/. Embryol. exp. Morph. Vol. 22, 3, pp. 505-10, November 1969
Printed in Great Britain
505
Fate of primordial germ cells in the transplanted
hind gut of mouse embryos
ByWACfcAW OZDZENSKI 1
From the Department of Embryology, University of Warsaw
During normal development of the mouse embryo, primordial germ cells
(PGCs) differentiate in the root of the allantois and in the hind region of the
embryo, then pass to the hind gut and through the mesentery to reach the
germinal ridges (Chiquoine, 1954; Bennett, 1956; Mintz & Russell, 1957;
Mintz, 1957; Ozdzenski, 1967). The number of PGCs increases greatly during
migration (Chiquoine, 1954; Mintz & Russell, 1957). After penetrating into
the gonads PGCs continue to divide mitotically for a certain time, then undergo
changes which differ in each sex: the meiotic prophase begins in the ovaries
(Brambell, 1927; Borum, 1961), gonial divisions are arrested in the testes and
the chromatin in the nuclei of the germ cells undergoes characteristic dispersion.
The behaviour of germ cells in the male embryos of the mouse is similar to that
described by Clermont & Perey (1957) and Beaumont & Mandl (1963) in the
rat.
The present study has been undertaken in order to investigate the fate of
PGCs which were prevented from penetrating into the germinal ridges. In
particular we were interested in the question as to whether PGCs remaining
outside the gonad are able to initiate differentiation into definitive germ cells,
i.e. to enter into meiotic prophase.
As an object of study the hind gut of 9-day-old embryos (= 10th day of
pregnancy) was chosen on account of the considerable number of PGCs
present in it at this time, and the ease with which it can be dissected out without
risk of the rudiments of germinal ridges being included in the transplant.
MATERIAL AND METHODS
The material consisted of 52 hind guts dissected from 9-day-old mouse
embryos (11-26 pairs of somites) of the CBA-p strain, A strain, and randomly
bred albino mice. The guts were transplanted to the anterior chamber of the
eye of adult female and male mice (mainly of the same strain) or to the chorioallantoic membrane of 7- to 8-day chick embryos.
Transplants, intended for staining for alkaline phosphatase, were fixed after
Author's address: Department of Embryology, University of Warsaw, Warsaw 64,
Poland.
506
W. OZDZENSKI
1-8 days in 75 % alcohol or Baker's fluid (90 ml distilled water, 10 ml formalin,
0-4 g CaCl2), embedded in paraffin and sectioned serially at 6 /i. The sections
were stained with Fast Red TR Salt (G. Gurr) according to the azo-dye-coupling
method of Gomori for alkaline phosphatase. PGCs were identified by virtue
of the greater activity of the enzyme compared with that in surrounding somatic
tissues (Chiquoine, 1954). The number of PGCs was established by counting
them on successive sections. Results obtained in this way are probably overestimated (some of the PGCs may have been counted twice) but satisfactorily
illustrate differences in the number of PGCs in different transplants.
Three guts, fixed directly after transplantation into the eye, served as a
control of the starting point of the experiment.
In order to discover the possible presence of PGCs undergoing differentiation
into definitive germ cells, 5- to 16-day-old grafts were fixed in Bouin's fluid,
sectioned at 6 n and stained with haematoxylin and eosin.
RESULTS
Thirty-six guts transplanted to the anterior chamber of the eye were examined:
19 guts developing for 1-8 days were stained for alkaline phosphatase, 17 guts
developing for 5-16 days were stained with haematoxylin and eosin. In the
majority of cases the transplanted guts remained in the anterior chamber of
the eye, adhered to the iris and underwent vascularization. Some of the grafts
escaped from the anterior chamber and attached themselves to the iris from
the inner side; their development proceeded in a similar way to that of grafts
in the anterior chamber of the eye. The growth of transplants was at first slow
and it was not until more than 12 days after transplantation into the eye
that growth was accelerated (cf. Figs. 1, 2). The older transplants were highly
differentiated and contained villi, glands and a layer of smooth muscle (Fig. 3).
In two cases contractions of the graft were observed. Numerous mitoses were
visible in the transplants and the general state of the tissues was good.
Thirteen guts were transplanted to the chorio-allantoic membrane: 10 guts
developing from 2 to 1 days were stained for alkaline phosphatase, 3 guts
developing for 5, 8 and 11 days were stained with haematoxylin and eosin.
After transplantation to the chorio-allantois the grafts sank into the tissue of
the membrane and complete fusion of the mesenchyme of the chorio-allantois
and of the transplant took place (Fig. 4). Although the grafts underwent
vascularization their growth was slight.
During the first 3 days after transplantation to the eye the number of PGCs
in the guts did not increase (Table 1). The majority of the PGCs exhibited
the normal reaction for alkaline phosphatase and did not show any signs of
degeneration (Fig. 6). This picture changed on the 4th day; the PGCs became
scanty, the majority displayed weak alkaline phosphatase reactions and exhibited signs of degeneration. No traces of PGCs could be seen by the 7th and
Primordial germ cells
Fig. 1. Hind gut developing for 5 days in the anterior chamber of the eye.
Haematoxylin + eosin. x 40.
Fig. 2. Hind gut developing for 16 days in the eye. The gut underwent considerable
twisting and fills the whole of the anterior chamber. Haematoxylin + eosin. x 20.
Fig. 3. A fragment of the transplant shown in Fig. 2. The gut has undergone
advanced differentiation. Haematoxylin + eosin. x 50.
Fig. 4. Transplant after 8 days of development on the chorio-allantois of a chick
embryo. The gut is completely surrounded by the mesenchyme of the chorioallantoic membrane. Haematoxylin + eosin. x 80.
Fig. 5. Hind gut directly after transplantation to the anterior chamber of the eye.
PGCs are located in the gut epithelium. Fast Red TR Salt, x 350.
Fig. 6. Transplant after 1 day of development in the anterior chamber of the eye.
Majority of PGCs are located in the mesenchyme of gut. Fast Red TR Salt,
x 150.
507
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W. OZDZENSKI
8th days of development in the eye. Inhibition of mitotic activity of PGCs was
not accompanied by loss of the capacity for active movement, since the PGCs
migrated from the gut epithelium to the surrounding mesenchyme during the
initial period after transplantation (cf. Figs. 5, 6).
Table 1. Numbers of PGCs in hind guts after transplantation
Estimate based on preparations stained for alkaline phosphatase
Age of
transplant
(days)
0
Anterior chamber
of the eye
Chorio-allantoic
membrane
102, 142, 186
(control)
1
2
3
4
5
6
7
8
243
—
—
0
77, 121, 189
4, 15, 30
0, 0, 0, 4, 15, 25
6,20
0, 0, 3, 60, 136
0, 150
0,0
0,0
0
0
—
—
In the guts transplanted to the chorio-allantoic membrane, disappearance
of the alkaline phosphatase reaction in PGCs and their degeneration were also
observed, but the timing of these processes was not as regular as in grafts
developing in the anterior chamber of the eye (Table 1).
No primordial germ cells or germ cells in meiotic prophase were found in
any of the grafts investigated histologically. Some degenerating cells were
observed in several transplants after 5-6 days; these may have been degenerating
PGCs.
DISCUSSION
Primordial germ cells in transplanted guts are capable of survival for a
certain time, but are not capable (at any rate the majority of them) of multiplying. PGCs stop multiplying immediately or soon after transplantation, i.e.
during the period when in normal development they are still in the hind gut
and increase greatly in number. From the time of their appearance to the
13th day of development, the number of PGCs increases from 100-150 to
2000-5500 according to Chiquoine (1954), from 'several' to over 5000 according
to Mintz & Russell (1957).
It could be postulated that the conditions of the transplant's environment
are responsible for inhibition of the divisions of PGCs and subsequently for
their degeneration. It would follow that the requirements of PGCs differ
from the requirements of the somatic cells of the gut, since the tissues of the
transplant grow considerably and undergo differentiation.
The present study does not provide a complete answer to the question as
Primordial germ cells
509
to what the final fate of PGCs in transplants is. It would seem certain that
some of them degenerate. Some, however, do not exhibit degenerative changes
up to the time of complete loss of phosphatase reaction. In such cases loss of
phosphatase reaction is not equivalent to the death of the cell. The subsequent
fate of such cells is unknown. It must be emphasized that the loss of phosphatase
reaction by PGCs in the transplants takes place during the period when the
germ cells in the gonads continue to exhibit a distinct reaction (Mintz, 1959;
W. Ozdzenski, unpublished observations).
No signs of differentiation of PGCs into definitive germ cells were observed.
It should be stressed, however, that PGCs in the transplanted guts had from
the very first behaved abnormally (absence of mitotic divisions followed by
disappearance of phosphatase reaction and degenerative changes). At the time
when the PGCs of the graft could have entered into the meiotic prophase, i.e.
at the time when meiotic prophase begins in embryonic ovaries, their number
was already very small and some of those which still remained were undergoing
degeneration.
SUMMARY
1. Transplantation of the hind gut of a 9-day-old mouse embryo to the
anterior chamber of the eye of an adult mouse or to the chorio-allantoic
membrane of a chick embryo inhibits the mitotic activity of the primordial
germ cells.
2. Some of the PGCs undergo degeneration, others lose the alkaline phosphatase reaction and become undetectable.
3. Abnormal behaviour of PGCs is probably connected more with the
transplantation itself and/or the influence of the environment in which the
transplant develops than with the fact that they remain in the tissues of the gut.
RESUME
Le sort des cellules germinates primordiales situees dans Vintestin
posterieur transplants d'embryons de souris
1. La transplantation de Fintestin posterieur d'embryons de souris de 9 jours,
dans la chambre anterieure de 1'oeil de la souris adulte ou sur la membrane
chorio-allantoidienne de l'embryon de poulet, inhibe l'activite mitotique des
cellules germinales primordiales.
2. Quelques cellules germinales primordiales degenerent; d'autres perdent
leur activite phosphatasique alcaline et ne sont plus identifiables.
3. Le comportement anormal des cellules germinales primordiales depend
davantage de la transplantation elle-meme et/ou de l'influence de l'environnement dans lequel le greffon se developpe que du fait que les cellules germinales
primordiales demeurent dans les tissus de l'intestin embryonnaire.
I wish to express my sincere thanks to Dr. Andrzej K. Tarkowski for his advice and
encouragement throughout every stage of this work.
510
W. OZDZENSKI
REFERENCES
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(Manuscript received 23 December 1968)