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Embryo |
School of Agricultural Biotechnology,3 Seoul National University, Suwon 441-744, Korea
Central Research Center, Hanmi Pharm Company Limited,4 Kyunggi Province 463-400, Korea
Avicore Biotechnology Institute,5 Kyunggi Province 437-020, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gamete biology, gametogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we further improved germline transmission in gPGC-derived chicken chimeras, which remained low level in our previous study [6]. We postulated that the culture of gPGCs would be one of the critical factors for increasing germline transmission of the produced chimeras, since gPGC culture for an extended period increased the incidence of germline transmission in produced chimeras (preliminary results; data not shown). On the other hand, it was reported Ficoll gradient separation before injection was essential for the collection of PGCs from embryonic blood [8, 9]. However, PGC collection from the embryonic gonad and in vitro culture of gPGC in our developed system increased the gPGC population, which may have lessened the necessity of Ficoll separation. In addition, we postulated that homo- or heterosexuality between transplanted gPGCs and recipient embryos might affect germline transmission, since the transplantation of opposite-sex PGCs could affect the gametogenesis in the gonads of recipient embryos [10, 11]. Accordingly, this study consisted of evaluating 1) whether in vitro culture of gPGCs could contribute to promoting the efficacy of germline transmission in produced chimeras, 2) whether gPGC separation by Ficoll gradient before transplantation could be effective for inducing germline transmission, and 3) how the sexuality of gPGC-derived germline chimeras affected the efficiency of germline transmission.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retrieval and Culture of gPGCs
Experimental animals provided for this experiment were maintained at the University Animal Farm, Seoul National University, and all experimental procedures were performed at the affiliated laboratories of the university. PGCs were retrieved from the gonads of Korean Ogol chicken (KOC) embryos at stage 28 (5.5 days of incubation) [12]. Chicken embryos developed to stage 28 were freed from yolk by rinsing with calcium- and magnesium-free PBS, and the gonads were then retrieved from the embryos by dissecting the abdomen with sharp tweezers under a stereomicroscope. Gonadal tissues were dissociated by gentle pipetting in 0.05% (v/v) trypsin solution supplemented with 0.53 mM EDTA. After centrifugation at 200 x g for 5 min, 1 x 104 cells isolated from the gonadal tissues were seeded onto a 96-well culture plate according to our standard protocol [13]. Since preliminary data showed that approximately 1% of seeded gonadal cells was gPGC, the number of gPGCs initially seeded into one well was within the range of 100–120. Seeded cells were then cultured in Dulbecco minimal essential medium (Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL); 2% (v/v) chicken serum (Gibco BRL); 1 mM sodium pyruvate (Sigma Co., St. Louis, MO); 55 µM ß-mercaptoethanol (Sigma); 20 ng/ml conalbumin (Sigma); 10 mM Hepes (Gibco BRL); 1x antibiotics-antimycotics (Sigma); 5 ng/ml human stem cell factor (Sigma); 5 U/ml murine leukemia inhibitory factor (Sigma); 10 ng/ml bovine basic fibroblast growth factor (Sigma); 0.04 ng/ml human interleukin-11 (Sigma); and 10 ng/ml human insulin-like growth factor-I (Sigma). The isolated gPGCs were then cultured for 5 or 10 days in a CO2 incubator maintaining 5% CO2 in an air atmosphere at 37°C, and medium change was only conducted in a 10-day-culture group at 5 days after culture.
Characterization of gPGCs
For the identification of gPGCs, stage-specific embryonic antigen-1 (SSEA-1) staining was employed. Anti-SSEA-1 antibody developed by Solter and Knowles [14] was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. Gonadal PGCs in the cell suspension were fixed with 1% (v/v) glutaraldehyde for 5 min and rinsed with 1x PBS twice. The anti-SSEA-1 ascites fluid diluted 1:1000 in PBS was added and subsequent steps were carried out using a DAKO universal LSAB kit, Peroxidase (DAKO, Carpinteria, CA), according to the manufacturer's instructions.
Transplantation of gPGCs into Recipient Embryos
Zero-, 5-, and 10-day-cultured KOC gPGCs were transferred into the dorsal aorta of White Leghorn chicken (WL) recipient embryos at stage 17 (2.5 days of incubation) [12]. According to the experimental design, Ficoll separation using 10% (v/v) and 16% (v/v) gradient concentrations was done prior to the transplantation, the protocol that was the most effective for gPGC separation in preliminary results (data not shown). For gPGC transfer, a small window was made on the sharp end of the recipient egg, and approximately 2–3 µl of cell suspension containing 150–200 gPGCs was injected into the dorsal aorta of the recipient embryo using a micropipette. The egg window of the recipient embryo was sealed twice with paraffin film and then laid down with the pointed end at the bottom until hatching.
Testcross Analysis
Possession of homozygous I/I or i/i gene in the WL and the KOC, respectively, simply enable germline transmission capacity of chimeric chickens to be evaluated by the feather color of produced progenies. Presumptive chimeras derived from gPGC transplantation were maintained for up to 6 months with our standard management program [6]. All sexually matured chickens producing egg or semen were then provided for a testcross analysis by artificial insemination with adult KOCs. It was considered that autosomal I gene, a pigmentation inhibitor, governs the genes of melanin pigment stimulation by epistatic effects. Accordingly, the genotype of both homozygous I/I in WL and heterozygous I/i in the hybrid progenies from testcross analysis results in white feather color. Hatched progenies of black feather coat has only the i/i gene, which represents complete germline transmission of transplanted gPGCs through the chimeras. This also shows normal proliferation and differentiation of transplanted gPGCs of KOC in the recipient WL embryos.
Experimental Design
Either prospective (culture duration and Ficoll treatment) or retrospective (chimera sexuality) analyses were undertaken to evaluate each treatment effect. For the prospective approach, gPGCs collected from KOC embryos were randomly allotted into five different treatments: 1) collection and transfer simultaneously after Ficoll separation (0 day culture) into recipient embryos; 2) culture for 5 days, then transfer after Ficoll treatment; 3) culture for 5 days, then transfer without Ficoll treatment; 4) culture for 10 days, then transfer after Ficoll treatment; and 5) culture for 10 days, then transfer without Ficoll treatment. In the prospective assignment, the transfer of 0-day-cultured gPGCs without Ficoll separation was excluded, since gPGC isolation from the gonadal stroma tissue immediately after collection could be made only after Ficoll treatment because of its low population in the collected tissue being less than 1%. A crossbred chimera production system in a testcross analysis was employed for evaluating each treatment effect, and retrospective data on chimera sexuality were subsequently combined with the prospective designation. Obtained values from the whole sets of experimental parameters were then subjected to ANOVA using the general linear model (PROC-GLM) in the SAS program (SAS, Inc., Cary, NC) [15]. When the significance of the main effects was detected in each experimental parameter, treatment effects were subsequently compared by the least squares method. Significant difference among treatments was determined where the P-value was less than 0.05. General experimental procedure conducted in this study is depicted in Figure 1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For the culture of gPGCs, chicken gonadal stroma cells were used as a feeder cell, and mixed population of gonadal stroma cells and gPGCs in the embryonic gonads were concomitantly seeded into a culture dish. The stromal cells quickly proliferated and formed a confluent monolayer within 5 days after seeding, whereas gPGCs proliferated slowly on gonadal stroma feeder cell layers and began to form colonies on the confluent stromal cell layer 7 days after culture (Fig. 2). The colonies firmly adhered to the confluent stromal cell layer, and no signs of cell degeneration such as dispatch from the cell layer could be seen throughout the culture. In contrast, gonadal stromal cells attached to the surface of the culture dish and spread over it. Gonadal PGC colonies expressed the stage-specific embryonic antigen-1 (SSEA-1) epitope during culture, which indicated typical characteristics of primordial germ cells.
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Treatment Effects on Germline Chimera Production
One hundred fifty to 200 gPGCs per recipient were injected, and a total of 683 chickens were hatched after gPGC injection. After management of up to 6 months, 301 (44.1%) were sexually matured and subsequently provided for testcross analysis. As shown in Table 1, 27 chickens consisting of 15 males and 12 females were proved to be a germline chimera in the progeny test. The proportion of the chimeras to sexually matured chickens was 9.0% (27/301). The range of germline chimerism in each treatment was within 5.1% and 14.4%, and no significant (P = 0.6831) treatment effect was found in chimera production. These results indicated that production efficiency of germline chimera was not affected by our experimental treatments.
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Treatment Effects on the Efficacy of Germline Transmission in Sexually Matured Chimeras
Another comparison was made for evaluating whether designated treatments affected the efficiency of germline transmission. Although the factorial design of each treatment consisting of both prospective and retrospective assignments was made, total treatments became 9; the groups of 0-day culture only include Ficoll treatment, and only male chimeras were produced by transfer of 0-day-cultured gPGCs after Ficoll treatment. Due to such incomplete factorial assignment, a completely block design was employed for statistical analysis.
As shown in Table 2, a significant (P < 0.0001) model effect was found. The efficacy of germline transmission was greatly improved as cultured duration increased (0.6%, 1.5%–7.8%, and 10.7%–49.7% in 0-, 5-, and 10-day-culture regime, respectively). Within the same in vitro culture duration, improved efficacy of germline transmission was found in the non-Ficoll-treated group compared with the Ficoll-treated group. Such an effect was prominent in the groups of 10-day culture; significant (P < 0.0001) increase in germline transmission, regardless of chimera sexuality, was found (44.7%–49.7% vs. 10.7%–25.4%). Immunohistochemical assay using anti-SSEA-1 antibody showed that Ficoll treatment might not affect gPGC characteristics. Therefore, additional effort to retrieve enough numbers of gPGCs for transplantation such as increasing the population of gPGCs was not necessary in the non-Ficoll-treatment groups. Sexuality of chimera did not affect the transmission efficacy; no significant (P = 0.6011) difference was found between different sexualities under the same culture duration and separation treatment, except for the group of 10-day culture with Ficoll treatment. Induction of germline transmission in each individual chimera originated from different gPGC preparation systems is also shown in Table 3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The migration pattern of avian PGCs is very different from that of mammals, and this unique mode allows the production of germline chimeras by the injection of PGCs into the blood vessel of recipient embryos. PGCs, which are the progenitors of functional gametes after sexual maturity, first originate from the epiblast in the blastoderm and translocate to the hypoblast of the area pellucida [12, 16]. During gastrulation stages, they circulate through the vascular system and finally colonize into the gonadal anlagen [17]. Previous reports showed that the transfer of PGCs collected from the germinal crescent [2, 4] or blood vessel [3] into recipient embryos could produce germline chimeras. However, the number of PGCs provided for transplantation was extremely limited when collected from those embryonic tissues.
Chang et al. [6] reported that PGCs from the embryonic gonads, which have passed beyond the migration stage, could also induce germline transmission after being transferred into the recipient embryos at the migratory stage. It has been known, however, that PGCs lose their migration activity after settling down into the gonad [17]. In our experiment, we used gPGCs already localized in the gonad. Our result showing that 0-day-cultured gPGCs had an extremely low capacity to induce germline transmission reflected the previous finding on the loss of migration activity in gonad-localized PGCs. Consequently, these data suggested that careful consideration regarding the potential of gPGCs on migration and pluripotency should be made with the use of such cells for improving germline transmission efficacy.
In our previous study [18], we found the possibility that gPGCs can obtain the properties of embryonic germ cells as a stem cell and localization activity into the embryonic gonads by extending in vitro culture under a suitable environment. Accordingly, we hypothesized that gPGCs themselves, which are collected from 5.5-day-old embryos, can recover migration capacity, chemotactic movement, and the ability to colonize in the recipient gonadal ridges after in vitro culture. A significant increase in migration and localization activity into the gonad, as well as improved germline transmission, was initially expected after transfer of gPGC cultured for extended periods. In the results, the efficiency of germline transmission was tremendously improved when 10-day-cultured gPGCs were transplanted (Table 2). However, in vitro culture did not affect migration activity of gPGCs after being transferred (Table 1). Revision of the initial hypothesis was necessary based on these results, and it was subsequently suggested that in vitro culture mainly affected the proliferation and differentiation of transplanted gPGCs after migration into the gonads of recipient embryos. In vitro culture probably might drive gPGCs reprogrammed for adapting to the gonadal environment after being transferred. It was further suggested that in vitro culture before transplantation might contribute gPGCs recovering from initial damage from environmental changes at the time of collection.
A steady but significant increase in germline transmission of produced chimeras was found by increasing the culture duration up to 10 days. This strongly suggests that in vitro preparation of gPGCs before transfer into recipient embryos is one of the important factors for improving the efficiency of germline transmission in produced chimeras. No decrease in the viability of gPGCs after being cultured for 10 days in our supplement experiment was detected (data not shown). Additionally, gPGCs even cultured for extended periods of up to 60 days under our optimal culture environment still retained the capacity to induce germline transmission [18]. Such an established system based on our series of studies could contribute to developing an innovative technology for gene manipulation and targeting during in vitro culture.
In the present study, an interstrain combination of donor gPGC and a recipient embryo was made for inducing germline chimerism, but we only established a transfer system of KOC gPGCs into WL recipient embryos because of potential applicability. Naito et al. [3] reported that the efficacy of germline transmission was increased more than three times after transfer of WL PGCs into Barred Plymouth Rock recipient embryos than after vice versa. This increase still remains unclear since no further reports support such a possibility to date [19], and no prospectively designed study to confirm such a hypothesis has been conducted in our system to date. In any case, it is possible that reverse combination of KOC and WL in our system either negatively or positively affects chimera production and the germline transmission of produced chimera.
It has been reported that Ficoll gradient separation contributes to increasing the population ratio of PGCs for effective injection into the recipient embryo [8, 9]. Several previous reports showed the positive effect of Ficoll treatment on the separation of PGCs from embryonic blood and its safety on maintaining cell viability [8, 9]. However, data in Table 2 demonstrated that, except for the transplantation of 0-day-cultured gPGCs, no beneficial effect of Ficoll separation was found in our developed system. At least, Ficoll treatment is no longer effective for separating gPGCs from in vitro-cultured embryonic gonadal tissue. Two factors could be considered to explain such a discrepancy. In our study, Ficoll was first used for the separation of PGCs from the embryonic gonad, in which cell population is much more intricate than in embryonic blood; therefore, it can reduce the availability of Ficoll separation shown in other reports using embryonic blood. On the other hand, we selected Ficoll concentrations of 16% and 10% for gPGC separation based on the results of our preliminary study. However, even these were too high compared with other reports [8, 9], and such concentrations might decrease the potency of collected gPGCs to induce germline transmission after transfer. More discrete examination using proper concentration of Ficoll for the separation of PGCs from various embryonic tissues is therefore needed.
Retrospective analysis was conducted to evaluate the effect of the sexuality on germline transmission, but no significant effect was found. For increasing the number of gPGCs transferred, gPGCs collected from 10–15 embryos were transferred to a recipient. This might interfere in detecting the treatment effect, and the inconsistency of our data with the previous results [10, 11] could be explained by such a procedure. Otherwise, it could be suggested that the transfer of gPGCs with mixed sexuality is not detrimental to germline transmission, regardless of in vitro culture duration (up to 10 days) and Ficoll treatment.
In conclusion, our established system using in vitro-cultured gPGCs greatly promotes the germline transmission in produced chimera. Therefore, this system contributes to the effective production of highly potent chicken germline chimeras and to developing an efficient transgenic chicken production system. An established system using avian gPGC in this study can further develop a transgenic technology for producing a capable bioreactor.
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FOOTNOTES |
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2 Correspondence: Jae Yong Han, School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea. FAX: 82 31 294 6543; jaehan{at}snu.ac.kr
Received: 24 April 2002.
First decision: 13 May 2002.
Accepted: 25 November 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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