J. Embryol. exp. Morph. Vol. 63, pp. 75-84, 1981
Printed in Great Britain © Company of Biologists Limited 1981
75
X-chromosome activity in foetal germ cells
of the mouse
By MARILYN MONK 1 AND ANNE McLAREN 1
From the MRC Mammalian Development Unit, London
SUMMARY
A cycle of inactivation and reactivation of one X chromosome in the female (XX) germ
line is shown by analysis of gene dosage effects on activity of an X-linked enzyme. The ratio
of activities of the X-linked enzyme HPRT and an autosomal enzyme APRT are determined
in XX and XY germ cells from embryonic gonads from the 12th to the 17th day of pregnancy.
Mitotic stages of XX and XY germ cells on the 12th day have similar HPRT: APRT ratios,
but on the 13th day the ratios are significantly higher in XX than XY germ cells. As the XX
germ cells enter meiosis they show a marked increase in HPRT:APRT ratio which is
primarily due to a rise in X-linked HPRT activity. Comparisons are made with XO germ
cells on the 12th and 14th day. On the 12th day, XO do not differ from XX and XY germ
cells, suggesting that only one X chromosome is active in XX germ cells at this stage. On
the 14th day, on the other hand, the HPRT:APRT ratios in XO and XY germ cells are
similar but in XX germ cells the ratio is significantly higher. The twofold difference between
the ratio in XX and XO germ cells suggests that by this stage both X chromosomes are
active in XX germ cells. The subsequent large increase of the ratio in XX relative to XY
germ cells is thought to reflect their differing cell states.
INTRODUCTION
The only cells in female mammals that are known to have both X chromosomes active are the pluripotential cells of the early embryo, up to the epiblast
stage (Monk & Harper, 1979), and the oocytes (Epstein, 1969; 1972; Gartler,
Liskay & Gant, 1973; Mangia, Abbo-Halbasch & Epstein, 1975; Kozak,
McLean & Eicher, 1974; Monk & Kathuria, 1977). A continuous cell lineage
might link these two developmental stages, with both X-chromosomes active in
the primordial germ cells as well as in their precursors in the epiblast and their
successors throughout oogenesis. If, on the other hand, one X chromosome is
inactivated in all cells before the emergence of the primordial germ cells, or in
the germ cells during their migratory phase or after entry to the genital fidges,
then a mechanism for X-chromosome reactivation during oogenesis must be
sought.
A little evidence exists in favour of the second alternative. Ohno (1964)
claimed to see a heteropyknotic X in primordial germ cells of female mouse
1
Authors' address: MRC Mammalian Development Unit, 4 Stephenson Way, London
NW1 2HE, U.K.
76
M. MONK AND A. McLAREN
embryos during their period of migration; and Semyonova-Tian-Shanskaya &
Patkin (1978) reported the presence of sex chromatin and heterochromatization
of the X chromosome in female germ cells of human embryos after they had
reached the genital ridge. Andina (1978) examined the level of activity of two
enzymes coded by the X chromosome, glucose 6-phosphate dehydrogenase
(G6PD, E.C. 1.1.1.49) and hypoxanthine phosphoribosyl transferase (HPRT,
E.C. 2.4.2.8). In foetal germ cells of XX and XO mouse embryos he found
that G6PD activity was similar in the two types of embryo before birth, but
the ratio of XX to XO activity increased to nearly two-fold after birth. For
HPRT, however, the ratio was between 1-5 and 2 for all prenatal stages
examined. If inactivation of the HPRT locus had occurred, then reactivation
must have taken place before 14^ days post coitum (p.c), the earliest stage
examined.
P. Johnston (personal communication), using an electrophoretic variant of
PGK, found evidence for only one active X-chromosome in mitotic germ cells
and reactivation of the second X-chromosome before entry into meiosis. A hybrid
band for dimeric G6PD, bearing witness to the activity of both X chromosomes,
has been reported by Gartler, Andina & Gant (1975) in ovarian extracts from
heterozygous human foetuses at 14-21 weeks, but no such band was found at
an earlier stage (12 weeks) although some of the germ cells were already in
early prophase of meiosis. Migeon & Jelalian (1977), on the other hand, were
able to detect a hybrid G6PD band as early as 8 weeks and are unconvinced
that X inactivation occurs in the female germ line at any stage.
We decided to reinvestigate this question in foetal germ cells of the mouse
with particular emphasis on the early period of germ-cell development within
the genital ridges (11^-13^ days p.c), since the study of Andina (1978) which
had yielded somewhat ambiguous results, did not begin until 14^ days p.c. We
used the very sensitive assay utilized by Monk (1978) in which X-chromosome
function is assessed by determining the relative activity of an X-chromosomecoded enzyme, HPRT, to an autosome-coded enzyme, adenine phosphoribosyl
transferase APRT (E.C. 2.4.2.7.). Daily determinations were made on normal
female (XX) and male (XY) germ cells, before, during and after the period
when meiosis is initiated in the female. Some determinations were also made
on XO germ cells in female embryos.
MATERIALS AND METHODS
Mouse embryos were obtained from randomly bred Q-strain XX females or
from XO females (Institute of Animal Genetics, Edinburgh) on successive days
from the 12th day of pregnancy (11^ days post coitum) to term. The gonads
were removed into PB1 (Whittingham & Wales, 1969) containing 0-4% polyvinylpyrrolidone instead of albumin (PBl-PVP)and sexed by their characteristic
morphology (developing testes show cords) from the 13th day onwards, and by
X-chromosome activity in foetal germ cells of the mouse
11
J2 0-3 -
01 -
Time (h)
Fig. 1. HPRT and APRT activities with increasing time of reaction in a sample of
XX germ cells collected 15£ days p.c. 100 germ cells were collected in 5 /il of
PB1-PVP and an extract prepared by freeze thawing. After centrifugation the
supernatant was added to 250 /*1 of reaction mix and incubated at 37 °C. The
reaction mix contained [3H]guanine (10 /IM specific activity 610 Ci/M), [14C]adenine
(10/*M, specific activity 276 Ci/M), phosphoribosyl pyrophosphate (2ITIM), magnesium chloride (5 min), and phosphate buffer (50 HIM pH 7-4). Aliquots of 50 fi\
were removed at times shown and added to ice cold lanthanum chloride (01 M)
containing adenine and guanine (100 /IM). The precipitates were collected on glassfibre filters and the reaction products, [3H]guanine monophosphate and [14C]adenine monophosphate, determined in a Packard scintillation counter.
chromosome sexing or by presence or absence of sex chromatin bodies in
amnion preparations for 12th-day embryos. XO embryos were distinguished
from XX embryos by chromosome counts or by amnion preparations. These
two methods were checked against one another on 14th-day embryos and
invariably gave the same result in that female embryos lacking sex chromatin
proved always to have 39 chromosomes. In embryos from XX females $exchromatin constitution (scored blind) was always concordant with phenotypic
sex.
78
M. MONK AND A. McLAREN
«9 and, 6, germ cells
.,
enter genital ridges
9 and
c$ gonads
dj j
uishable
W "1
PRE-SPERMATOGONIA
— • enter mitotic arrest
r
10
11
12
i
13
i
i
i
14
i
i
i'
15
t
'1
16
(
^
17
Mitotic-OOGONIA
Leptotene zygotene
Pachytene
>•
Entry into meiosis-OOCYTES
Days gestation
Fig. 2. Stages of germ cell development in mouse embryos of the Q strain. Entry
into meiosis and the progression of female germ cells through the first stages of
meiotic prophase are shown with reference to number of days' gestation. The times
given are a guide to when the majority of the germ cells are in a particular stage.
In fact cells enter into meiosis continuously over a period of at least 2 days.
Individual germ cells are in leptotene or zygotene for periods of several hours and
in pachytene for several days (see also Borum, 1961; Peters, 1970).
Cells were released from the gonad by mashing it with a needle, the residue
of the gonad was then discarded. The large round germ cells were easy to
recognize; their identity was confirmed initially by testing for alkaline phosphatase activity (Brinster & Harstad, 1977). The germ cells were washed in
PB1-PVP, collected in batches of 50 or 100 in approximately 5 /i\ of supporting
medium in 10 /d microcaps, and stored at - 7 0 °C. Somatic cells released from
the gonad in the same way included a high proportion of foetal blood cells.
Some samples of these somatic cells were also collected in microcaps. Extracts
were prepared by freeze-thawing in liquid nitrogen three times, followed by
centrifugation. Approximately 4 jA of supernatant of each sample was transferred to 50 jitl of reaction mix (see legend to Fig. 1) at 37 °C for 3 h to assay
the X-linked enzyme HPRT and the autosomally-linked enzyme APRT. The
enzyme reactions were linear over this period (Fig. 1). Details of the reaction
have been previously described (Monk & Kathuria, 1977; Monk & Harper,
1978). The results are expressed as a ratio of HPRT: APRT per 100 germ cells
thus monitoring X-chromosome:autosome dosage and eliminating sample
errors.
RESULTS
In the Q-strain of mice, primordial germ cells first enter the genital ridges at
10-11 days post coitum (Figure 2); at \2\ days/7051/ coitum the gonads can for
the first time be sexed by their appearance, since under a dissecting microscope
cords can be seen in the developing testes; 24 h later some of the germ cells in
the ovary have entered the leptotene stage of meiotic prophase. At 14^ days
zygotene stages are the most common, and at 15^ and 16^ days p.c. pachytene
X-chromosome activity in foetal germ cells of the mouse
79
40 -
35
30
XX germ cell
25
20
15
10
11
12
13
14
15
16
17
Days gestation
Fig. 3. HPRT: APRT ratios as a function of gestational age in germ cells from
female XX and male XY embryos and in gonadal (somatic) cells. Duplicate
samples of 100 germ cells were collected from between two to six male or female
embryos from pregnant females on successive days p.c. Extracts were prepared,
added to 50 [A of reaction mix and assayed as described in the legend to Fig. 1 for
HPRT and APRT activities ([3H]guanine, specific activity 700 Ci/M, [)4C]adenine,
287 Ci/M). The reaction time was 3 h. Samples of somatic cells taken from male and!
female embryonic gonads showed no significant difference in HPRT: APRT ratios
and are pooled in thefigure.A repeat of this experiment using embryonic germ cells
at \\\, 12£, 13£, 15£ and 16£ days p.c. gave similar results.
stages prevail. In the male, by \5% days p.c, most of the XY germ cells in the
testis are T-prospermatogonia and are in a state of mitotic arrest.
Figure 3 shows the HPRT:APRT ratio for XX female and XY male germ
cells as a function of gestational age of the embryo. XX germ cells have
equivalent HPRT:APRT ratios to XY germ cells at 11£ days but show
approximately seven times the XY value by 15^ days. The ratios of HPRT: APRT
from gonadal somatic cells show little difference between male and female
embryos (pooled in Figure 3) and no significant change with gestational age.
80
M. MONK AND A. McLAREN
12 r
10
^
6
0
0-4
» 0-3
8
0-2
0-1
0L
12
13
14
15
16
17
Days gestation
Fig. 4. HPRT and APRT activities in XX and XY germ cells as a function of
gestational age of the embryos. Assays were performed for 3 h as described in the
legend to Fig. 1. The results for the two enzymes looked at separately rather than
as a ratio show considerable scatter due to sampling errors introduced at some
stage of germ cell collection or preparation of extracts.
Figure 4 shows that the difference between XX and XY HPRT: APRT ratios
in XX and XY germ cells in Figure 3 is a consequence of changes in activity
of the X-linked enzyme HPRT rather than in the activity of the autosomallylinked APRT.
Four additional experiments were done to see whether the significant
difference between XX and XY germ cells at \2\ days post coitum (Figure 3)
could be confirmed. Table 1 shows that the divergence is indeed significant by
daysp.c.
XX and XY germ cells have a similar HPRT:APRT ratio at 1H days p.c
X-chromosome activity in foetal germ cells of the mouse
81
Table 1. HPRT/APRT ratios in XX and XY foetal germ cells
12\ days post coitum
Exp.
9 XX (n)
6 XY (n)
1
2
3
4
1-45(4)
1-40(5)
1-92(5)
1-79(4)
1-08(5)
1-29(6)
1-55(6)
1-67(4)
Table 2. HPRT/APRT
Difference
(mean ±S.E.)
0-37±0-16
011±015
0-37±0-16
0-12±0-31
Mean difference
0-243 ±00872
(P < 001)
ratios in foetal germ cells from an XO mother
11\ days post coitum
9
A
XX
XO
<?XY
211
1-97
1-45
104
1-68
1-68
1-67
106
1-40
1-33
0-92
F 2 i 8 = 1-12, P > 0-2
Table 3. HPRT/APRT
ratios in foetal germ cells from an XO mother
1S\ days post coitum
xx
xo
<? X Y
406
5-33
—
2-57
2-86
3-28
2-53
3-97
—
XX v. X O : t3 = 3-30, P < 0 0 5
XO v. XY: t3 = 0-58, P > 0-5
If we assume that XX and XY germ cells at this stage are equivalent in all
respects other than sex chromosome constitution, the results strongly suggest
that only one X chromosome is active in the XX germ cells.
This interpretation is confirmed by comparison of XX, XO and XY germ
cells in embryos from an XO mother at 11£ days p.c. (Table 2). A statistical
analysis of the data in Table 2 shows no significant difference in HPRT:APRT
ratios for XO and XX female and XY male germ cells.
A similar comparison of XX, XO and XY germ cells in embryos from an
XO mother at 13£ days p.c. is given in Table 3. The XO still resemble the XY
82
M. MONK AND A. McLAREN
germ cells, but now differ significantly from the XX germ cells. Indeed, the
difference between XX and XO germ cells in HPRT:APRT ratio approaches
the twofold value that we would expect to see on reactivation of the silent
X chromosome in the XX cells.
DISCUSSION
The ratio of HPRT to APRT activity gives a good relative index of Xchromosome function when essentially similar cell types are being compared,
for example male and female embryos at the same stage of cleavage (Monk &
Kathuria, 1977; Monk, 1978). The more dissimilar the cell types, the less valid
the comparison becomes, since changing enzyme activities may reflect some
functional requirement characteristic of a particular cell type. In the present
work, the comparison of XX and XY germ cells may be considered to give a
reasonable indication of X-chromosome function at 11^ and 12£ days p.c,
when both are proliferating mitotically as gonia, but is unlikely to be valid
later in gestation, when XX germ cells have entered the prophase of meiosis
and XY germ cells are in mitotic arrest. A more reliable picture is given by the
comparison of XX with XO germ cells, since the developmental pathway is
the same.
We have shown that the HPRT:APRT ratio in XX germ cells is similar to
that in XY germ cells at 11^ days p.c. The conclusion that only one Xchromosome is expressed in the XX oogonia is confirmed by the observation
that XO oogonia at this stage also have a similar HPRT:APRT ratio. We
cannot say whether X inactivation occurred in germ cell precursors at the same
time as in other epiblast cells (i.e. by 6 days/?.c, Monk & Harper, 1979) or at
some later stage, between 6 and 11^ days. An alternative explanation that
HPRT activity is regulated independently of functional gene dosage, is made
less likely by the absence of such regulation in early embryogenesis and later
stages of oogenesis.
By 12^ days/7.c. the HPRT:APRT ratio in XX germ cells has increased to
a level significantly higher than that in XY germ cells, suggesting that the
second X chromosome is now being expressed. This conclusion is supported by
the two-fold difference in ratio seen between XX and XO germ cells at 13^ days
p.c. The significantly lower values shown by the XO germ cells cannot be
accounted for by developmental retardation: with respect to the onset of
meiosis XO germ cells are delayed at most a few hours (McLaren unpublished
observations), and at 13^ days/J.c. there has been little change in HPRT: APRT
ratio in the previous 24 h.
The very rapid increase in HPRT:APRT ratio in XX relative to XY germ
cells after 13^ days p.c. is due to an increase in X-linked HPRT activity. This
probably corresponds to some functional requirement of meiotic prophase
rather than reflecting X-chromosome dosage.
X-chromosome activity in foetal germ cells of the mouse
83
With respect to HPRT activity, reactivation of the silent X chromosome has
occurred by 12£ days p.c. We cannot say exactly how much earlier it occurs,
because we are ignorant of the kinetics of HPRT and APRT synthesis and
degradation in germ cells. However, one may speculate, as did Gartler et al
(1975), that it is linked to the onset of meiosis. There is some evidence (McLaren,
1981) that germ cells with two X chromosomes (XX) enter the leptotene stage
of meiotic prophase in response to the influence of Meiosis-Inducing Substance
(Byskov & Saxen, 1976; O & Baker, 1976) more readily than do those with only
a single X (XO or XY). This would imply that the second X in XX germ cells
is already expressed at the time that this response occurs. It may be that
reactivation of the inactive X in female embryos is initiated by the same event
that initiates the meiotic process, and indeed that reactivation is an integral
part of this process, but precedes the entry into meiotic prophase. This
reactivation during oogenesis is the only known example of the reversal of the
non-functional state of the silent X chromosome in female mammals.
REFERENCES
ANDINA, R. J. (1978). A study of X-chromosome regulation during oogenesis in the mouse.
Expl Cell. Res. I l l , 211-218.
BORUM, K. (1961). Oogenesis in the mouse. A study of the meiotic prophase. Expl Cell Res.
24, 495-507.
BRINSTER, R. L. & HARSTAD, H. (1977). Energy metabolism in primordial germ cells of the
mouse. Expl Cell Res. 109, 111-117.
BYSKOV, A. G. & SAXEN, L. (1976). Induction of meiosis in fetal mouse testis in vitro. Devi
Biol. 52, 193-200.
EPSTEIN, C. J. (1969). Mammalian oocytes: X-chromosome activity. Science 163, 1078-1079.
EPSTEIN, C. J. (1972). Expression of the mammalian X-chromosome before and after
fertilisation. Science 175, 1467-1468.
GARTLER, S. M., ANDINA, R. & GANT, N. (1975). Ontogeny of X-chromosome inactivation
in the female germ line. Expl Cell. Res. 91, 454-457.
GARTLER, S. M., LISKAY, R. M. & GANT, N. (1973). Two functional X-chromosomes in
human fetal oocytes. Expl Cell Res. 82, 464-466.
JOHNSTON, P. G. (1979). Mouse Newsletter, no. 61, July 1969.
KOZAK, L. P., MCLEAN, G. K. & EICHER, E. M. (1974). X-linkage of phosphoglycerate
kinase in the mouse. Biochem. Genet. 11, 41-47.
MANGIA, F., ABBO-HALBASCH, G. & EPSTEIN, C. J. (1975). X-chromosome expression during
oogenesis in the mouse. Devi Biol. 45, 366-368.
MCLAREN, A. (1981). The fate of germ cells in the testis of fetal Sex-reversed mice. / . Reptod.
Fert., in press.
MIGEON, B. R. & JELALIAN, K. (1977). Evidence for two active X-chromosomes in germ cells
of female before meiotic entry. Nature, Lond. 269, 242-243.
MONK, M. (1978). Biochemical studies on mammalian X-chromosome activity. In Development in Mammals, vol. in (ed. M. H. Johnson). Amsterdam: North Holland.
MONK, M. & HARPER, M. (1978). X-chromosome activity in preimplantation mouse embryos
from XX and XO mothers. / . Embryol. exp. Morph. 46, 53-64.
MONK, M. & HARPER, M. I. (1979). Sequential X-chromosome inactivation coupled with
cellular differentiation in early mouse embryos. Nature 281, 311-313.
MONK, M. & KATHURIA, H. (1977). Dosage compensation for an X-linked gene in preimplanation mouse embryos. Nature, Lond. 270, 599-601.
84
M. MONK AND A. McLAREN
O, W. & BAKER, T. G. (1976). Initiation and control of meiosis in hamster gonads in vitrol.
J. Reprod. Fert. 48, 399-401.
OHNO, S. (1964). Life history of female germ cells in mammals. 2nd Intern. Conf. Congenita
Malformations, 1963. pp. 36-40. Intern. Med. Congr., Ltd., New York.
PETERS, H. (1970). Migration of gonocytes into the mammalian gonad and their differentiation.
Phil. Trans. R. Soc. Lond. B 259, 91-101.
SEMYONOVA-TIAN-SHANSKAYA, A. G. & PATKIN, E. L. (1978). Changes in gonocyte nuclei at
different stages of their differentiation in early human female embryos. Arkh. Anat. Gistol.
Embriol. 74, 91-97. (Tn Russian.)
WHITTINGHAM, D. G. & WALES, R. G. (1969). Storage of two cell mouse embryos in vitro.
Aust. J. Biol. Sci. 22, 1065-1068.
(Received 6 June 1980, revised 11 December 1980)