:: Asian Journal of Andrology ::

Proliferation of exogenously injected primordial germ cells (PGCs) into busulfan-treated chicken embryos

H. Furuta, N. Fujihara

Animal Resource Science Section, Division of Bioresource and Bioenvironmental Sciences Graduate School Kyushu University, Fukuoka 812-8581, Japan

Asian J Androl 1999 Dec; 1: 187-190

Keywords: chick embryo; germ cells; proliferation; busulfan

Abstract

Aim: This study was designed to investigate the effect of busulfan treatment on the proliferation of chicken primordial germ cells (PGCs) in vivo, focusing on the preferential settlement of PGCs onto the germinal ridges of chicken embryos. Methods: Busulfan (250 ng/egg) was injected into the egg white of freshly oviposited fertilized eggs, which were then incubated. Embryonic development and viability were examined, and exogenous PGCs collected from embryonic blood vessels were injected into the germinal crescent region of recipient embryos. The number of PGCs resided onto germinal ridges of the right and left sides were compared. Results: Busulfan had a slight harmful effect on the embryo viability and the PGCs proliferation. The number of PGCs resided onto the left side of germinal ridges was slightly higher as compared with the right side. Conclusion: Busulfan suppressed the viability of embryos and the proliferation of endogenous PGCs in the recipient embryos. However, the number of exogenous PGCs proliferated was higher in embryos treated with busulfan than those without busulfan. Data also suggest the possibility of a preferential residence of PGCs toward the left side of the germinal crescent region as compared with the right, which may be due to a more advanced functional development of the left gonad than the right.

1 Introduction

Chicken primordial germ cells (PGCs) are important resources to produce transgenic offspring^[1] and successful transfer of exogenous genes to PGCs has been carried out with blastodermal cells^[2,3]. It is a common technique to establish germ line chimeric chickens using PGCs transferred between embryonic blood vessels^[4-8]. Recently, busulfan (1,4-butanediol dimethanesulfonate) has been often used to introduce exogenous PGCs into chicken embryos. The drug has been said to suppress the proliferation of PGCs in chicken^[9-12]. The present study was designed to investigate the effect of busulfan treatment of fertilized eggs, and the migration and proliferation of PGCs subsequent to the drug treatment.

2 Materials and methods

2.1 Experiment I

In this experiment, there were 4 Treatments.

Treatment 1: Busulfan 625 g were dissolved in 100 mL of phosphate buffered saline (PBS). A circular window about 1.0 cm in diameter was opened on the sharp edge of the recipient eggs. Around 40 L of PBS solution containing 250 ng busulfan was injected into the egg white through the window just before incubation. Embryonic blood samples were collected from vessels of the embryos after 72 hours of incubation (stage 12-15)^[13]. Approximately 1.0 L blood was smeared on a glass slide and the number of PGCs was then counted under microscope after staining with PAS solution.

Treatment 2: Embryonic blood samples were collected from the donor embryos incubated for 72 hours (stage 12-15). The donor PGCs were isolated by the ficoll density centrifugation method^[13-15]. For recipient embryos, busulfan-treated eggs were also incubated for 48 hours (stage 9-10) as in Treatment 1. Fifty to sixty donor PGCs were introduced into the germinal crescent of recipient embryos through the window. After an additional 24-hour incubation (stage 14-16), blood samples were collected from the recipient embryos and PGCs number per 1 L blood was counted.

Treatment 3: Donor PGCs were prepared as in Treatment 1; recipient eggs were prepared without busulfan treatment. The introduction of PGCs from donor embryos into recipient embryos and the subsequent procedures were the same as in Treatment 2.

Treatment 4: Control eggs without busulfan injection. Experimental procedures were similar to those in Treatment 1.

2.2 Experiment II

Donor and recipient eggs were prepared as in Treatment 2 of Experiment I with or without busulfan injection. PGC proliferation in the recipient embryos with or without busulfan treatment was compared. Donor PGCs isolated from the blood samples were labeled with PKH26 red fluorescent cell linker (Sigma Aldrich Japan, Tokyo) to distinguish the exogenous PGCs from the endogenous ones originated from the recipient embryos. The labeled PGCs were introduced into the germinal crescent of the recipient embryos, which were then incubated for 4 more days (total incubation time: 6 days; stage 23-26). The labeled donor PGCs at the region of germinal ridges of recipient embryos were examined under microscope equipped with TE-FM-Epi-fluorescent attachment (Nikon, Tokyo).

2.3 Statistical analysis

Data were presented as means, if applicable. Statistical analysis was performed using the chi square test and analysis of variance^[17]. Significance of differences was set at P<0.05.

3 Results

The development of the control embryos was normal, displaying around 24 PGCs per 1.0 L of blood sample (Figure 1). On the contrary, busulfan injection (Treatments 1 and 2) resulted in significantly fewer numbers of PGCs as compared with those without drug treatment (Treatments 3 and 4) (Table 1). The viability of the embryos in Treatments 3 and 4 was higher than those in Treatments 1 and 2 (P<0.05), while no significant difference was observed in the viability of embryos and the number of PGCs between the control and Treatment 3 (Table 1).

Figure 1. PGCs at stage 12-15, stained with PAS. Bar indicates 50 m.
Table 1. Number of embryos survived and PGCs detected. ^bP<0.05, compared with the values of Treatments 3 or 4.

Treatment	n	Embryo survived (%)	PGCs detected/L
1. Busulfan	19	11 (57.9)^b	1.52.4^b
2. PGCs and busulfan	16	4 (25.0)^b	12.36.5^b
3. PGCs	7	5 (71.4)	29.48.2
4. Non-treatmentcontrol	9	9 (100)	24.42.8

The labeled donor PGCs introduced into the recipient embryos in Experiment II could be found at the germinal ridges of the recipient embryos (Figure 2). It was shown that the exogenous PGCs presented preferential residence toward the left side of the germinal ridges in comparison with the right side (Table 2).

Figure 2. PKH26-positive PGCs (stage 23-26) at recipient germinal ridge. Bar indicates 50 m.
Table 2. Preferential residence of exogenous PGCs.

Treatment	Number of embryos		Number of exogenous PGCs in recipient embryo
Treatment	Treated	Survived	Right	Left
PGC	18	11	13.412.9	19.316.6
PGCs and busulfan	26	9	18.020.1	27.128.3

4 Discussion

When busulfan was injected into eggs, the viability of the treated embryos was reduced significantly (P<0.05), and the number of endogenous PGCs was also significantly lower (P<0.05) as compared with the control embryos. In Treatment 2, a similar tendency was observed in the proliferation of exogenously injected PGCs in recipient embryos. Busulfan has been reported to suppress the viability of embryos and the proliferation of PGCs in the recipient embryos^[9-12]. In this experiment, the number of exogenous PGCs proliferated in the germinal ridges of drug-treated recipient embryos was higher than that in embryos without drug-treatent. It is considered that proliferation of exogenous PGCs occurred successfully in the embryos where the endogenous PGCs proliferation was suppressed by busulfan. Injection of exogenous PGCs into the embryonic blood vessel^[4-8] appears to be harmful to the development of embryos. Interestingly, the present results suggest the possibility of a preferential residence of PGCs toward the left side of the germinal crescent region as compared with the right, which may be due to a more advanced functional development of the left gonad than the right. A slightly dominant development of the left gonad compared with right one has generally been accepted in birds.

5 Acknowledgments

The authors are grateful to Dr. T. Kuwana for his kindest guidance to collect avian PGCs from early embryos with special techniques, and also thanks the highly skilled technical assistance by Mr. Yoshihiro throughout the experiments. Special financial support was provided grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, Japanese Society for the Promotion of Science (JSPS), Toyota Foundation, Sumitomo Foundation and Nissan Science Foundation.

References

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Supported by grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, Japanese Society for the Promotion of Science (JSPS), Toyota Foundation, Sumitomo Foundation and Nissan Science Foundation.
Correspondence to Dr. Noboru Fujihara, Animal Resource Science Section, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Fukuoka 812-8581, Japan.
Tel & Fax: +81-92-642 2938
E-mail: nfujiha@agr.kyushu-u.ac.jp
Received 1999-11-27 Accepted 1999-12-22