:: Asian Journal of Andrology ::

Percoll fractionation of adult mouse spermatogonia improves germ cell transplantation

Kyu-Bom Koh¹, Masatoshi Komiyama^{1,
2}, Yoshiro Toyama³, Tetsuya Adachi⁴, Chisato Mori^{1, 5}

¹Department of Bioenvironmental Medicine, ³Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
²Center for Environment, Health and Field Sciences, Chiba University, Kashiwa 277-0882, Japan
⁴Department of Genomic Drug Discovery Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
⁵Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Asian J Androl 2004 Jun; 6: 93-98

Keywords: germ cell transplantation; green fluorescent protein; percoll fractionation; spermatogenic cells; mice

Abstract

Aim: To isolate and transplant germ cells from adult mouse testes for transplantation. Methods: In order to distinguish transplanted cells from endogenous cells of recipients, donor transgenic mice expressing green fluorescent protein (GFP) were used. Germ cells were collected from the donors at 10-12 weeks of age and spermatogonia were concentrated by percoll fractionation and transplanted into recipient seminiferous tubules that had been previously treated with busulfan at 5 weeks of age to remove the endogenous spermatogenic cells. Results: Twenty weeks after the transplantation, a wide spread GFP signal was observed in the recipient seminiferous tubules. The presence of spermatogenesis and spermatozoa was confirmed in sections of 12 out of 14 testes transplanted (86 %). However, when germ cells were transplanted without concentration the success rate was zero (0/9). Conclusion: Germ cells from adult mouse testes can be successfully transplanted into recipient seminiferous tubules if the cell population is rich in spermatogonia and the percoll fractionation is useful in obtaining such a cell population.

1 Introduction

Germ cell transplantation is a technique to transplant spermatogenic stem cells, spermatogonia, into semini-ferous tubules devoid of endogenous spermatogenesis. The technique was first introduced by Brinster and his co-workers, they indicated that spermatogenic cells transplanted into seminiferous tubules of infertile mice commenced spermatogenesis [1, 2]. Since then it has been used in certain studies, including analysis of spermatogenesis [3-5], vertical transmission of genetic modification to progeny [6] and treatment of male infertility [7].

The key to the success of this technique is to transplant a cell population rich in spermatogonia, since in various stages of spermatogenic cells, only the spermatogonia have the ability to reside onto the seminiferous tubules and initiate spermatogenesis [1, 2]. In order to obtain such a cell population, neonatal testes or artificially cryptorchid testes have been mainly used as the source of transplanted germ cells [1-6]. Although there are several reports using adult testes as a source of donor cells, the success rates were highly variable, ranging from 11 % to 73 % and the number of donor colonies per testis were reported to be from 1 to more than 12 [2, 8-10]. Thus, the lack of adult germ cell population may have restricted the application of this technique.

In this study we transplanted germ cells from adult mouse testes after concentrated by percoll fractionation to obtain a spermatogonia-rich cell population.

2 Materials and methods

2.1 Animals

As donors of germ cells, the adult GFP mice kindly provided by Mitsubishi Kagaku Institute of Life Science (Tokyo, Japan) were used. Recipients of the germ cells were C57BL/6 mice of the same strain as the donors and were purchased from the Japan SLC (Shizuoka, Japan). The endogenous germ cells of the recipient mice were destroyed by ip administration of 50 mg/kg busulfan (Sigma-Aldrich, St. Louis, USA) at 5 weeks of age as suggested by Brinster & Zimmermann [1] and Brinster & Avarbock [2]. Transplantation was performed at week 5 after the busulfan treatment. All animal experiments were approved by the Laboratory Animal Care Committee and conducted in accordance with the Guideline for Animal Experimentation of the Graduate School of Medicine, Chiba University.

2.2 Germ cell transplantation

For preparation of germ cells, testes of 10-12 weeks old GFP mice were excised and put into phosphate buffered saline (PBS) with the tunica albuginea removed. Seminiferous tubules were treated with 1 mg/mL collagenase type IV (Sigma-Aldrich, St. Louis, USA) in PBS for 20 minutes at 37 and then centrifuged at 100 for 5 minutes at 18 . Resulting pellet was suspended with 0.25 % trypsin in Hank's balanced salt solution (Invitrogen Co., Tokyo, Japan) and incubated for 20 minutes at 37 . The cell suspension was mixed with a half volume of fetal bovine serum (FBS, Invitrogen Co.) and filtered through a 60 m nylon mesh in order to remove cell clumps. After centrifugation (100 for 5 minutes, 18 ), the cells were washed twice with Dulbecco's modified Eagle's medium (D-MEM; Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 0.75 % bovine serum albumin (D-MEM-BSA). The cells were fractionated on a discontinuous density gradient prepared with the following percoll (Pharmacia Biotech, Tokyo, Japan) concentrations: 65 %, 50 %, 40 %, 36 %, 33 %, 30 %, 25 % and 20 % in D-MEM-BSA [12]. After centrifugation (800 for 30 minutes, 18 ), fractions of 33 % and 36 % percoll density were collected and washed twice with D-MEM-BSA. Finally, the collected cells were suspended in D-MEM supplemented with 1 % FBS at a concentration of 10⁸ cells/mL. Germ cells prepared in a similar way without the percoll fractionation step were used as the controls.

Upon transplantation the cell suspension was mixed with equal volume of 0.4 % trypan blue in PBS so that the injected cell suspension was visible. Recipient mice were anesthetized with 50 mg/kg pentobarbital sodium solution (Abbott Laboratories, North Chicago, USA). Both testes were exposed and the tunica albuginea near the rete testis was cut by a razor blade. The cell suspension was injected into the rete testis by a glass pipette (outside diameter 50 m, Narishige Co., Tokyo, Japan) coated with silicon (Sigma-Aldrich). The injected volume was approximately 7 L for each testis. The success of injections was confirmed by the spread of the trypan blue in the seminiferous tubules. The germ cells concentrated by percoll fractionation were injected into 14 testes and the control germ cells into 9 testes.

2.3 Histological observation

Twenty weeks after transplantation, recipient mice were euthanized and the testes collected. In order to detect spermatogenesis derived from exogenous sperma-togonia, the testes were embedded in O.C.T. compound (Miles Inc., Elkhart, USA) and frozen in 2-methylbutane cooled with liquid nitrogen. Sections were cut at 20 m in a cryostat and mounted on slides. After fixation in 4 % paraformaldehyde (Sigma-Aldrich, Tokyo, Japan) in phosphate buffer (pH 7.4), the sections were observed under an Axioplan fluorescent microscope (Carl Zeiss Japan, Tokyo, Japan), equipped with a digital camera (SENSYS, Photometrics, Tucson, USA). The same sections were then stained with hematoxylin and eosin (HE) and examined under a conventional light microscope (Olympus PX-51, Olympus Co., Tokyo, Japan), equipped with a digital camera HC-2500 (Fujifilm Co., Tokyo, Japan). Sections of intact testes derived from 12-week-old GFP mice and C57BL/6 mice were prepared in the same way as the controls.

3 Results

Figure 1A & 1B were the frozen sections of testes derived from intact adult GFP mice and Figure 1C & 1D, those from C57BL/6 mice. In the sections of GFP mice, green fluorescence was observed in each seminiferous tubule (Figure 1A), however, in C57BL/6 mice it was not found (Figure 1C). Normal spermatogenesis in the testes of both mice was evident (Figure 1B and 1D).

Figure 1. Spermatogenesis in intact adult (12-week-old), GFP mice (A and B) and C57BL/6 mice (C and D). A and C are fluorescent micrographs of seminiferous tubule sections showing GFP signal in GFP mice (A), but not in C57BL/6 mice (C). B and D are photomicrographs of HE-stained sections of seminiferous tubules showing normal spermatogenesis in both mice. Scale Bar = 50 m.

In 12 of 14 testes, the GFP signal was observed in the seminiferous tubules of intact testis, as well as when the tubules were isolated (Table 1 and Figure 2A & 2B). The fluorescence of GFP-positive cells was widely spread to a considerable extent along the seminiferous tubules. At higher magnification, GFP-positive cells were visible through the wall of the tubules (Figure 2B). When cross sections were observed, the seminiferous tubules were filled with GFP-positive cells (Figure 2C). In HE-stained sections, spermatogenesis occurred in the GFP-positive tubules with the presence of spermatozoa (Figure 2D). However, when germ cells had not been concentrated prior to transplantation, neither GFP fluorescence nor spermatogenesis of transplanted cells was seen (Table 1 and Figure 2E).

Table 1. Spermatogenesis 20 weeks after transplantation with concentrated or non-concentrated germ cells.

	Concentrated	Non-concentrated
Number of testes transplanted	14	9
Number of testes with spermatogenesis	12	0
Success rate (%)	86	0

Figure 2. Spermatogenesis of transplanted cells in recipient seminiferous tubules at 20 weeks after transplantation.
A and B: Fluorescent photomicrographs of seminiferous tubules showing GFP fluorescence derived from transplanted cells.
C and D: Photomicrographs of seminiferous tubule section showing normal spermatogenesis from transplanted cells (C: Fluorescent microgragh; D: HE staining).
Arrows indicate seminiferous tubules with spermatogenesis and presence of spermatozoa; asterisks indicate seminiferous tubules lacking germ cells.
E: Photomicrograph of testis section at weeks after transplantation with non-concentrated germ cells, showing no spermatogenesis in seminiferous tubules.
Scale Bar = 500 m (A), 200 m (B), 100 m (C, D and E).

4 Discussion

In the present study, GFP mice were used as the spermatogenic cell donors. Since cells of these mice carried the GFP gene under the control of a chicken b-actin promoter and cytomegalovirus enhancer [11], it was expected that the transplanted cells and their descendants would express GFP and be easily distinguished from the endogenous cells of the recipient mice. In most previous studies, transgenic mice expressing LacZ gene were used as the donors. As a result, it was necessary to stain with X-Gal in order to discriminate transplanted from the endogenous cells [1-3, 6, 8, 9]. The present study and that of Ogawa et al [10] suggested that compared with the previous method, GFP mice have the following advantages as a donor: unnecessary to stain for visualization of transplanted cells and capable of real-time observation on transplanted cells without killing animals.

Unfortunately we could not observe the GFP fluorescence in the spermatozoa, because the chicken b-actin promoter for the GFP gene in the transgenic mice used in the present study was inactive in spermatozoa [11]. However, neither spermatogenesis nor spermatozoa was found in the seminiferous tubules of mice when non-concentrated cells were transplanted or in busulfan-treated mice that did not receive transplanted germ cells (data not shown). These data suggest that the spermatozoa observed in the present study are derived from the transplanted spermatogonia. Thus, we had an 86 % success rate of transplantation when using an enriched spermatogonia preparation isolated from adult mouse testis and it was 0 % when cells were transplanted without concentration. These results confirm the findings of previous investigators who suggested that the key to success in the germ cell transplantation is to transplant sufficient number of spermatogonia [1, 2].

We used discontinuous density gradient centrifugation with percoll to concentrate the germ cells. Percoll and centrifugation did not seem to damage the germ cells. Other methods, for instance flow cytometry, may also be applicable, however, percoll fractionation appears to be easier as labeling and special equipment are not needed.

This technique may also be a useful tool to identify the type of testicular cells damaged by certain chemicals so far not clearly elucidated, including methoxychlor, flutamide and environmental estrogens [13-16]. The use of adult mice as the germ cell donor may greatly extend the application of the technique.

In conclusion, germ cell population rich in spermatogonia collected from adult testes can be successfully transplanted to recipient seminiferous tubules and the discontinuous density gradient centrifugation with percoll is useful in obtaining such a cell population from adult testes.

Acknowledgements

The research was supported by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of the Environment, Japan.

References

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Correspondence to: Professor Chisato Mori, Department of Bioenvironmental Medicine (A3), Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670, Japan.
Tel: +81-43-226 2017, Fax: +81-43-226 2018
E-mail: cmori@faculty.chiba-u.jp
Received 2003-12-24 Accepted 2004-3-23