/. Embryol. exp. Morph. Vol. 27, 3, pp. 515-524, 1972
515
Printed in Great Britain
A study of meiotic germ cells in the
gonads of foetal mouse chimaeras
By ANNE McLAREN, 1 ANN C. CHANDLEY 2
2
A N D S U S A N A KOFMAN-ALFARO
from the A.R.C. Unit of Animal Genetics
and the M.R.C. Clinical and Population Cytogenetics Unit, Edinburgh
SUMMARY
The gonads of 53 foetuses (16$, 37$) derived from fusion of embryos at about the 8-cell
stage were examined. Ovotestes were found in two of the male foetuses. When gonads were
sectioned 16£ days post coitum, germ cells in meiotic prophase were seen in 6/6 female and
4/14 male foetuses. When air-dried preparations were made from gonads 15^-17^ days post
coitum, germ cells in meiotic prophase were seen in 7/7 females and 4/13 males. At 18^ days
post coitum, 2/2 female but only 2/10 male foetuses showed any meiotic cells, and these were
degenerating.
[3H]Thymidine injected into the mother 13-^ days post coitum resulted in labelling of the
meiotic germ cells, but the sex vesicle characteristic of meiotic cells in normal males was never
seen. The timing of DNA synthesis and meiosis was identical to that seen in the germ cells
in foetal ovaries.
We conclude that the meiotic cells seen in the testes of foetal chimaeras are probably XX
in constitution, and rarely survive beyond the 19th day of gestation.
INTRODUCTION
The occurrence and timing of meiosis in the germ cells of the foetal ovary of
the mouse were described by Brambell (1927), and later analysed in more detail
by Borum (1961), Peters, Levy & Crone (1962), and Crone, Levy & Peters (1965).
The pre-meiotic DNA synthesis takes place 13-15 days post coitum, and the
germ cells enter meiosis from 13 days onwards. At 16-18 days post coitum, many
oocytes are seen in zygotene and pachytene, and at birth (19-20 days post coitum)
the ovary contains about equal numbers of oocytes in pachytene and in early
diplotene. Many degenerating pachytene oocytes are seen before and shortly
after birth (Borum, 1961).
In contrast, germ cells in the male mouse do not enter meiosis until after
birth: the foetal testis therefore contains large numbers of spermatogonia, many
of which are undergoing mitosis, but no meiotic cells are seen.
1
Author's address: A.R.C. Unit of Animal Genetics, West Mains Road, Edinburgh 9,
U.K.
2
Authors' address: M.R.C. Clinical and Population Cytogenetics Unit, Western General
Hospital, Edinburgh 4, U.K.
33-2
516
A. MCLAREN, A. C. CHANDLEY AND S. KOFMAN-ALFARO
An exceptional situation occurs in experimental chimaeras, formed by fusion
of cleaving mouse embryos. Given an equal sex ratio, half on average of such
fusions will be between male and female embryos. On the basis of the low proportion of hermaphrodites and the predominantly male sex ratio in his material,
Tarkowski (1961, 1963) postulated that most XX/XY chimaeras develop as
fertile males, a suggestion which was supported by evidence obtained later by
Mystkowska & Tarkowski (1968). An examination of primary spermatocytes in
adult male XXjXY chimaeras revealed no germ cells of XX chromosome constitution (Mystkowska & Tarkowski, 1968). No oocytes were found in the testes
of 12 male chimaeras examined at 8-12 days of postnatal life, but some oocytes
as well as spermatogonia were seen in a single 5-day-old animal (Mystkowska &
Tarkowski, 1968, 1970). Prenatally (15-16 days post coitum), five out of 11 male
chimaeras contained germ cells in meiotic prophase, as well as gonocytes (Mystkowska & Tarkowski, 1970). Three out of these five animals were examined
cytologically and all proved to be XX/XY chimaeras.
It is tempting to assume that the meiotic germ cells seen in the testes of foetal
XX/XY chimaeras are of XX constitution, behaving autonomously in the sense
that they enter meiosis at the same chronological time as they would in the
foetal ovary. However, another possibility exists. The somatic tissue in the testis
of a sex chromosome chimaera consists of patches of XX and patches of XY
constitution. It is possible that any germ cell located in the neighbourhood of
predominantly XX tissue will enter meiosis before birth, in which case the
meiotic germ cells observed in the testes of foetal chimaeras will be indifferently
XXandXY.
The aim of the present experiments was: (a) to confirm the observations of
Mystkowska & Tarkowski (1970) on the occurrence of meiotic germ cells in the
testes of foetal chimaeras; (b) to collect further information on the fate of such
germ cells; and (c) to make use of the very striking late-labelling characteristic
of the mouse Y chromosome (contained in the sex vesicle) in the DNA synthesis
preceding the first meiotic division (Kofman-Alfaro & Chandley, 1970), in order
to determine the chromosome constitution of the meiotic germ cells.
MATERIAL AND METHODS
The mice were from the randomly bred Q strain. Fusion, culture and transfer
of embryos were as described by Bowman & McLaren (1970). The first series
of foetuses were those derived by Bowman & McLaren (1970) from fused
embryos. The recipient foster-mothers were killed 16^ days post coitum, the
foetuses were fixed in formol saline, and their gonads later dissected out and
serially sectioned at 8 /.im, stained with haematoxylin and eosin and examined
for the occurrence of meiotic germ cells. Although the quality of fixation was
poor, meiotic cells were in general easy to recognize. In the second series, the
foster-mothers received an intraperitoneal injection of 0-25 mCi [3H]thymidine
Meiosis in the germ cells of mouse chimaeras
517
Table 1. The occurrence of germ cells in meiotic prophase in the gonads of14 male
and 6 female foetuses derived from pre-implantation fusion {sectioned material)
Gonad
Test is
Ovotestis
Ovary
No.
examined
No. containing
meiotic germ cells
23
1
11
6
1
11
(Radiochemical Centre, Amersham; specific activity 22-8 Ci/mM) 13^ days post
coitum, and were killed 2-5 days later. Some of the foetal gonads from these
mothers were fixed in Bouin and processed as before for histological examination, but most were placed in a 1 % solution of sodium citrate for 30 min, fixed
in 3:1 methanol:glacial acetic acid for at least 15 min and transferred to 45%
acetic acid for 1-2 min prior to spreading on slides pre-heated at 60 °C. Air-dried
preparations were stained with carbol fuchsin and filmed for autoradiography
using llford L.4 liquid emulsion with an exposure period of 3 weeks at 4 °C.
RESULTS
The first series consisted of 20 foetuses, 16-^ days post coitum, derived from
embryo fusion. From the position and external appearance of the gonads, the
foetuses were classified as 6 females and 14 males. Histological examination
revealed that one of the males possessed an ovotestis very similar to that described
by Mystkowska & Tarkowski (1970). The distal end of the gonad consisted of
clearly differentiated sex cords, but the proximal end was ovarian in appearance,
with no sex cords and the majority of the germ cells in meiotic prophase (Figs.
1, 2).
Histological examination (Table 1) showed germ cells in meiotic prophase
not only in the ovotestis, but in 6/23 of the otherwise normal testes examined
(Fig. 3). The total, 7/24 (or 4 out of 14 males examined), does not differ significantly from the proportion (5/11) reported by Mystkowska & Tarkowski (1970).
In the second series, 16/33 foster-mothers became pregnant, and 33/104 fused
embryos transferred to them survived to the time of autopsy. Ten were females
and 23 males, giving a sex ratio very similar to that found in the first series.
For the two series combined, the sex ratio deviated significantly from a 1:1
ratio (P < 001) but not from the 3:1 ratio expected if all XXIXY chimaeras
had developed as males. In this series also, one of the males proved on histological examination to possess an ovotestis.
Air-dried preparations were made from the ovaries of nine female foetuses
derived from fused embryos and two control female foetuses derived from
transfer of non-fused embryos, 15^-18^ days post coitum. At 15^ days the germ
cells were typically in leptotene and zygotene, at 16-^- and \1\ days most were in
518 A. MCLAREN, A. C. CHANDLEY AND S. KOFMAN-ALFARO
Meiosis in the germ cells of mouse chimaeras
519
Table 2. The occurrence of germ cells in meiotic prophase in the testes of foetuses
derived from fused embryos, at different stages of pregnancy (air-dried material)
Days
post coitum
No. of male
foetuses
No. with
meiotic
germ cells
6
1
6
10
2
0
2
No. of meiotic
germ cells
Abundant*; 36, —
—
5, 14; 2, 3
4, 3; 2,1
2t
* In this foetus, the opposite gonad was identified histologically as an ovotestis.
t Degenerating.
zygotene and pachytene and at 18^ days post coitum zygotene, pachytene and
some diplotene stages were seen. The ovaries of female foetuses derived from
transferred embryos (whether fused or single) resembled those of controls of
the same developmental stage.
Air-dried preparations were also made from one or both gonads of the 23
male foetuses. The results are given in Table 2. Combining the data from 15£,
16^ and 17-J- days post coitum, four out of thirteen males contained germ cells
in meiotic prophase, closely resembling the meiotic cells seen in female foetuses
of the same developmental age (Figs. 4-6). By contrast, at 18^ days post coitum,
no normal germ cells in meiotic prophase were seen, though small numbers of
cells believed to be degenerate pachytene stages were found in two foetuses out
of ten (Fig. 7).
When meiotic cells were found in one testis of a foetus, they were invariably
present in the other testis also. No similar cells were seen in air-dried preparations
from the testes of control male foetuses 15-J—18-^- days post coitum.
A total of 164 meiotic cells were examined after autoradiography, of which
94 were from the male with an ovotestis. None of the meiotic cells contained a
sex vesicle, nor did any show a pattern of incorporation of [3H]thymidine
characteristic of XY cells.
FIGURES
1-3
8/<m sections of foetal gonads, 16^ days post coitum, stained with haematoxylin
and eosin.
Fig. 1. Ovotestis from male foetus derived from embryo fusion, showing sex cords
in distal region (T) and ovarian tissue proximally (O). x 110
Fig. 2. Higher magnification of area outlined in Fig. 1, to show germ cells in meiotic
prophase (pachytene) (arrowed), x 1100
Fig. 3. Testis from phenotypically normal male foetus derived from embryo fusion,
showing germ cell in meiotic prophase (pachytene) (arrowed), x 1100
520
8a
A. MCLAREN, A. C. CHANDLEY AND S. KOFMAN-ALFARO
Meiosis in the germ cells of mouse chimaeras
521
Table 3. Expected numbers of embryos of different chromosome constitutions on
the assumption of 21 % non-chimaerism
Fused embryos
Chromosome
constitution
XX
XX/XY
XY
Total
Chimaeras
19-75
39-50
19-75
7900
>
•
Non-chimaeras
10-50
10-50 /
21 00
% (estimated)
30-25
69-75
10000
DISCUSSION
The results of the present study amply confirm Mystkowska & Tarkowski's
(1970) finding of meiotic germ cells in the testes of foetal chimaeras. In the
earlier study and in the two series reported in the present paper, the proportion
of 15-^—17^ day male foetuses possessing such cells was 5/11, 4/14 and 4/13
respectively, in good agreement with each other. Cells in meiosis have never been
seen in the testes of male foetuses not derived from embryo fusion, and the
results of Mystkowska & Tarkowski (1970) suggest that they occur only in the
testes of sex chromosome chimaeras (XXjXY). We may therefore inquire what
proportion of male foetuses derived from embryo fusion may be expected to
be sex chromosome chimaeras.
Not all foetuses derived from embryo fusion are chimaeric, since one of the
two component cell populations may fail for a number of reasons to become
incorporated in the developing foetus (for discussion, see McLaren & Bowman,
1969). In the strain combination described by McLaren & Bowman (1969), for
example, 28 mice have been raised to an age when they could be classified for
chimaerism in respect of a number of characters involving different tissues, and
FIGURES
4-8
Air-dried preparations from the testes of foetuses derived from embryo fusion.
No sex vesicles visible.
Fig. 4. Group of six germ cells in the zygotene and early pachytene stages of meiotic
prophase, 15^ days post coitum.
Fig. 5. Two labelled pachytenes, 15^ days post coitum.
Fig. 6. Labelled meiotic cell thought to be in the diplotene stage, 11\ days/w.?/ coitum.
Fig. 7. Degenerate meiotic cells, 18+ days post coitum.
Fig. $a. Germ cell in the pachytene stage of the first meiotic prophase, from an
air-dried preparation of the testis of a normal male mouse 13 days old, to show the
prominent sex vesicle (arrowed) containing the Xand Y chromosomes.
Fig. Sb. Germ cells in the pachytene stage of meiotic prophase from an adult male
mouse showing the characteristic late-labelling pattern of the sex vesicle and centric
heterochromatin. (From Kofman-Alfaro & Chandley, 1970.)
522 A. MCLAREN, A. C. CHANDLEY AND S. KOFMAN-ALFARO
six (21 %) failed to show chimaerism in any tissue (A. McLaren, unpublished
observations). If a similar frequency be assumed in the present study, 69-75 %
of foetuses derived from embryo fusion will be expected to be of either XY or
. 0 7 AT constitution (Table 3). Since 69-8 % of our foetuses (37/53) were male, it is
reasonable to conclude that, in the present study, all XX/XY chimaeras developed as males. Mystkowska & Tarkowski (1970), on the other hand, found a
more nearly equal sex ratio (57% males) and detected two XXIXY foetuses
which were developing as normal females.
From Table 3 it will be seen that the best estimate that can be derived for the
expected proportion of XXjXY chimaeras among our male foetuses is 57 %
(39-50/69-75). During the period 15^—17^ days post coitum, the proportion of
male foetuses showing meiotic germ cells in our material is only 30 % (8/27),
significantly lower than the expected 57% (P < 001), indicating either that
some of the meiotic cells eluded detection, or that not all XXjXY chimaeras have
meiotic cells in the testis. Probably both explanations are true: if meiotic cells
were present in very low frequency they could well have been missed, particularly
in the sectioned material, while Mystkowska & Tarkowski (1970) identified one
, 0 7 ^ 7 chimaera without any meiotic cells in the testis.
At 18^ days post coitum, the few remaining meiotic cells appeared very degenerate. Borum (1961) reports large numbers of germ cells degenerating at the
pachytene stage in squash preparations of foetal ovaries 19^ days post coitum.
We therefore wondered whether the degeneration of meiotic cells in chimaera
testes 18y days post coitum might represent part of a normal process of atresia,
which would also affect meiotic cells in the foetal ovary. However, the air-dried
preparations of ovaries at this stage of development showed no cells comparable
to the degenerating meiotic cells seen in the chimaera testes. It would seem that
the environment of the foetal testis is hostile to the development of meiotic germ
cells beyond the pachytene stage, and that few are likely to survive further than
the nineteenth day of gestation. The presence of oocytes in the testis of a 5-dayold male chimaera (Mystkowska & Tarkowski, 1968) may have been an exceptional occurrence. In mules, where meiosis also fails to be completed, it is again
at the pachytene stage that the germ cells degenerate (Short, 1972).
Are the germ cells in meiotic prophase which we have observed in the testes
of foetal chimaeras XX in constitution, or do they include both XX and XY
germ cells which have been induced to enter meiosis under the influence of XX
somatic cells? The second alternative raises the further question of the ultimate
fate of those XX germ cells in an XX\XY chimaera which do not enter meiosis
prenatally, since no XX germ cells are seen at the primary spermatocyte stage.
The fact that we were unable to detect a sex vesicle in any of the meiotic germ
cells in the foetal testes strongly supports the first alternative, since the first male
germ cells to enter meiosis after birth in the mouse show, by the late zygotene
stage, a large sex vesicle (Fig. 80). Also, the pattern of incorporation of
[3H]thymidine into AT meiotic cells is very characteristic in the mouse, with parts
Meiosis in the germ cells of mouse chimaeras
523
of the X and Y chromosomes giving rise to late-labelling regions (Fig. Sb)
(Kofman-Alfaro & Chandley, 1970). This labelling pattern was never seen in
the meiotic cells of the chimaera testes. Another piece of circumstantial evidence
suggesting that the germ cells in meiotic prophase are indeed XXcells is that the
meiotic cells in the chimaeric testes label on precisely the same day and develop
at the same rate as do female germ cells in the foetal ovaries of normal and
chimaeric mice. Spontaneous sex reversal, observed in the goat, pig and mouse
(for references, see Short, 1972), can also lead to .Of germ cells developing in a
testis, and it is perhaps relevant that in each instance the germ cells have been
found to degenerate before birth.
However, if it is the germ cell's own genetic constitution which alone determines the timing of its entry into meiosis, so that all XX germ cells in sex chromosome chimaeras enter meiosis prenatally, it is hard to see why the meiotic germ
cells should constitute such a small minority of the total germ cell population of
the testis. Primordial germ cells presumably reach the gonadal ridge in equal
numbers regardless of their sex, and if subsequent development is autonomous,
one might expect 50 % of the germ cells in a sex chromosome chimaera to enter
meiosis prenatally. Yet Mystkowska & Tarkowski's (1970) estimates of the
proportion of meiotic germ cells in phenotypically normal chimaeric testes
range from 0-08 to 16-3 %, averaging only just over 5%. Because of this finding,
and because of the large differences in the frequency of occurrence of meiotic
cells in the two testes of a single foetus which they sometimes observed, these
authors incline to the view that it is the genetic constitution of the somatic tissue
of the testis rather than of the germ cell itself which determines entry of the germ
cell into meiosis. Their 'environmental' and our 'genetic' model could both be
satisfied, and all existing data accommodated, on the assumption that the
meiotic germ cells that are seen in the foetal testis are in fact XX cells, but that
such cells only enter meiotic prophase under the influence of neighbouring XX
somatic tissue.
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{Manuscript received 5 November 1971)