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BOR - Papers in Press, published online ahead of print October 4, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.055863
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BIOLOGY OF REPRODUCTION 76, 55–62 (2007)
DOI: 10.1095/biolreprod.106.055863
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Leukemia Inhibitory Factor Enhances Formation of Germ Cell Colonies in Neonatal Mouse Testis Culture1

Mito Kanatsu-Shinohara 3 4, Kimiko Inoue 7, Narumi Ogonuki 7, Hiromi Miki 7, Shosei Yoshida 5, Shinya Toyokuni 6, Jiyoung Lee 4, Atsuo Ogura 7, and Takashi Shinohara 2 4

Horizontal Medical Research Organization,3 Departments of Molecular Genetics,4 Pathology and Tumor Biology,5 and Pathology and Biology of Diseases,6 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan The Institute of Physical and Chemical Research (RIKEN),7 Bioresource Center, Ibaraki 305-0074, Japan

ABSTRACT

Spermatogonial stem cells continuously divide in the testis to support spermatogenesis throughout the life of adult male animals. Although very few spermatogonial stem cells are present in vivo, we recently succeeded in expanding these cells in vitro. Germ cells from postnatal testes were able to proliferate in the presence of several types of cytokines, and they formed uniquely shaped colonies of spermatogonia (germline stem or GS cells). These cells reinitiated normal spermatogenesis when transplanted into seminiferous tubules. However, much remains unknown about the contributions of cytokines to successful stem cell culture. In the present study, we examined the role of leukemia inhibitory factor (LIF) in GS cell culture. We found that the addition of LIF to newborn testis cell culture enhances the formation of germ cell colonies. Ciliary neurotrophic factor, but not oncostatin M, had the same effect, although they both bind to the IL-6ST (gp130) receptor. On the other hand, GS cells could be established from pup or adult testes in the absence of LIF. No phenotypic or functional difference was found between GS cells established from different stages, and normal offspring were born from pup-derived GS cells that had been maintained in the absence of LIF, indicating that LIF per se is not involved in the self-renewal of GS cells. These results demonstrate that LIF is useful in the initiation of GS cell culture and suggest that LIF or a related cytokine is involved in the maturation of gonocytes into spermatogonia.

developmental biology, gametogenesis, Sertoli cells, spermatogenesis, testis

INTRODUCTION

Male germline development is a long and complex process [1]. In mice, precursors of fetal germ cells originate from the epiblast and can be detected at the base of the allantois by alkaline phosphatase activity at about 7.5 days postcoitum (dpc) [2]. In males, the fetal germ cells, called primordial germ cells (PGCs), increase in number and migrate to the gonad by 12.5 dpc. When PGCs enter the gonad, they become enclosed in seminiferous cords and are then called gonocytes. These gonocytes proliferate for a couple of days and then become mitotically quiescent by about 16.5 dpc. At 1–3 days postpartum (pp), they enter the cell cycle. Although they are uniformly round in the fetal stage, they form pseudopods and begin to migrate toward the basement membrane. By Days 3–7 pp, type A spermatogonia appear in the seminiferous tubules, and a small number of these spermatogonia exhibit the unique proliferative potential both to self-renew and differentiate. In differentiation, the spermatogonia produce spermatocytes, and the latter cells go through meiosis to produce spermatozoa, thereby supporting spermatogenesis throughout adult life [3]. Although the morphological aspects of the dynamic development of the male germ cell system have been well described, much remains unknown about the developmental mechanisms involved and their regulation.

Leukemia inhibitory factor (LIF) plays an important role in the regulation of stem cells. LIF binds to a heterodimeric receptor (LIF receptor) that includes the LIF-specific binding subunit and the transmembrane signal-transducing subunit IL-6ST. Binding of LIF to this receptor complex results in the phosphorylation of several tyrosine residues in IL-6ST by the activated Janus kinase (JAK) tyrosine kinases, which then activates signal transducer and activator of transcription (STAT) 3 transcription factor [4, 5]. This signaling cascade has been shown to be involved in the maintenance or self-renewal of stem cells in several tissues. For example, a decrease in the number of neural stem cells was found in LIF receptor-deficient mice [6], and the introduction of dominant negative STAT3 molecules was found to negatively influence hematopoietic stem cell potential [7]. In embryonic stem (ES) cells, LIF acts to activate STAT3 and maintain the undifferentiated state and allows their efficient propagation in vitro [8, 9]. LIF also promotes the survival or proliferation of PGCs, from which the ES-like pluripotent cells, known as embryonic germ (EG) cells, have been derived from in vitro experiments [10, 11]. In contrast, although LIF receptors are also found on gonocytes and spermatogonia at all postnatal ages [12, 13], the involvement of LIF in postnatal germ cell development has been unclear.

We recently developed a culture system for spermatogonial stem cells. As we originally reported, gonocytes from neonatal mouse testes proliferated to form uniquely shaped colonies of germ cells when cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) and LIF [14]. Similarly, when spermatogonia from pup or adult testes were directly seeded on LIF-secreting feeder cells or in LIF-containing medium with the addition of GDNF, the cultured cells continued to proliferate logarithmically [15, 16]. Even after 2 yr of in vitro culture, the cultured cells initiated long-term spermatogenesis and produced normal offspring when transplanted into the seminiferous tubules of infertile recipient mice [17]. On the basis of these properties, we designated these cells germline stem or GS cells [14]. Although GS cells were originally established from neonatal gonocytes, they express spermatogonia markers and show functional characteristics of spermatogonia. The observation that, under the same culture conditions, similar cells could be established from spermatogonia of pup and adult testes indicates that gonocytes can differentiate into spermatogonia during culture [16].

Although GDNF has been shown to be a self-renewal factor for spermatogonial stem cells, the role of LIF in GS cell culture is not well understood. In the present investigation, we cultured germ cells collected at different stages of testicular development, and the stage-specific effects of LIF were analyzed by phenotypic and functional analysis of spermatogonial stem cells.

MATERIALS AND METHODS

Cell Culture

For the initiation of testis cell culture, Institute for Cancer Research (ICR) mice were purchased from Japan SLC (Shizuoka, Japan). In experiments to visualize spermatogonia cells, we used Neurog3 (ngn3)/enhanced green fluorescent protein (EGFP) transgenic mice from a DBA/2 background [18]. In long-term culture experiments, we used the transgenic mouse line C57BL6/Tg14(act-EGFP-OsbY01) bred into the DBA/2 background (designated Green; provided by Dr. M. Okabe [Osaka University]) [19].

Testis cells were collected at various time points by two-step enzymatic digestion with collagenase and trypsin [20], and cell culture was performed according to the previously published protocol [14]. In some experiments, anti-CD9 antibody was used to enrich spermatogonial stem cells from pup and adult testes [21]. The growth factors used were mouse epidermal growth factor (20 ng/ml; BD Biosciences, Franklin Lakes, NJ), human basic fibroblast growth factor (10 ng/ml; BD Biosciences), and recombinant rat GDNF (10 ng/ml; R&D Systems, Minneapolis, MN). In some cases, murine LIF (103 U/ml; Invitrogen, Carlsbad, CA), rat ciliary neurotrophic factor (CNTF; 100 ng/ml), or mouse oncostatin M (10 ng/ml [OSM]; both from R&D Systems) was added to the medium. Cells were cultured in the presence of 1% fetal bovine serum (JRH Biosciences, Lenexa, KS). After the second or third passage, cells were maintained on mouse embryonic fibroblasts.

Germ cell colonies were quantified by analyzing individual wells of the culture plate. A cluster of germ cells was defined as a colony when it contained >16 cells. In the fetal and neonatal cell cultures, cells were plated in a 12-well plate (2 x 105 cells/3.8 cm2), and the cultures were assayed at Day 10 after the initiation of culture. In the pup and adult cell cultures, CD9-expressing cells were plated in a six-well plate (2 x 105 cells/9.5 cm2), and the cultures were assayed at Days 6 and 9, respectively, because of the extensive proliferation of testicular somatic cells.

Transplantation Procedure

For testicular injection, cultured cells were dissociated by trypsin and suspended in Dulbecco modified Eagle medium with 10% fetal calf serum, supplemented as described [20]. Approximately 3 µl of single-cell suspension containing 1.5 x 104 cells was introduced into the seminiferous tubules of 4- to 10-wk-old WBB6F1-W/Wv (W) recipient mice, which are congenitally infertile because of mutations in the c-kit tyrosine kinase receptor [22]. Microinjection was performed by the efferent duct injection method [20], which filled 75%–85% of the tubules in each recipient testis. Because the recipient mice were not histocompatible with the transplanted cells, they were treated with anti-CD4 antibody to induce tolerance to the donor cells [23]. The Institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols.

RT-PCR Analysis

Total RNA was isolated from the cultured cells or testis cells with Trizol reagent (Invitrogen). First-strand cDNA was synthesized with Superscript II (RNase H– reverse transcriptase, Invitrogen). PCR was carried out with specific primers (5'-TCAGGACTTCAAGGATAGTGAGG-3' and 5'-AAGGCATGAGAGAGAGCACC-3' for Osm, 5'-TCCGTTGTGAGATGGAAGG-3' and 5'-GAATCATCCGGTGTACTGCC-3' for Cntf, and 5'-CCTCTTCCCATCACCCCTGTAAAT-3' and 5'-ACTTGGTCTTCTCTGTCCCGTTGC-3' for Lif). Other primers were as previously described [17].

Analysis of Testes

Donor cell colonization was analyzed by UV fluorescence microscopy [14]. Because the host testis does not have endogenous fluorescence, this method allows the specific identification of transplanted cells. 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. The efficiency of colonization was evaluated by counting the total number of colonies under a stereomicroscope equipped with UV light. For histological analysis, the recipient testes were also fixed in 10% neutral-buffered formalin and processed for paraffin sectioning. All sections were stained with hematoxylin and eosin. Statistical analysis was performed by the Student t-test.

Flow Cytometry

The primary antibodies used were as follows: rat anti-EpCAM (G8.8) and mouse anti-SSEA-1 (MC-480; Developmental Studies Hybridoma Bank, University of Iowa, Ames), rat anti-human INTA6 (CD49f) (GoH3), biotinylated hamster anti-rat INTGB1 (CD29) (Ha2/5), biotinylated rat anti-CD9 (KMC8), allophycocyanin (APC)-conjugated rat anti-mouse c-kit (CD117) (2B8) (BD Biosciences), and rat anti-TDA (EE2) (provided by Dr. Y. Nishimune, Osaka University). APC-conjugated goat anti-rat immunoglobulin G (IgG; Cedarlane Laboratories, Ontario, TN, Canada), APC-conjugated streptavidin (BD Biosciences), or Alexa Fluor 633-conjugated goat anti-mouse immunoglobulin M (IgM; Molecular Probes, Eugene, OR) was used as the secondary antibody. Cells were stained as previously described [24] and analyzed with a FACS-Calibur system (BD Biosciences), with 10 000 events acquired.

Western Blot Analysis

Samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Hybond-P; Amersham Pharmacia Biosciences, Buckinghamshire, U.K.), and probed with rabbit polyclonal anti-STAT3-P (tyrosine 705) antibody (Cell Signaling, Danvers, MA). Peroxidase-conjugated anti-rabbit IgG antibody was used as the secondary antibody (Cell Signaling).

Microinsemination

The seminiferous tubules of recipient testes were dissected under UV illumination when the recipient mice were 7 mo old. The EGFP-expressing seminiferous tubules were recovered, and the germ cells were collected mechanically from the tubules with fine forceps. Microinsemination was performed as described previously with round spermatids [25]. Round spermatids were used in the present experiments because they were more readily recovered from the recipient testes. Embryos that reached the two-cell stage after 24 h in culture were transferred to the oviducts of Day 1 pseudo-pregnant ICR female mice. Fetuses that were retrieved on Day 19.5 were raised by ICR foster mothers.

RESULTS

LIF Promotes Formation of GS Cell Colonies from Neonatal Testes

To examine the effect of LIF on gonocytes, we cultured fetal or newborn testis cells in the presence or absence of LIF. The only germ cells present at this stage are gonocytes, which are separated from the basement membrane. Although they are mitotically quiescent and differ morphologically from spermatogonia, they have spermatogonial stem cell potential and the ability to colonize empty seminiferous tubules after germ cell transplantation [26, 27]. In ICR strains, gonocytes are found in the center of the seminiferous tubules at the time of birth (Fig. 1A), and they migrate to the basement membrane by 3 days after birth (Fig. 1B). Genital ridges or testes were collected from 14.5-, 16.5-, and 18.5-dpc embryos, and testes were collected from 0- to 2-day-old newborn animals. The tissue fragments were dissociated by enzymatic digestion to obtain single cells.


Figure 76011801
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  		FIG. 1. Initiation of germ cell culture from neonatal and pup testes. A, B) Histological appearance of mouse testes on Day 0 (A) and Day 3 (B). Note the absence of proliferation on Day 0 pp. Arrows indicate germ cells. C, D) Neonatal testis cells cultured in the absence (C) or presence (D) of LIF for 9 days. Note the growth of germ cells in the presence of LIF (D). E, F) Pup testis cells cultured in the absence (E) or presence (F) of LIF for 5 days. G) Proliferation of GS cells cultured from pup testes in the absence of LIF. Small gold objects are magnetic beads that were used to isolate spermatogonial stem cells. Bar = 20 µm (A, B) and 50 µm (C–F).

To enrich germ cells, the dissociated cells were plated at a density of 2 x 105 cells/3.8 cm2 on gelatin-coated plates. After overnight incubation, some of the plated cells had adhered to the culture dish. The floating cells were recovered by pipetting the next day and transferred to a new tissue culture plate at 1:1 dilution. In 2–3 days, floating gonocytes began to attach to the somatic cells, and the germ cells started to form colonies. The formation of germ cell colonies from newborn testes was significantly reduced in the absence of LIF (Fig. 1, C and D). Proliferating cells were observed in cultures from genital ridges or testes from 18.5-dpc embryos and newborn mice in 7–10 days, but very few proliferative cells were observed in cultures from the genital ridges of 14.5- or 16.5-dpc embryos, even in the presence of LIF.

To quantify the effect of LIF on germ cell colony formation, we counted the number of germ cell colonies containing >16 cells. Although LIF did not have a significant effect on colony formation in fetal germ cell culture (Fig. 2A), the addition of LIF increased the number of germ cell clusters by 3.1-fold when the cultures were analyzed 10 days after culture initiation (Fig. 3A). To examine whether the germ cells differentiated into spermatogonia, we used ngn3-EGFP transgenic mice that expressed EGFP in Asingle, Apaired, and Aaligned spermatogonia [18]. In the presence of LIF, cells in the colonies displayed EGFP fluorescence, indicating that the gonocytes had already differentiated to spermatogonia by the time of analysis. On the other hand, few EGFP-positive cells were present in the culture when LIF was absent (Fig. 4).


Figure 76011802
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  		FIG. 2. The effect of LIF on the formation of germ cell colonies from (A) fetal (14.5 dpc), (B) pup, and (C) adult mouse testes. Means ± SEM for at least six samples in two to three experiments are shown. No statistical difference could be found by t-test analysis (P < 0.05).


Figure 76011803
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  		FIG. 3. The effect of LIF (A), CNTF (B), and OSM (C) on the formation of germ cell colonies from a neonatal mouse testis. Means ± SEM for at least 11 samples in three experiments are shown. All values were statistically significant by t-test analysis (P < 0.05).


Figure 76011804
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  		FIG. 4. Derivation of GS cell colonies from neonatal ngn3-EGFP transgenic mouse testes. A, B) Neonatal testis cells cultured in the absence of LIF for 23 days. No fluorescence was detected under UV light (B). C, D) Neonatal testis cells cultured in the presence of LIF for 23 days. EGFP-positive ngn3-expressing spermatogonia could be detected under UV light (D). Bar = 200 µm.

In contrast, LIF did not have a significant effect on colony formation from spermatogonia. Testis cells from 5-day-old pups and 4-wk-old adults were dissociated by enzymatic digestion, and anti-CD9 antibody was used to select CD9-expressing spermatogonia, which contain spermatogonial stem cells [21]. When the selected cells were directly plated on a tissue culture plate, spermatogonia from both the pup and adult testes proliferated and formed colonies in the absence of LIF (Fig. 1, E and F). We did not observe a significant difference in the morphology or frequency of colony formation between LIF-treated and -untreated cultures (Fig. 2, B and C), indicating that LIF does not influence the initiation of culture with spermatogonia. In agreement with these results, the effect of LIF on GS cell colony formation was limited to the initiation phase of neonatal testis cell culture. The GS cell colonies derived without LIF from neonatal testes could be passaged, and the GS cells derived with LIF continued to proliferate when LIF was removed at 2 or 3 wks after the initiation of culture (data not shown).

Because LIF is a member of the interleukin 6 (IL-6) superfamily, we examined whether other IL-6 family members could enhance germ cell colony formation from gonocytes. We chose to test CNTF and OSM, both of which belong to the IL-6 superfamily and can bind to the IL-6ST receptor [4, 28]. The CNTF and OSM are expressed in neonatal and pup testes (Fig. 5A). Interestingly, the effect of LIF was mimicked by CNTF, which increased the number of colonies 2.9-fold, whereas the addition of OSM did not have a significant effect on colony formation, decreasing to only 0.7-fold in 10 days (Fig. 3, B and C). Although the morphology or proliferation patterns of germ cell colonies in these cultures were similar to those that developed in the presence of LIF, in the presence of OSM, germ cell colonies disappeared within 2–3 wk because of the prolific growth of somatic cells. Phosphorylated STAT3, which transmits signals from the LIF receptor [4, 5], was found not only in the GS cells but also in the neonatal and pup testes by Western blotting (Fig. 5, B and C), suggesting that ligand binding and activation of downstream molecules also occurs in vivo.


Figure 76011805
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  		FIG. 5. Expression of LIF and other members of the IL-6 family in the neonatal and pup testes. A) RT-PCR analysis of neonatal and pup testes. B) Western blot analysis of GS cells cultured on a laminin-coated dish. Phosphorylation of STAT3 was detected in the presence of LIF. C) Western blot analysis of neonatal and pup testes. Phosphorylation of STAT3 was detected in the testes of 1-day-old neonates. The signal was weaker in the testes of 5-day-old pups.

Phenotypic and Functional Analysis of Cultured Cells

We next examined the phenotypic characteristics of GS cells that were established from 5-day-old Green mouse pup testes in the absence of LIF. When these cells were examined for the expression of cell surface markers by flow cytometry (Fig. 6A), they were found to express several spermatogonial markers strongly, including INTGB1 and INTA6, CD9, EpCAM, and EE2 [21, 24, 29, 30]. KIT, a marker for differentiated spermatogonia [31], was expressed in varying amounts. However, the cultured cells never expressed SSEA-1 or Forssman antigen, each of which is a marker of PGCs [32, 33]. Results from RT-PCR also confirmed the spermatogonia phenotype of the pup testis-derived GS cells (Fig. 6B). The cells expressed spermatogonial markers, such as Pou5f1 (Oct-4), Zfp42 (Rex-1), Ret (c-ret), Neurog3 (ngn3), Ddx4 (Mvh), and Zbtb 16 (PLZF) [18, 34–39], but did not express Nanog, a marker of pluripotent cells [40, 41]. Overall, these features are similar to those of GS cells cultured in the presence of LIF [14]. In addition, neither the colony morphology nor the rate of proliferation was influenced by the addition of LIF, OSM, or CNTF, and the cells were passaged at a ratio of 1:3 to 1:5 every 5–6 days. No effect was observed for LIF, even when the cells were cultured in feeder-free or suspension culture conditions. Similar results were found for GS cells established from adult testes in the absence of LIF (data not shown).


Figure 76011806
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  		FIG. 6. Phenotypic analysis of GS cells cultured in the absence of LIF. A) Characterization of cell surface antigen by flow cytometry. EE2 and EpCAM are surface antigens that are expressed on spermatogonia. Black line, specific antibody; broken line, unstained control. B) RT-PCR analysis of GS cells. Specific primers were used to amplify cDNA from mouse embryonic fibroblasts (MEFs), GS cells, and multipotent germline stem (mGS) cells.

To address whether the cultured cells contained spermatogonial stem cells, spermatogonial transplantation was used to assess the cells for their ability to colonize and reinitiate spermatogenesis in empty seminiferous tubules [42]. Cells were collected at three different time points after the initiation of culture and microinjected into the seminiferous tubules of infertile W recipient mice. The recipient testes were analyzed 2 mo after transplantation by detection of EGFP-positive colonies under UV light (Fig. 7A). While the total cell number increased 4.8 x 106-fold during the 65-day period after initial transplantation, the number of spermatogonial stem cells, as assessed by the functional transplantation assay, increased 2.6 x 106-fold. Because the number of stem cells in the pup testis is 5/105 cells [43], this result also indicates that stem cells expanded at least approximately 1.4 x 1013-fold from the initiation of culture to 149 days, while the total cell number increased by 1.7 x 1012 after stem cell purification and in vitro culture (Table 1 and Fig. 1G). When histological sections of the recipient testes were examined, these colonies contained normal-appearing spermatogenesis (Fig. 7B).


Figure 76011807
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  		FIG. 7. Production of fertile offspring from GS cells cultured in the absence of LIF. A) Recipient testes transplanted with EGFP-expressing GS cells that were cultured in the absence of LIF. B) Histological analysis of the recipient testis. Normal-appearing spermatogenesis was observed. C, D) Spermatogenic cells released from a segment of seminiferous tubule that was colonized by EGFP-positive donor cells. Nomarski (C) and fluorescent (D) images are shown. The arrow indicates a donor-derived round spermatid that was used in microinsemination. E) Offspring that developed from microinsemination. Bar = 1 mm (A), 50 µm (B).


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  		TABLE 1. Spermatogonial stem cell expansion in LIF-free culture.*

Finally, to determine whether the germ cells that developed from LIF-free GS cells were functionally normal, we used in vitro microinsemination, a technique commonly used to produce offspring from infertile animals and humans [25, 44]. Germ cells were recovered from the EGFP-positive seminiferous tubules by repeated pipetting (Fig. 7, C and D). Oocytes derived from C57BL/6 x DBA/2 F1 females were microinjected with round spermatids collected from two different recipient W mice. A total of 49 eggs were cultured for 24 h, and 30 embryos were transferred into the oviducts of pseudo-pregnant ICR host mothers. Ten embryos were implanted successfully in the uteri, and five offspring were born, four males and one female (Fig. 7E). The average body and placental weights of the offspring were 1.63 and 0.15 g, respectively, both of which are within the reference range. Of the five offspring, four displayed EGFP fluorescence, indicating their donor origin. Three of the offspring grew up to be normal fertile adults.

DISCUSSION

The immature testis is a good source for spermatogonial stem cell culture because it lacks differentiated germ cells and is enriched for spermatogonial stem cells. In particular, in the neonatal testis, germ cells are separated from the basement membrane and are located in the lumen of immature seminiferous tubules. The only germ cells present at this time are early differentiating spermatogonia and spermatogonial stem cells [45]. Hence, germ cells can be efficiently recovered by simple gelatin selection. In contrast, in the mature testis, spermatogonia are attached to the basement membrane, and the relative frequency of spermatogonial stem cells is very low (usually 0.2%–0.3%) [1, 3]. Thus, the recovery of spermatogonial stem cells from the mature testis requires cell sorting or other purification procedures, which often result in a significant loss or reduced stem cell viability [21, 24]. The favorable characteristics of the neonatal mouse testis were exploited in the initial establishment of the GS cell culture system from neonatal testes [14]. In this system, a cocktail of several cytokines was shown to stimulate germ cell proliferation, but the roles of the individual components have not precisely been evaluated.

In the testis, LIF is produced by the peritubular cells of the seminiferous tubules, and its receptor is expressed on germline cells from fetal stages to spermatogonia in mature testes [12, 13, 46]. On the basis of this information, LIF has often been used in the culture of germline cells. In testis cell culture, LIF was originally shown to promote the survival or proliferation of rat gonocytes; it stimulated the proliferation of quiescent gonocytes isolated from Day 1 testes [47]. However, the proliferation stopped within ~1 wk under those conditions, and LIF did not promote the survival of proliferating gonocytes from 3-day-old rat testes. In another study, a similar clonogenic assay in mice did not show any enhancement of gonocyte proliferation by LIF [48]. These conflicting results may be because of from the suboptimal culture conditions used in these studies. In particular, the lack of GDNF, a self-renewing factor for spermatogonial stem cells [36], in previous studies may have adversely influenced the stem cell conditions or viability. In the present study, we examined the role of LIF by a GS cell culture system in which a self-renewal division of spermatogonial stem cells stably occurs in vitro. In contrast to previous methods that do not maintain spermatogonial stem cells, this culture system allows a more precise estimation of the effects of growth factors on germ cells, because the germ cells are viable long term.

The results of the present study indicate that the effect of LIF on testis culture is stage-specific. Although LIF was able to stimulate the proliferation of gonocytes in 18.5-dpc to neonatal mice, we were unable to derive GS cells from early gonocytes from 14.5- to ~16.5-dpc fetal gonads by the same protocol, suggesting that late gonocytes more readily convert into spermatogonia. Although gonocytes proliferate until about 16.5 dpc, several functional differences between the early and late stages of gonocytes are indicated. For example, while gonocytes from 12.5-dpc embryos failed to colonize seminiferous tubules after spermatogonial transplantation, those from 18.5-dpc embryos produced normal spermatogenesis [26]. In addition, male germ cells acquire androgenetic imprint patterns during this period [49]. Much information remains unknown about how these functional changes occur in gonocytes, and our results indicate that LIF is apparently insufficient to reconstitute the changes that occur during development.

On the other hand, we found that GS cells can be established and expanded from pup and adult testes in the absence of LIF and, furthermore, that the established cells can be maintained long term in the absence of LIF and contribute to the germline. These features contrast with those of the ES cell system, in which the removal of LIF results in the differentiation and loss of germline potential [8, 9]. Although both types of stem cells can contribute to the germline lineage, ES and GS cells apparently differ in their differentiation potentials and regulatory mechanisms. Our results suggest that the effectiveness of LIF is limited to perinatal gonocytes and that LIF signaling per se does not promote proliferation or maintenance of the undifferentiated state of GS cells once the cells are established. Consistent with this idea, GS cells that were initially established from gonocytes in the presence of LIF could later be maintained in the absence of LIF, suggesting that conversion from gonocyte to spermatogonial stem cells had occurred in the culture.

The results of our experiments raise several questions about the mechanism of GS cell colony formation. We think that at least two factors are important for the success of GS cell culture. One factor is the removal of testicular somatic cells. GS cells do not require somatic cells for their survival and can grow in the complete absence of feeder cells [50, 51]. On the contrary, in the initial phase of the culture, the growth of GS cells is often very slow and is easily overwhelmed by actively proliferating somatic cells. Particularly in pup or adult testis cell culture, since the somatic cells tend to proliferate more actively than the neonatal testes, the establishment of GS cells from these stages requires the highly enriched germ cells. LIF appears to stimulate the proliferation of neonatal somatic cells to some degree, but it does not appear to have an apparent effect on the culture of the pup or adult testis, in which somatic cells proliferate more actively, regardless of LIF. In addition, although we have shown that mouse embryonic fibroblasts have beneficial effects on GS cell growth after their establishment [50], the negative effects of testicular somatic cells on gonocytes and spermatogonia have previously been reported [52, 53]. Therefore, we do not think that LIF acts by promoting the attachment of germ cells to somatic cells.

Another factor is the identification of the appropriate growth factor. It is well established that GDNF is essential for the self-renewal of spermatogonial stem cells. However, it is still unknown when the male germline cells begin to acquire responsiveness to GDNF. Even in the presence of GDNF and LIF, we could not establish the GS cell from the 14.5- to ~16.5-dpc fetal gonocyte, suggesting that gonocytes have different growth requirements, depending on their developmental stage. While LIF may be only one of the molecules that enhance the differentiation of gonocytes to spermatogonial stem cells, there are probably other unknown growth factors that coordinate with LIF to promote the maturation of gonocytes into self-renewing spermatogonia. The identification of these molecules may be required to derive GS cells from different stages of testicular development.

Interestingly, the effect of LIF was mimicked by CNTF, which belongs to the IL-6 superfamily. However, OSM, another member of this family, did not have a similar effect, suggesting that these molecules have differential effects in the testis. Although these IL-6 family molecules are expressed in the neonatal testis, their functions are poorly understood. Previously, OSM was reported to be involved in the regulation of Sertoli cells [28]. The addition of OSM in vitro enhanced the proliferation of Sertoli cells derived from the neonatal testis, but LIF did not exhibit a similar activity [28]. Although both LIF and CNTF were able to enhance the formation of germ cell colonies in the present study, their mechanisms of action are unclear. They have been thought to transmit signals through the IL-6ST receptor, but a recent study showed that the IL-6ST receptor is dispensable in the male germline and that a male lacking IL-6ST retained normal fertility [46]. However, because the deletion efficiency was not assessed in that study, it remains possible that deletion did not occur in all of the male germ cells owing to incomplete cre-mediated deletion. Alternatively, another unknown receptor for LIF or CNTF may compensate for the loss of IL-6ST. Further studies are clearly required to determine which molecules trigger the gonocyte-spermatogonium transition in vivo.

The success of spermatogonia culture has opened up new possibilities in spermatogonia research. While GS cells can be cultured in serum- or feeder-free conditions, they can also be propagated in the absence of substrata [50, 51]. Furthermore, knockout animals can be produced by gene trapping and homologous recombination in GS cells [54], and these cells can potentially convert into pluripotent ES-like cells [55]. The conditions presently used for culture are not optimal, however, and many improvements are needed to increase the usefulness of the technology. In particular, the initiation phase of culture is probably the most critical, because the overgrowth of somatic cells often interferes with the growth of germ cells. We have demonstrated here that LIF is one of the molecules that enhance the initiation phase, but much remains to be studied about the process of GS cell derivation and its relation to male germline development. Our present experimental system may prove valuable for identifying other factors involved in the functional differentiation of PGCs to gonocytes and spermatogonial stem cells.

ACKNOWLEDGMENTS

We are grateful to Ms. A. Wada for her technical assistance.

FOOTNOTES

1Financial support for this research was provided by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan and by grants from CREST and the Human Science Foundation (Japanese). This study was also supported in part by Special Coordination Funds for Promoting Science and Technology from MEXT. Back

Correspondence: 2 Takashi Shinohara, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 751 4169; e-mail: tshinoha{at}virus.kyoto-u.ac.jp

Received: 24 July 2006.

First decision: 4 August 2006.

Accepted: 3 October 2006.

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M. Kanatsu-Shinohara, T. Muneto, J. Lee, M. Takenaka, S. Chuma, N. Nakatsuji, T. Horiuchi, and T. Shinohara
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