/. Embryol exp. Morph. Vol. 26, 1, pp. 31-36, 1971
Printed in Great Britain
31
Laser irradiation of the chick embryo
germinal crescent
By MARTHA FEARON MIMS 1 AND
ROBERT GILMORE McKINNELL 2
From the Department of Biology, Newcomb College of
Tulane University, New Orleans
SUMMARY
The germinal crescent of head-fold and one-somite-stage chicken embryos was irradiated
with multiple pulses of a microbeam ruby laser. Primordial germ cells were not detected in
the gonad primordium of six laser-irradiated 5-day embryos; ten laser-irradiated embryos
had varying numbers of primordial germ cells. Ten control embryos had gonad primordia
populated with many primordial germ cells.
INTRODUCTION
Primordial germ cells have an extra-gonadic origin in the chick embryo. They
may be found in the primitive streak or head-process stage, in an area known as
the germinal crescent, located in the endoderm anterior and antero-lateral to the
primitive streak at the junction of the area opaca and area pellucida (Swift,
1914; Willier, 1937; Reynaud, 1967; Clawson & Domm, 1969). Primordial germ
cells are distinctively large and thus are easy to identify (Goldsmith, 1928).
Primordial germ cells are migratory. They begin to separate from the endoderm
at the definitive primitive streak stage (Clawson & Domm, 1967), are soon found
in the lumina of blood vessels (Abdel-Malek, 1950), and are in the tissues of the
embryo proper by the cranial flexure stage (Meyer, 1961, 1964). Most primordial germ cells become situated in the gonad primordium, some appear to
degenerate, and others are thought to form extra-gonadic islands of germ tissue
(Clawson & Domm, 1968).
The fate of these migratory giant cells has been in dispute because extirpation
of the germinal crescent has generally resulted in the death of the host embryo.
The fate of primordial germ cells would be revealed if they could be selectively
removed with negligible or no damage to embryonic tissues. The purpose of the
present study is to report preliminary results that suggest laser irradiation of the
1
Author's address: 245 Harmon, San Antonio, Texas 78209, U.S.A.
Author's address: Department of Zoology, University of Minnesota, Minneapolis,
Minnesota 55455, U.S.A.
2
32
MARTHA F. MIMS AND R. G. MCKINNELL
germinal crescent may in some instances be an effective method of preventing
the appearance of primordial germ cells in the gonad primordium without
causing the death of the host embryo.
MATERIALS AND METHODS
The laser
A Hadron/TRG Model 513 Biolaser System mounted on a Leitz Ortholux
microscope was the source of laser radiation. The instrument produces a highintensity monochromatic source of energy that is focused through the microscope
to the target area. The site to be irradiated is aligned with cross-hairs in the
optical system of the microscope. The laser has a pulse length of 150 /*sec with
a wavelength of 6943 A. The diameter of irradiation used in the present study
was approximately 100-150 /an, utilizing 120-180 J energy input with the
x 3-5 scanning lens of the microscope.
Chicken embryos
Head-fold and one-somite embryos (stages 6 and 7, Hamburger & Hamilton,
1951) were obtained by incubating fertile chicken eggs for 24 hat 37 °C. Windows
were cut above the embryos in the shell with a rotating carborundum disk
(Drexel moto-tool). The shells of control embryos were opened in the same
manner. The windows were closed in experimental and control embryos with
two layers of cellophane tape. Experimental embryos were supported on a small
platform on the substage condenser during laser irradiation. The germinal
crescent area was irradiated with an average of 18 laser pulses to adjacent areas.
The number of laser pulses depended upon the size and clarity of the crescent
and varied from 8 to 28. Pilot experiments indicated that energies higher than
120-180 J resulted in a high mortality of the experimental embryos. The
experimental embryos had a mortality of 55 % after 5 days, which was about
the same mortality (53 %) as that of the control embryos. Mortality in experimental and control embryos was high probably because the experiments were
conducted during the hot New Orleans summer. Mortality in chicken embryos
is highest during warm weather. Control embryos were maintained at room
temperature for the same time as the laser-irradiated embryos and both were
returned to the incubator at the same time.
Histology
Live 5-day-old embryos were fixed whole in Bouin's solution and were
sectioned serially. The sections were stained with Harris hematoxylin and eosin.
Slides were labelled and arranged so that the experimenter was unaware whether
or not the embryo had been laser-irradiated. The sections were examined for
the presence of primordial germ cells in the gonad primordium.
33
Laser irradiation of chick embryos
RESULTS
Serial microscopic sections of ten unirradiated control embryos were examined. The gonad primordia of the control embryos appeared normal. The
germinal epithelium was populated by many primordial germ cells that were
recognized by their large size, round shape, and hyaline cytoplasm (Fig. 1).
Sixteen laser-irradiated experimental embryos were studied histologically. We
were unable to find primordial germ cells in the germinal epithelium in 6 of the
Fig. J. Germinal epithelium of a control (unirradiated) 5-day chick embryo.
Primordial germ cells are large, round and have hyaline cytoplasm.
Fig. 2. Germinal epithelium of a 5-day chick embryo after microbeam laser
irradiation to its germinal crescent 4 days earlier.
16 experimentally irradiated embryos (Fig. 2). Primordial germ cells were found
in varying numbers in the other irradiated embryos. Although some of the
irradiated embryos appeared to contain the normal number of primordial germ
cells, at least four of them had primordial germ cells in substantially reduced
numbers. Presence or absence of primordial germ cells did not appear to be
related to the laser energy used within the range of 120-180 J. Laser-irradiated
embryos that contained no, or few, primordial germ cells were histologically
normal except for a reduction in the size of the germinal ridges.
K M
n ?.Ci
34
MARTHA F. MIMS AND R. G. MCKINNELL
DISCUSSION
This study shows that microbeam laser-irradiation to the germinal crescent
can be an effective means of preventing the appearance of primordial germ cells
in the gonad primordium of 5-day chicken embryos. The efficacy of the procedure is witnessed by the six laser-irradiated embryos whose germinal epithelium appeared to be devoid of primordial germ cells. However, the procedure as developed thus far is imperfect because ten of the treated embryos
contained from a few to many primordial germ cells in the germinal epithelium.
The presence of primordial germ cells in some of the laser-irradiated embryos
may be attributed to one or more of the following possibilities. (1) Primordial
germ cells sometimes appear to originate in parts of the blastoderm other than
the germinal crescent (Rawles, 1936; Romanoff, 1960; Fargeix, 1969). (2)
Primordial germ cells may begin their migration as early as the definitive streak
stage (stage 4, Hamburger & Hamilton, 1951). We irradiated first somite
(stages 6-7, Hamburger & Hamilton, 1951) embryos; accordingly, some
primordial germ cells may have already begun migration. (3) The area of
irradiation, in some instances, may have been insufficient to affect all of the
primordial germ cells in the crescent. Despite the fact that some irradiated
embryos contained germ cells, it is significant that six laser-irradiated embryos
were devoid of detectable primordial germ cells in contrast to their consistent
presence in control embryos.
Our data are in harmony with many experimental studies of the germinal
crescent in the chicken embryo. Excision (Reagan, 1916; Goldsmith, 1935;
Simon, 1957), cauterization (Dantchakoff, 1932), and ultraviolet irradiation
(Benoit, 1930; Reynaud, 1969) of the germinal crescent result in gonad primordia
that are sterile. Unfortunately, these procedures result in trauma to the embryo
sufficient to cause the death of most experimental animals.
Methods of reduction of primordial germ cells in other vertebrates include
treatment with drugs, irradiation other than laser, and loss of specific cytoplasm. Treatment of frog eggs with actinomycin, an inhibitor of RNA transcription, is reported to result in larvae with reduced numbers of primordial
germ cells (Wolsky & L'Estrange, 1969). Ultraviolet irradiation of vegetal
hemisphere cytoplasm (Smith, 1966) and pricking with loss of germinal cytoplasm (Buehr & Blackler, 1970) are procedures that result in sterile anurans.
Abnormally small germinal ridges were observed in laser-irradiated embryos
that contained no, or few, primordial germ cells. The reduced size of these
germinal ridges seems to suggest an interaction between primordial germ cells
and the normal development of the germinal ridge. Further studies are suggested because this result in our, admittedly preliminary, observations conflicts
with the study of Reynaud (1969).
Our results do not rule out the germinal epithelium as a source of primordial
germ cells in embryos older than 5 days (Firket, 1920; Asayama, 1967). Our
r
Laser irradiation of chick embryos
35
results are consistent with the theory that primordial germ cells originate in the
germinal crescent, migrate to the gonad primordia, and persist there for at least
5 days. The site of the origin of the definitive sex cells in the chicken is not yet
definitely known (Hamilton, 1952; Nelsen, 1953; Asayama, 1967). It is possible
that laser-irradiated embryos that are sterile may be reared to maturity in future
experiments. Sterile chickens that were laser-irradiated would comment cogently
on the developmental significance of primordial germ cells.
Microbeam laser irradiation is emerging as a useful tool in experimental
embryology. Parts of cells such as chromosomes and nucleoli can be selectively
irradiated with significant biological effect (Moreno, Lutz & Bessis, 1969; Berns,
Olson & Rounds, 1969; McKinnell, Mims & Reed, 1969; Berns & Rounds,
1970). The present study is probably the first^ study of laser-irradiated chick
embryos in vivo although chick pigmented retinal cells have been irradiated
in vitro (King & Geeraets, 1968).
No vital dye was used on the germinal crescent to facilitate absorption of
laser irradiation. We sought to avoid the use of any agent that might decrease
the vitality of the embryo. Evidently primordial germ cells or other adjacent
structures absorb sufficient energy to result in death or altered migration of
many primordial germ cells such that sterile germinal ridges ensue.
This study was supported by a research grant (DRG-990-A) from the Damon Runyon
Memorial Fund for Cancer Research, Inc., New York, N.Y., U.S.A. The authors thank
Professor Franklin Sogandares of Tulane University for the use of the Hadron/TRG Biolaser
System.
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_,„<„,_
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{Manuscript received 16 November 1970)