Culture of mouse germ cells isolated from fetal gonads

Experimental Cell Research 154 (1984) 530-S36

Culture

of mouse BARBARA

germ cells isolated

WABIK-SLIZ*

from fetal gonads

and ANNE McLAREN**

MRC Mammalian Dewlopment Unit, Wolfson House, University College London, London NW1 2HE, UK

Mouse germ cells isolated from male or female genital ridges at 12% days post coitum were cultured at room temperature for up to 6 days, with [“Hlthymidine present in the culture medium for either the first 24 h or the last 24 h of each culture period. Germ cells were also isolated 13%--16% days post coitum and cultured for 24 h in the presence of [3H]thymidine. The proportion of cells in metaphase, and the proportion of labelled interphase and metaphase nuclei, was recorded. The labelling index declined from 13% days onwards, after development either in vivo or in vitro. No labelled metaphase plates were seen after 24 h in the presence of [3H]thymidine, suggesting that under these culture conditions the duration of the G2 phase exceeded 24 h. The results showed that the culture system, in spite of the low temperature, allowed the germ cells to replicate their DNA and undergo mitosis for up to 6 days. Addition of db-CAMP to the culture medium proved highly toxic to male germ cells, and did not markedly increase the proliferation rate of female germ cells.

In the mammalian gonad, germ cells exist in close association with somatic cells, from which they may obtain both nutrients and developmental signals (for review see 111).An adequate system for the culture of isolated germ cells might reveal the extent to which processes such as migration, proliferation, differentiation and metabolic change are autonomous properties of the germ cells themselves, rather than being dependent on interactions with somatic cells. In addition, proliferating cultures of a pluripotent stem cell population would present many features of interest. So far there are few reports of culturing isolated germ cells. Heath [2] maintained rat primordial germ cells in vitro for up to one week but they did not divide and their viability progressively decreased. De Felici & McLaren [3, 4] devised a method of isolating and culturing fetal mouse germ cells: over a period of 5-6 days the germ cells showed both mitotic and meiotic activity, although their viability decreased, Germ cells isolated from early genital ridges would only survive at temperatures of less than about 30°C. Using the same culture system, the present paper examines the mitotic activity of mouse germ cells, and their capacity for DNA replication as judged by the uptake of [3H]thymidine. Since dibutyryl cyclic adenosine monophosphate (db* Present address: Department of Genetics and Evolution, Jagiellonian University, M. Kavasia 6, 30-060 Krakow, Poland. ** To whom offprint requests should be sent. Copyri&r @ 1984 by Academic Press. Inc. All righIs of reproduction in any form reserved 00144827/&4 $03.00

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CAMP) has been reported by Mayer [5] to increase the proliferation rate of cultured mouse embryonic pigment cells, we also examined the effect of dbCAMP on our germ cell cultures. MATERIALS AND METHODS Collection of Germ Cells Embryos were obtained from female mice of the randomly bred strains Q and MFl (OLAC), 12 %16% days post coitum (dpc). The gonads were dissected into phosphate-buffered saline (PBl; [7]) and sexed by their characteristic morphology. Germ cells were released into culture medium by pricking the gonads with a fine needle after incubation for 10-15 min in 0.02% EDTA [3]. Contamination with somatic cells, mainly red blood cells, did not amount to more than lO-20% of the total number of cells.

Culture Conditions The culture medium was Medium 199(M 199, Modified) with Earle’s Salts and 20 mM Hepes buffer (Flow Laboratories), supplemented with 1 mM r.-glutamine, 0.5 mM sodium pyruvate, 20 mM sodium lactate and 10% heat-inactivated fetal calf serum (FCS, Gibco). De Felici & McLaren [4] failed to identify any culture conditions that would maintain viability of 11% and 12% dpc germ cells at 37°C. We made further attempts, e.g., using McCoy’s 5 A medium, by adding db-CAMP to the culture medium, by culturing at room temperature for l-2 days and then transferring to 37”C, or by culturing on agar blocks, which allows survival and development in vitro of germ cells in 11% and 12%~day genital ridges [6], but these were uniformly unsuccessful. We therefore cultured germ cells from 12% dpc embryos in droplets of medium under liquid paraflin in air at room temperature and germ cells from 13’/2-16% dpc embryos either in similar droplets in a humidified atmosphere of 5 % CO2 in air at 37°C or in air at room temperature. Sodium bicarbonate was added to the medium at a concentration of 5 mM for cultures maintained in air or 15 mM for those maintained in 5 % CO2 in air [4]. The following components were added to the culture medium as necessary: 1 mM db-CAMP (Sigma); 25 yCi/ml i3H]thymidine (Amersham, UK); 0.2X 10’ M colcemid.

Examination of Cells The viability of cells in culture was determined using Erythrosin B in a dye exclusion test [3]. After culture, samples of cells were air-dried [8] and examined autoradiographically. Slides for autoradiography were covered with Kodak R-10 emulsion, exposed for one week, developed and stained with Giemsa. On each slide at least 100 nuclei were examined: a nucleus over which five or more grains were counted was scored as labelled. Cells in mitotic metaphase were also counted and scored for autoradiographic labelling of chromosomes; non-viable cells were not included.

Plan of Experiments Four series of experiments were carried out, as follows: Series I. Germ cells from 12% to 16% dpc embryos were cultured at room temperature in medium containing [‘Hlthymidine for 24 h; germ cells from 13% to 16% dpc embryos were similarly cultured at 37°C (fig. 1 a). Series ZZ.Germ cells from 12% dpc embryos were cultured in culture medium alone; at 24 h intervals, samples of cells were transferred to medium with [3H]thymidine for 24 h (fg 1b). Series ZZZ.Germ cells from 12% dpc embryos were cultured in medium with [3H]thymidine for 24 h and then transferred to culture medium alone. On each subsequent day samples were taken for making preparations; in some cases colcemid was added for the last 5 h (fig. 1c). Series IV. Germ cells from 12% dpc embryos were cultured in medium with db-CAMP for 24 or 48 h and then transferred to culture medium alone. On each subsequent day samples of cells were transferred to medium with [3H]thymidine for 24 h (fig. 1d). Exp Cell Res 154 (1984)

532 Wabik-Sliz and McLaren

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Fig. 1. Diagram of the plan of experiments. Germ cells: 4 , placed in culture; 8 , exposed to new component in medium; -, in vivo. Culture 111 medium: =; alone; ---, with [3H]thymidine; W, with colcemid; =, with db-CAMP. 0, Cells air-dried.

RESULTS

Germ cells showed clear differences according to age and sex in incorporation of [3H]thymidine; the labelling index was also affected by the temperature of culture. Fig. 2 a shows the proportions of labelled cells in samples from male and female embryos of various ages after 24 h in culture with [3H]thymidine at room temperature. Germ cells from 12% dpc male gonads showed a labelling index of about 60 %; this dropped sharply on subsequent days, reaching less than 5 % for germ cells from gonads 14% dpc or older. Germ cells from female gonads also showed about 60 % labelling at 12% dpc; the labelling index fell more slowly than with male germ cells, reaching less than 5% only at 16% dpc. The difference between male and female was statistically significant for germ cells from 13% and 14% dpc gonads (p
Cell Res 154 (1984)

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Fig. 2. Proportions of labelled germ cells (points) and cells in metaphase (columns) from embryos of

various ages, after 24 h of culture in medium with [3H]thymidine. (a) Room temperature; for 13% to 16% dpc, points represent results of one experiment only; for 12% dpc, as in (b). (b) 37°C. Each point here and in later figures represents the average of at least three experiments with 0, female or 0, male cells. Vertical range bars, SE.

male and female (fig. 4). Comparison with the labelling of germ cells taken from embryos 13% dpc (fig. 2) shows that 24 h in culture does not alter [3H]thymidine incorporation in female but appears to stimulate it in male germ cells (~~0.001). Some cells in metaphase were still seen after 3-4 days of culture of germ cells from 12% dpc embryos of both sexes (fig. 4), although the chromosomes were not labelled.

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Fig. 3. Viability of 12% dpc germ cells after various times of culture in medium alone (0, female; 0, male) and in medium with db-CAMP for 24 h, followed by culture in medium alone (H, female; Cl, male). Points and vertical range bars as in fig.

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Exp Cell Res 154 (1984)

534 Wabik-Sliz and McLaren

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Fig. 4. Proportions of labelled cells (points) and cells in metaphase (columns) in 12% dpc germ cells after different times in culture before [3H]thymidine was added. Points and vertical bars as in fig. 2 b. Fig. 5. Proportions of labelled 0, female; 0, male germ cells from 12% dpc embryos after 24 h of culture in medium with [3H]thymidine followed by 24, 48, 72 and % h of culture in medium alone. Points and vertical bars as in fig. 2 b.

Series III When germ cells from 12% dpc embryos were exposed to [3H]thymidine for the first 24 h of culture and then transferred to culture medium alone, the proportion of labelled cells stayed high (fig. 5). No significant differences were found between male and female germ cells. Mitotic metaphases were seen on each day of culture; their number increased after 5 h incubation in colcemid (fig. 6). The proportion of cells in metaphase was higher in germ cells from male than from female embryos, significantly so after 2 days in culture (t=2.6, ~~0.05). Some of the metaphase plates on each day of culture in both female and male cultures contained chromosomes labelled with [3H]thymidine (fig. 6). Series IV The survival of female germ cells was higher after 24 h incubation with dbCAMP than in control cultures, but male germ cells showed markedly decreased viability (fig. 3), so that only very few viable cells could be found 4 days after exposure to db-CAMP. When male germ cells were exposed to db-CAMP for 48 h, their viability was so low that no further experiments could be carried out. Female germ cells survived prolonged exposure to db-CAMP better, showing about 50 % viability after 48 h and a labelling index of about 30 % after subsequent culture with r3H]thymidine for 24 h. Exposure to db-CAMP for 24 h did not significantly affect the proportion of germ cells incorporating [3H]thymidine in either sex (fig. 7). Male and female germ cells showed similar numbers of metaphase plates; the proportion tended to be lower than in cultures not exposed to db-CAMP, but the difference was not significant. Exp Cd Res 154 (1984)

Mouse germ cell culture

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Fig. 6. Proportions of cells in metaphase when 12% dpc germ cells were cultured for 24 h in medium with [3H]thymidine followed by 0,24,48 and 72 h of culture in (a) medium alone, and (b) for the last 5 h of culture in medium with colcemid. Hatched columns, metaphase plates with labelled chromosomes. Vertical range bars, SE.

DISCUSSION Between 11% and 14% dpc, germ cells in the mouse testis undergo a last round of DNA replication and a last mitosis before entering mitotic arrest in the Gl (or GO) phase of the next cell cycle [6], the next DNA replication not taking place until the time of birth. In the mouse ovary, the last mitotic division of the germ cells takes place at about the same time as in the testis, but it is followed by a further round of DNA replication before the germ cells enter the prophase of meiosis. It is therefore not surprising that the labelling index for germ cells taken from fetal gonads 13%16% dpc and maintained for 24 h in culture in the presence of [3H]thymidine is consistently higher for female than for male embryos (fig. 2). However, more metaphase plates were observed among male than among female germ cells (figs 4, 6), suggesting that mitotic arrest in prospermatogonia may be delayed when germ cells are removed from the testis. The effect of culture temperature on the labelling index (fig. 2) suggests that fewer germ cells undergo DNA replication at the lower temperature. On the other hand, the apparent stimulation of [3H]thymidine incorporation when 12% dpc male germ cells are first maintained for 24 h in culture at room temperature, rather than being left for the equivalent period in vivo and then recovered as 13%

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Fig. 7. Proportions of cells labelled (points) and in metaphase (columns) after 24 h of culture of 12Y2 dpc germ cells in medium with db-CAMP followed by 0,24,48 and 72 h (days 1,2,3 and 4, respectively) of culture in medium atone before [3H]thymidine was added. Points and vertical range bars as in fig. 2 b.

Exp Cell Res 154 (1984)

536 Wabik-Sliz and McLaren dpc germ cells (cf figs 2,4), is probably due to the culture conditions retarding the development of the germ cells over a period when their labelling index is declining rapidly. The decline in labelling index over a period of several days in culture at room temperature (fig. 4) may reflect merely the comparable decline that takes place in vivo over the same period. The decline in viability that we observed (fig. 3) suggests that the culture system is suboptimal; however, DNA replication and mitosis was still occuring in male germ cells after 4 days of culture, and in female germ cells after 6 days. Since no labelled metaphases were observed when [3H]thymidine was added to the cultures for 24 h only (series I, II, IV), the duration of the G2 interval between DNA replication and mitosis must have been at least 24 h. Although in series III, [3H]thymidine was present only for the first 24 h of culture, some labelled metaphases were observed on each subsequent day; their number was not increased by the presence of colcemid for the last 5 h, so they may represent a population of cells that underwent DNA replication shortly after entering culture and was subsequently blocked in metaphase. On the other hand, the much larger number of unlabelled metaphase plates that we observed was markedly increased on each day of culture by the addition of colcemid (fig. 6) suggesting that these metaphases represented new cells entering mitosis throughout the culture period. However, in the absence of cell counts or grain counts, we cannot be certain to what extent the germ cell population was capable of proliferation in our culture system. db-CAMP in the concentrations that we used proved highly toxic to germ cells from male embryos. Female germ cells were less adversely affected but only showed a slight stimulation of [3H]thymidine incorporation, rather than the dramatic increase in proliferation rate produced in embryonic pigment cells [5]. We conclude that our culture system for 12% dpc germ cells, in spite of the relatively low temperature at which it operates, does not merely maintain the germ cells in a static condition, but allows them to replicate their DNA and to undergo mitosis for up to 6 days. REFERENCES 1. McLaren, A, Germ cells and soma: a new look at an old problem. Yale University Press, New Haven (1981). 2. Heath, J K, Development in mammals (ed M J Johnson) vol. 3, p. 267. North-Holland Publ. Co., New York (1978). 3. De Felici, M & McLaren, A, Exp cell res 142 (1982) 476. 4. - Ibid 144 (1983) 417. 5. Mayer, T C, Dev biol94 (1982) 509. 6. McLaren, A, Controlling events in meiosis. Sot exp biol symp 38 (ed H C Dickinson) Cambridge University Press (1984). In press. 7. Whittingham, D G & Wales, R G, Aust j biol sci 22 (1969) 1065. 8. Tarkowski, A K, Cytogenetics 5 (1966) 394. Received March 23, 1984 Exp Cell Res 154 (1984)

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