/. Embryol. exp. Morph. Vol. 68, pp. 59-67, 1982
59
Printed in Great Britain © Company of Biologists Limited 1982
Regulation of meiosis in the foetal mouse gonad
ByCLIVE W. EVANS, 1 DIANA I. ROBB, 2
FIONA TUCKETT 3 AND SUSAN CHALLONER 1
From the Department of Anatomy and Experimental Pathology,
The University of St Andrews, St Andrews
SUMMARY
In vitro culture of male and female gonads was found to have significant effects on
gonadal structure and development. Culture resulted in a reduction of testicular cord diameter and a reduction in the number of Sertoli cells lining each cord in cross section. In the
female, culture increased the percentage of pyknotic oocytes and fewer germ cells per unit
of ovary volume reached diplotene. Mixed sex co-culture using different culture methods
showed that day 14 p.c. testes inhibited meiosis in day 14 p.c. ovaries when the cultures were
continued until the equivalent of day 21 p.c. Day 15 p.c. and mixed age co-cultures of mixed
sex provided more equivocal data since meiosis was inhibited in some preparations but not in
others. The possibility is suggested that prophase I may proceed irrevocably to diplotene after
about day 15 p.c. and thus the inhibitory effects of foetal testes may be a function of female
gonadal age. No evidence was found to support the hypothesis that mixed sex co-culture
may stimulate meiosis precociously in foetal testes.
INTRODUCTION
,
In the mouse, as in other vertebrates, all fertile germ cells must undergo
meiosis which consists of two cell divisions each composed of four phases
(prophase, metaphase, anaphase and telophase). The first division is relatively
complicated with an extended prophase divided into four stages (leptotene,
zygotene, pachytene and diplotene). Although meiosis is fundamentally the
same in both sexes, there are three significant differences between meiosis in the
l
male and in the female:
(1) Meiosis in the male does not commence until puberty, whereas in the
female meiosis begins during foetal life or just after birth.
(2) Meiosis in the male is an uninterrupted process which occurs throughout
life after puberty. In the female, meiosis is arrested at diplotene and is not
resumed until shortly before ovulation. Meiosis is arrested in the female for a
1
Authors' address for all correspondence: Department of Anatomy and Experimental
Pathology, The University of St Andrews, St Andrews, Fife KYI6 9TS, Scotland.
2
Present address: Department of Zoology, The University of Edinburgh, West Mains
Road, Edinburgh EH9 3JT, Scotland.
3
Present address: Department of Human Anatomy. The University of Oxford, South
Parks Road, Oxford 0X1 3QX, England.
60
C. W. EVANS AND OTHERS
second time at metaphase II and is not completed unless the oocyte has been
fertilized.
(3) Cytoplasmic divisions during meiosis in the male are equal, whereas in
the female such divisions are unequal.
What mechanisms underly these differences in the meiotic pathways of the
two sexes ? Analysis of in vitro experiments on meiotic progression in mammals
has suggested a number of factors which may regulate meiosis in both males
and females. Byskov (1974) suggested that a factor derived from the foetal rete
ovarii (the Meiotic Inducing Substance, MIS) may be involved in initiating
meiosis in the female while, according to Ohno & Smith (1964), contact with
follicular (i.e. granulosa) cells may be responsible for arresting oocytes at
diplotene. Since Byskov (1975,1978 a) has suggested that the rete ovarii contains
precursors of the definitive granulosa cells, the possibility exists that meiotic
control may lie within one cell type arising relatively early in gonadal development. In the male, the delay in the initiation of meiosis might involve some
interaction between the germ cells and the Sertoli cells which are in close
proximity within the confines of the testicular cords. It has been suggested that
the Sertoli cells may secrete a substance which inhibits meiosis in the male until
puberty (Byskov & Saxen, 1976; Byskov, 1978 b). Indeed, the interplay of such a
substance (the Meiotic Preventing Substance, MPS) with the postulated inducing
substance MIS in the presence of other components such as the endocrine
hormones may contribute to the mechanism underlying the inhibition and
onset of meiosis (Byskov, 1978/?). However, at this stage the presence of
inducing and preventing substances in both sexes has not been demonstrated
conclusively. The present study was designed to test the effects of co-culturing
testes and ovaries from foetal mice of differing ages on both male germ cells (in
which meiosis has not been initiated) and on female germ cells (after or at the
time of the initiation of meiosis but before arrest at diplotene).
METHODS
Pregnant mice of the C57BL strain were killed by exposure to ether vapour
on the appropriate day after coitus. The day when a vaginal plug was found
was recorded as day 1 post coitum (day 1 p.c). The foetuses were placed in
Dulbecco's phosphate-buffered saline (PBS) and the gonads (including their rete
systems) were removed. The foetuses were sexed using morphological criteria or
following examination of chromosome preparations (Evans, Burtenshaw &
Ford, 1972).
Organ cultures consisted of two types (Fig. 1):
(a) Rafts: these were either blocks of 2 % agar (Baker & Neal, 1973) or
squares of Millipore filter (0-22 ptm pore size) supported on expanded stainlesssteel grids. In raft cultures, the gonads were placed on top of the agar or filter
61
Regulation ofmeiosis
Fig. 1. Organ culture methods. Organs were cultured using either the raft or the
vertical techniques. In the raft technique, cultures were supported on either 2 %
agar (a) or Millipore filters (b). In the vertical technique (c), gonads of appropriate
combinations were placed on either side of a Millipore filter (0-2 /on pore size) to
test for the effects of soluble factors released by each gonad. In practice, only one
preparation (either single gonads or combinations of gonads) was maintained per
dish.
support in Petri dishes and the medium level was adjusted to just approach the
junction between the gonads and their support.
(b) Vertical cultures: the gonads were placed on either side of a square of
Millipore filter held vertically in a specially designed stainless steel grid (Fig. 1;
Robb & Evans, 1981). Enough medium was added to the Petri dish containing
the culture system to reach the bottom of the filter and pass up to the gonads by
capillary action.
The medium used for all cultures was Eagle's Minimal Essential Medjum
with Earle's Salts (Flow Labs.) at pH 7-3, supplemented with 20% newborn
calf serum, 2 IDM glutamine, 50 i.u. ml" 1 penicillin and 50 fi%. ml" 1 streptomycin
sulphate. Cultures were maintained at 37 °C in a humidified atmosphere of
95 % air and 5 % CO2 in the presence or absence of additional pressure (5 p.$.i.)
as indicated in Results. The medium was changed every second day and after
the appropriate length of time the gonads and their supporting surfaces were
removed from the Petri dishes, fixed in aqueous Bouin's solution and prepared
for histological examination using routine techniques.
RESULTS
The percentages of cells in different stages of meiosis in non-cultured control
ovaries were determined for each age and the results are presented in Table 1.
These results are in general agreement with those of Borum (1961) and confirm
slight differences in the rate of progression of germ cells through meiosis in
different strains of mice.
3
EMB 6$
62
C. W. EVANS AND OTHERS
Table 1. Percentages of cells in different stages of meiosis in
control ovaries
Age (days)
14 p.c.
15 p.c.
16 p.c.
11 p.c.
IS p.c.
19 p.c.
lp.p.
Oogonia
Leptotene
Zygotene
Pachytene
80-5
19-5
—
23-5
76-5
3-8
13-3
51-4
—
—
13-4
—
—
—
—
—
—
—
—
—
Day 20 p.c. not done. Pyknotic germ cells not
—
31-5
86-6
870
460
80
included.
Diplotene
—
—
—
—
130
540
920
Table 2. Variations in testicular cord structure in control testes
Age (days)
14 p.c.
15 p.c.
16 p.c.
np.c
18 p.c.
19 p.c.
lp.p.
Mean±s.d. N = 10. Day
cross-section.
Cord diam. (/tm)
No. Sertoli cells
35-2±4-5
9±1
42-2 + 4-2
13±1
46-4 ±3-5
14±1
50-5 ±6-7
16±1
55-4 ±4-0
17±1
560 ±4-1
18±1
58-2±3-3
19±1
20 p.c. not done. No. of Sertoli cells is per testicular cord
Variations in testicular cord structure in non-cultured control male gonads
are shown in Table 2. It can be seen that there is an increase in cord diameter
and in the number of Sertoli cells lining each testicular cord with increasing
gestational age.
Table 3 summarizes the effects of culture on the structure of testicular cords
in males and on meiotic progression in females. Culture has considerable
effects on gonadal structure and development, significantly reducing the
testicular cord diameter and the number of lining Sertoli cells in males and
increasing the percentage of pyknotic oocytes in females.
From counts of the number of germ cells in diplotene and the volume of the
ovaries in cultured and non-cultured (control) females, we have determined
the effects of culture on germ cell progression through meiosis (Table 4).
Culture of day 14p.c. and day \5p.c. ovaries results in about a 50% decrease in
oocyte density.
Table 5 illustrates the inhibitory effect of day \4p.c. testes on the progression
through meiosis of day 14 p.c. oocytes. In day \4 p.c. mixed-sex co-cultures,
26-4 ± 0*5 % of all germ cells remained as oogonia while the rest were pyknotic.
The variations in testicular cord structure in co-cultures prepared using the
Regulation of meiosis
63
Table 3. Effect of culture on testicular cord structure in males and meiotii;
development in females
Preparation (m)
Cord diam. (/*m)
Day 1 p.p. uncultured
Day 14 p.c. cultured
Day 15 p.c. cultured
58-2±3-3
39-0 + 4-7**
37-2±2-7**
No. Sertoli cells
19±1
10±l**
10±l**
Oogonia Leptotene Zygotene Pachytene Diplotene PyknOtic
Preparation (f)
(%)
(%)
(%)
(%)
(%)
(%)
—
—
—
Day 1 p.p. uncultured
14-2 ±4-8 76-9 ±6-9 8-9 ± H
—
—
—
61 ±2-5* 75-7±5-8 18-3±3-5*
Day 14 p.c. cultured
—
—
—
Day 15 p.c. cultured
2-2 ±0-2* 86-4±0-3 ll-4±0-5
Data from Millipore raft cultures and controls. All cultures were continued until they
reached the equivalent of day 1 post partum (p.p.) i.e. day 21 p.c. Meiotic stages were identified
according to Challoner (1974). Data presented as mean ± standard deviation. A^ =* 10.
m = male, f = female.
* = P < 005; ** = P < 0001 by Student's t test.
Table 4. Density offemale germ cells in diplotene
Preparation (f)
No. diplotene
Vol. ovary (mm3)
Density (mm"3)
1126±50
O-38±OO3
8105± 1088
Day 1 p.p. uncultured
Day 14p.c. cultured
345±34
0-08±0-03
4158±34
Day 15p.c. cultured
524±91
012±001
4519±91
Mean±s.d. N= 10. Diplotene count determined according to Abercrombie (1946).
Volume of ovary = mid-sectional area x total section no. x section thickness.
Millipore raft technique are shown in Table 6. The diameter of testicular cords
and the number of Sertoli cells per cord cross-section are not significantly
different when similar and mixed-sex co-cultures prepared from day 14 p.c, are
compared. However, similar data from cultures prepared from day 15 p.c.
could not be compared because testicular cord structure degenerated in day
15 p.c. mixed-sex co-cultures. Likewise, in all but one of the mixed-sex cocultures of different ages the cord structure also degenerated (Table 6). In the
single case where the day 14 p.c. testicular cords did not break down it was
apparent that the germ cells of the day 15 p.c. female did not progress into
leptotene (result not shown).
Results from cultures prepared using the vertical culture system at 5 p.s,i. or
agarose rafts were essentially similar to those obtained using the Millipore raft
technique (summarized in Table 7). Again it was apparent that day \4p.c. male
gonads inhibited meiosis in day 14 p.c. oocytes. Cultures prepared from day
15p.c. testes also inhibited meiosis in the day \4p.c. female providing male germ
cells were present and providing the testicular cords were more or less intact.
Day 14 p.c. testes affect day 15 p.c. oocytes in a similar manner, However, the
3-2
64
C. W. EVANS AND OTHERS
Table 5. Percentages of oocytes in different stages of meiosis in similar and
mixed sex co-cultures
Preparation
Day 14p.c.
Day 14 p.c.
N Oogonia Leptotene Zygotene Pachytene Diplotene Pyknotic
f
f
l9
)
_
_
_
21-2±6-9 561 + 5-8 22-7±2-7
~
73-6 + 5-4*
_
_
_
28-7±40 45-4±8-l 26-3±4-9
Day 14p.c. f | 9
Day 15 p.c.
m )
Dayl5/>.c. f l 5
_
_
_
3-7±3-3 83-4±9-2 12-9±6-4
f )
Day 15 p.c.
Dayl5/>.c. f l 6
_
_
_
2-0±2-8 86-6±4-7 ll-5±l-9
Day 15 p.c.
m )
Day 15p.c. f U
_
_
_
io-O±3-4 70-6±4-l 19-5±2-l
Day 14 p.c.
m )
Mean %±s.d.
* = P < 0-001 using Student's / test. Cultures were maintained until the female gonad
reached the equivalent of day 1 p.p. Data from Millipore raft cultures, f = female; m = male.
Table 6. Variations in testicular cord structure in similar and mixed-sex co-cultures
Preparation
N
Cord diam. (jim)
No. Sertoli cells
Day 14 p.c. m
} 20
31-4±4-7
9±1
Day 14 p.c. m
Day 14 p.c. m
\ 20
33-4±3-5
10±2
Day 14 p.c. f .
Day 14 p.c. m
| 20
—
Broken down
Day 15 p.c. f .
Day 15 p.c. m
\ 20
36-8 ±4-7
Day 15 p.c. m
Day 15 p.c. m
\ 20
Broken down
—
Day 15 p.c. f
Day \5 p.c. m
| 20
—
Broken down
Day 14 p.c. f .
Mean ± s.d. Data from Millipore raft cultures. No. of Sertoli cells is per cord cross-section.
results from day \5 p.c. mixed-sex co-cultures are somewhat more equivocal
(Table 7). Thus, in agreement with earlier results, in six cultures meiosis in the
ovary proceeded in the absence of both intact testicular cords and germ cells
in the male portion of the co-culture. On the other hand, in nine cultures meiosis
was able to proceed even in the presence of intact testicular cords and germ cells
in the male.
Regulation of meiosis
65
Table 7. Summary of conditions found in foetal gonad cultures
Preparation
Day
Day
Day
Day
Day
Day
Day
Day
Day
14 p.c.
14 p.c.
14 p.c.
14 p.c.
14 p.c.
15 p.c.
15 p.c.
15 p.c.
15 p.c.
Day 15/J.C.
Day 15 p.c.
Day 14/J.C.
N
f
f
f
m
f
m
f
f
f
m
f
m
Testicular cords intact
Spermatogonia
} 13
Female meiosis
+
} 21
+
+
/ 19
I 2
+
-
+
-
} 9
- \
+ /
+
/ 6
-
±
+ \
\ 9
/ 6
19
+
+
-
+
+
-
+ )
- \
+ /
Data from agarose rafts and vertical culture systems.
Examination of testes in single and mixed-sex co-cultures provided no
evidence for precocious meiosis in the germ cells of the foetal male gonad.
DISCUSSION
Reliable culture conditions are obviously a prerequisite for examining the
events following the co-culture of mixed-sex gonads since apparent inhibitory
effects may be due to the relatively greater sensitivity of the gonads of one sex
to in vitro conditions. We find that during culture spreading of the gonads tends
to occur over the supporting substrate although in cultured single organs the
capsules remain intact. This spreading leads to the eventual fusion of the gotiads
in all co-cultures if the gonads are initially placed within a few millimetres of
each other. As well as changes in the external structure, there are also changes in
the internal structure at the cellular level. Thus, in the male, the testicular cords
fail to reach their potential diameter during culture and the number of lining
Sertoli cells is significantly decreased (Table 3). Comparison of the data in
Tables 2 and 3 shows that in cultured testes (unlike control testes) there is no
significant growth in the diameter of testicular cords and nor is there any
increase in Sertoli cell number from the age at which the cultures were e$tablished. In the ovary, culture leads to an increase in pyknosis which is reflected
in a smaller percentage of cells in pachytene (Table 3). In real terms, however,
there is a 50 % loss in germ cells reaching diplotene when cultured ovaries are
compared to uncultured ovaries of equivalent age (Table 4). These variations
reflect the need to seek improved conditions for the culture of foetal gotiads.
However, we believe the present experimental regimes to be more than adequate
to justify our conclusions following the co-culture of gonads for a number of
66
C. W. EVANS AND OTHERS
reasons. First, although fewer oocytes progress through prophase I, enough
germ cells reach meiosis in control cultures to allow us to assess the effects of
experimental regimes designed to modulate germ cell development. Secondly,
meiotic inhibition in the female partner of mixed-sex co-cultures is seen when
the male gonad is healthy with intact testicular cords encompassing germ cells
but has not been seen when the structure of the male portion is degenerating
(Table 7). Thirdly, inhibition of meiosis is not due to the effects of culturing
more than one gonad on the same substrate since control experiments show no
specific effects on meiosis following co-culture of similar sex gonads (Tables 5-7).
From our studies of mixed-sex co-cultures we conclude that day 14/7.c. testes
inhibit meiosis in day 14 p.c. ovaries when the gonads are cultured to the
equivalent of day 1 p.p., i.e. 7 days (Tables 5, 7). It is unlikely that the high level
of pyknotic oocytes (Table 5) reflects a feature of the culture system per se since
other gonad combinations show that cultures can be maintained for this length
of time without significant cell death. Meiotic inhibition requires intact testicular
cords and the presence of germ cells in the male and is able to act across a
vertical Millipore filter barrier (100 jum thick, 0-22 fim pore size) when appropriate gonads are placed on either side (Table 7). Histological sections of
Millipore filters from vertical cultures failed to reveal the presence of direct
physical contact across the filter, but the presence of a soluble inhibitory factor
has not been proven because of the possibility of cellular microprojections
across Millipore filters (Saxen et al. 1970).
In day 15 p.c. co-cultures and mixed-age co-cultures the results are less
straightforward. The absence of significant numbers of male germ cells and the
disruption of testicular cords again correlates consistently with progression
through meiosis in the co-cultured female (Table 7). In two out of three combinations the presence of intact cords and germ cells in the male correlates with
meiotic inhibition in the female. The set of results (day \5p.c. female/day \5p.c
male) at variance with the others may reflect difficulties in assessing true foetal
age (which will vary firstly with the exact time of fertilization of oocytes and
secondly, with the time of experimentation, i.e. a variation in the order of 6-9 h)
and could suggest that progression through the early stages of meiosis may be
irrevocably committed in the female around day 15 p.c. The concept of a
critical time beyond which meiosis in female germ cells cannot be inhibited by
the testes gains circumstantial support from other studies (Ozdzenski et al. 1976)
but further experiments with more accurate assessment of foetal age will be
required to fully evaluate this possibility.
We found no evidence in any of our day 14 and day 15 p.c. cultures to
support the possibility that mixed-sex co-culture may lead to the precocious
stimulation of meiosis in spermatogonia. Similarly, Ozdzenski et al. (1976)
failed to detect the initiation of meiosis in spermatogonia from the mouse
when foetal testes were co-cultured in vivo with mouse ovaries. O and Baker
(1976, 1978), however, have suggested that isolated spermatogonia which are
Regulation of meiosis
67
not enclosed by the testicular cords may enter meiosis precociously. Byskov
(19786) has also suggested that extra-tubular germ cells may enter meiosis* It
would seem from these conflicting lines of evidence that the early induction of
meiosis in foetal spermatogonia may be a relatively rare event and that the role
of the postulated meiotic-inducing substances in the precocious initiation of
meiosis in the male requires further study.
We conclude, in agreement with Byskov (1974), that cellular activities
associated with foetal testes may inhibit meiosis in foetal ovaries. This inhibitory
effect may be mediated by a soluble meiosis-preventing substance and our
results suggest a possible means of regulating the meiotic pathway in mammals.
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