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research-article |
Department of Food and Animal Biotechnology,3 Seoul National University, Seoul 151-921, Korea
Avicore Biotechnology Institute Inc.,4 Gunpo, Gyeonggi-Do 435-824, Korea
Research Institute for Agriculture and Life Sciences,5 Seoul National University, Seoul 151-921, Korea
ABSTRACT
We recently succeeded in inducing germline transmission by transferring chicken testicular cells into heterologous testes. This study was designed subsequently to identify pluripotent cells in the testicular cells, which would induce the germline transmission. Testicular cells retrieved from juvenile (4-wk-old) or adult (24-wk-old) White Leghorn (WL) chickens were stained with germ cell-specific markers anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, and anti-ITGB1 antibodies; 2C9; and lectin-Solanum tuberosum agglutinin (STA). The percentages of the cells that were positive for each marker were within the ranges of 0.33%–0.44% and 0.029%–0.072% of the total testicular cell population in the juvenile and adult, respectively, and significant (P < 0.0002) differences were detected between the ages. When 1 x 106 testicular cells were cultured in Dulbecco minimum essential medium-based medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF2), and/or insulinlike growth factor 1 (IGF1), colony formation was detected only in LIF++FGF2-containing or LIF+FGF2+IGF1-containing medium during primary culture, and the supplementation of LIF+FGF2+IGF1 was the most efficient for maintaining the colony-forming cells through subculture. The established cells retrieved at the end of the primary culture or the 20th subpassage were positive for chicken germ cell-specific periodic acid-Schiff (PAS), EMA1, 2C9, SSEA1, SSEA3, SSEA4, ITGA6, and ITGB1; and lectin-STA markers (evaluated after 11th subpassage). Double staining of lectin-STA with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, and anti-ITGB1 also was possible. They differentiated spontaneously into embryoid bodies after being cultured in LIF-free medium. We conclude that germline stem cell-like cells are present in chicken testicular cells retrieved from both juvenile and adult testes, which can be identified with the specific markers for primordial germ cells or embryonic germ cells.
chicken, culture, cytokines, developmental biology, gamete biology, germ cell-specific marker, germline stem cell, spermatogenesis, testis
Since the last decade, we have made great efforts to develop an embryo-mediated germline transmission system using primordial germ cells or embryonic germ cells in chickens [1, 2], and we have succeeded subsequently in producin germline chimera [3–5]. To further improve germline transmission efficiency and to develop a case-specific germline chimera production system, we directed our attention toward developing an alternative germline transmission system. Testis-mediated germline transmission by direct transfer of heterologous testicular cells into juvenile or adult testes then was suggested. Recently, we produced the progenies with different coat colors after the transfer [6], which implied the induction of heterogenic spermatogenesis after the transplantation. In other words, these results suggested the presence of cells being able to induce heterogenic spermatogenesis in the transplanted testicular cells. This hypothesis was confirmed by our finding that some testicular cells were positive for several chicken germ cell-specific markers and that the marker-positive cells have an ability to form colonies or colony-like cell mass during primary culture.
Consequently, we designed a series of experiments to identify putative pluripotent cells in testicular cells and to culture and characterize the identified cells. We first undertook an experiment to count the number of testicular cells positively stained with germ cell-specific markers. We selected an optimal culture system to maintain the identified cells in vitro and characterized the established (colony-forming) cells with chicken germ cell-specific markers reported previously [2].
All procedures for animal management, reproduction, and surgery were performed in accordance with the standard operation protocols of Seoul National University, Seoul, Korea. Appropriate management of experimental samples and quality control of the laboratory facility and equipment also were conducted. All chickens were maintained at the University Animal Farm, College of Agriculture and Life Sciences, Seoul National University.
In experiment 1, testicular cells retrieved from juvenile (4-wk-old) or adult (24-wk-old) testes of White Leghorn (WL) chickens were stained with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, anti-ITGB1, 2C9 antibodies, and lectin-Solanum tuberosum agglutinin (STA). The number of positive cells for each marker was counted, and percentile value per total cell number was evaluated. On the other hand, immunohistochemical assay was undertaken subsequently to detect SSEA1- and EMA1-positive cells in the testicular tissue. In experiment 2, 1 x 106 testicular cells retrieved from the testes of juvenile WL chickens were cultured in modified Dulbecco minimal essential medium (DMEM), to which leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF2), and/or insulinlike growth factor 1 (IGF1) were added. Cell proliferation and colony formation were monitored during primary culture, and colony-forming cells were subcultured with gonadal stroma cell feeder in different culture media. In experiment 3, isolated cells retrieved at seeding or colony-forming cells retrieved at the end of the primary culture or after subculturing 20 times (cultured for 155 days) were characterized with the chicken germ cell-specific markers alkaline phosphatase (AP); periodic acid-Schiff (PAS; evaluated after subculturing 3 times); anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, anti-ITGB1, and 2C9 antibodies; lectins (tested after subculturing 11 times): lectin-STA, lectin-wheat germ agglutinin (WGA), and lectin-concanavalin A (ConA). Double immunostaining with lectin-STA and anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, or anti-ITGB1 antibodies also was performed. In experiment 4, the colony-forming cells were cultured in LIF-free medium, and the embryoid bodies that formed 5–7 days after the culture were analyzed by three germ layer-specific markers (desmin and muscle actin for mesoderm, 
-1-fetoprotein (AFP) for endoderm and neural adhesion molecule, S100 and TROMA1 for ectoderm).
Retrieval of Testicular Tissue and Spermatogonia
The retrieved tunica albuginea and connective tissue of the testes were removed mechanically, and the collected testes were trimmed under a stereomicroscope with a surgical blade and forceps before enzymatic digestion. After being trimmed, the decapsulated testes were treated with Hanks buffer salt solution containing 0.25% trypsin (Gibco Invitrogen, Grand Island, NY) and 1 mg/ml collagenase IV (Sigma, St. Louis, MO) for 25 min in a shaking water bath, and the dispersed cells were filtered through 70-µm nylon cell strainer (Falcon, Franklin Lakes, NJ) [7, 8].
In Vitro Culture of Testicular Cells
Approximately 1 x 106 dissociated cells were placed in a 100-mm culture dish with modified DMEM (Gibco Invitrogen) containing 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT), 2% (v/v) chicken serum (Gibco Invitrogen), 1x antibiotic-antimycotics (Gibco Invitrogen), 10 mM nonessential amino acids (Gibco Invitrogen), 10 mM HEPES buffer (Gibco Invitrogen), and 0.55 mM ß-mercaptoethanol (Gibco Invitrogen). Subsequently, 10 ng/ml LIF (L5283; Sigma), 10 ng/ml FGF2 (F3133; Sigma), and/or 100 ng/ml IGF1 (I3769; Sigma) were added to the base medium according to the experimental design. The seeded cells were cultured in an incubator at 37°C and 5% CO2 in an air atmosphere with 60%–70% relative humidity. The proliferation of cultured cells was assessed using a cell proliferation assay kit (Chemicon, Temecula, CA) at the end of the culture (on Day 15 of the culture), and changes in cell morphology also were monitored under an inverted microscope. Subculture of colony-forming cells with feeder cells in the optimal culture medium was conducted at intervals of 6 to 10 days.
Culture of Mouse Embryonic Stem Cells
Mouse embryonic stem (ES) cells (E14 cell line of 129 strain; ATCC, Manassas, VA) were used as positive controls for the AP assay. To culture the ES cells, a feeder cell layer was established by growing 2 x 105 STO cells in each well of a 6-well plate. When the cells reached 80% confluence, they were inactivated for 2 h with 10 µg/ml mitomycin-C (Sigma). Mouse ES cells were thawed, seeded (2 x 105/well) onto the STO feeder cells, and cultured with DMEM supplemented with 15% (v/v) FBS, 1.7 mM L-glutamine (Gibco Invitrogen), 0.1 mM ß-mercaptoethanol, 1x antibiotic-antimycotic supplement, and 2 ng/ml LIF. For immunostaining, 1 x 104 ES cells were seeded onto 1 well of a 24-well plate containing an inactivated feeder layer of STO cells.
Colony-forming cells were fixed with 3.7% buffered paraformaldehyde for 10 min and rinsed with PBS three times. The cells then were immersed in periodic acid solution (Sigma) for 5 min and were subsequently treated in Schiff solution (Sigma) for 5–10 min. All procedures were performed at room temperature, and stained cells were observed under an inverted microscope (TE2000-U; Nikon, Tokyo, Japan). For AP staining, fixed cells were immersed in filtered AP staining solution (2 mg naphtol AS-MX phosphate, 200 µL N,N-dimethylformamide, 9.8 mL of 0.1 M Tris [pH 8.2], and 10 mg Fast Red TR salt) for 30 min and then rinsed three times with PBS.
Reagents for Immunocytochemical and Immunohistochemical Analysis
Mouse anti-SSEA1 monoclonal immunoglobulin M (IgM) antibody (MC-480), mouse anti-SSEA3 monoclonal IgM antibody (MC-631), mouse anti-SSEA4 monoclonal IgG antibody (MC-813–70), and mouse anti-EMA1 monoclonal IgM antibody were obtained from the Developmental Studies Hybridoma Bank (DSHB; University of Iowa, Iowa City, IA). Mouse anti-chicken ITGB1 monoclonal IgG antibody (Sigma) and mouse anti-chicken ITGA6 monoclonal IgG antibody (Chemicon) were tested. A monoclonal 2C9 antibody (IgM chi-light chain) [9] was provided by T. Nishida (Nihon University, Japan). For the detection of the primary antibodies, either rhodamine (TRITC)-conjugated goat anti-mouse IgG and IgM (H+L) antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) or AP-conjugated antibodies and a detection kit (DAKO Universal LSAB2 kit; DAKO Cytomation, Carpinteria, CA) were used. Three types of fluorescein isothiocyanate (FITC)-conjugated lectins—STA, WGA, and ConA agglutin—were used (Sigma).
Hematoxylin and Eosin Staining and Immunohistochemical Analysis with Anti-SSEA1 and Anti-EMA1 Antibodies
Juvenile and adult testes were fixed in 3.7% buffered paraformaldehyde and were embedded subsequently into the paraffin block. The paraffin-embedded testicular tissue was sectioned with 6 µm in thickness and was hydrated after being deparaffinized. The deparaffinized tissue was incubated in hematoxylin (Sigma) for 2 min and incubated subsequently in eosin (Sigma) for 20 min. The deparaffinized tissues also were heated in a microwave for 10 min after immersing in a sodium citrate buffer solution at pH 6.0, and the heated epitope was reacted subsequently with anti-SSEA1 or anti-EMA1 antibody. The reaction was blocked by incubation in 3% H2O2 in blocking buffer (3% BSA/0.1% Tween-20 in PBS) for 10 min. A DAKO LSAB2 system was employed for final detection of antibody reaction according to the manufacturer's instructions.
Immunocytochemical Analysis with a Single Antibody or Lectin
The AP detection system was used for the immunocytochemical analyses using anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, anti-ITGB1, and 2C9 antibodies. Briefly, testicular cells, colony-forming cells, and/or embryonic germ (EG) cells were fixed in 3.7% paraformaldehyde solution for 10 min. To minimize nonspecific binding, the fixed cells were treated for 30 min with 5% (v/v) goat serum before immunostaining. The optimal concentration of each antibody was selected based on the results of preliminary experiments (40 µg/ml for anti-SSEA1 and anti-EMA1 antibodies and 20 µg/ml for all others).
After treatment for 1 h with the primary antibodies, the cells were sequentially reacted for 10 min each with biotinylated anti-mouse immunoglobulins, AP-conjugated streptavidin, and the substrate chromogen. To avoid interference by the potential endogenous activity of AP, the cells provided for each staining were treated with 0.02 M levamisole.
The fixed cells also were incubated with FITC-conjugated STA, WGA, or ConA for 1 h and then washed three times in PBS. The concentrations of the conjugated lectins without nonspecific binding were within the range of 20–60 µg/ml based on the results of preliminary study.
Colony-forming cells were collected using a trypsin and EDTA solution (0.05% trypsin in 0.5 mM EDTA) and were fixed subsequently in 4% paraformaldehyde. After storage at 4°C for at least 24 h, 2 x 105 fixed cells were stained for 15 min at room temperature with a diluted solution containing FITC-conjugated STA, WGA, ConA, and BSA as a negative control. The staining intensity of the cells treated was measured using a fluorescence-activated cell sorting scan (EPICS XL; Beckman Coulter Inc., Fullerton, CA) and further compared with those of gonadal stroma cell feeder and colony-forming cells of a mixed population.
Densitometric Analysis for Quantification
A densitometer (LAS-3000; Fujifilm, Stanford, CA) for measuring image scanning and the MultiGauge software program (version 2.0; Fujifilm) for analyzing signal intensity were used for quantification of the reaction with each marker reagent. Photographs were taken after immunocytochemical staining, and five different areas with images of both colonized cells and background (feeder layer) were randomly selected in each photograph. The colony and the background in each area occasionally had different staining intensities with tested substrates. The difference in the intensity of staining between the colony-forming cells and the background feeder cells then was measured, and the average of the five areas was taken as one replicate. The feasibility of this quantification method was confirmed by the preliminary results. The densitometric difference between the colony and background was measured in all colonies of one picture, and different aspects were not detected compared with the results obtained from the method suggested. More than three replicates were made for the quantification of differential staining and submitted for statistical analysis.
Double Immunofluorescent Staining
Double staining of colony-forming cells was performed with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, or anti-ITGB1 antibodies in combination with lectin-STA. After fixation, the cells were incubated with the primary antibodies for one of the five candidate markers and were treated subsequently with rhodamine-labeled anti-mouse IgG or anti-mouse IgM secondary antibodies. After washing several times with PBS, colony-forming cells were treated with FITC-conjugated lectin-STA for 1 h and DAPI (4'-6'-diamidino-2-phenylindole) for 1 min. The stained cells were observed under an inverted fluorescence microscope (TE-300; Nikon).
In Vitro Differentiation of Chicken Germline Stem Cell-Like Cells
To examine whether the chicken colony-forming germline stem cell-like cells could form embryoid bodies, colonies (subpassage P20) were gently agitated and harvested by centrifugation. Colony-forming cells were resuspended in culture media without LIF and placed in a nonadhesive bacterial Petri dish. The medium was changed every other day, and morphology was moniotored daily for embryoid body formation. After 7 days, floating masses of cells were collected for immunohistochemical analysis. Antibodies for mesoderm (actin [DAKO] and desmin [Santa Cruz Biotechnology Inc., Santa Cruz, CA]), endoderm (AFP [DAKO]), and ectoderm (S100, neural adhesion molecule [DAKO], TROMA1 [DSHB]) were used [10, 11].
The PROC-GLM model of the SAS program, which employs ANOVA and a least square method, was employed for statistical analysis of the numerical data. A significant difference was determined when P value was less than 0.05. In the first set of experiments, the number of marker-positive cells was compared between juvenile and adult testes. In experiment 3, the specific immunocytochemical reactivities on the markers were determined by analyzing the statistical differences in the densitometric levels between the cells stained and the feeder cell background, whereas both densitometric analysis and flow cytometric analysis were employed for analyzing lectin reactivities.
Experiment 1: Detection of the Cells Positively Stained with Germ Cell-Specific Markers
Among the testicular cells retrieved from the juvenile and adult testes, 0.33%–0.44% and 0.029%–0.072% were positive for germ cell-specific markers, respectively (Table 1). Overall, the number of positive cells for each germ cell-specific marker was 6.3 times higher in the juvenile than in the adult (P < 0.0002). As shown in Figure 1, seminiferous tubules in juvenile testes did not completely develop compared with those in adult testes. However, there were SSEA1- and EMA1-positive cells in the tubules in both testes. The layers positive for each marker in seminiferous tubules were wider (more dense) in the adult than in the juvenile testes.
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Experiment 2: Optimization of a Culture System
Regardless of culture media, testicular stoma cells proliferated rapidly during the primary culture and formed a confluent monolayer within 10–15 days after seeding. However, several of the testicular cells proliferated to form colonylike cell mass on the testicular stroma cell monolayer during primary culture and, as shown in Figure 2, the cells that formed colonylike cell mass were found only after culture in DMEM supplemented with LIF+FGF2 or LIF+FGF2+IGF1. Different levels of cell proliferation were detected among medium supplements (Table 2); the cells cultured in DMEM supplemented with LIF+FGF2+IGF1 had higher levels of proliferation (1.771–3.059) than the cells cultured in DMEM supplemented with LIF+FGF2 or in DMEM without supplements (0.974–1.755). Colony formation after the supplementation of LIF+FGF2 or LIF+FGF2+IGF1 was observed mainly in the later stages (Days 10–13) of the 15-day culture. Early cell colonies adhered firmly to the stroma cell monolayer, and no signs of degeneration were detected.
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Colony-forming cells established in DMEM with LIF+bFGF or DMEM with LIF+bFGF+IGF1 during primary culture were subcultured on gonadal stroma feeder cells. Colony formation and expansion during the second subculture were maximal when the cells were cultured in DMEM with LIF+FGF2+IGF1 for both culture stages. Two types of cells developed in the subsequent subculture. Some of the cells formed well-delineated colonies, whereas others consisted of single cells. Under the optimal conditions (use of DMEM with LIF+FGF2+IGF1 and culture with feeder cells), colony-forming cells were subcultured at intervals of 6–10 days and maintained for up to 22 passages.
Experiment 3: Characterization of Isolating and Colony-Forming Cells
In the instances of AP staining, there was a different reactivity between mouse ES cells and chicken EG cells (positive for the mouse and negative for the chicken). As shown in Figure 3, colony-forming cells did not react to AP, which was similar to chicken EG cells. Regardless of the cell type or source, colony-forming cells were positive for PAS (147.1 ± 8.3 vs. 101.7 ± 0.6 in primary, P = 0.0006; 156.2 ± 2.7 vs. 101.3 ± 0.8 in the third passage, P < 0.0001), anti-SSEA1 (161.0 ± 23.5 vs. 102.4 ± 1.8 in primary, P = 0.0118; 127.3 ± 3.1 vs. 101.2 ± 0.6 in the 20th subpassage, P = 0.0001), anti-SSEA3 (132.8 ± 14.6 vs. 102.4 ± 1.8 in primary, P = 0.0234; 146.9 ± 4.4 vs. 101.7 ± 1.5 in the 20th passage, P < 0.0001), anti-SSEA4 (148.0 ± 9.2 vs. 100.6 ± 0.3 in primary, P = 0.0010; 128.3 ± 7.3 vs. 101.7 ± 0.4 in the 20th passage, P = 0.026), anti-EMA1 (122.8 ± 7.2 vs. 101.0 ± 0.5 in primary, P = 0.0001; 137.2 ± 6.6 vs. 102.0 ± 0.8 in the 20th subpassage, P < 0.0001), anti-ITGA6 (167.6 ± 3.8 vs. 102.0 ± 0.3 in primary, P < 0.0001; 125.3 ± 3.5 vs. 100.5 ± 0.8 in the 20th subpassage, P = 0.0003), anti-ITGB1 (149.3 ± 12.7 vs. 101.0 ± 0.8 in primary, P = 0.0034; 132.5 ± 4.0 vs. 101.3 ± 1.1 in the 20th subpassage, P = 0.0002), and 2C9 (130.4 ± 4.5 vs. 100.8 ± 0.3 in primary, P < 0.0001; 136.9 ± 3.1 vs. 100.4 ± 0.3 in the 20th subpassage, P < 0.0001; Figs. 2, 4–6 and Table 3). The cells also were positive for lectin-STA staining, which was confirmed by both densitometry (236.2 ± 43.6 vs. 101.5 ± 0.7 in primary, P = 0.0059; 313.1 ± 31.0 vs. 102.0 ± 2.2 in the 11th passage, P = 0.0003) and flow cytometric peaks (Figs. 7 and 8 and Table 3).
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Nonspecific binding with the colony-forming cells and gonadal stroma feeder cells also was detected after lectin-ConA (103.5 ± 7.3 vs. 102.6 ± 0.5 in primary, P = 0.8809; 103.8 ± 1.7 vs. 102.8 ± 1.6 in the 11th subpassage, P = 0.3739) or lectin-WGA (105.0 ± 4.1 vs. 108.1 ± 9.5 in primary, P = 0.6792; 104.9 ± 1.4 vs. 102.0 ± 2.2 in the 11th subpassage, P = 0.0907) treatment (Figs. 7 and 8). Colony-forming cells were characterized by double immunostaining with FITC-conjugated STA and anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, or anti-ITGB1 antibodies. No competitive binding was detected in these cases (Fig. 9).
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Experiment 4: Assessment of Colony-Forming Cells to Spontaneously Differentiate In Vitro
In LIF-free media, embryoid bodies were formed successfully for 5–7 days. The embryoid bodies formed from colony-forming cells were positive for the specific markers for three germinal lineages: desmin and muscle actin for mesoderm, AFP for endoderm, and neural adhesion molecule, S100, and TROMA1 for ectoderm (Fig. 10).
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Through our study we demonstrated the presence of positive cells for chicken germ cell-specific markers in testicular cells retrieved from both juvenile and adult testes. The positive cells proliferated to form colonies on testicular stroma cells during primary culture, and the colony-forming cells could be subcultured in DMEM supplemented with LIF, FGF2, and IGF1. The established cells had an activity to differentiate in vitro in a spontaneous manner, as well as the reactivity for chicken germ cell-specific markers. Considering our previous study indicating that the testicular cells containing the marker-positive cells induced germline transmission, we suppose there are germline stem cell-like cells in testicular cells, which may have similar properties to chicken EG cells.
In vivo, primordial germ cells differentiate into gonocytes after migrating into embryonic gonads, and the gonocytes subsequently transformed into spermatogonia. It has been reported in the mouse that germline stem cells are identified from testicular cells; of those, stem cell activities are confirmed later [12, 13]. Our previous report on inducing germline transmission by heterologous testicular cells may demonstrate the presence of putative germline stem cells in the chicken [6] as well as in the mouse testicular cells [13, 14]. This hypothesis is confirmed clearly in our present results: chicken germ cell marker-positive cells are mixed in testicular cells and form homogenous colonies by subculture. Apparently, germline cells, including migrated primordial germ cells, gonocytes, and spermatozoa, involve the germline transmission, but we need to further clarify which cells are responsible for the germline transmission. In other words, the testicular cells containing germline stem cell-like cells could be used as inducers of heterologous germline transmission after being transferred into the testes of recipients.
In experiment 1, we estimated the number of positive cells for germ cell-specific markers in the testicular cells. The number of marker-positive cells was more than six times greater in the juvenile than in the adult testicular cells. This probably is due to close contact between marker-positive cells, as well as basal layer or testicular mesenchyme in adult testes compared with juvenile testes. The positive cells may already be programmed or may enter spermatogenesis with developing physical and biological communication with Sertoli cells, which prevents mechanical isolation. Otherwise, the undeveloped seminiferous tubules in the juvenile testes may have more undifferentiated cells that have germ cell activity.
It was reported that the percentage of germline stem cells in the chicken adult testes was 1.9 times higher than that in the mouse (approximately 0.03% of the total testicular cells) [15, 16]. On the other hand, we retrieved 4.07 x 104 germ cell-like cells of the 1.13 x 107 mixed population of testicular cells. Considering that only an average of 790 primordial germ cells are routinely retrieved from one embryo having a mean of 9.88 x 104 gonadal cells, juvenile or adult chicken testes are an excellent reservoir of cells that may have germ cell activity. These results confirm the feasibility of a testis-mediated germline transmission system using testicular cells in the chicken [6].
To date, the culturing of germline stem cells has not been successfully undertaken. In mammalian species, successful culture of germline stem cells has been reported only in the mouse [13, 14] and in the bovine with great limitations [17]. In this study, the use of the medium supplemented with LIF, FGF2, and IGF1 allowed some testicular cells (presumably having germ cell activity) to form homogenous colonies during primary culture, and the colony-forming cells were subsequently subcultured on gonadal stroma cell feeder. Our finding that the culture medium used in this study has similar composition with the medium used for primordial germ cell (PGC) culture, except for the supplementation of stem cell factor, indirectly supports the fact that there are germ cell-like cells in the testicular cells. LIF and FGF2 are known to promote the maintenance or proliferation of PGCs without inducing differentiation in vitro [18, 19], whereas IGF1 is absolutely required for the in vitro survival and proliferation of chicken primordial germ cells [20]. Considering the results of immunostaining with germ cell-specific markers and spontaneous differentiation, we conclude these are germline stem cell-like cells.
PAS reacting with glycogen in the cytoplasm has frequently been used for detecting chicken PGCs and EG cells [1, 21]. Both isolated and colony-forming cells were positive for PAS staining (Fig. 2), and similar results also were obtained from bovine studies [17]. The SSEA family has been used for the characterization of pluripotent cells in several species. SSEA1 is strongly expressed in mouse ES cells [22, 23]. Mouse and human EG cells also are positive for anti-SSEA1 antibody staining. SSEA3 (Galß-globoside) and SSEA4 (sialyl-Gal-globoside) are epitopes localized on the cell surface that associate with globoseries glycolipid [24]. These epitopes are expressed in cleaving mouse embryos [24, 25]. SSEA3 and SSEA4 also bind to monkey and human ES cells and human EG cells, but not mouse ES and EG cells. Regarding chicken PGCs and EG cells, some chicken testicular cells were positive for anti-SSEA1, anti-SSEA3, and anti-SSEA4 antibody staining in this study, suggesting that properties of chicken germ cell-like cells mixed in the testicular cells are much closer to those of the human than those of the mouse.
ITGA6 and ITGB1, heterodimeric transmembrane mediators, are involved in cell attachment to extracellular matrix molecules. These molecules had a critical role in signal transduction between the inner and outer membrane. In spermatogenesis they are surface markers on the gonocyte and spermatogonia in the mouse. Specific antibodies for the integrins have been used routinely to isolate spermatogonial stem cells [26]. In the case of PGCs, ITGB1 plays a key role in the migration of mouse primordial germ cells into the embryonic gonads [27]. ITGA6 and ITGB1 were expressed and localized on the plasma membrane of primordial germ cells [28]. Our previous study [2] demonstrated that primordial germ cells are responsible mainly for the positive signal of the genital ridge on ITGA6 and ITGB1. From this viewpoint, the integrins may have crucial roles in spermatogenesis and cell-to-cell interactions with Sertoli cells during spermatogenesis.
Lectins are carbohydrate-binding proteins that interact with sugar residues included in glycoproteins located at other cell surfaces. Previous reports have shown that lectin-WGA, which has a specific affinity for N-acetyl-glucosamine, labeled spermatogonia or spermatocytes in the toad Bufo calamita [29], lizards [30], rats [31], and humans [32–34]. Lectin-STA is a chitotriose (GlcNAc3)-binding protein from potato tubers and contains about 50% carbohydrate, composed of arabinose and galactose. The carbohydrate-binding specificity of the lectin is similar in WGAs and STAs that recognize GlcNAc3, but WGA binds (GlcNAc)n and Neu5Ac. In addition, lectin-ConA, a globular protein isolated from the jack bean, has been considered an important probe for studying the structure of cell surfaces. Lectin-ConA staining was effective for detecting spermatogonia and spermatocyte in various species [29–37]. The results of this study might imply that only lectin-STA could be used for the detection of in vitro-cultured chicken pluripotent cells that could induce germline transmission. Our results on the positive reactivity of colony-forming cells to PAS further confirmed the availability of lectin-STA, because lectin-STA can be identified as a glycoprotein interacting with PAS.
Phosphate binding with active AP was accomplished by the interaction of 2-nitrosoacetophenone with AP-induced structural changes [38]. AP is a unique characteristic of mouse-human ES and EG cells [39–42]. However, chicken primordial germ cells and embryonic germ cells do not react with AP [1], and the same reactivity also was detected in some testicular cells in this study. These results suggest that chicken pluripotent cells have different channels to activate AP during cell differentiation, and other factors are involved in increasing the AP activity.
In conclusion, our results identifying germline stem cell-like cells in chicken testicular cells indirectly confirm the establishment of testis-mediated germline transmission using germline stem cell-like cells. A testicular cell culture system to isolate the stem cell-like cells apparently reinforces the infrastructure of avian transgenic biotechnology for germline chimera and transgenic bioreactors. Our newly developed system helps overcome the current limitation of the embryo-mediated germ line transmission method by improving the efficiency of germline transmission through securing a sufficient number of the transmission inducers.
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ACKNOWLEDGMENTS
We thank T. Nishida (Nihon University, Japan) for kindly providing the 2C9 antibodies.
FOOTNOTES
1Supported by a graduate fellowship from Brain Korea 21 project, Republic of Korea. 
Correspondence: 2 Jae Yong Han, Division of Animal Genetic Engineering, School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. FAX: 822 874 4811; e-mail: jaehan{at}snu.ac.kr
Received: 7 August 2006.
First decision: 10 September 2006.
Accepted: 5 October 2006.
REFERENCES
6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 1999; 96:5504–55096 and ß1 integrin subunits during oogenesis. Dev Biol 1998; 200:27–34[CrossRef][Medline]This article has been cited by other articles:
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  M. Naito, T. Minematsu, T. Harumi, and T. Kuwana Testicular and ovarian gonocytes from 20-day incubated chicken embryos contribute to germline lineage after transfer into bloodstream of recipient embryos Reproduction, October 1, 2007; 134(4): 577 - 584. [Abstract] [Full Text] [PDF]
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