for the evaluation.

For observations of eGFP fluorescence, recipient testes were fixed in 4% paraformaldehyde at 4°C overnight and then embedded in glycol methacrylate (Technovit 8100; Kulzer, Wehrheim, Germany). Histological 5- µm-thick serial sections at 50-µm intervals were prepared and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO). After observation of the eGFP fluorescence, the sections were stained with hematoxylin and observed under a photomicroscope for detailed analysis.

Intracytoplasmic Sperm Injection

Mature oocytes were collected from 8- to 12-wk-old CDF1 hybrid (BALB/c x DBA/2) mice. Briefly, CDF1 females were induced to superovulate by intraperitoneal injection of 5 IU eCG (Teikokuzoki Co., Tokyo, Japan) followed 48 h later by injection of 5 IU hCG (Teikokuzoki Co.). Mature oocytes were collected from the ampullary region of the oviducts 13–15 h after the hCG injection. Oocytes were freed from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (Sigma) in Hepes- CZB medium. Oocytes were rinsed and kept in fresh CZB medium at 37°C under 5% CO2 until sperm injection.

Sperm were collected from the recipient testis at 10 wk after gonadal tissue transplantation. Briefly, W/Wv recipient testes, transplanted with 12.5-dpc male gonad tissue (C57BL/6 background), were placed in PBS, and the donor testicular tissue was dissected from the recipient testis using fine forceps. The selected tissue was minced with fine scissors and suspended in PBS. About 2 µl of the sperm suspension was mixed with a drop of Hepes-CZB medium containing 12% (w/v) polyvinylpyrrolidone (PVP; Mr 360 000; Wako Pure Chemical Industries, Tokyo, Japan). The sperm head was separated from the tail by applying a few piezo pulses to the sperm neck region, and was injected into an oocyte as described by Kimura and Yanagimachi [19]. Oocytes that received intracytoplasmic sperm injections (ICSI) were incubated in CZB medium at 37°C under 5% CO2 in air. Two-cell embryos were transferred into the oviducts of 0.5- dpc pseudopregnant CD-1 females.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Commitment of Fetal Germ Cells to Differentiate into Spermatogonial Stem Cells Was Demonstrated by Their Transplantation into Seminiferous Tubules of Adult Testis

Donor cell fractions prepared from the gonads of embryos at 12.5, 14.5, 16.5, and 18.5 dpc (Fig. 1A) were transplanted into the seminiferous tubules of W/Wv mice. The number of transplanted germ cells was estimated from the percentage of germ cells in the donor cell suspension as determined by staining with the germ cell-specific monoclonal antibody TRA98 (Fig. 1B). As the endogenous differentiated germ cells are completely absent from adult W/ Wv mice but the transplanted spermatogonia can initiate normal spermatogenesis (Fig. 1C) [20, 21], the transplantation of fetal germ cells into the seminiferous tubules of adult W/Wv mouse testis should be an effective method for the investigation of spermatogonial stem cell activity.



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  		FIG. 1. Development of mouse male gonad. A) Stereomicroscopic picture of male gonads with mesonephric duct at 12.5, 14.5, 16.5, and 18.5 days postcoitum (dpc). B) A seminiferous cord of a 12.5-dpc male gonad. Arrows indicate male germ cells. Inset in B shows immunocytochemistry of donor cells prepared from 12.5-dpc male gonads stained with the germ-cell-specific monoclonal antibody TRA 98. C) Photograph of a recipient seminiferous tubule of W/Wv testis transplanted adult spermatogonia after 10 wk. Scale bars = (A) 1 mm, (B) 50 µm, inset in B 25 µm, (C) 100 µm

No spermatogenesis was observed in the recipient testis when donor germ cells were derived from the gonads of 12.5-dpc embryos (Fig. 2A), even though there were as many TRA98-positive germ cells present in the donor fraction as in other donor cell fractions (Table 1). In contrast, fetal germ cells prepared from embryos at 14.5–18.5 dpc differentiated into mature sperm in the recipient testes by 10 wk after transplantation (Fig. 2B). This observation clearly demonstrated that gonocytes or fetal male germ cells had committed to differentiate into spermatogonial stem cells by at least 14.5 dpc. These results indicate that the fetal germ cells of 12.5-dpc embryos do not yet have the potential of spermatogonial stem cells and that the commitment of fetal germ cells to differentiate into spermatogonial stem cells takes place between Embryonic Days 12.5 and 14.5.



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  		FIG. 2. Transplantation of fetal male germ cells into the seminiferous tubules of adult W/Wv mutant testis. A, B) Testicular cross sections of recipient W/Wv testes ten weeks after transplantation of donor gonadal cells prepared from embryos at 12.5 (A) or 14.5 (B) dpc. In B, the arrows indicate the seminiferous tubules containing donor cell-derived spermatogenesis. Scale bars = 100 µm


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  		TABLE 1. Transplantation of fetal male germ cells into the seminiferous tubules of adult W/Wv testis

Spermatogonial Stem Cell Activity of Fetal Germ Cells

Although fetal gonadal cells have spermatogonial stem cell activity after 14.5 dpc (Table 1), the activity seems to be much lower than that of adult spermatogonia, as indicated by the very limited number of tubules with spermatogenesis as compared with adult germ cells, even when 18.5-dpc fetal germ cells were transplanted. To investigate the stem cell activity of fetal germ cells in more detail, we transplanted fetal germ cells along with infant germ cells labeled with GFP and compared their stem cell activities by differentially counting fluorescence-positive and -negative spermatogenesis in the transplanted seminiferous tubules of the same testis. By transplantation of 2.8 x 104 fetal germ cells at 12.5 dpc, no spermatogenesis was observed, whereas 270 seminiferous tubules with spermatogenesis were observed by cotransplantation of 2.2 x 104 infantile germ cells labeled with GFP into the same testes (Table 2). This was also confirmed by the data in Table 1, which show that no testis transplanted with 12.5-dpc germ cells exhibited spermatogenesis and that this was not due to any harmful effects of the germ cell transplantation itself. Cotransplantation of fetal germ cells later than 14.5 dpc with infant germ cells produced spermatogenesis derived from both fetal and infant germ cells, which could be differentially counted in the recipient testis. A cotransplantation assay revealed spermatogonial stem cell activity of fetal germ cells older than 14.5 dpc, but the activity was much lower than that of the infant spermatogonia (Fig. 3, A–C; Table 2). These results indicate that fetal germ cells start to acquire the ability of spermatogonia between 12.5 and 14.5 dpc, but the full determination of fetal germ cells into spermatogonial stem cells is not completed during embryonic development.


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  		TABLE 2. Cotransplantation of fetal male germ cells with infant male germ cells.*



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  		FIG. 3. Cotransplantation of fetal male germ cells with GFP-labeled infant male germ cells. Donor male germ cells prepared from 14.5-dpc and 4- dpp gonads were cotransplanted into the seminiferous tubules of W/Wv mutant testis. A testicular cross section at 10 wk after germ cell transplantation showing (A) the fluorescence microscopy picture of GFP and (B) the DAPI staining of (A). C) A merged photograph of (A) and (B). Asterisks show seminiferous tubules containing spermatogenesis without green fluorescence, indicating that spermatogenesis was derived from fetal germ cells. Scale bars = 100 µm

Interaction Between Germ Cells and Supporting Cells in the Fetal Gonad Is Important for Spermatogonial Cell Determination

Germ cells derived from 12.5-dpc gonads did not function as spermatogonial stem cells in the recipient adult seminiferous tubules (Fig. 2A; Tables 1 and 2), indicating that the fetal germ cells at this age do not have the potential to differentiate into sperm in adult recipient testis. In contrast, when gonadal tissue from 12.5-dpc embryos was transplanted into recipient testes, normal spermatogenesis was observed at 10 wk after transplantation (Fig. 4, A and B; spermatogenesis occurred in 9/12 recipient testes). These results indicate that the germ cell-somatic cell interaction is important for the commitment of germ cells to spermatogonial stem cells in the fetal gonad at 12.5 dpc. In this case, interaction with fetal somatic cells, and not with adult Sertoli cells, is required for the commitment of fetal germ cells at 12.5 dpc.



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  		FIG. 4. Transplantation of 12.5-dpc gonadal tissue into adult testis. A) Ten weeks after transplantation of male gonadal tissue prepared from 12.5 dpc mouse into W/Wv mouse testis. Arrow indicates grafted tissue. B) A recipient testicular cross section transplanted with 12.5-dpc male gonadal tissue after 10 wk. C) Normal progeny produced by ICSI using sperm obtained from the grafted 12.5-dpc male gonad. Scale bars = (A) 1 mm; (B) 100 µm

To investigate whether the sperm obtained from 12.5- dpc fetal germ cells have the ability to support normal development, intracytoplasmic sperm injection (ICSI) was performed. Of the 146 embryos constructed, 125 (85%) developed to the two-cell stage within 24 h in culture. After transfer of the embryos into the oviducts of nine pseudopregnant females, a total of 55 live pups were born. The ICSI experiment clearly showed that the sperm that differentiated from 12.5-dpc fetal germ cells of the whole gonadal transplant in the adult testes had the ability to support normal development (Fig. 4C). These results indicate that normal development of fetal germ cells occurred in the adult testicular environment with the support of fetal somatic cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Male germ cells undergo several complex processes during embryonic development: proliferation and migration to the genital ridges from 7.2 to 11 days postcoitum (dpc), a quiescent state in the embryonic gonad from 15.5 to 18.5 dpc, and the initiation of spermatogenesis shortly after birth [8]. The time at which fetal germ cells acquire the abilities of spermatogonial stem cells has been unclear. In this study, we demonstrated that the commitment of fetal germ cells to differentiate into spermatogonial stem cells takes place during Embryonic Days 12.5–14.5, i.e., fetal germ cells from 12.5-dpc male gonad could not initiate spermatogenesis when transplanted into the adult seminiferous tubules, whereas fetal germ cells after 14.5 dpc could. Furthermore, the interaction of fetal germ cells with somatic cells was demonstrated to be critical for germ cell commitment during these gestation days. To our knowledge, this is the first report of the detailed analysis of fetal male germ cells committed to differentiate into spermatogonial stem cells, although a transplantation of fetal male germ cells was once reported in rat testis [22].

It has been reported that male germ cells between 12.5 and 14.5 dpc have unique properties that might be vital for male germ cell development. Male germ cells at 12.5 dpc are actively undergoing cell cycles, but at 14.5 dpc, male germ cells are in a transitional state and entering into quiescence [23]. Although the mechanisms of the cell-cycle regression have not yet been elucidated, this process might be important for the commitment of fetal germ cells. At around 11.5–12.5 dpc, fetal germ cells are also known to undergo demethylation at imprinting loci [24, 25], which erases parental imprinting marks; the methylation is reestablished at around 15.5–16.5 dpc [26, 27]. The difference between genomic imprinting at 11.5 dpc and 16.5 dpc matches well with our results of the difference between spermatogonial stem cell activity of germ cells at 12.5 dpc and 14.5 dpc. Genomic imprinting from 11.5 to 16.5 dpc may play a role in the commitment of fetal germ cells to differentiate into spermatogonial stem cells. This idea is also supported by recent studies in which mice with mutations in DNA methyltransferase genes, such as Dnmt3L [28, 29] or Dnmt3a [30], displayed a lack of spermatogenesis in the testis, indicating that genomic imprinting is critical for male germ cell differentiation.

Normal spermatogenesis was observed when 12.5-dpc whole gonad tissue was transplanted into adult recipient testis, unlike the results of transplantation of gonadal cells into seminiferous tubules, even though the donor cell fraction contained both gonadal somatic cells and germ cells (Fig. 1B). The somatic cell environment of the adult intraseminiferous tubule did not support the commitment of 12.5-dpc fetal germ cells, but did support the proliferation and differentiation of committed fetal germ cells later than 14.5 dpc (Tables 1 and 2). These results indicate that interaction with fetal supporting cells in complete cell-cell organization is important for the commitment and development of 12.5-dpc male germ cells, suggesting that fetal somatic cells, which are precursors of Sertoli cells, affect male germ cell development through cell-cell interactions.

When fetal gonadal cells of 12.5–18.5 dpc were transplanted, an abnormal proliferation of transplanted cells was observed in the recipient testis (data not shown). This is quite different from the transplantation of testicular cells from the adult mouse, in which all proliferated cells have a germ cell phenotype [16]. Not all of the proliferated cells in the recipient testis were germ cells; some were gonadal somatic cells not stained with TRA98 (data not shown). In some cases, small seminiferous tubule-like minitubules were also formed in the recipient seminiferous tubules, consistent with a previous report [31]. After transplantation of 12.5-dpc gonadal cells along with infant spermatogonia from GFP transgenic mouse testis, fluorescence-positive normal spermatogenesis was observed (Table 2); fluorescence-negative differentiated germ cells derived from a 12.5-dpc donor were never found in the recipient testis (Table 2), but some somatic cell clusters were present. In contrast, both fluorescence-positive and -negative spermatogenesis was found in the recipient testis when 14.5-dpc gonadal cells were transplanted along with infant GFP transgenic spermatogonia (Fig. 3). In all cotransplantation experiments, substantial numbers of infant spermatogonial stem cells made colonies in seminiferous tubules (Table 2). These results indicate that the abnormal proliferation of transplanted embryonic somatic cells does not affect the stem cell activity of transplanted male germ cells.

With the transplantation of fetal gonad tissue into adult testis, we did not find any teratoma formation, only normal spermatogenesis (Fig. 4). The apparent lack of teratomas in our experiments may be because we used a low-responder mouse strain, C57BL/6, as opposed to a high-responder strain with a high incidence of spontaneous testicular teratomas, such as the 129 strain [32, 33]. Thus, testicular transplantation of fetal gonad tissue mimicked the commitment of fetal germ cells to differentiate into spermatogonial stem cells under our experimental conditions.

We did not observe the settlement or proliferation of 12.5-dpc fetal male germ cells transplanted into adult seminiferous tubules. The transplanted noncommitted germ cells present in the 12.5-dpc gonadal cells seemed to be discarded from the seminiferous tubules. For a more detailed analysis, it may be best to use donor cells that are labeled with a marker protein, such as GFP or LacZ, although that may make it difficult to collect a sufficient number of fetal germ cells for transplantation.

Finally, although the germ cell transplantation technique has become a powerful method for not only understanding male germ cell development but also for advancing the field of animal reproduction [11–13], our study indicated technical limitations. The differentiation of fetal germ cells transplanted into seminiferous tubules was not as efficient as that of transplanted adult spermatogonial stem cells. To obtain mature sperm from fetal gonad, gonadal tissue transplantation functions better than the transplantation of gonadal cell fractions in the seminiferous tubules. Using the ICSI technique, differentiated sperm produced from 12.5- dpc male germ cells were of sufficient quality to result in normal pups. To our knowledge, this is the first report of progeny obtained from fetal male germ cells. The ability to produce functional sperm from fetal male gonad transplanted into adult testis will allow us to obtain the offspring from mutant animals with embryonic lethality. Recently, mature sperm have been obtained from xenogeneic or cryopreserved neonatal testicular tissue transplantation under the skin or into the testis of the recipient mouse [34, 35]; sperm production from fetal gonad tissue, as used in our study, may also be possible by transplantation under the skin or after cryopreservation of donor gonad. Future findings using these reproductive techniques may provide a further understanding of male germ cell development.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. M. Okabe (Osaka University) and Dr. S. Yamada (Kyoto University) for kindly providing the GFP transgenic mouse lines.


   FOOTNOTES
 
1 Supported by a Research Fellowship to H.O. from the Special Postdoctoral Researchers Program. Back

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

Received: 22 October 2003.

First decision: 14 November 2003.

Accepted: 23 December 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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