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Research Article |
Department of Food and Animal Biotechnology,3 Seoul National University, Seoul 151-921, Korea
Bioresources Institute,4 EasyBio System, Chonan, Choongcheongnam-Do 330-822, Korea
Avicore Biotechnology Institute Inc.,5 Gyeonggi-Do 435-824, Korea
College of Bioresources Science,6 Dankuk University, Chonan, Choongcheongnam-Do 330-714, Korea
ABSTRACT
In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 x 107 cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 x 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.
chicken, chimera, gamete biology, germline, male reproductive tract, spermatogenesis, testicular cells, testis
INTRODUCTION
Because of their unique physiological characteristics and genetic attributes, birds are considered to be one of the most suitable organisms for developing transgenic bioreactors and experimental models [1–3]. To exploit this potential of birds, we endeavored to develop an effective chicken transgenic system for establishing embryo-mediated germline transmission methods [4–7] as the basis for transgenic manipulations. Germline chimeras yielding a high rate of germline transmission (up to 49.7%) [7] were produced by transfer of in vitro-cultured primordial germ cells (PGCs) retrieved from 5.5-day-old embryos into 2.5-day-old recipient embryos. However, these procedures are time-consuming and laborious. In addition, it has been difficult to retrieve sufficient numbers of PGCs that have retained pluripotency from developing embryos. The development of an alternative system of germline chimera production is therefore necessary, which will subsequently optimize the efficiency and feasibility of the chicken transgenic system.
In this study, we attempted to develop an alternative system of germline chimera production that operates via the testes rather than through developing embryos. This system consisted of isolation and in vitro culture of chicken testicular cells, transfer of in vitro-maintained cells into heterologous testes, production of germline chimeras, and confirmation of germline transmission for evaluating production of heterologous, functional spermatozoa. To ensure efficient germline chimera production, this system creates a local chimeric state in the testes by the direct transfer of heterologous testicular cells, a mixture of cells containing pluripotent cells, into the seminiferous tubules of recipients. Accordingly, the time-consuming germline chimera production, which is central to the embryo-mediated method, is not necessary.
MATERIALS AND METHODS
Experimental Design
All procedures for animal management, reproduction, and surgery were performed in accordance with standard protocols of the Division of Animal Genetic Engineering, Seoul National University. The appropriate management of experimental samples, animal care and use, and quality control of the laboratory facility and equipment were performed. The review board of the University Animal Farm, Seoul National University, approved our research procedure in June 2003.
Two series of experiments were conducted to develop the testis-mediated system, one for transplantation of testicular cells for germline chimera production and the other for in vitro culture of the testicular cells. In the first set (experiment 1) we retrieved testicular cells from 4-wk-old juvenile (sexually immature but ready to start spermatogenesis) or 24-wk-old adult (sexually mature and undergoing spermatogenesis) chickens using three different methods (methods 1, 2, and 3). After culturing for 0, 5, 10, or 15 days, 2 x 107 testicular cells retrieved by an optimal method were injected directly into the testes of juvenile or adult chickens (in a 2 x 2 factorial arrangement) using the modified method of Nagano et al. [8]. We verified the presence of transplanted testicular cells and spermatogonia in the seminiferous tubules using Trypan blue and PKH-26. We evaluated germline chimera production by progeny testing. In the second set (experiment 2), we monitored the changes in the morphology and properties of testicular cells during in vitro culture. Chicken germ-cell-specific markers, i.e., periodic acid Schiff (PAS) solution, anti-stage specific embryonic antigen (SSEA)-1, anti-SSEA-3, and anti-SSEA-4 antibodies [9], and a general stem-cell-specific marker that is negative for chicken germ cells, alkaline phosphatase (AP), were used for the characterization. The PROC-GLM model in the SAS program, which incorporated ANOVA and a least-squares method, was used for statistical comparison of retrieval methods (experiment 1) and markers' reactivity (experiment 2); the level of significance was set at P < 0.05.
Retrieval of Testicular Tissue
All chickens used in this study were maintained at the University Animal Farm, Seoul National University. Testicular tissues were retrieved from sexually immature 4-wk-old (juvenile) or sexually mature 24-wk-old (adult) Korean Ogol Chicken (KOC) or White Leghorn (WL) males. The tunica albuginea and connective tissue of the testes were removed mechanically, and the collected testes were trimmed under an inverted microscope with surgical blades and forceps before enzymatic digestion.
In this experimental design, we used three methods to dissociate testicular cells; these methods were modified from Bellve et al. [10] and van Pelt et al. [11]. In the two-step enzymatic method [10] the decapsulated testes were placed in enriched Krebs-Ringer bicarbonate (EKRB) medium [11] containing 0.5 mg/ml collagenase for 15 min at 37°C in a shaking water bath. The dissociated seminiferous tubules were collected by decanting the sediment after treatment; this collagenase treatment was repeated three times. The collected sediments were incubated in EKRB containing 0.5 mg/ml trypsin and 1 µg/ml DNase for 15 min, and dispersed cells were filtered with Nitex filter cloth (40 mesh) after washing twice in EKRB containing 0.5% BSA (method 1). In the method of van Pelt et al. [12], the decapsulated testes were subsequently treated with 1 mg/mL collagenase, 1 mg/mL trypsin, 1 mg/mL hyaluronidase II, and 5 µg/ml DNase I in Hanks balanced salt solution (HBSS) for 15 min. After eliminating interstitial cells, the testicular cells were retrieved by a second dissociation with the same solution for 20 min (method 2). In the one-step enzymatic method, decapsulated testes were treated with HBSS containing 1 mg/ml of collagenase (Collagenase type I; C0130, Sigma-Aldrich Corp., St. Louis, MO), and 0.25% trypsin (Trypsin; T8003, Sigma-Aldrich) for 25 min in a shaking water bath, and the dispersed cells were filtered through Nitex filter cloth (method 3). Method 3 was used both for experiment 1 and for experiment 2, while the other methods were used for a comparison of retrieval methods in experiment 1. To optimize the retrieval protocol, the viability of the cells retrieved by different methods was assessed with a cell proliferation assay kit (Chemicon, Temecula, CA). Briefly, 1 x 105 cells dissociated from the testes were placed in one well of a 96-well plate and subsequently treated with 10% (v/v) water-soluble tetrazolium salt-1 (WST-1)/Electro Coupling Solution (ECS) for 3 h at 37°C, 5% CO2 in air atmosphere. Absorbance of the solution was measured with a microplate reader (Biotrak, Amersham, Buckinghamshire, UK) at 450 nm.
In Vitro Culture of Testicular Cells
Based on the results of our previous experiments (data not shown), dissociated cells (1 x 106) were placed in a 100-mm culture dish with modified DMEM (Gibco Invitrogen, Grand Island, NY) that contained 10% (v/v) 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), 0.55 mM ß-mercaptoethanol (Gibco Invitrogen), 2 ng/ml human leukemia inhibitory factor (LIF; L5283, Sigma-Aldrich), 0.5 ng/ml human basic fibroblast growth factor (FGF2; F3133, Sigma-Aldrich), and 10 ng/ml human insulin-like growth factor-I (IGF1; I3769, Sigma-Aldrich). The seeded cells were cultured in a CO2 incubator at 37°C and 5% CO2 in air atmosphere with 60%–70% relative humidity.
Transplantation and Testcross Analysis
Testicular cells were immediately transferred into recipients or cultured for 5, 10, or 15 days before transfer. Cultured cells of any types were placed in 0.25% (v/w) trypsin-EDTA solution for 5 min. Surgical injection of the cell suspension (2 x 107 testicular cells in 0.5–1 ml trypsin-EDTA-free DMEM supplemented with 10% FBS and 0.01% Trypan blue) was conducted after anesthetizing the recipients with 50 mg/kg ketamine (Yuhan Pharmaceutical Corp., Seoul, Korea). The injection of testicular cells was performed with a Hamilton syringe (Hamilton, Reno, NV) and a 33-gauge needle, and the cells were injected into the testes located in the ventral abdominal cavity close to the kidney. To confirm injection site (the seminiferous tubule), Trypan blue was generally added to the cell suspension shortly before the injection. The results of preliminary experiment showed no harmful effect of the addition on cell viability (data not shown). For a single injection, the testicular cell suspension was loaded into the syringe and inoculated at 10 different sites in the testis.
We further evaluated the localization of testicular cells (presumably spermatogonia or germline cells) by labeling with PKH26. Trypan blue-free cell suspension was centrifuged at 200 x g for 5 min, and the supernatant was subsequently discarded. The pellet containing testicular cells was suspended with gentle pipeting in 1 ml of a serum-free working solution containing 4 x 10–3 mM PKH26 fluorescent. The labeling was stopped by the addition of 125 µl FBS 5 min after the treatment and Trypan blue was added before the injection. The recipient chickens were killed to evaluate the localization of PKH-labeled cells 24 h after the injection. DAPI (1:10 000) staining for cryosection of 20 µm thickness was conducted for detecting PKH26-labeled cells under a fluorescent stereomicroscope.
As approximately 17 days are required for the completion of spermatogenesis in the chicken [13], testcross analysis was conducted on a weekly basis by artificial insemination of adult KOC females by the WL recipients' semen for up to 12 mo (on average, 10 KOC females were used for one WL male recipient, starting 2 wk after injection). To avoid data fluctuation, 10 females were used to testcross with each presumptive chimera (WL recipient) throughout the examination period. A conventional heterologous system using two different strains [5] was employed for evaluating the capacity of the transplanted cells (presumptively spermatogonia, dedifferentiated cell or germline stem cells) to initiate spermatogenesis leading to fertile sperm: black-feathered KOCs with a recessive pigmentation inhibitory gene and white-feathered WL chickens with the dominant pigmentation inhibitory gene. Black-feathered progeny that hatched from WL recipients therefore demonstrated that the transplanted KOC spermatogonia in the mixed population proliferated normally and subsequently underwent spermatogenesis in the seminiferous tubules of the heterologous recipients.
PAS and AP Staining
Cells were fixed with 50 mM phosphate buffer that contained 2% (v/w) glutaraldehyde (G7651; Sigma-Aldrich), 2% (v/v) formaldehyde, and 2 mM MgCl2 for 10 min, and rinsed three times with PBS. The fixed cells were immersed in periodic acid solution (395-1; Sigma-Aldrich) for 5 min and then treated with Schiff Solution (395-2; Sigma-Aldrich) for 15 min. All procedures were performed at room temperature, and the 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), 10 mg Fast Red TR salt) for 15 min and then rinsed three times with PBS.
Immunostaining
For the immunocytochemical analysis, the anti-SSEA-1 (MC-480) and anti-SSEA-3 (MC-631) monoclonal IgM antibodies, and anti-SSEA-4 (MC-813-70) monoclonal antibody were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). For immunocytochemical analysis using the anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies, the Universal DAKO Cytomation Labeled streptavidin biotin 2 system and AP (LSAB2 System) were applied. All cells provided for immunostaining were retrieved at seeding or 15 days after seeding onto a 24-well plate. The cells were fixed for 10 min in 50 mM phosphate buffer that contained 2% (v/w) glutaraldehyde, 2% (v/v) formaldehyde, and 2 mM MgCl2. The fixed cells were treated with 5% (v/v) goat serum for 30 min to minimize nonspecific secondary antibody binding. The optimal concentration was 40 µg/ml for the anti-SSEA-1 antibody and 20 µg/mL for the anti-SSEA-3 and anti-SSEA-4. After incubation with the primary antibodies for 1 h, the cells were first treated with biotinylated goat anti-mouse immunoglobulins for 10 min, then with AP-conjugated streptavidin; finally, the substrate-chromogen (3,3-diaminobenzadine) was added for 10 min. Because endogenous AP activity may yield false-positive results, the cells were treated with 0.02 M levamisole, which is an inhibitor of endogenous AP.
RESULTS
Experiment 1: Germline Chimera Production by Testicular Cell Transfer into Heterogeneous Testes
In the first experiment, a significant (P = 0.0096) treatment effect was detected among different retrieval methods. As shown in Figure 1, greater viability was detected in testicular cells retrieved by the one-step method (method 3) than in cells retrieved by other methods (0.234 ± 0.012 vs. 0.151 ± 0.026 to 0.158 ± 0.022). Accordingly, method 3 was employed for subsequent experiments. In the second experiment, the Trypan blue staining of testicular cells showed that the transplanted cells were successfully injected into the seminiferous tubules within 24 h after the transfer in all cases (Fig. 2). PKH-labeled cells were present in the inner space of the seminiferous tubules, but did not localize into the basal membrane.
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Subsequently, 64 recipients were used for testicular cell transplantation; among the 64 recipients, germline transmission was detected in five cases (7.8%) based on testcross analysis (Table 1 and Fig. 3). Three cases involved the injection of adult cells into adult recipients, one case involved the injection of cells from adult into juvenile, and one case involved the injection of cells from juvenile into juvenile. This pattern of transmission was observed after the transfer of spermatogonia that were injected immediately or cultured for 5 or 10 days. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days in the case of adult into adult, 188 days in the case of adult into juvenile, and 137 days in the case of juvenile into juvenile. The percentage of germline transmission (spermatogenesis of transplanted cells), which was calculated as the proportion of black-feathered progeny to total progeny, was 0.4%–0.9%.
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Experiment 2: In Vitro Culture and Characterization of Testicular Cells
Because both WLs and KOCs were employed as sources of pluripotent spermatogonial stem cells in our conventional embryo-mediated system, we attempted to develop a culture system for the testicular cells of these two strains. The testicular cells retrieved from juvenile or adult male KOCs were cultured in modified DMEM, which was supplemented with LIF, FGF2, and IGF1. Regardless of donor age, testicular stroma cells proliferated rapidly and formed a confluent monolayer 5–6 days after seeding (Figs. 4 and 5). Both colony-forming (probably spermatogonia or germline cells) and monolayer-forming (probably stroma cells) cells were derived from the culture of the testicular cells. The colonies adhered firmly to the monolayer, and no signs of degeneration that was determined by both morphological criteria and cell viability assay were detected. Two types of colonies developed after the culture. Under microscopic observation, some of the colonies formed well-delineated cell masses, while others consisted of isolated cells that were linked by cell-to-cell bridges. The bridge formation within colonized cells became eminent during subculture of colony-forming cells and the isolated colonies formed predominantly in subcultures of the KOC testicular cells (supplementary data not shown).
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Similar to KOC, the culture of WL testicular cells also yielded both colonies and monolayer during primary culture. Retrospective comparison showed rapid proliferation of WL testicular cells relative to KOC testicular cells.
In the second experiment, several cells at seeding and colony-forming testicular cells on Day 15 of culture (at the end of primary culture) were positive for PAS, anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibody staining (Figs. 4 and 5). However, both cells did not react to AP staining. The age of donor cells did not affect staining affinity patterns. As shown in Table 2, a densitometric analysis for quantifying staining affinity showed a significant (P < 0.05) difference between spermatogonia and background feeder cells after staining with PAS, anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies on Days 0 and 15 of culture, regardless of retrieval source (juvenile or adult).
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DISCUSSION
We conclude from our results that heterologous, fertile spermatozoa could be produced by transfer of testicular cells into juvenile or adult testes. Others have demonstrated the capability of intact or in vitro-cultured spermatogonia to induce germline transmission after transfer into recipients [14, 15]. Transplanted cells (presumptively spermatogonia, dedifferentiated spermatogenic cells, or germline stem cells) may become established in the spermatogonial cell layer of the seminiferous tubules of the recipient testes. The testicular tissue of the recipients supported spermatogenesis of the injected spermatogonia that subsequently yielded functional spermatozoa.
The testicular cells retrieved by the optimized methods of this study consisted of various types of germline cells, Sertoli cells, and stroma cells. Presumably, spermatogonia, germline stem cells, and/or dedifferentiated spermatogenic cells of the transplanted testicular cells might participate in the spermatogenesis of the recipient testes. It has generally been presumed that spermatogonial (germline) stem cells continuously proliferate to finally become undifferentiated type A spermatogonia [16–19]. Brinster and Zimmerman [20] and Ogawa et al. [21] demonstrated that the transplanted donor spermatogonial stem cells can participate in spermatogenesis in the seminiferous tubules of recipient mice and generate donor-derived progenies. Live births have been achieved after transplantation of spermatogonia into adult testes, confirming the stemness of the spermatogonia in the mouse [22]. Considering short-term culture of the testicular cells before transfer, however, it might be a very small possibility that dedifferentiate spermatogenic cells participated in germline chimera production in this study.
Mature spermatozoa were not observed among the transferred cells of the cell suspension. It is possible that hatched progeny with black feathers were derived from primary or secondary spermatocytes, or from round spermatids, which could have been transplanted. However, this possibility might be very small in this case. In general, spermatogenic cells in the early stages of spermatogenesis do not stay in the testicular tissues for longer than 17 days [10]. Considering the fact that the hatch of the progeny with black feathers was detected at more than 38 days after transfer in this experiment, this possibility may almost certainly be excluded. To date, there have been no reports on the potential feasibility, pluripotency, and gene targeting of spermatogenic cells in domestic animals.
The biochemical and molecular markers for detecting and characterizing chicken pluripotent cells in the testes have yet to be developed. Probably, several chicken germ-cell-specific markers can be applied for the characterization. Our supplementary results have further demonstrated the similarity of staining affinity between chicken colony-forming testicular cells and embryonic germ cells: the colony-forming cells were positive for all chicken germ-cell-specific markers such as anti-integrin 
6 and anti-integrin ß1 antibodies, and lectin-STA and lectin-DBA, in addition to PAS, and anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies (data not shown; unpublished data). These results suggest that the heterologous testicular cells can be used as sources of the mature spermatozoa and that colony-forming cells of the testicular cells may participate in spermatogenesis in transplanted tissue [14]. Therefore, testis-mediated germline transmission might be developed on the basis of exploiting the potential of some testicular cells to undergo spermatogenesis.
The testis-mediated germline transmission system involves less time and labor for creating chimeric status than does the embryo-mediated system [15]; the procedures for pluripotent cell transfer to the developing embryo, embryo development and hatching (21 days), and sexual maturation of the chimera (5–6 mo) are omitted in the new system [4]. Our study suggests the possibility of developing this alternative, testis-mediated system that allows the use of testes as a reservoir for new stem cells. Sufficient numbers of the cells that give rise to functional spermatozoa are simply retrieved from this newly discovered reservoir in young and adult chickens, which may provide for transgenesis for bioreactor production. We are now undertaking the experiment on in vitro maintenance of colony-forming cells and subsequent characterization and differentiation of the established cells. Nevertheless, our results are preliminary, as a total of only five cockerels had chimeric testes as shown by a low frequency of donor-derived sperm shown by progeny test. More detailed information on the population of pluripotent cells in the testicular cells and accessibility of transgenic technology for testicular cell-derived, chicken pluripotent cells is necessary, as well as the improvement of the transmission efficiency by modifying various subprotocols.
FOOTNOTES
1 Supported by a grant from the BioGreen 21 program, Rural Development Administration, Korea and a graduate fellowship provided by the Korean Ministry of Education through the Brain Korea 21 project. 
2 Correspondence: FAX: 822 874 4811; jaehan{at}snu.ac.kr
Received: 26 February 2006.
First decision: 19 March 2006.
Accepted: 17 May 2006.
REFERENCES
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