In vitro approach to fertility research: Genotoxicity tests on primordial germ cells and embryonic...

Reproducuve Toxicology, Vol 7, pp 69-73, 1993 0890-6238/93 $6 00 + 00 Pnntcd m the U S A All rights reserved Copynght © 1993 Pergamon Press Ltd

• Female Reproductive Toxicity

I N V I T R O A P P R O A C H T O F E R T I L I T Y R E S E A R C H :

G E N O T O X I C I T Y T E S T S O N P R I M O R D I A L G E R M C E L L S A N D

E M B R Y O N I C S T E M C E L L S

RICHARD VOGEL Max von Pettenkofer-Instltute, Federal Health Office, Berhn, Germany

Abstract - - In vitro screening tests for reproductive toxicology are required by the 7th amendment of the directive 67/548 EEC and the OECD-programme on existing chemicals. Unfortunately, appropriate methods for testing developmental toxicity or impairment of fertility are not at hand. Therefore, we have tried to design test method based on mutagenic effects in germ cells that may be used as a test of fertility impairment as well. Embryonic stem cells (ESC) derived from mouse blastocysts can be kept in culture routinely. Establishment of ESC and improvement of their culture conditions are described and special properties of ESC are discussed in relation to germ cells. Because some properties of germ cells and pluripotent embryonic stem cells (ESC) are found to be comparable, permanent lines of ESC hold promise to be used as an in vitro test of impairment of fertility.

Key Words fertility, genotoxlclty, pnmordml germ cells, embryonic stem cells, in vitro mouse

THE NEED FOR IN VITRO TESTING ON REPRODUCTIVE TOXICITY

Impairment of fertility was defined m a recent draft of the EEC-commission on general classification and labelling requirements for dangerous substances and preparations: "Effects on male or female fertility Include effects on libido, sexual behaviour, any as- pect of spermatogenesis or oogenesis or on hor- monal activity or physiological response which would interfere with the capacity to fertllise, fertlllsatlon Itself or the development of the fertilised ova up to and including implantation." Special risk phrases are provided for chemicals that Impair fertility. These chemicals, as well as those that are terato- genlc, will be classified in the EEC as "toxic to reproduction."

Information on fertlhty of both male and female parent animals can be obtained from multlgeneratlon studies for which standard protocols are available [for example, OECD 1983 (1)]. The cost and the duration of this kind of Investigation are consider- able and great numbers of animals are necessary. Therefore, competent authorities call for multigen-

Address correspondence to Richard Vogel, Max von Pet- tenkofer-Instltute, Federal Health Office, D-14195 Berlin, Germany

eration studies only when a new chemical will be placed on the market. However, with respect to the forthcoming international programmes on existing chemicals (EEC, OECD), short term in vitro tests should be established to screen for reproductive toxicity. In consideration of the great number ofexlstmg chemicals to be tested, full-time in vivo studies would consume too many animals and too much time and money. Some aspects of the above defined effects on fertility could be covered by investigations of adverse effects on male and female gametes. In the field of genetic toxicology a variety of germ cell tests are available. These test systems, for example the ooycte test, the zygote test, and the dominant lethal test, would provide informations on female fertility as well. In mutagenicity testing, however, germ cell tests are rarely used because only very few cells (embryos) can be scored per female and, thus, an enormous number of females is required. Additionally, chromosome abnormatities and lethal gene mutations are the only endpoints that can be determined. Therefore, we have designed a new approach to establish a screening test on germ cell mutageniclty that may be used as a test on reproductive t o x i c i t y as well. Because some cellular and biochemical properties of germ cell cells and pluripotent embryonic stem cells (ESC) are comparable, permanent lines of ESC should be used as an in vitro

69

70 Reproductive Toxicology Volume 7, Supplement 1. 1993

model provided that the culture conditions can be simplified.

CULTURE OF PLURIPOTENT EMBRYONIC STEM CELLS

Since the estabhshment of routine culture methods of embryonic stem cells derived from mouse blastocysts (ESC) by Evans and Kaufmann in 1981 (2), ESC became a tool to study gene expression and cell differentiation during early embryonic development (3,4). ESC have a number of advantages compared to embryonic derived carcinoma cells (ECC), which had been used until then ESC retain the euploid chromosome constitution and they are able to differentiate into a variety of embryonic tissues. The differentiation potential of ECC turned out to be limited and was variable between the different lines used (5,6).

In our laboratory blastocysts were obtained from mice of the strain NMRI. Animals were housed at 22 °C in an atmosphere of 60% humidity. The day after an overnight mating penod was designated day 0 of pregnancy. ESC lines were established as described by Robertson (7) using blastocysts flushed from the uteri on the third day of pregnancy. Cells can be frozen in DMEM with 10% FCS and 10% DMSO and stored under liquid nitrogen until use. The basic medium for culture of ESC was DMEM supplemented with penicillin (50 unlts/mL), streptomycin (50 txg/mL), glutamine (1 mM), non-essential amino acids, and 2-mercaptoethanol (0.1 mM). Ad- ditionally, the medium contained fetal calf serum (FCS) and newborn calf serum (NCS) in a final con- centratlon of 10% each. This medium was designated as ESC-medlum and used for ESC growing on a feeder layer.

Each of the three cell lines used In the present study has a male euploid karyotype: 40 (XY). The euploid chromosome complement has not changed to date in more than 100 passage generations. Poly- plold cells were detected only randomly (-<5%). An increase in polyploid cells has always pointed to unfavourable culture conditions, e.g., exhausted medium or when cells were plated at too high a density. As most polyploid cells grow slower they are quickly overgrown by diploid cells when conditions improve.

Feeder-dependent culture of ESC The difficulty of keeping pluripotent stem cells

in an undifferentiated state has limited the use of these cells. ESC can be grown on mitotically inactivated feeder cells, which are prepared from STO fibroblasts (8) or from primary embryonic fibroblasts

(9) Flbroblasts are thought to produce a factor to inhibit differentiation (I0), which can be substituted by an inhibitory factor found in buffalo rat liver (I I) and also by myelold leukaemia inhibitory factor (12). Once inactivated, feeder cells can be used for ap- proximately ten days Embryomc fibroblasts have a limited hfespan m culture. Consequently, fresh preparations have to be made regularly Moreover, the source of feeder influences the morphology of ESC and their ablhty to differentiate (13). Feeder cells are established and maintained as follows:

Primary embryonic fibroblasts are prepared according to Robertson (7). Embryos are taken on the fourteenth day of pregnancy. Flbroblasts are cultivated In 80 cm 2 plastic flasks m DMEM supplemented with penicillin (50 unlts/mL), streptomycin (50/xg/mL), glutamine (I mM), and 5% NCS. During the first 24 h of culture 10% FCS is used instead of 5% NCS because this regime improves cell adhe- sion. Sera are heat-inactivated at 56 °C for 40 min Cells are placed in a humidified incubator at 37 °C in an athmosphere of 5% CO2. They are subcultivated once a week using 0.05% trypsin, 0 02% EDTA for disaggregatton Medium is changed twice a week. Embryomc fibroblasts for feeder cells or conditioned medium are used after the first passage up to four weeks Redundant fibroblasts are frozen after the first passage as described for ESC and stored at -80 °C for several weeks Feeder layer fibroblasts were rendered mitotically inactwe by treatment w~th m~- tomycin C (10 /zg/mL) After 2 5 to 3 h cells are harvested and plated on gelatine coated tissue flasks (25 cm2). Inactivated fibroblasts are used at the last for one week.

Feeder-independent culture of ESC Because feeder cells of different origin may in-

fluence ESC differently and because they may interfere when biochemical differentiation is investigated, we have improved culture conditions to maintain ESC in the undifferentiated state even in the absence of feeder cells or other sources of inhib- iting factors. ESC-medlum was varied as follows to keep ESC without feeder cells: a) Conditioned medium was mixed at equal shares

of a medium in which embryonic fibroblasts had grown and a double concentrated stock solution, so that all ingredients were as concentrated as in stem cell medium

b) Semtdefined medium contained 5% FCS, 5 tzg/ mL bovine insulin, 10 ~g/mL transferrin, and 0.03/zM sodium selenite.

Medium was prepared freshly every week. ESC were cultivated in 25 cm 2 plastic flasks in an athmosphere of 5% COz at 100% humidity For feeder-

Ferhhty stu&es m wtro • R VOGEL 71

Table 1. Rate of undifferentlal colonies m different media after one week of culture.

Number of Number of ES % ES cell

Medium colomes cell colomes colomes

ES-medmm 542 99 18 3 Condmoned

medmm 604 87 14 4 Semldefined

medmm 310 201 64 8

independent cultivation, flasks were coated with 0.1% gelatine in PBS. Cells were subcultivated three times a week.

To compare cell proliferation ESC were taken from the feeder layer and plated in equal densities on plastic dishes (24-well), fed with the medium under test and incubated for one week. Colonies were then fixed and counted. For staining, ESC were washed twice in PBS and subsequently fixed in 20% formaldehyde in PBS (5 to 10 min) and methanol (5 to 10 min) and stained in 2 to 5% Giemsa in distdled water (5 to 15 min).

When cells were plated m equal (low) densihes, the number of colonies sigmficantly differed in the three media after one week (Table 1). Using "defined" medium the majority of colonies stdl con- sisted of ESC and ESC colonies were much larger compared to the other medm. It in&cates that semidefined medium inhibited differentiation or at least growth of differentiated cells. After up to 25 passage gener/atlons plating was repeated to investigate whether differentiation has proceeded. Fortunately, the portion of differentiated colonies never exceeded 6% m ESC populations grown in semidefined medium. Evidently, these culture conditions are most appropriate to maintain ESC without feeder cells.

The new procedure of culturmg ESC m the absence of feeder-cells still reqmres feeder-dependent establishment of ESC from blastocysts as the first step. Although this study was performed only on three ESC lines, preliminary results with other ESC lines suggested that they can also be adapted and cultured in semidefined medium. Feeder-independent culture of ESC offers many advantages for studying special problems; evaluation of genetic and biochemical parameters are no longer interferred with by a second cell species.

PLURIPOTENT ESC AS A MODEL FOR GERM CELLS?

Special properties of ESC Differential staming of the identical (sister)

chromatids of a chromosome enables the determina-

tion of two different endpoints: sister chromatid exchanges (SCE) and cell proliferation. SCE represent DNA double-strand breakage and reunion at appar- ently homologous loci between the two chromatids of a chromosome. SCE is believed to reflect DNA damage (14). Therefore, SCE assay is routinely em- ployed to detect the mutagenic potential of specific agents although httle is known about the molecular basis of SCE (15). Investigation of SCEs in non-ring chromosomes requires differential incorporation of thymldme analogues like bromodeoxyuridlne (BrdU) into the DNA of the sister chromatids (16). However, the mutagen BrdU reduces SCE itself and calculation of a spontaneous SCE level is difficult. Therefore, experimental conditions must be similar for comparison of the baseline SCE frequency between species, or tissues or cells of different developmental stages.

To date, BrdU incorporation into the DNA of preimplantahon mouse embryos is only possible during in vitro culture at conditions and BrdU con- centrations quite different from those used for fetal or adult cells. Furthermore, data from SCE assays on prelmplantatlon mouse embryos obtained from non-hormone treated mother ammals are not con- Stlstent: whde Elbling and Color (17) reported an extremely low SCE frequency, other investigators found an unusually high SCE background rate as compared to other cell types (18,19). There is some evidence for an unusual SCE frequency in pluripotent cells, however, SCE-rates of preimplantatlon mouse embryos and tissues from adult mtce may not be comparable directly.

SCE-frequencies were determined m permanent lines of pluripotent embryonic stem cells (ESC lines B, D, and F) derived from preimplantation mouse embryos. ESC are maintained in an undifferentiated stage without feeder cells using semidefined medium. BrdU-labelhng was carried out m exactly the same way as m any other cell culture. Therefore, after induction of differention, ESC-derlved cell types could be investigated m comparison to ESC. After changing the pH of the culture medium by reduction of the CO2 atmosphere from 5% to 2%, differentiation of the hne ESC-F was induced. Three weeks after initiation a portion of the cells differentiated from ESC-F showed an epitheloid morphology. These cells (Diff-F) were separated by subcloning and kept for 20 passages in 5% CO2 atmosphere using the same medium as described for stem cells. For differential staining of sister chromatids, BrdU was added to the cultures to give a final concentration of 10 .5 M. ESC were prepared on slides after 24 h and fibroblasts after 48 h of culture m the pres- ence of BrdU. Diff-F cells were harvested at both

72 Reproductive Toxicology Volume 7, Supplement 1, 1993

Table 2 SCE frequencies m cells of different origins

SCE per Cell type metaphase

24 h 48 h

Range M 1 M2 M3 M 1 M2 M3

ESC-B 225-+46 ESC-D 247-+44 ESC-F 236-+ 39 Dlff-F 112-+24" Flbroblasts 9 5 -+ 2 2*

17-31 0 70 30 not done 18-44 8 92 0 not done 15-40 4 92 4 0 8 92 6-22 63 37 0 3 75 22 3-16 97 3 0 7 54 39

*Stgmficantly different from ESC, Student's t test (P -< 0 001) At least 50 complete metaphases were scored per entry

time points. Slides were stamed according to the Fluorescence-Plus-Glemsa technique (17).

First division metaphases (M1) are evenly stained, second division metaphases (M2) show re- ctprocal, and third division metaphases (M3) nonre- ciprocal sister chromatld exchanges. At least 50 complete M2 were scored for SCE. Prohferation kinetics were determined on 200 metaphases per cell type by the relation of M1 to M2 to M3.

SCE-rates obtained with three different ESC- lines were found to be unusually high (Table 2), but comparable to SCE-rates in blastomeres of prelmplantation mouse embryos. Under identical culture conditions using the same BrdU concentration, SCE frequency decreased drastically when plunpotent cells of line ESC-F start to differentiate (Table 2). This change was obviously lndependant of differences in cell cycle kinetics, since prolongation of the cell cycle did not influence SCE frequencies in Diff-F cells (Table 2). Similar results on SCE-levels in relation to cell cycle length had been reported for preimplantatlon mouse embryos (19) In general, SCE frequencies were shown to be elevated by an increased uptake of BrdU. With respect to the very short cell cycle of the pluripotent cells compared to diffentlated cells in vitro, an extremely high BrdU uptake is unlikely. This is supported by the fact that the BrdU concentration used was near the resolution limit for sister chromatld differentiation. The unusually high SCE frequency m ESC, therefore, seems to reflect a special property of plunpotent cells

Induced SCE's are found to persist In cells from prelmplantatlon mouse embryos (20) as well as in other cell types (21). Therefore, most of the DNA lesions leading to SCE are obviously not repaired but by-passed according to a model postulated by Shafer (22). By-passing of DNA lesmns results in a still damaged daughter cell and a normal one. Thus, SCE enables regular replication of even damaged DNA. In contrast, activation of cellular DNA repair mechanisms often leads to chromosomal aberrations followed by death of the aberrant cell. Loss of a

critical number of plurlpotent cells, however, may be lethal for prelmplantation embryos. Data from the present study and previous investigations (18-20) demonstrate that plunpotent embryonic cells seem to prefer SCE-management of DNA damage, which allows normal proliferation thus avoiding the dan- gers of misrepair, cell death, and loss of early embryos in vivo This assumption is in good accord with results from experimental embryotoxlclty studies that point to far less sensitivity for developmental disruption during the pretmplantatlon period compared to organogenesls (23-25). Embryolethal and teratogenic effects archieved after treating male or female animals prior to fertlllSatlon have been quite similar to those after treatment during the prelmplantatlon period. It is likely that similar mechanisms of repair or reaction to DNA-damage are active m blastomeres as well as in germ cells.

Further biochemical markers of ESC that can be related to germ cells have been reported: a tran- scription factor (Oct 4) was identified recently It is expressed only in primordial germ cells, unfertdized oocytes, and the inner cell mass of prelmplantation embryos. Additionally, the alkaline phosphatase was described as another enzyme occurring only in primordial germ cells, zygotes, and blastomeres.

Future aspects So far, it has not been possible to determine

in greater detail the basic cellular and biochemical properties of germ cells as well as undifferentiated cells derived from early mammalian embryos. Due to the improved culture condttIons we are encour- aged to characterize ESC by investigating a variety of genetic and biochemical endpolnts. Fortunately, primordial germ cells (PGC) can be kept in vitro as primary cultures (26,27). Therefore, PGC should be the reference system for the future experiments with ESC. Consequently, the background frequencies of chromosome aberrations and sister chromatid exchanges, as well as alkahne phosphatase activity and repair capacity will be compared. Additionally, the

Fertthty studies m vitro • R VOGEL 73

reaction of the two cell types upon treatment with germ cell mutagens should be tested and compared to published data obtained by in vtvo germ cell experiments. Properties similar to those of the germ cells and more simple culture conditions hold prom- tse that ESC can serve as an appropriate indicator test system for reproductive toxicity, in particular as a screen for adverse effects on fertility.

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11 Smith AG, Hopper ML Buffalo rat liver cells produce a diffusible actavlty wMch inhibits the dlfferentlatmn of murlne embryonal carcinoma and embryomc stem cells Develop- mental Blol 1987,121 1-9.

12 Williams RL, Hilton DJ, Pease S, Geanng BP, Wagner EF, Metcalf D, Nlcola NA, Gough NM. Myelold leukaemia mhlb- ltOry factor mamtaans the developmental potential of embryomc stem cells Nature 1988,336.684-687

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14 KatoH Inductionofslsterchromatldexchangesbychemlcal mutagens and its possible relevance to DNA repair Exp Cell Res 1974,85 239-247

15 Latt SA, Allen J, Bloom SE, Carrano A, Faike A, Kram D, Schneider E, Schreck R, Tlce R, WMtfield B, Wolff S. Sister chromatld exchanges' a report of the GENE-TOX program Mutat Res 1981,87:17-62

16 Perry P, Wolff S New Giemsa method for the differential staining of sister chromatids Nature 1974,251 156-158

17 ElbhngL, ColotM Amethodforanalysmgstster-chromatld exchange m mouse prelmplantatlon embryos Mutat Res 1985,147 23-28

18 Porter AJ, Slngh SM Toxicity and sister chromatld exchanges in cultured prelmplantation mouse embryos exposed to serum from cyclophosphamlde-treated rats possible lmph- cations for testing maternal serum genotoxlclty Mutagene- sis 1988,3 45-49

19 Vogel R, Splelmann H Cytotoxlc and genotoxlc effects of bromodeoxyundlne during in vitro labelling for sister chromatld differentiation m prelmplantatlon mouse embryos Mutat Res 1988;209 75-78

20 Vogel R, Granata I, Spmlmann H Cytogenetic studies on prelmplantatlon mouse embryos exposed to methylm- trosourea m vlvo. Reprod Toxlcol 1989,3.23-26

21 Endo A, Watanabe T Individual and strmn differences in patterns of long-term persistence of urethane-mduced sister chromatld exchanges (SCEs) m mouse lymphocytes and their relatmn to carcinogen susceptlblhty Environ Moi Mutagen 1988,12 375-383

22 Shafer DA. Replication bypass model of sister chromatld exchanges and implications for Bloom's syndrome and Fan- conl's anemm Hum Genet 1977,39 177-190

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