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BOR - Papers in Press, published online ahead of print March 21, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.056879
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BIOLOGY OF REPRODUCTION 77, 127–137 (2007)
DOI: 10.1095/biolreprod.106.056879
© 2007 by the Society for the Study of Reproduction, Inc.

Identification, Isolation, and In Vitro Culture of Porcine Gonocytes1

Sandeep Goel 3, Miki Sugimoto 4, Naojiro Minami 3, Masayasu Yamada 3, Shinichi Kume 4, and Hiroshi Imai 2 3

Laboratories of Reproductive Biology3 and Anatomy and Cell Biology,4 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

ABSTRACT

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

developmental biology, gamete biology, gametogenesis, spermatogenesis, testis

INTRODUCTION

Before sexual differentiation, primordial germ cells (PGCs), which originate from the proximal epiblast, migrate from the base of the allantois via the gut mesentery and populate the genital ridge [1, 2]. Shortly afterwards, these bipotential cells colonize the primitive undifferentiated gonads that are beginning to form and become gonocytes [1, 2]. In rodents, gonocytes and undifferentiated spermatogonia are primitive cells that reside in the seminiferous tubules of the neonatal and prepubertal testes and give rise to spermatogonia, thereby initiating spermatogenesis. In pigs, gonocytes are the primary germ cells in neonatal testes, and spermatogonia are only seen at 2 mo of age [3]. Little is known about the stages and timing of the transition from gonocytes to spermatogonia in this species. Male germline stem cells have gained a great deal of attention in recent years, as it is possible to isolate and culture them in vitro [4–12]. These cells can be genetically modified and they further differentiate to spermatozoa following transplantation into a recipient testis, eventually producing transgenic offspring [13–17]. In the case of livestock species, in which no embryonic stem (ES) cell line with germline characteristics has been reported to date, a male germ stem cell line could be used to produce transgenic animals in a way that overcomes the numerous shortcomings of the conventional pronuclear injection method. If these cell lines could be established, homologous recombination techniques could be applied to create targeted mutated animals, the generation of which is presently achieved inefficiently by the somatic cell nuclear transfer (SCNT) technique [18–21]. However, no male germ stem cell line has yet been established for any livestock species. The most probable reason for this is a dearth of knowledge regarding their specific markers and culture conditions. In order to purify efficiently germline stem cells from the numerous types of cells in the testis and to develop a robust culture system, it is mandatory to identify markers that are specifically expressed by these cells. This would facilitate their identification during purification and in culture. In cattle, the binding of the lectin Dolichos biflorus agglutinin (DBA) [22] and the expression of UCHL1 (previously known as PGP 9.5) [23], which is a ubiquitin C-terminal hydrolase, have been identified as specific markers for spermatogonia. These markers have been used to identify spermatogonia in xenografted bovine testis [24] and during isolation and culture [25]. However, most of the isolated cells underwent differentiation during prolonged culture, which suggests that a more elaborate analysis of culture conditions is needed [26].

During purification of germ cells from 3-wk-old pig testis, morphological identification of germ cells and the use of markers, such as vimentin, to rule out somatic cell contamination have not been sufficiently successful to identify germ cells in a specific manner [27]. Although an anti-KIT antibody (previously known as c-kit antibody) has been used as a marker to identify and purify porcine type A spermatogonia [28], KIT is also expressed by differentiated spermatogonia [29, 30]. Therefore, in two previous studies [27, 28], it remains unclear as to whether the purified and cultured cells contained stem cells that were capable of initiating spermatogenesis. Recently, sheep [31] and pig [32] spermatogonia have been identified using an antibody directed against UCHL1. Furthermore, in the pig, UCHL1 expression could be used to distinguish spermatogonia in culture from somatic cells, although UCHL1 expression was also detected in Sertoli cells after long-term culture [32].

DBA has specific affinity for {alpha}-D-N-acetyl-galactosamine. The anti-SSEA-1 and EMA-1 monoclonal antibodies and DBA bind specifically to the large round PGCs of the genital ridge in pigs [33]. In the postpubertal boar testis, DBA binds weakly to the apical cytoplasm of Sertoli cells [34]. In neonatal human testis, DBA binds to spermatogonia and Leydig cells [35].

In the present report, we show that DBA is a specific marker for pig gonocytes and primitive spermatogonia in neonatal pig testis. Unlike UCHL1, which is expressed by porcine undifferentiated germ cells at all ages, DBA was found to mark only primitive germ cells in neonatal pig testes. We found that DBA binding was specific for germ cells in culture. Isolated gonocytes proliferated in vitro for at least 7 days in culture and differentiated further to SSEA-1-expressing colonies.

MATERIALS AND METHODS

Collection of Testes

Testes were collected from crossbred piglets (Landrace x Large White Yorkshire x Duroc) aged from 2 days to 3 wk that were obtained from a local farm. Pigs are castrated at this age to improve meat quality and growth rate. Testes from 5-mo-old pigs were collected from the abattoir. Testes tissues for histochemical analysis were fixed immediately after collection. For the isolation and cultivation of gonocytes, collected testes were washed with PBS and transported on ice within 2 h to the laboratory in DMEM/F12 (Gibco, Invitrogen) that was supplemented with antibiotics and 15 mM Hepes (Wako, Japan). Animals were handled according to the guidelines of the Institutional Animal Care and Use Committee of Kyoto University.

Histochemistry

Testicular samples were fixed in Bouin fixative, dehydrated, embedded in paraffin, and sectioned (8-µm thickness). Dilutions of lectin DBA and the primary and secondary antibodies were performed in PBS with 1% (v/w) BSA (Sigma). Sections were dewaxed, rehydrated, and stained with DBA, as described previously [25]. Briefly, each testis section was incubated with 3% H2O2 (Merck, Japan) for 10 min, washed with PBS, incubated in 5% BSA in PBS for 15 min, incubated with DBA-conjugated horseradish peroxidase (DBA-HRP, 1:100; EY Laboratories) for 1 h at 37°C in a moist chamber, rinsed three times with PBS, incubated for 3–5 min in a substrate-chromogen mix that contained 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Dojindo, Japan) and 20 µl of 30% H2O2 in PBS, rinsed thoroughly in distilled water, counterstained with hematoxylin, mounted with Aquatex (Merck, Germany), and observed under an Olympus BX 50 microscope. Negative control sections were incubated in 1% BSA in PBS without lectin.

Sections from animals of up to 3 wk of age were also stained with rabbit anti-UCHL1 (1:100; Biomol, UK), mouse anti-vimentin (clone V9, 1:100; Sigma), goat anti-GATA-binding protein 4 (GATA4, clone C-20, 1:100; Santa Cruz Biotechnology), mouse anti-{alpha}-smooth muscle actin (clone 1A4, 1:500; Lab Vision Corporation), mouse anti-ZBTB16 (clone 2A9, 1:100; Calbiochem), rat anti-KIT (anti-CD117, clone ACK45, 1:100; BD Biosciences), and mouse anti- SSEA-1 (anti-human CD15, 1:50; DAKO), to assay their specificities in the neonatal testis. Testis sections from 5-mo-old pigs were also analyzed for UCHL1, SSEA-1, and ZBTB16 expression. Briefly, after deparaffinization and rehydration, sections were blocked with 10% normal goat serum (rabbit serum for GATA4 immunostaining) in PBS for 30 min, incubated with the above-mentioned primary antibodies overnight at 4°C, washed several times with PBS, incubated with 3% H2O2 for 10 min, washed three times with PBS, incubated with the corresponding HRP-conjugated secondary antibody, i.e., sheep anti-rabbit IgG or sheep anti-mouse IgG (1:100; Amersham Biosciences, UK), goat anti-rat (Vector Laboratories), and rabbit anti-goat IgG (1:200; Abcam) for 30 min at 37°C, rinsed three times with PBS, incubated for 3–5 min with the substrate-chromogen mix, rinsed thoroughly in distilled water, counterstained with hematoxylin (Wako), mounted with Aquatex (Merck), and observed under an Olympus BX 50 microscope. For the negative controls, the primary antibody was omitted and instead the section was incubated with 1% BSA in PBS.

The alkaline phosphatase (AP) activity in each section was determined using the Alkaline Phosphatase Substrate Kit III (Vector Laboratories), as described by the manufacturer. The specificity of staining was determined by incubating a few sections with substrate that contained levamisole (Vector Laboratories), which is an inhibitor of AP activity. Since the microscopic morphologies of the testis were quite similar until 3 wk of age, serial cross-sections of the testes of 3-, 7-, 14-, and 21-day-old pigs were counted in ten random fields, to estimate the average numbers of UCHL1-, DBA-, and AP-positive cells per tubule section. Positive cells in 150 to 200 tubules from the testes sections of at least three animals in each age group were counted for tabulation of the data.

To confirm the specificity of DBA binding to germ cells, sections were double-stained with DBA and for germ-cell specific markers, i.e., UCHL1, KIT, SSEA-1, and ZBTB16 (previously known as PLZF). Briefly, after deparaffinization and rehydration, the sections were blocked with 10% goat serum and 5% BSA in PBS for 30 min. The sections were incubated separately at 4°C overnight with one of the following antibodies: anti-UCHL1 (1:100), rat anti-mouse KIT (1:100), anti-ZBTB16 (1:100), and anti-SSEA-1 (1:50). The sections were then washed several times with PBS, incubated with the corresponding secondary biotinylated antibody, i.e., goat anti-rabbit, goat anti-mouse (1:200; both from DAKO), and goat anti-rat IgG (1:200; Vector Laboratories) for 30 min at 37°C, incubated with streptavidin-FITC (1:200; DAKO) and DBA-rhodamine (1:100; Vector Laboratories) for 1 h at 37°C, rinsed three times with PBS, stained with 1 µg/ml Hoechst 33342 (Sigma) for 10 min, mounted in SlowFade (Molecular Probes), and observed under an Olympus BX 50 microscope that was fitted with an epifluorescent lamp. For the negative controls, the primary antibody and lectin were omitted and instead the section was incubated with 1% BSA in PBS.

Preparation of Cell Suspensions

All chemicals used for cell culture were from Sigma-Aldrich (St. Louis, MO), unless noted otherwise. Testes collected from piglets aged 2–4 days were treated as described previously [27, 28], with some modifications. Briefly, after washing several times with PBS, the tunica and other visible connective tissues were removed. The testes were minced with scissors and incubated in DMEM/F12 medium that was supplemented with 15 mM Hepes, 100 IU/ml penicillin, 50 µg/ml streptomycin, 40 mg/ml gentamycin, 1.5 mg/ml collagenase, and 5 µg/ml DNase at 35°C for 15 min in a shaking water bath operated at 100 cycles/min. After three washes with DMEM/F12 and removal of most of the interstitial cells, seminiferous tubules fragments were incubated in DMEM/12 medium that contained 1.5 mg/ml collagenase, 1.5 mg/ml hyaluronidase, 0.5 mg/ml trypsin, and 5 µg/ml DNase for 30 min under the conditions described above. The dispersed cells were washed twice with medium, suspended in DMEM/F12 with 10% FBS (JRH Biosciences) and filtered successively through 80-µm and 40-µm nylon meshes (Kyoshin Rykou, Japan). The collected cells were subjected to discontinuous Percoll density gradient for further purification.

Cell Separation in a Discontinuous Density Gradient

Cells were separated as described by van Pelt et al. [36], with minor modifications. Briefly, an iso-osmotic Percoll suspension (Pharmacia Biotech) was prepared by the addition of nine parts (v/v) Percoll to one part (v/v) 1.5 M NaCl (Wako). A discontinuous density gradient was made by diluting the iso-osmotic Percoll suspension with Hepes-buffered DMEM/F12. The gradients were created by layering 2 ml each of 60%, 50%, 40%, 30%, and 20% Percoll in a 15-ml centrifuge tube (Iwaki, Japan). The filtered cell suspension were layered on top of the gradients in 2 ml DMEM/F12 and centrifuged at 800 x g for 30 min at 20°C. Cells located at the interface between the different density suspensions were collected as fractions 1–5 and examined for gonocytes by DBA binding.

Gonocytes Among Separated Cells

The cells collected from each fraction after Percoll separation were washed twice with PBS and attached to poly-L-lysine (Sigma)-coated slides (Superfrost, Matsunami, Japan). Attached cells were fixed with Bouin fixative. Cells from each fraction were stained with DBA, to identify gonocytes. Briefly, fixed cells were washed three times with PBS, incubated with 3% (v/v) H2O2 for 10 min, incubated with DBA-HRP (1:100) for 1 h at 37°C in a moist chamber, incubated with the substrate-chromogen mix for 3–5 min to develop the peroxidase, mounted with Aquatex (Merck), and observed under an Olympus BX 50 microscope. To evaluate the average number of DBA-positive cells, 10 random fields from each fraction were counted. Approximately 1 x 104 (n = 3) cells from each fraction were counted.

Culture of Isolated Gonocytes

Cells collected from the gonocyte-enriched Percoll fractions were analyzed for viability by trypan blue dye exclusion before being seeded into a 60-mm tissue culture dish (Nunc) at a density of 2 x 105 cells/cm2 for differential plating. Cells were cultured simultaneously in 24-well culture dishes (Nunc) with or without a microcover glass (Matsunami, Japan), for either double labeling with DBA plus an antibody or for DBA alone. Cover glasses were coated with 0.01% poly-L-lysine (Sigma) for 15 min before cell seeding. Floating cells were collected from the 60-mm culture dish after 4 h of seeding and stained with DBA and the anti-UCHL1 antibody. The culture medium used was DMEM/F12 supplemented with 10 µg/ml insulin, 10 µg/ml apo-transferrin, 100 U/ml penicillin, 50 µg/ml streptomycin, 40 µg/ml gentamycin sulfate, single-strength non-essential amino acid solution (Gibco, Invitrogen), 1 mM pyruvate, and 10% FBS. Cells were cultured at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. The medium was changed twice per week. Cells were analyzed histochemically after 3 days and 7 days of culture in the 24-well culture dish.

UCHL1 and DBA Staining of Floating Cells

Floating cells collected after differential plating were analyzed for viability by trypan blue dye exclusion. Cells were fixed on poly-L-lysine-coated slides and fixed with Bouin fixative for 10 min. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 (v/v) (Sigma) in PBS for 20 min. Immunofluorescent localization with anti-UCHL1 antibody and lectin DBA-HRP staining was performed as described above. Ten random fields were counted and the average number of positive cells was determined. Approximately 200–300 cells were scored in each experiment (n = 5).

Gonocyte Viability and Proliferation

Cultured cells attached to a poly-L-lysine-coated cover glass were cultured for 3 days and 7 days, incubated with single-strength BrdU (Roche) for 6 h, fixed in Bouin solution for 10 min at room temperature, washed several times with PBS, permeabilized with 0.1% Triton X-100 (Sigma), washed with PBS several times, incubated in 2 N HCl for 30 min at 37°C, neutralized with 0.1 M borate buffer (pH 8.5), incubated in 10% normal goat serum with 5% BSA in PBS for 30 min to block nonspecific binding, incubated with mouse anti-BrdU antibody (1:100; Chemicon) for 1 h at 37°C, rinsed several times with PBS, incubated with goat anti-mouse Alexa Fluor 546 (1:200; Invitrogen) for 30 min at 37°C, incubated with DBA-FITC (1:100) for 1 h at 37°C, washed several times with PBS, mounted in SlowFade, and counted in 10 random fields to evaluate the average number of proliferating gonocytes that were stained with both DBA and anti-BrdU antibody. Approximately 250–300 DBA-positive cells were scored in each experiment (n = 4) to evaluate gonocyte proliferation.

Characterization of Germ Cell Colonies

Germ cell colonies that grew as focal colonies and as three-dimensional colonies were double-stained with anti-GATA4 antibody (1:100) and DBA, to rule out DBA binding to Sertoli cells. Colonies were also double-stained with anti-{alpha}–smooth muscle actin antibody (clone 1A4, 1:1000; Lab Vision) and DBA, to specify DBA binding to germ cells in culture. To ascertain the presence of germ cells in culture, the cells were double-stained with DBA and the anti-ZBTB16 (1:100), anti-KIT (1:100), and anti-SSEA-1 (1:50) antibodies in separate culture dishes. Briefly, after fixing in Bouin solution and permeabilizing with 0.1% Triton X-100, the cells were incubated with 10% goat serum (rabbit serum for anti-GATA4) and 5% BSA in PBS. Cells were double-stained using the earlier described procedure for the double-staining of sections. For the negative controls, the primary antibody and lectin were omitted and instead the cells were incubated with 1% BSA in PBS.

Statistical Analysis

Testes samples from at least three animals from each age group were used for immunohistochemistry. For the isolation of gonocytes (n = 8), cell culture and proliferation assays (n = 4), and characterization of germ cell colonies (n = 5) (n = number of trials), the results are presented as mean ± SEM. The statistical analysis was conducted using the Statview 4.0 (Abacus Concepts Inc.). Differences were determined using the Fisher PSLD test for significance between means. The level of significance was set at P ≤ 0.05.

RESULTS

Histochemistry

Lectin DBA affinity in the neonatal pig testis was restricted to germ cells, which were identified based on size, topological position, and morphology (Fig. 1A). In neonatal pig testes, the seminiferous tubule consists of spindle-shaped myoid cells that surround the tubules, Sertoli cells, and germ cells. The germ cells are much larger in size than the Sertoli cells and thus, are easy to identify. DBA staining was localized to the surfaces of the germ cells. The stained cells contained a large nucleus with 1–3 nucleoli. DBA binding was restricted to germ cells; no somatic cells were stained. However, a few germ cells did not show any affinity for DBA (Fig. 1A). Interestingly, the intensity of DBA staining in individual germ cells varied. Sections from 3-day-old and 7-day-old testes had 6.2 ± 1.3 and 5.8 ± 1.2 DBA-positive cells/tubule, respectively (Fig. 2). The number of DBA-positive cell decreased significantly with age. At 14 days and thereafter, very few cells showed strong binding to DBA (Fig. 1B).


Figure 101
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  		FIG. 1. Immunostaining and alkaline phosphatase activities of testis sections. A, B) Testis sections from 3-day-old (A) and 2-wk-old (B) pigs were stained with DBA. Arrows indicate DBA-positive germ cells. The arrowhead in A indicates a DBA-negative germ cell. Note that DBA-stained germ cells are significantly fewer in the 2-wk-old section, and that no somatic cells are stained with DBA. C) DBA affinity in a testicular section from a 5-mo-old pig. The arrow indicates a rare, weakly stained germ cell. D) AP staining of a neonatal testicular section from a 3-day-old pig. The main AP activity is observed in peritubular cells (arrowhead). Germ cells also showed weak staining (arrow). E, F) UCHL1 staining of testes from 2-wk-old (E) and 5-mo-old (F) pigs. UCHL1 expression is constantly present in germ cells until 5 mo of age. G, H, I) ZBTB16 staining of testis sections from 3-day-old (G), 2-wk-old (H), and 5-mo-old (I) pigs. Arrows indicate ZBTB16-positive germ cells that are located in the seminiferous tubules. The ZBTB16-expressing cells become progressively sparser in the seminiferous tubules with age, a pattern similar to that seen for DBA binding. Vimentin (J) and GATA4 (K) staining of a 3-day-old testis section. Note that vimentin expression is also present in germ cells (arrows, J), although the germ cells are not stained by GATA4 (arrow, K) and its expression is present in Sertoli cells and interstitial cells. L) KIT staining of a testis section from a 3-day-old pig. KIT expression is weak and variable among the germ cells; most cells show expression (arrows) and a few do not (arrowhead). Bar = 50 µm.


Figure 02
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  		FIG. 2. Average numbers of UCHL1-, DBA-, and AP-positive germ cells in the seminiferous tubules of testes at different ages. The numbers of DBA- and AP-staining cells decreased significantly by 2 wk of age (asterisk, P < 0.0001), although the number of UCHL1-positive germ cells was the same in all age groups.

At 2 wk of age (Figs. 1B and 2), the number of DBA-positive cells/tubule (2 ± 1.1) was significantly lower than the number found in younger testes (P < 0.0001) (Fig. 2). Testes from 5-mo-old animals rarely contained germ cells that were positive for DBA, and no DBA-positive somatic cells were observed (Fig. 1C). UCHL1 expression was restricted to germ cells in all the age groups studied (Fig. 1, E and F). The average number of germ cells that expressed UCHL1 ranged from 6.8 ± 0.2 to 7.0 ± 0.2 per tubule from 3 days to 3 wk of age. The number did not differ significantly among the age groups studied (Fig. 2). However, the average number of DBA-positive germ cells was significantly lower than the average number of UCHL1-positive germ cells from 14 days of age onwards (P < 0.0001) (Fig. 2). Unlike DBA binding, 5-mo-old testes contained numerous spermatogonia that were positive for UCHL1 expression, and differentiated germ cells, such as spermatocytes and spermatids, were negative for UCHL1 expression (Fig. 1F).

Intense AP staining was observed in the peritubular cells. In testis sections until 3 wk of age, the germ cells also had AP activity, although the staining intensity was weak and variable among the stained cells (Fig. 1D). The average number of AP-positive germ cells was 2.6 ± 0.4 cells/tubule at 3 days of age and 1.7 ± 0.3 at 1 wk of age (Fig. 2). The number of AP-positive germ cells decreased significantly at 14 days of age (P < 0.0001), at which time the number of AP-positive germ cells/tubule was 0.75 ± 0.3. At 3 wk of age, the number of AP-positive germ cells/tubule was 0.25 ± 0.22. In the 5-mo-old testicular samples, none of the germ cells were AP-positive, while many of the peritubular myoid cells stained intensely for AP (data not shown). Immunostained sections fro animals of various ages showed progressive loss of ZBTB16 expression from germ cells, a pattern that resembled that of DBA staining. ZBTB16-expressing germ cells were abundant in the seminiferous tubules of 3-day-old testes (Fig. 1G) but became progressively sparser by 2 wk of age (Fig. 1H). In the sections of 5-month-old testes, ZBTB16 expression was restricted to a few undifferentiated spermatogonia (Fig. 1I). SSEA-1 expression was not seen until 2 wk of age, at which time it was localized to the surfaces of germ cells (data not shown). In the 5-month-old testis, no SSEA-1-positive cells were seen. Vimentin staining was primarily restricted to the somatic cells, although the germ cells were also stained in neonatal pig testis sections (Fig. 1J). GATA4 expression was present in the Sertoli and interstitial cells, while the germ cells were not stained by the anti-GATA4 antibody (Fig. 1K). The expression of {alpha}-smooth muscle actin was restricted to the peritubular myoid cells (data not shown). KIT expression was weak and variable in germ cells until 3 wk of age, whereas some of the germ cells were stained with the anti-KIT antibody (Fig. 1L).

Sections that were double-stained with DBA plus an antibody (anti-UCHL1, anti-KIT, anti-ZBTB16 or anti-SSEA-1 antibody) confirmed that DBA binding was restricted to germ cells (Fig. 3). In the double-stained sections, DBA binding was always present in germ cells that were positive for UCHL1 expression, although the UCHL1-expressing cells were not always DBA-positive (Fig. 3, A-C). By 2 wk of age, most of the DBA-positive cells were lost, while UCHL1-expressing cells were still abundant. KIT expression was weak in the sections and was inconsistent in germ cells from neonatal testes. Some of the DBA-positive cells and some of the DBA-negative germ cells expressed KIT (Fig. 3, D-F). ZBTB16 expression was weak and was localized to the nuclei of all the DBA-positive germ cells but not to any of the nuclei of the DBA-negative germ cells (Fig. 3, G-I). In a few weakly DBA-positive cells, ZBTB16 expression was not detected due to weak staining intensity. SSEA-1 expression was not detected in germ cells until 2 wk of age. In double-stained sections from 2-wk-old and 3-wk-old testes, DBA-negative cells and weakly DBA-positive cells that were losing DBA binding expressed SSEA-1 (Fig. 3, J-L). Comparatively strongly DBA-positive cells in 2-wk-old and 3-wk-old sections showed weak SSEA-1 expression (Fig. 3, J-L). However, few of the germ cells that were negative for DBA binding did not express SSEA-1.


Figure 03
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  		FIG. 3. Fluorescence double-staining of testes sections. A, B) A 3-day-old testis section that was double-stained with UCHL1 and DBA. A UCHL1-positive germ cell is also positive for DBA binding (arrows), although the DBA-negative germ cells also stain with UCHL1 (arrowheads). The same section was stained with Hoechst 33342 (C). D, E) A 3-day-old testis section that was double-stained with KIT and DBA. KIT expression is observed in DBA-positive germ cells (arrows) and occasionally in DBA-negative cells (arrowhead). Some DBA-positive germ cells are also KIT-negative (asterisk). F) The same section stained with Hoechst 33342. G, H) ZBTB16 and DBA double-staining of a testis section from a 2-day-old pig. ZBTB16 expression is visible in a DBA-positive germ cell (arrows) but is lost in a weakly DBA-positive germ cell (arrowhead). DBA-negative germ cells do not express ZBTB16 (asterisk). The same section stained with Hoechst 33342 (I). J, K) Double-staining with SSEA-1 and DBA of a testis section from a 2-wk-old pig. All of the strongly SSEA-1-positive germ cells are negative for DBA binding (arrows). Strongly stained DBA-positive cells show weak SSEA-1 expression (arrowheads). The same section stained with Hoechst 33342 (L). Note that DBA binding is weak in 2-wk-old pig sections. Bar = 50 µm.

Cell Separation

Having found a specific marker, we next attempted to isolate a pure population of gonocytes and undifferentiated germ cells from the neonatal pig testis. After two-step enzymatic digestion, most of the interstitial cells were removed by repeated washing, and the dissociated tubular cells were subjected to discontinuous density Percoll centrifugation with five density layers (Supplemental Fig. 1, available online at www.biolreprod.org). The cell population collected in fraction 5 (between the 50% and 60% Percoll layers) was rich in germ cells that consisted of 69.6 ± 3.4% DBA-positive cells. In addition, germ cells were morphologically distinguishable from somatic cells, as they were much larger in the neonatal testis. DBA staining intensity was variable within the cells and a few DBA-positive cells were aggregated as clumps. DBA-negative cells were comprised of undifferentiated germ cells, which did not bind to DBA, and somatic cells.

Gonocyte Culture and Proliferation

The viability of the purified cells was greater than 98%, as determined by trypan blue exclusion. Most of the cells attached to the culture dish. Four hours after seeding the plates with 2 x 105 cells/cm2, a low number of the cells (equivalent to 1200 ± 154 cells/cm2) was collected as floating cells. Almost all (~95%) of the floating cells that were collected were viable. Of these cells, 15.2 ± 0.8% were DBA-positive (although most were stained weakly) and 91.3 ± 2.3% were UCHL1-positive (Supplemental Fig. 2, available online at www.biolreprod.org). The attached cultured cells showed a high nucleus to cytoplasm ratio and each cell was found to have 2–3 nucleoli when observed at high magnification. These cells formed flat focal colonies within 2–3 days of culture (Fig. 4A). The colonies grew in size, reaching an average diameter of 145 ± 3.6 µm by 4–5 days of culture, and some of the colonies reached 250 µm in diameter. These focal colonies contained pairs and chains of DBA-positive cells (Fig. 4B). After 4–5 days of culture, raised three-dimensional colonies that resembled murine ES cells appeared, and by 7 days of culture, these colonies were larger and denser (Fig. 4C). Some of these colonies stained positively for DBA with variable staining intensity (Fig. 4D). Double-labeling with lectin DBA and anti-BrdU antibody revealed that the gonocytes could proliferate in culture. After 3 days and 7 days of culture, 53.0 ± 1.5% and 54.7 ± 2.1% of the DBA-positive cells were also BrdU-positive, respectively (Supplemental Fig. 2A, available online at www.biolreprod.org). Although DBA affinity was slightly lost by 7 days of culture (Supplemental Fig. 2B, available online at www.biolreprod.org), the average number of DBA-BrdU-positive cells did not differ from that at 3 days of culture. However, it was not possible to count the DBA-BrdU-positive cells in the three-dimensional colonies due to their compact nature, making it difficult to estimate the number of proliferating cells at day 7 of culture. Nevertheless, the coimmunolocalization of DBA and BrdU indicated that these colonies contained proliferating germ cells.


Figure 04
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  		FIG. 4. Gonocytes in culture. A) Gonocytes grow as focal colonies of adherent cells with epithelial morphology. B) The focal colonies are positive for DBA binding after 3 days in culture. Note the variable staining intensity and the chain of DBA-positive cells (arrow). C) After 7 days, three-dimensional colonies appear. D) The colonies also show DBA affinity. Bar = 50 µm.

Characterization of Germ Cell Colonies

The DBA-positive colonies of germ cells that grew as foci and as three-dimensional colonies were negative for GATA4 and {alpha}-smooth muscle actin expression (Fig. 5, A, B, F, and G). The germ cell colonies that had epithelial morphology initially and three-dimensional colonies later were surrounded by Sertoli cells, peritubular myoid cells, and interstitial cells. Peritubular myoid cells always grew as elongated spindle-shaped cells and were easily distinguishable in culture. These cells grew closely around the foci and around the three-dimensional germ cell colonies, and they stained positively with the anti-{alpha}-smooth muscle actin antibody (Fig. 5, B and G). Sertoli cells had a fibroblastic appearance and their nuclei stained positively with the anti-GATA4 antibody (Fig. 5, A and F). Although the DBA-positive germ cells grew in close proximity to somatic cells, none of the DBA-positive cells stained positively with the GATA4 or {alpha}-smooth muscle actin antibody. Similarly, we did not observe DBA binding to GATA4- or {alpha}-smooth muscle actin-expressing cells. KIT expression was detected in DBA-positive germ cells at 3 days of culture (Fig. 5C). These colonies had both strongly and weakly KIT-expressing cells and a few KIT-negative cells. KIT expression was detected in the three-dimensional colonies after 7 days of culture, and KIT expression was also seen in the DBA-negative germ cells within these colonies (Fig. 5H).


Figure 05
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  		FIG. 5. Immunohistochemical characterization of cultured gonocytes. Overlapping images after double-fluorescence staining with DBA and antibodies of 3-day (A–E) and 7-day (F–J) cell cultures. DBA-positive cells stain red and antibody-positive cells appear green. A and F) GATA4 and DBA double-staining. DBA-stained cells (arrow) are negative for GATA4 expression (green, nuclear). GATA4 expression is evident in the cells that surround the colony (arrowheads). B and G) Double-staining of cells with {alpha}-smooth muscle actin and DBA. DBA-positive germ cells (arrow) are surrounded by peritubular myoid cells (arrowheads). The peritubular myoid cells never stain positively for DBA. C and H) KIT-DBA coimmunostaining. The DBA-positive germ cells stain positively for KIT (arrow). Note that KIT expression is localized in the cytoplasm (green), and that DBA is localized to the cell surface (red). A few DBA-positive cells show weak KIT expression (arrowhead), and some DBA-positive cells are negative for KIT expression (asterisk). A three-dimensional colony (H) shows yellow fluorescence due to the colocalization of KIT and DBA (arrowhead), while cells that are negative for DBA binding and positive for KIT expression appear green (arrow). D and I) ZBTB16 and DBA double-staining. After 3 days of culture (D), ZBTB16 expression by DBA-positive cells is lost from most of the cells (arrow) or shows perinuclear or cytoplasmic localization (arrowhead). However, some DBA-positive cells show nuclear localization of ZBTB16 (asterisk). At 7 days of culture (I), most of the DBA-positive cells have lost ZBTA16 staining (arrow) and if present, it is localized to the cytoplasm (arrowhead). E and J) SSEA-1 and DBA coimmunolocalization. At 3 days of culture (E), germ cells that were either weakly positive or negative for DBA binding and that had begun to form three-dimensional colonies show positive staining for SSEA-1 (arrow). An SSEA-1-expressing colony at 7 days of culture (J) exhibits markedly low DBA binding. Some cells appear yellow due to the colocalization of DBA and SSEA-1 (arrowhead). A'–J') Cells stained with Hoechst 33342. Bar = 50 µm.

ZBTB16 expression was lost from most of the DBA-positive cells at 3 days of culture (Fig. 5D). However, a few DBA-positive cells showed nuclear, perinuclear, and cytoplasmic expression of ZBTB16 (Fig. 5D). In the DBA-positive three-dimensional colonies at 7 days of culture, ZBTB16 expression was very weak and mostly localized to the cytoplasm (Fig. 5I). After 3 days of culture, SSEA-1 expression was detected in germ cells that showed weak or no DBA binding and that had begun to form three-dimensional colonies (Fig. 5E). Similarly, after 7 days of culture, SSEA-1 expression was observed in only a few of the three-dimensional colonies that had lost DBA binding (Fig. 5J).

DISCUSSION

Histochemistry

Lectin DBA binds to gonocytes in the neonatal pig testis. Lectin DBA and the monoclonal antibodies against anti-SSEA-1 and EMA-1 have previously been found to bind to the surfaces of large, round porcine PGCs [33]. It is likely that this affinity is maintained in gonocytes and primitive spermatogonia. SSEA-1 expression was not detected in DBA-positive germ cells and was present only in weakly positive or DBA-negative germ cells in 2- wk-old and 3-wk-old testes. In the sections of 5-mo-old testes, SSEA-1-expressing cells were not found. This suggests that SSEA-1 expression disappears in gonocytes and reappears when the gonocytes further differentiate. Since SSEA-1 expression was not seen in spermatogonia, it is likely that gonocytes go through some intermittent cell stage(s) before differentiating into spermatogonia. The developmental stage at which SSEA-1 expression reappears is unclear, as little information is available regarding the development of germ cells in neonatal pig testis.

The fact that the DBA binding in the germ cells of the neonatal porcine testis was variable indicates the presence of germ cells at different developmental stages, with strongly binding ones being more primitive than the weakly binding ones. This also indicates that the unstained germ cells are in more advanced stages. Germ cells also lost DBA affinity with age, providing further evidence that DBA affinity is restricted to germ cells of a primitive nature, such as gonocytes and primitive spermatogonia. As the primitive cells differentiated, DBA staining was lost, which explains why the spermatogonia showed no affinity at all. Similarly, DBA does not bind to spermatogonia in postpubertal boar testes [34]. UCHL1 expression was restricted to germ cells, as reported previously [32]. The average number of UCHL1-expressing cells was not different from the number of DBA-binding cells until 1 wk of age. However, the number of DBA-positive cells started to decline significantly at 2 wk of age, while the number of UCHL1-positive cells remained constant until 3 wk of age. Double-immunostaining with UCHL1 and DBA supported this finding, and further confirmed that DBA binding was indeed restricted to germ cells. These findings suggest that UCHL1 is expressed by both primitive germ cells and advanced-stage germ cells in the testis, while DBA binding is restricted to germ cells of a primitive nature in the neonatal testis, and clearly indicate that up to 1 wk of age, the neonatal pig testis contains a significant number of primitive germ cells.

KIT, which is a transmembrane tyrosine-kinase receptor for stem cell factor (SCF), has been reported as a marker for type A spermatogonia in pig testes [28]. In the present study, we found that KIT expression was weak and inconsistent and that KIT was expressed by germ cells in the pig testes of all the age groups studied. KIT was detected in DBA-positive and DBA-negative germ cells in the neonatal pig testis. Few of the DBA-positive cells were also negative for KIT expression. KIT protein expression has been reported in the gonocytes of the rat testis [37] and in differentiated spermatogonia [29, 30]. However, we conclude that KIT is expressed in early germ cells in the neonatal pig testis.

In each age group studied, the ZBTB16 expression profile and DBA-binding pattern in the pig testis were similar. That is, DBA binding and ZBTB16 expression were localized to the same germ cells and the number of these cells decreased progressively with age. ZBTB16 is a transcriptional repressor that is reported to be expressed only in the gonocytes and SSCs of the mouse testis [38, 39]. Similar to our finding, it has been shown that the ZBTB16-positive cells in the seminiferous tubules of the mouse testis become progressively sparser with age [39]. This finding strongly suggests that DBA mainly has affinity for quiescent and nondifferentiating cells, such as progenitor-type stem cells. However, it is unclear that at what stage DBA loses its affinity for germ cells in the neonatal pig testis. In 5-mo-old pig testes, DBA did not bind to somatic cells, and the rare DBA-positive cells could be primitive or progenitor-type spermatogonia, as indicated by their location at the basement membranes of seminiferous tubules. Similarly, ZBTB16-expressing cells were scarce and were located in the basement membranes of the seminiferous tubules. Since the number of cells that were both ZBTB16-positive and DBA-positive cells was low and DBA binding in the germ cells was very weak in 5-mo-old testes, we could not confirm that the DBA-positive cells were indeed ZBTB16-positive. Our finding differs from that in a previous report [22], in which DBA was found to bind specifically to gonocytes and spermatogonia in bull testes during the first 30 wk after birth. This discrepancy may be due to the species difference. In postpubertal boar testis, DBA binds weakly to Sertoli cells at the apical cytoplasm [34]. However, in the present study, DBA affinity was not observed in Sertoli cells in any of the age groups studied.

A previous report [27] used visual identification and markers for somatic cells, such as vimentin, to ensure the purity of the germ cells from 3-wk-old pig testes. In the present study, we found that vimentin was occasionally expressed by germ cells in the testes of neonatal pigs. A similar observation was made for the prepubertal sheep testis, in which vimentin was present in the cytoplasm at prespermatogonia-I and disappeared when the cells changed to prespermatogonia-II [40]. Therefore, vimentin could not be used to identify Sertoli cells in cultures of porcine gonocytes. GATA4 has been reported as a specific marker for porcine Sertoli cells [41]. We detected GATA4 expression also in interstitial cells. However, this could be a characteristic of the breed we used, which was different from that used in the earlier study. Nevertheless, GATA4 and {alpha}-smooth muscle actin expression was undetected in the germ cells of neonatal testes, and this could be used to distinguish somatic cells from germ cells in culture.

Although we detected weak AP activity in the testicular germ cells of the neonatal pig testis, the numbers of AP-positive germ cells were significantly lower in testicular samples taken at 2 wk and 3 wk of age. The decrease in AP-positive germ cells was correlated with a significant decrease in germ cells that were strongly positive for DBA, which suggests that the AP activity was associated with primitive germ cells that were strongly positive for DBA. Previously, AP staining has been used as a marker for undifferentiated PGCs isolated from pig embryos at 25 days of pregnancy [42, 43]. Since the AP staining of germ cells was quite weak and was not specific for germ cells in the neonatal pig testis, it could not be used as a marker for germ cells.

Gonocyte Purification

Two-step enzymatic digestion of the testicular tissue followed by filtration was not sufficient for purifying gonocytes from neonatal pig testis. Somatic cells, such as Sertoli cells and myoid cells, need to be removed to obtain a pure gonocyte population. We used Percoll discontinuous density gradient to purify the testicular cell population, as described for the rat [36]. In the present study, the Percoll fraction that had the richest gonocyte population (70% of the cells were DBA-positive) was obtained at the interface between the 50% and 60% Percoll layers. However, in another report [27], most of the germ cells were found at the interface between the 20% and 40% Percoll layers. This inconsistency may be due to the age of the testes used in our study. We also found that differential plating was not useful for the purification of gonocytes, as most of the gonocytes attached to the culture dish within 4 h of seeding, possibly because the high serum content of the medium used for differential plating facilitated the attachment of gonocytes. The tendency of porcine gonocytes to grow as adherent cells on a culture dish is another possibility. A similar characteristic had been observed previously for rat gonocytes [5]. In the present study, most of the floating cells examined were negative or weakly positive for DBA and more than 90% of these cells were positive for UCHL1. Thus, the floating cells appear to be more advanced-stage germ cells, such as primitive spermatogonia. These data are in concordance with previous reports that porcine spermatogonia could be purified by differential plating [27, 28]. Recently, it has been found that a small population of attached spermatogonia has better viability than that in suspension after differential plating [32]. This may be due to the time required for differential plating and the age of the testicular samples used. Spermatogonia could only be seen at two months of age in the case of pigs [3], and in the present study, testes from young piglets (less than 7 days old) were used for the isolation of germ cells. The predominant germ cells in testis at this age are gonocytes and primitive spermatogonia.

Gonocyte Cultivation and Proliferation

Purified gonocytes were cultured in DMEM/F-12 that contained 10% FBS and other supplements but without any supplemental growth factors. Due to the high serum content of the culture medium, somatic cell proliferation was also significant by 7 days of culture. However, it was still possible to distinguish gonocytes from contaminating somatic cells based on DBA binding. Attached gonocytes formed flat colonies that showed epithelial-like morphology during the first 3–5 days of culture. The flat colonies grew in size, suggesting that they were composed of actively proliferating cells and not merely aggregations of germ cells. Intriguingly, the cells morphologically resembled differentiated PGC colonies described previously [42]. Furthermore, these flat colonies transformed to three-dimensional colonies by Days 4–5, and became compacted by Day 7 of culture. DBA binding was observed in the three-dimensional colonies, suggesting the presence of germ cells. Isolated germ cells remained viable during culture and could proliferate, as determined by DBA labeling and BrdU incorporation. The high-level viability of the DBA-positive cells can be attributed to the presence of FBS, which contains numerous undefined factors. As the isolated gonocytes were also contaminated with somatic cells, we could not rule out the possibility that somatic cells affected the proliferation of germ cells.

Characterization of Cultured Cells

Isolated gonocytes in culture were characterized using double-immunostaining with DBA and markers specific for somatic cells (GATA4 and {alpha}-smooth muscle actin) and germ cells (KIT, ZBTB16, and SSEA-1). DBA-expressing germ cells were negative for the Sertoli cell-specific marker GATA4. DBA is known to bind weakly to the apical cytoplasm of Sertoli cells in postpubertal boar testis [34]. In the present study, GATA4 was never expressed by DBA-positive cells and DBA binding was specific for germ cells. The {alpha}-smooth muscle actin marked specifically the peritubular myoid cells. These findings strongly suggest that DBA can be used as a marker for gonocytes for the first 7 days of culture. In contrast, UCHL1 is expressed by Sertoli cells and spermatogonia during long-term culture [32]. KIT expression was variable in cells at 3 days of culture. At 7 days of culture, the DBA-negative germ cells in the colonies also expressed KIT. These cells may be more differentiated than the DBA-positive cells. However, it is unclear whether these cells are differentiated spermatogonia, since type A spermatogonia in the pig are known to express KIT [28]. ZBTB16 expression progressively decreased in cultured DBA-positive germ cells, and in most cells after 3 days of culture, it was lost or localized to either the perinuclear region or cytoplasm. After 7 days of culture, ZBTB16 expression was no longer detected in most of the DBA-positive cells in the colony. Even if it was present, it was weakly expressed and restricted to the cytoplasm of DBA-positive germ cells. ZBTB16 has been reported to be a transcriptional repressor of cyclin A and it suppresses cell growth by inhibiting entry or progression to the S-phase of the cell cycle [44, 45]. Gene expression controlled by transcriptional regulators occurs in the nucleus, and nuclear export of these factors results in loss of this regulation. In the present study, we found that DBA-positive germ cells that also expressed ZBTB16 in the sections either lost ZBTB16 expression or showed cytoplasmic or perinuclear localization of ZBTB16 in cultured cells. This finding, that cultured cells lose ZBTB16 expression, correlates with the results of proliferation assays in which around 50% of the DBA-positive cells progress to the S-phase. This provides further evidence that DBA-positive germ cells can proliferate in vitro. SSEA-1 expression was detected in the focal colonies of germ cells in which DBA affinity was lost or weak. The SSEA-1-positive cells in these foci appeared closely aggregated and had begun to form three-dimensional colonies. After 7 days of culture, compact colonies with DBA-negative germ cells were positive for SSEA-1 expression. SSEA-1 is known to be expressed in mouse embryos and ES cells [46] and in mouse germline stem cells isolated from the neonatal testis [47]. This finding supports our immunohistochemical finding that weakly DBA-positive or DBA-negative germ cells express SSEA-1. This also indicates that DBA-positive cells can differentiate into the next germ cell stage, in which they may acquire germline stem cell potential. However, further investigations of these issues are needed.

In conclusion, the lectin DBA has a specific affinity for germ cells in the neonatal pig testis, which declines progressively with age, indicating that DBA affinity is specific for primitive germ cells and/or progenitor-type stem cells. By exploiting this property, we were able to isolate and culture gonocytes. The gonocytes could survive, proliferate, and form colonies in culture for up to 1 wk. The development of an in vitro method for long-term culturing of gonocytes would provide insights into gonocyte development and spermatogenesis. Once such a method is achieved, the gonocytes could be used to generate transgenic pigs, as well as knockout animals for therapeutic and research purposes.


Figure 102
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  		FIG. 1. Continued.

ACKNOWLEDGMENTS

We thank Kazuo Kishimoto and Sachiko Kishimoto for providing testis samples and Dr. Deepali Garg for critical reading of the manuscript.

FOOTNOTES

1Supported in part by the grant (17658123) from the Ministry of Education, Science and Culture (to H.I.). Back

Correspondence: 2FAX: 81 75 753 6329; e-mail: imai{at}kais.kyoto-u.ac.jp

Received: 29 August 2006.

First decision: 16 October 2006.

Accepted: 20 March 2007.

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