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Isolation of chick primordial germ cells from stages 4 У8 embryos.

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THE ANATOMICAL RECORD 235:604-610 (1993)
Isolation of Chick Primordial Germ Cells From
Stages 4-8 Embryos
GEORGE MATSUMURA AND MARJORIE A. ENGLAND
Department of Anatomy No. 2, Hokkaido University, Sapporo 060, Japan (G.M.);
Department of Anatomy, University of Leicester, Leicester, Great Britain (M.A.E.)
ABSTRACT
Chick embryo primordial germ cells (PGCs) stages 4 4
were manually isolated for the first time from the late hypoblast layer. They
were confirmed to be PGCs by periodic acid-Schiff (PAS) staining and examination by scanning electron microscopy (SEM). They were subsequently introduced onto a variety of artificial substrata. On two dimensional substrata, the cells change from a spherical shape covered with
numerous microvilli to a rounded cell with a “skirt” of cytoplasm. Eventually a process projects from one side of the smooth cell.
On a three dimensional substrate the PGCs change from a spherical
shape covered with numerous microvilli to a smooth surfaced cell with a
long single process.
It is concluded that the PGCs which are originally spherical in situ in
stage 4 alter their morphology both in vivo during their migration and in
vitro studies. o 1993 Wiley-Liss, Inc.
MATERIALS AND METHODS
Primordial germ cells (PGCs) in the early chick embryo have been extensively studied since 1870
Fertilized White Leghorn chicken eggs were incu(Waldeyer, 1870). The focus of numerous studies has bated at 37°C until stages 4-8 (Hamburger and Hamilbeen the origin, location, and migration of these cells ton, 1951). The embryos were then mounted as for New
into the gonads (Swift, 1914; Goldsmith, 1928; Rogul- Culture (New, 1955) and all excess yolk particles
ska, 1968; Fujimoto et al., 1976a, b; Fargeix et al., washed off.
1980; Eyal-Giladi et al., 1981; England, 1983; England
et al., 1986; Kuwana et al., 1986, 1987; Nakamura et
PGCs in Normal Stage 4 Embryos
al., 1991; Ukeshima et al., 1991). The PGCs originate
Embryos
mounted by New Culture were fixed in
in the epiblast (Eyal-Giladi et al., 19811, translocate to
Karnovsky’s
fluid (Karnovsky, 1965) for 3 hours and
the hypoblast, and finally during the primitive streak
and headfold stages separate from the hypoblast. They transferred to cacodylate buffer (Plumel, 1948). The
migrate along a fibrous band (Wakely and England, ectoblast layer was dissected off over the anterior area
1979) which is rich in fibronectin, sulphated glycosami- pellucida/area opaca border to expose PGCs within the
noglycans (Critchley e t al., 1979), and collagen type I underlying hypoblast layer. The specimens were then
(England et al., 1982). This band forms a n arc located prepared for scanning electron microscopy (SEM).
a t the cephalic border of the area pellucidalarea opaca
Isolation of PGCs for Culture
and its position exactly corresponds with the location of
The New Culture glass ring was filled with saline.
the PGCs described by many investigators (Swift,
1914; England, 1983; Kuwana et al., 1987). As the The PGCs are known to populate a n area along the
PGCs move along this fibrous band they physically de- cephalic area pellucida/area opaca border. This region
form and rearrange the fibers (England, 1983; England was identified (Fig. 1)and the hypoblast overlying this
et al., 1986). Subsequently, the PGCs enter the devel- border was carefully dissected away in a single piece
oping vascular supply (Dubois, 1969) and ultimately using Rebutia hybrid cactus needles mounted on
wooden cocktail sticks (England, 1981). As the hypopopulate the gonads (Ukeshima et al., 1991).
Although the PGCs are large, round, periodic acid- blast was separated up to 30-40 PGCs floated free and
Schiff (PAS)-positive cells they have not been isolated came to settle on the underlying ectoderm layer. In
and cultured for in vitro studies in the early chick em- addition to PGCs separating, yolk granules also float
bryo. Fujimoto et al. (1976a) isolated chick PGCs from free and come to lie with the PGCs. The only distinthe circulating blood a t stage 8 and cultured them in guishing features we were able to recognize between
vitro for a short time (unspecified) to study their mode
of migration and morphology. To date these have been
the youngest chick PGCs isolated for in vitro culture.
In the present study, we report the isolation, culture,
Received April 1, 1992; accepted September 11, 1992.
and examination of PGCs of the much earlier stages
Address reprint requests to Dr. G. Matsurnura, Department of
4-8 in the chick embryo.
Anatomy No. 2, Hokkaido University, Sapporo 060, Japan.
0 1993 WILEY-LISS, INC.
ISOLATION OF PRIMORDIAL GERM CELLS
0
/.
0
.\*
foo
Fig. 1. A diagram to illustrate the position of the chick PGCs at
stages 4-8.
the yolk and the living PGCs were a n elliptical shadow
on the PGCs and their size.
Preparation of Substrates for PGCs
PGCs were removed for in vitro studies using fine
glass pipettes attached to a mouthpiece and rubber tubing. They were transferred to a variety of substrata and
media. All of the equipment used was sterilized by
overnight ultraviolet radiation. The substrata included
glass coverslips, plastic (Thermonox Nunc., Inc.) coverslips, Sterispon (Allen & Hambury Ltd., London), and
Spongostan (Ferrosan, Denmark). Sterilized coverslips
(Thermonox) were used as supplied by the manufacturer. Glass coverslips were washed in distilled water,
some were further washed in acid alcohol (0.5%HC1 in
70% ethanol) and then dried in a plastic Petri dish and
placed overnight in the ultraviolet radiation cabinet.
The media used included normal physiological saline
and Medium 199 (Gibco). In addition to using plain
glass and plastic surfaces, these same substrata were
coated with purified collagen or 1% agar or rat tail
collagen. These conditions provide a direct comparison
with the work described by Kuwana et al. [1986,1987].
Purified collagen as supplied by the manufacturer, or
diluted, was placed on the coverslip the day before use
and allowed to dry overnight in a covered Petri dish a t
room temperature. This produced either a thick or a
thin layer of collagen on the coverslip. These coated
coverslips were used a s follows: a drop of medium was
placed on the coverslip and the PGCs added immediately. They were either left at room temperature for 0
minutes, 15 minutes, and 30 minutes before fixation or
incubated at 37°C for 30 minutes before fixation in
Karnovsky’s fluid.
Alternatively the coated coverslips were dried overnight and the next day the entire coverslip was added
to either saline or Medium 199 (Gibco) and left overnight a t room temperature. The PGCs were added to
the coverslip and incubated for 15 and 30 minutes at
room temperature. They were then fixed in Karnovsky’s fluid for SEM.
A solution of rat tail collagen (0.4%)was prepared by
adding distilled water to a bottle of collagen and shak-
605
ing it until the collagen dissolved. It was stored at 4°C
in the refrigerator. Before use the collagen was allowed
to warm to room temperature. Then it was added to the
coverslip and either left a s a drop or spread across the
coverslip. Coverslips were dried overnight at room
temperature in a Petri dish. The PGCs were then added
for 0 hours, 3 minutes, 15 minutes, 30 minutes, and 40
minutes.
Rat tail collagen with acetic acid (Kuwana et al.,
1987) was also used by adding 1 mg of collagen/ml of
1% acetic acid. Either a single drop was used or spread
across the coverslip and this was dried overnight in a
Petri dish at room temperature. These coverslips were
then used as the coverslips above.
Another substrate was prepared by dissolving agar
in distilled water to make a 1% solution (at 60°C) and
then using a warmed pipette placing a drop of agar on
the coverslip. After cooling, PGCs in saline were placed
on the agar and incubated for 15 minutes a t room temperature. They were then fixed in Karnovsky’s fluid.
The stages 4-8 PGCs were removed from the embryos and placed in contact with one of the above substrates and left for either 0, 10-15, or 30 minutes a t
room temperature. They were subsequently prepared
for SEM by carefully introducing drops of Karnovsky’s
fluid (Karnovsky, 1965) onto the medium used. Comparisons were also made with yolk granules.
Sterispon and Spongostan
Sterispon and Spongostan were used a s supplied by
their manufacturers. A very thin slice of either was cut
using a razor blade. The thin slice of gelatin was attached to a Cambridge stub using double-sided Scotch
tape (3M). PGC preparations were added directly to the
gelatin and left in contact for 15 and 30 minutes and
prepared for SEM.
Scanning Electron Microscopy
Specimens were fixed for 3 hours and then in buffered cacodylate (Plumel, 1948) overnight. The PGCs
were postfixed in 1%0, 0, in 0.1 M cacodylate buffer
for 30 minutes and dehydrated in a n ascending series
of ethanol/water from 30%-100%. They were transferred to 100% acetone and stored in sealed vials. The
specimens were critical point dried in a Polaron critical
point drier by acetone replacement. They were then
mounted on Cambridge stubs using silver Dag and
coated with 20 nm of gold in a Polaron coating unit.
The PGCs and their substrata were viewed in a
DS130 International Scientific Instruments scanning
electron microscope a t a n operating voltage of 9-15
KV. Photographs were taken using a fine grain ultraslow film (Kodak Technical Pan 2415).
Periodic Acid-Schiff Staining
Isolated PGCs and wholemount chick embryos were
also stained with PAS stain to determine the position
and appearance of PGCs in vivo. Isolated PGCs were
also stained with PAS to prove they were PGCs and not
somatic cells. Both the wholemount embryos and the
isolated PGCs were fixed in Karnovsky’s fluid for 3
hours, buffered in 0.1 M cacodylate overnight. They
were then stained in a 1% aqueous periodic acid solution (BDH Poole) for 20 minutes. They were transferred to Schiff‘s solution for 30 minutes, rinsed in dis-
606
G. MATSUMURA AND M.A. ENGLAND
Fig. 3. An isolated PGC. Note the nucleus (n). x 750
to their substrate often exhibit after 15-20 minutes a
sheet of cytoplasm projecting from the cell which conFig. 2. Chick PGCs in vivo in the stage 4 embryo as viewed by
scanning electron microscopy. The surrounding hypoblast cells (h) tacts the substrate (Fig. 6). This sheet resembles a
remain in contact with the PGCs. Note the presence of microridges (r) “skirt” projecting in a complete circle from the cell
and short microvilli (v). x 3,125.
base. Numerous microvilli are present on this skirt.
The upper regions of the cells retain their spherical
shape and microvilli. Some blebs also appear on the
tilled water three times, mounted on a slide in water, upper surface.
PGCs placed in contact with thick layers of collagen
and the coverslip sealed with nail varnish. These were
compared with living PGCs mounted on a glass slide in initially exhibit a similar appearance to those cells
distilled water without staining. Photographs were placed on glass or plastic coverslips. After 15-30 mintaken immediately on Kodak Ektachrome EPY135 or utes blebs are present (Fig. 7) and the cells appear to be
deforming the surrounding fibers of collagen.
on Kodak Technical Pan 2415.
The PGCs adhere best to the gelatin substrate SterRESULTS
ispon or Spongostan. Although 30-50 PGCs were
PGCs In Situ and PAS Staining
placed on a single coverslip (or substrate) often only 10
PGCs in situ in the hypoblast layer of the stage 4 PGCs or less adhered and were subsequently examined
chick embryo have a characteristically spherical shape. by SEM. When 30-50 PGCs were introduced on the
By SEM their surfaces are covered with numerous three dimensional gelatin latticework, approximately
small microridges and some punctate microvilli (Fig. the same numbers were examined by SEM. At zero
minutes, the PGCs resemble those cells in situ and
2).
In those wholemount stage 4 embryos stained by those cells introduced on other substrata (Fig. 8). By
PAS a large number of PGCs were evident in the re- 15-30 minutes, however, the cells often exhibit a broad
gion anterior to the primitive streak and concentrated process which varies from specimen to specimen in its
in the region of the area opacatarea pellucida border. length and breadth (Figs. 9,101. The spherical shape of
PGCs isolated from these specimens or PAS stained the PGC has altered to a more flattened corpus with a
following isolation confirmed that these large cells are broad projecting process. Few or no microvilli are
PGCs. By light microscopy, the cells are large and present on the upper surface of the germ cell. By 40
rounded with numerous refractile lipids in their cyto- minutes some of the processes are longer than those
plasm and a n identifiable nucleus (Fig. 3). They are present a t 15-30 minutes.
PAS positive. They often have a blunt process evident
DISCUSSION
bulging from one side of the cell.
PGCs and Artificial Substrata
With the exception of the two gelatin substrata, Sterispon and Spongostan, few PGCs adhere to the combinations of substrata employed in this study. The findings are summarized in Table 1.
Those stages 4-8 PGCs placed on glass coverslips or
Thermonox plastic initially resemble those PGCs observed in situ (Fig. 4). They are spherical but covered
with numerous long microvilli and punctate blebs.
Only a small number of microridges are present. There
are additionally, however, within 15 minutes some
small blebs present (Fig. 5). Those cells which adhere
/solation of PGCs
In the present study, stages 4-8 living chick PGCs
were isolated for the first time from the late hypoblast
layer. It should be emphasized that this method is only
achieved with lengthy experience. The average size of
chick PGC is 11.4 pm (Kuwana et al., 1987). As they
are in a mixed population of yolk and hypoblast cells
their separation by hand is difficult. The two criteria
used for separation were size and a n elliptical shadow
on the PGC edge.
The isolated cells were proven to be PGCs by PAS
staining and examination by SEM. The cells were subsequently introduced onto a variety of artificial sub-
607
ISOLATION OF PRIMORDIAL GERM CELLS
TABLE 1. A summary of combinations of in vitro
substrata tried in culturing stages 4-8 PGCs'
Substrate used
Plain glass coverslip
Thermonox plastic coverslip
Glass coverslip plus 1%agar
Glass coverslip plus purified
collagen
Glass coverslip plus rat tail
collagen
Thermonox plus 1% agar
Thermonox plus purified
collagen
Thermonox plus rat tail
collagen
Sterispon
Spongostan
Stages 4-8
PGCs +
Saline
__
\
Stages 4-8
PGCs +
Medium 199
__
\
\
\
\
\r
L
\+
\+
I+
\+
'--\, no attachment; - \, majority of specimens not attached; ,, approximately half specimens attached; ,+ , approximately three-quarters of
all specimens attached.
strata. On glass, plastic, or thin collagen layers, the
cells change from a spherical shape covered with numerous microvilli to a rounded cell with a skirt of cytoplasm. Eventually a process projects from one side of
the cell which loses its microvilli. On thick collagen, or
three dimensional gelatin lattices, the PGCs project
broad processes and lose their microvilli.
Cell Movement
Some features of the present study closely resemble
the morphological stages of attachment on glass, plastic, and collagen as described by Kuwana et al. (1987).
There are, however, several major differences between
the present work and the study by these authors. They
describe the PGCs isolated from the circulating blood
somite stage 19 in vitro as migrating on a thin collagen
layer either in isolation or in conjunction with gonadal
ridge tissue. Initially the cells are covered by numerous
microvilli which adhere only with the tips of their microvilli. If no gonadal ridge tissue was present, the
PGC elongated on its substrate without movement. If
gonadal ridge tissue was present, the PGC contacted
the substrate with a cytoplasmic skirt. The cell then
extended a long pseudopodium which swelled gradually. Eventually the main cell body moved through the
pseudopodium toward the gonadal ridge. In the present
report, the initial attachment and migration stage are
similar to that reported by Kuwana et al. (1987). However our PGCs were never placed in the vicinity of
gonadal tissue, but they assumed the morphology of
those cells moving toward ridge tissue. We never ob-
Fig. 4.A stage 5 PGC isolated on a glass coverslip and viewed by
scanning electron microscopy. x 3,072.
Fig. 5.A scanning electron micrograph of a stage 5 PGC cultured on
a glass coverslip for 30 minutes. Often by 15-30 minutes some small
blebs (b) are present. x 3,658.
Fig.6.A scanning electron micrograph of a stage 4 PGC cultured on
Thermonox plastic and rat tail collagen for 15 minutes. Note the sheet
of cytoplasm on the substrate (arrowheads). x 4,225.
608
G. MATSUMURA AND M.A. ENGLAND
Fig. 7. A stage 8 PGC incubated in Medium 199 for 30 minutes at
37°C on rat tail collagen. Note the blebs (b). X 7,310.
served the flattened morphology described for PGCs
not exposed to gonadal tissue. These reported differences may be due to the very different ages of the PGCs
used in the two studies. Kuwana et al. (1987) isolated
19 somite stage embryo PGCs, while ours were from
the stages 4-8. It would not be surprising to discover
there are some cell changes between the pre- and early
migratory stages 4-8 and the late migratory 19 somite
stage PGCs. In fact, while some of their features are
known to remain stable, others change dramatically
over this period. Fujimoto et al. (197613) demonstrated
that lipids (as stained with Oil red 0 or Sudan black B)
remain even during late stages of development. They
also demonstrated that PAS positive glycogen is
present in the cytoplasm of PGCs from stage 4- day 5.
Changes which occur have also been reported. Swartz
and Domm (1972) found that PGCs undergo mitotic
division during migration and the initial population of
PGCs is therefore not necessarily the same population
migrating in the vascular network. Fujimoto et al.
(1976a) also reported that yolk granules are almost
completely dissipated by the time the PGCs reach the
germinal ridge.
In the mouse embryo, changes have been reported
between migratory and post-migratory PGCs in vitro
(Donovan et al., 1986). Migratory cells were shown to
be motile in vitro and demonstrated characteristically
invasive behavior. Gonadal PGCs were no longer invasive and showed little locomotory activity. Similar
changes could be occurring between the pre- and early
migratory PGCs and the much older migrating PGCs.
Our method of isolation is dependent upon the chick
PGCs separating naturally from the late hypoblast
layer. These PGCs could be described as at the early
end of their migratory phase. Their next phase of development includes actively migrating along a fibrous
substrate rich in fibronectin (England, 1983). The morphology of the PGCs changes dramatically a s the
spherical cells project long pseudopodia and move toward and along this substrate. The newly isolated
stage 4 PGC movements are reminiscent of those described on a cellular substrate (England, 1983) rich in
fibronectin.
Fig. 8. A stage 5 PGC isolated and introduced onto Sterispon for 10
minutes. x 5,814.
Culturing Techniques
The culture of stages 4-8 chick PGCs using the routine laboratory substrata and culture media is
achieved only with difficulty. With the exception of the
two gelatin lattices, Sterispon and Spongostan, the
other substrata used were clearly failures in this study
(Table 1). This failure rate is similar to attempts by
other authors to culture PGCs in other species. DeFelici and McLaren (1983) found the mouse PGCs to be
consistently nonadherent to glass, plastic, or gelatin
coated substrata. Greater success in the survival of proliferating mouse PGCs has been achieved with the introduction of a cellular substrate (Donovan e t al., 1986)
and growth factors (MGF-SCF). The leukemia inhibitory factor (LIF) in conjunction with feeder layers of an
established Sertoli cell line TM, (De Felici and Dolci,
1991) has recently been shown to sustain the survival
of mouse PGCs from 10.5 dpc embryos for at least 3
days.
Sterispon and Spongostan are routinely used in surgical procedures and act as scaffolding for tissue reconstruction. Their advantage in tissue culture systems is
the provision of a three dimensional substrate for cell
movement and architecture similar to the normal morphology encountered in vivo. It is known that the cell
morphology alters between two and three dimensional
substrata (England and Wakley, 1979) and the changes
in this report would confirm this observation. The morphology of the cells on glass, plastic, and collagen is
different from that on the gelatin lattices. The morphology of the PGCs on the two dimensional substrata
closely resembles that of the migrating PGCs described
in the early primitive streak stage chick embryo (England, 1983). Their shape changes on the gelatin lattices most closely resemble the changes observed in
amphibian PGCs introduced and cultured on chick embryos (England et al., 1986). This variation in appearance of the same aged PGCs is probably also a n indication that the cell spreading behavior is influenced by
609
ISOLATION OF PRIMORDIAL GERM CELLS
LITERATURE CITED
Critchley, D.R., M.A. England, J. Wakely, and R.O. Hynes 1979 Distribution of fibronectin in the ectoderm of gastrulating chick embryos. Nature, 280:498-500.
De Felici, M., and S. Dolci 1991 Leukemia inhibitory factor sustains
the survival of mouse primordial germ cells cultured on TM,
feeder layers. Dev. Biol., 147:281-284.
DeFelici, M., and A. McLaren 1983 In vitro culture of mouse primordial germ cells. Exp. Cell Res., 144t417-427.
Donovan, P.J., D. Stott, L.A. Cairns, J . Heasman, and C.C. Wylie 1986
Migratory and postmigratory mouse primordial germ cells behave differently in culture. Cell, 44:831-838.
Dubois, R. 1969 Le mecanisme d' entree des cellules germinales primordiales dans le reseau vasculaire, chez l'embryon de Poulet. J.
Embryol. Exp. Morphol. 21:255-270.
England, M.A. 1981 Application of the SEM to the analysis of morphogenetic events. J . Microsc 123:133-146.
England, M.A. 1983 The migration of primordial germ cells in avian
embryos. In: Current Problems in Germ Cell Differentiation. A.
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Developmental Biology, Cambridge University Press, Cambridge, pp. 91-114.
England, M.A.. D.R. Critchlev, and G. Shellswell 1982 Collagen Type
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England, M.A., and G. Matsumura 199213 Primordial germ cells in the
primitive streak stages chick embryo as studied by scanning electron microscopy. J . Anat., submitted.
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from a 2-dimensional to a 3-dimensional substrate. Experientia,
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Eyal-Giladi, H., M. Ginsburg, and A. Farbarov 1981 Avian primordial
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the primordial germ cells in blood samples from the chick embryo. Dev. Biol., 49:278-282.
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I
Fig. 9. A stage 5 PGC isolated and introduced onto Sterispon for 30
minutes. Note the cellular process (arrowhead). x 5,172.
Fig. 10. A stage 5 PGC introduced onto Sterispon for 30 minutes.
Compare this Figure with Figure 8. X 2,610.
the substratum (Fisher and Solursh, 1979). Interestingly, the chick PGCs populating the gonads lost their
morphology of migration and become rounded
(Ukeshima e t al., 1991). They are similar in appearance to premigratory PGCs (England and Matsumura,
1992a,b; Matsumura and England, 1992).
ACKNOWLEDGMENTS
The authors are grateful to Miss D. Meecham for
printing the scanning electron micrographs produced
by Mr G.L.C. McTurk of the Leicester Scanning Electron Microscope Unit. We are also pleased to thank
Mrs. L. Bradshaw for excellent secretarial services.
Dr. Matsumura is very grateful to the Wellcome
Trust for the travel grant awarded to him to support
these studies.
610
G. MATSUMURA AND M.A. ENGLAND
Swift, C.D. 1914 Origin and early history of the primordial germ cells
in the chick. Am. J. Anat., 15:483-516.
Ukeshima, A,, K. Yoshinaga, and T. Fujimoto 1991 Scanning and
transmission electron microscopic observations of chick primordial germ cells with special reference to the extravasation in their
migration course. J. Electron. Microsc., 40:124-128.
Wakely, J., and M.A. England 1979 Scanning electron microscopical
and histochemical study of the structure and function of basement membranes in the early chick embryo. Proc. R. SOC.Lond.
(B). 206:329-352.
Waldeyer, W. 1870 Eierstock und Ei. Els Beitrag zur anatomie und
entwicklungs geschichte der sexualorgane. W. Engelmann
Leipzig.
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