J. Cell Sd. 65, 249-263 (1984)
249
Printed in Great Britain © The Company of Biologists Limited 1984
MORPHOLOGICAL AND TEMPORAL SEQUENCE OF
MEIOTIC PROPHASE DEVELOPMENT AT PUBERTY
IN THE MALE MOUSE
P. GOETZ', ANN C. CHANDLEY AND R. M. SPEED
MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh
EH4 2XU, U.K.
SUMMARY
The correct sequence of meiotic prophase development in the male mouse has been established
by the use of pubertal males. The first wave of spermatogenesis at this time provides a unique
opportunity to study progressive meiotic development in a direct way. Air-dried and micro-spread
analyses have been carried out. Temporal and morphological progression at this time is entirely
consistent with that occurring in the later waves of meiosis of the adult male. Morphological detail
shows delayed pairing of the X and Y chromosomes relative to the autosomes. The longest XY
synaptonemal complex is seen in early pachytene cells, occupying up to 72% of the length of the
Y and 22% of the length of the X axis. By late pachytene, end-to-end pairing in the XY bivalent
is established, the autosomal axes remaining fully paired. Desynapsis of the autosomes commences
at early diplotene. A 'diffuse' diplotene stage in the male, comparable to the dictyate stage of the
female, could not be found. Marked lengthening of the XY and autosomal axes did, however, occur
through the diplotene stage.
INTRODUCTION
The simple and rapid technique by which primary spermatocytes of mammals can
be prepared by micro-spreading (Counce & Meyer, 1973; Moses, 1977a,b) has led,
in recent years, to a great expansion of knowledge concerning cellular morphology and
chromosome pairing at meiotic prophase in a variety of species (see Moses, 1980, for
a review). Complemented by serial reconstructions of whole nuclei (see Gillies, 1975,
for a review), for accurate identification of individual stages in meiosis, much has been
learned of the behaviour of chromosomes during normal and abnormal synapsis and
desynapsis, and the role of the synaptonemal complex (SC) in meiosis. Sequential
meiotic development has to be inferred, however, from relationships between the XY
pair, nucleolar development and autosome behaviour (Moses, 1980; Solari, 1980)
since, among spermatocytes from mixed testicular suspensions of adult males, there
is no means of knowing the correct sequence of meiotic staging.
A recent attempt to overcome this problem was made by Oud, de Jong & de Rooij
(1979), who described a method for restricting the spermatocyte population in adult
male mice by the creation of two large gaps in the spermatogenic line using
hydroxyurea and triaziquone to kill spermatogonia. The two gaps enclosed a small,
•Permanent address: Department of Medical Genetics, 151 12 Prague 5, V uvalu 84, Czechoslovakia.
CEL65
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P. Goetz, A. C. Chandley and R. M. Speed
well-defined cohort of surviving spermatocytes in pre-meiotic interphase. The
development of this restricted spermatocyte population was then followed day by day
as it progressed through meiotic prophase, and the correct morphological sequence,
as seen in air-dried preparations, was recorded.
An alternative approach, and one not requiring the application of drugs, would
simply exploit the natural sequence of development at puberty in the male. By
systematic sampling around the peri-pubertal period the morphological and temporal
sequence of the first wave of meiotic development can thus be determined directly.
This paper describes our results for the pubertal male mouse. Both air-dried and
spread preparations have been analysed, and descriptions of cell stages seen at both
light- (LM) and electron-microscope (EM) levels are given. The advantages of correlated light and EM studies have been emphasized by Dresser & Moses (1980).
Particular attention has been focussed on the behaviour and pairing sequence in the
XY bivalent and on the possible existence of a 'diffuse' stage at diplotene.
MATERIALS
AND
METHODS
Testicular sampling
Mice of the Swiss (Schofield) strain were used throughout. Females in the late stages of pregnancy
were checked twice daily and births of litters were recorded. Litter sizes were then reduced to ten
by destruction of excess females in order to allow vigorous development and to minimize growth
differences between males. Over days 8—20 (the peri-pubertal period) males were killed daily and
testes removed. To obtain a sufficient number of cells in suspension, it was found necessary over
days 8-13, to pool the testes of 4-6 males from one litter. From day 13 today 20, preparations were
made from three individual males on each day. Because it was found that critical events in XY
pairing were occurring over days 11 to 13 of development, additional males were killed at half-daily
intervals over this period. A check on inter- and intra-litter variation in male meiotic development
was also carried out. Testicular suspensions prepared from all ten males in two different litters on
day 14 post-partum were analysed. Six males were obtained from one litter and four from the other.
Cell preparation
For analysis at the light-microscope (LM) level. Air-dried preparations were made according to
Evans, Breckon & Ford (1964), and stained with carbol fuchsin (Carr & Walker, 1961). In our
experience, this is the best possible combination for spermatogenic stage identification in air-dried
preparations of the mouse.
Spread preparations were generally made according to Fletcher (1979), with minor modification,
but an alternative technique was also sometimes employed. This entailed mixing the testicular cell
suspension, drop for drop, with 0-15 M-sucrose on the slide. After 1 h of drying on the bench, the
slide was fixed with 4 % formaldehyde and washed with 0-4% buffered Photoflo. Staining was
carried out overnight using 50 % AgNCh (aqueous) at 60 °C, or by the rapid colloidal silver method
of Howell & Black (1980) counterstained for 1 min with 5 % Giemsa (pH 9).
For analysis at the electron microscope level. EM grids were prepared according to the method of
Felluga & Martinucci (1976) with minor modification. Spermatocytes were spread on Parlodioncoated slides, stained with AgNC>3 (Howell & Black, 1980), and examined at LM level. The best
spreads were selected, and cut from the film using a Leitz diamond slide marker, the small round
film discs being floated off the slides onto distilled water and picked up on G 200 HS Cu(Gilder)
grids. The operation was facilitated by coating the slides in Victawet (Emscope Laboratories Ltd)
in a vacuum evaporator prior to dipping in Parlodion. Grids were examined with a Philips EM300
at60kV.
Meiosis in pubertal male mice
251
Analytical methods
For a sequential analysis of cell morphology, an LM analysis of 300 meiotic cells was made at each
sampling time, both from air-dried and spread preparations. In air-dried preparations, cells were
followed in development from preleptotene to diakinesis/metaphase I. In spreads, it is only possible
to examine cells up to late diplotene as the SCs (synaptonemal complexes) after that stage are broken
down and no longer visible by silver staining. Spread preparations provide a considerably greater
degree of detail by which stages and sub-stages can be identified than do those prepared by air-drying
(see Results).
Differential counts of meiotic stages on successive days were made in both series, and the most
advanced cell type observed at each sampling time was recorded. For the inter- and intra-litter
comparisons, carried out on day 14, differential analyses were made on 100 cells from each male of
the two litters sampled again using both air-dried and spread preparations.
To complement the LM analysis, morphological detail was also studied in photographic enlargements of at least 20 clear cells obtained by spreading at each stage of meiotic prophase. Using these,
the XY SC was measured in order to determine the sequence and extent of XY pairing, and the
relative degrees of contraction and elongation of the XY and autosomal bivalents throughout
pachytene and diplotene. Measurements were made using the cursor of a Digiplan electronic
measurer (Reichert-Jung) to trace the silver-stained axes on the prints. Each measurement was made
three times and a mean calculated. Prints made from cells on EM grids were also examined for
morphological detail and, in particular, were studied critically for detail of XY behaviour between
zygotene and late diplotene.
RESULTS
Morphological analysis
Air-dried preparations. The principal prophase stages identified in air-dried
preparations as progressive sampling took place, are shown in Fig. 1.
At preleptotene, the nucleus is small and round (approx. 6/im in diameter), the
chromatin being generally homogeneous (Fig. 1A). Leptotene and zygotene cannot
readily be distinguished from each other, but cells in these early prophase stages are
slightly larger than preleptotene nuclei, and show a somewhat 'serrated' outline (Fig.
1B). Early and mid-pachytene can be distinguished on the basis of size and degree of
chromatin condensation. Early pachytene cells are small with dense chromatin and
occasionally show the beginnings of the sex vesicle (Fig. lc); mid-pachytenes are
much larger and show a prominent sex vesicle against a paler background of autosomal
elements (Fig. ID).
Cells in late pachytene may easily be confused with those in mid-pachytene and
early diplotene. Nuclei are only slightly larger than those in mid-pachytene but the
autosomal bivalents are more clearly delineated (Fig. 1E).
By early diplotene (Fig. IF), the nucleus is very large and pale-staining. Careful
examination shows a double structure to the autosomal bivalents and a very prominent
sex vesicle. Several clusters of darker staining bodies are present. In the past, this
stage has been incorrectly interpreted by us to be late pachytene. Oud et al. (1979)
were the first to recognize in air-dried preparations that such cells were, in fact, in the
early diplotene stage, the autosomal bivalents having already commenced separation,
at least in the interstitial regions. They named this stage 'pre-diffuse' diplotene. By
late diplotene, the longitudinal separation of homologues has continued further and
252
P. Goetz, A. C. Chandley and R. M. Speed
1A
Fig. 1. The sequence of meiotic prophase progression as seen in air-dried preparations.
A. Preleptotene; B, leptotene/zygotene; c, early pachytene; D, mid-pachytene; E, late
pachytene; F, early diplotene; G, late diplotene. The sex vesicle is arrowed. Bar, 10/im.
the beginnings of a diakinesis-like appearance are first seen (Fig. 1G). This stage is
referred to by Oud et al. (1979) as 'post-diffuse' diplotene. Between pre-diffuse and
post-diffuse diplotene, these authors also identified a 'diffuse' diplotene stage in which
the autosomal bivalents were completely despiralized and the nucleus was filled with
a network of fine threads. Only the condensed sex vesicle and heterochromatic regions
of bivalents (seen as small dots) were distinguishable. In our air-dried preparations
Meiosis in pubertal male mice
253
2A
I
\
H
Fig. 2. The sequence of meiotic prophase progression as seen in spread preparations (LM
level). A. Preleptotene; B, leptotene; c, early zygotene; D, late zygotene; E, midpachytene; F, late pachytene; G, early diplotene; H, late diplotene. The sex bivalent is
arrowed. Bar, 10 /Mm.
(made by a method different from the one used by Oud et al. 1979) we were, however,
unable to find a stage answering to that description.
Spread preparations. As stated earlier, a degree of detail of cellular morphology that
quite exceeds that seen in air-dried preparations, is found when spread preparations
254
P. Goetz, A. C. Chandley and R. M. Speed
are examined. Stages can be more accurately identified and sub-stages defined. The
critical stages in development, as seen in progressive samplings over days 8 to 19 are
shown in Figs 2 (LM) and 3 (EM). Much of the information given in the succeeding
¥;
Fig. 3. Selected prophase stages from spread preparations (EM level), A. Late zygotene
showing unpaired X and Y axes; B, mid-pachytene; C, early diplotene; D, late diplotene.
The sex bivalent in the latter three is arrowed. Bar, 10 fim.
Meiosis in pubertal male mice
255
paragraphs confirms observations recorded in earlier publications dealing with
meiotic prophase in the adult male mouse (Solari, 1970; Tres, 1977; Moses, 1980;
Dietrich & Mulder, 1981). We feel justified, however, in describing our findings,
since a description of the correct meiotic sequence obtained from the pubertal male
mouse has not, as far as we can ascertain, been given before.
Fig. 2A shows the spread nucleus at preleptotene (LM level). The chromatin is
dispersed, but scattered in it are many small silver-positive bodies and a number of
larger such bodies. At leptotene (Fig. 2B), these can still be seen, but now the first
fine silver-stained threads are discerned. In early zygotene (Fig. 2c), paired elements
are first seen but the X and Y cannot yet be identified. By late zygotene (Fig. 2D),
autosomal pairing is almost complete, only some ends remaining unpaired. The X
and Y, although recognizable as long single dark-staining elements in a few late
zygotene nuclei (see Figs 3A, 4A), cannot generally be identified at this stage but
where they can be recognized, they appear to be delayed in pairing relative to the
autosomes. By early pachytene (Fig. 2E), pairing has occurred between X and Y,
and the XY SC at this earliest stage of pachytene is seen to be longer than at any
other time (see later). The individual lateral elements of the autosomal and XY SCs
can be clearly seen at the EM level, while the unpaired segments of the X and Y axes
are more darkly stained than the paired autosomal lateral elements. This darker
staining is even more pronounced at mid-pachytene, especially in EM spreads (Fig.
3B). By now, a shorter XY SC is present (see also Fig. 4c). The Y appears thinner
than the X. Darker staining now also begins to characterize the ends of autosomes
(Fig. 3B).
By late pachytene, end-to-end pairing of X and Y is seen in most cells (Figs 2F,
4D), and in only a few does a minute remnant of SC remain. Splitting of the X and
Y axes into two or three filaments also characterizes cells in mid/late pachytene
(Tres, 1977). At the same time, more prominent staining of the terminal segments
of autosomal SCs is seen, while differential thickening appears in the XY bivalent for
the first time. Such thickenings become ever more pronounced as fusiform swellings
that increase in size throughout diplotene (Fig. 2G, H; Fig. 3c, D; Fig. 4E, F) (Tres,
1977). In early diplotene (Fig. 2G), autosomal desynapsis commences, taking place
first in the sub-terminal regions (see also Fig. 3c). The dark-staining ends of bivalents
become ever more prominent. By late diplotene (Figs 2H, 3D), autosomal desynapsis
is extensive in the interstitial regions as the silver axes of separating homologues
balloon out. Terminal segments are still associated for the most part, either end-toend or side-by-side (Tres, 1977), one end of each bivalent showing greater thickening
(the centromeric end associated with heterochromatin) than the other (telomeric)
end.
Diplotene is a stage that also shows clear association of the smooth, dark-staining
round body (Fletcher, 1979) with the XY bivalent. (Fig. 3c, D; Fig. 4E, F). This was
seen in 95 % of cells examined at this time. Earlier stages also showed it but with
declining frequency (85% at late pachytene, 65% at mid-pachytene, 50% at early
pachytene). Prior to early pachytene, it could not be found. Its function remains
unknown.
256
P. Goetz, A. C. Chandley and R. M. Speed
Fig. 4. The pairing sequence of X and Y (EM spreads), A. Late zygotene, X and Y still
unpaired (composite photograph from cell in Fig. 3A); B, early pachytene, XY SC
occupies more than one-third the length of the Y axis; c, mid-pachytene, XY SC occupies
less than one-third the length of the Y axis; D, late pachytene, X and Y now in end-to-end
association; E, early diplotene, end-to-end pairing and fusiform swellings of X and Y; F,
late diplotene, end-to-end pairing and more pronounced fusiform swellings of X and Y.
The round dense silver-stained body is seen in D, E and F, in association with the XY
bivalent. Bar, 1 jim.
Meiosis in pubertal male mice
257
Table 1. Meiotic progression in air-dried preparations. Differential analysis of
prophase cells observed on each day sampled
Prophase stage distribution (%)
A
t
Pachytene
Days postpartum
Preleptotene
Leptotene/
zygotene
8
Sporadic
98-3
46-3
25-0
19-3
18-0
11-3
13-3
7-0
7-0
6-7
7-0
4-0
1-7
52-0
55-0
45-0
40-7
39-5
25'7
26-7
22-0
12-7
14-7
12-7
9
10
11
12
13
14
15
16
17
18
19
20
A
Early
Mid
Late
Diplotene
Diakinesis/
MI
1-7
18-0
27-7
28-3
28-6
30-3
25-0
28-3
13-0
18-0
19-0
2-0
7-3
13-0
19-7
29-0
40-7
21-3
30-0
21-7
29-0
0-9
1-7
0-7
7-7
12-3
13-3
13-7
13-7
22-0
21-0
20-0
3-0
4-0
1-6
r
On day 14, numbers of cells analysed (n) = 1000; on all other days, n = 300. MI, metaphase I.
Table 2. Meiotic progression in spread preparations. Differential analysis ofprophase
cells observed on each day sampled
Prophase stage distributor i(%)
A
r
Days postpartum
8
9
10
11
12
13
14
15
16
17
18
19
20
Preleptotene Leptotene
Sporadic
98-0
64-3
34-0
24-0
20-7
14-1
12-3
13-0
9-0
8-3
10-7
10-7
1-7
21-7
39-3
30-0
27-3
29-6
24-0
18-0
16-7
11-0
12-7
15-7
Zygotene
,
'k
^
Early
Late
0-3
10-0
16-0
170
9-7
15-3
9-7
7-0
7-0
7-3
5-0
9-7
3-7
8-0
14-0
14-0
13-4
9-3
8-3
6-3
3-7
3-7
6-3
Pachytene
A
t
Early
Mid
Late
Diplotene
0-3
2-6
12-0
20-0
8-8
15-0
10-0
7-3
40
3-7
6-3
3-0
7-6
18-5
26-6
31-3
13-7
5-0
7-0
12-0
0-6
0-3
3-0
12-3
18-3
20-0
31-7
27-7
21-7
40-7
25-4
11-7
Meiotic progression
The daily progression of cells, as seen from air-dried preparations, over days 8-20
post-partum, is given in Table 1. That seen in spread preparations is given in Table 2.
The overall time taken for cells to progress from preleptotene to diakinesis/metaphase
258
P. Goetz, A. C. Chandley and R. M. Speed
I was 10 days. Comparison of Table 2 with Table 1 shows that a greater degree of
precision in sub-staging could be obtained using spread preparations, which accounts
principally for the conspicuous differences in the frequencies of different stages for
the two sets of data. Overall timings were, however, the same: early zygotene was
reached on day 9, early pachytene on day 10 and late pachytene on day 13. Diplotene
first appeared on day 17, and diakinesis/metaphase I (only seen in air-dried preparations) on day 18.
For the inter- and intra-litter variations between males, it was found that no significant timing differences existed. Thus, all the ten males analysed in two different
litters on day 14 after birth showed the same differential distributions of cell types.
XYpairing sequence
Confirmation of the XY pairing sequence through the pachytene stage, described
by Tres (1977) and Moses (1980) for the adult male mouse, was obtained in our study.
Pachytene cells were simply followed as they progressed from day 11 (when
pachytenes in sufficient number first appeared on the slides) to day 20. The proportions of pachytenes showing either unpaired XY axes, more than one-third of the Y
paired with the X, less than one-third of the Y paired with the X, or XY end-to-end
pairing, were recorded. The result of the analysis is given in Table 3 and the morphological detail is shown in Fig. 4B-D. Whilst at days 11 and 11-5, 64-5% of all
pachytene cells observed showed an XY SC occupying more than one-third the total
length of the Y axis, by day 14 only 31-3% did so. Over days 16-20, the figure
declined further as these early pachytene cells became diluted in the total pachytene
population. By day 20, the majority of pachytene cells showed XY end-to-end pairing,
the numbers showing more than one-third XY pairing now having declined to 8 %.
From these observations, it was clear that the stage of pachytene showing the most
extensive pairing between X and Y was indeed the earliest pachytene stage, as stated
previously by Tres (1977) and Moses (1980).
Measurements made of the actual length of XY SC found over the early, mid and
late pachytene stages, and early and late diplotene stages showed that at early
pachytene, the XY SC occupied between 39 % and 72 % of the total length of the Y
Table 3. Proportions of pachytene cells with different types of XY association in
successive daily samples (expressed as percentages)
Days post-partum
11-11-5
12-12-5
14
16
18
20
31
27
32
25
26
25
X + Yf
SO1/3Y
SC<1/3Y
End-to-end
19-3
3-7
6-2
0-0
7-7
0-0
64-5
630
31-3
160
7-7
161
33-3
56-3
40-0
15-4
36-0
0-0
00
6-2
44-0
69-2
56-0
80
• Numbers of analysed cells in photographic prints (LM spread preparations).
f Unpaired.
Meiosis in pubertal male mice
259
axis: the mean was 58%. The paired region at this stage occupied from 16-28%
(mean 22%) of the X axis. By mid-pachytene, the XY SC had shortened and now
occupied only 24 % of the length of the Y and 9-6 % of the X. Between late pachytene
and late diplotene, only a minute synaptic region remained.
Measurements of the whole SC complement (autosomal plus sex chromosomal),
made on photographic prints, showed that the mean overall SC length at early
pachytene was 156-3/im (range 123-4—179-2/im) and at mid-pachytene was 156-5 /im
(range 127-3-189-3/im). Gradual lengthening took place through late pachytene
(mean 186-9/im, range 158-7-234-3 /im), early diplotene (mean 195-0/im, range
144-7-217-9/im), to late diplotene (mean 200-9/im, range 182-6-213-2/im). The
relative degree of elongation observed in the XY axes was the same as that found in
the autosomal axes throughout these stages.
DISCUSSION
The use of pubertal males to define the correct sequence of meiotic prophase
development has two distinct advantages over other methods. The use of noxious
drugs to produce a cohort of developing cells (Oud et al. 1979; Dietrich & Mulder,
1981) is obviated, and direct observation can be made on a natural population of
maturing cell types without the need for cell-stage identification based on deduction.
The one reservation in our minds when we embarked on the study was that the initial
wave of meiosis at puberty might not be typical of later waves occurring in the adult
male. Our findings, however, indicate that in both timing and morphological
progression, this first wave of spermatogenesis is entirely representative. Cells at
puberty in our Swiss males progressed from preleptotene to diakinesis/metaphase I
(MI) in 10 days, a finding consistent with earlier studies carried out in this laboratory
on adult males of the random-bred Q-strain (Kofman-Alfaro & Chandley, 1970) using
tritium autoradiography to trace the advancing front of cells labelled at the premeiotic 5-phase (preleptotene). Also within this 10-day period of development at
puberty, the time taken for cellular progression through individual prophase stages
was consistent with Oakberg's (1957) early estimations for the adult male, based on
histological sections. Thus, leptotene and zygotene each lasted approximately 1 day,
pachytene approximately 6 days and diplotene approximately 1 day. These timings
could not, however, be established with any greater precision in the present study
since only 24-h sampling intervals were used.
The overall timing of prophase development found for our Swiss pubertal males
was also consistent with that recorded previously by Nebel, Amarose & Hackett
(1961) for pubertal males of the CF strain. Furthermore, there was a remarkable
constancy in temporal progression in the Swiss strain for males of the same age, both
within and between litters. Spermatogenic timing differences between strains, between pubertal and adult males and between different males of the same age appear,
therefore, to be minimal, if indeed they occur at all.
This is not to say, however, that inconsistencies in timing are not to be encountered
in the literature when the results of different authors using different preparative
260
P. Goetz, A. C. Chandley and R. M. Speed
methods are compared. For example, in studies on pubertal males, and when radioactive tracers in adults are used, it is the faster rate of development of certain spermatogenic cells that is recorded. In cytological studies on testicular sections, on the
other hand, it is average timings that are given (see discussion by Sirlin & Edwards
(1957), following Oakberg (1957)). Also, differences in the duration of stages within
prophase are to be found, the diplotene stage being notable in this respect. According
to Oakberg (1957), diplotene in the male mouse is of 21 h duration. Kofman-Alfaro
& Chandley (1970), however, believed that diplotene was a very brief stage, lasting
only 6 h. Oud et al. (1979) reported it to be of 2 days duration while Dietrich & Mulder
(1981), using a different treatment and SC spreads, found it to last 2-3 days. These
disparate results have arisen mainly out of the problem of recognition of the diplotene
stage in air-dried preparations. In the study of air-dried material by Kofman-Alfaro
& Chandley (1970), the full extent of the diplotene stage was not realized, and only
very late diplotene cells, i.e. those approaching diakinesis, were recorded as such.
WhenOude/a/. (1979) published their findings on air-dried material, however, many
of the largest spermatocytes, which had been recorded by Kofman-Alfaro & Chandley
(1970) as late pachytenes, were in fact seen to be early-mid diplotenes. In the light of
knowledge gained from the studies of Oud et al. (1979), it would now seem that the
diplotene stage in our pubertal males is of approximately 24h duration: early
diplotenes first appeared in spread preparations on day 17 post-partum, while
diakinesis was first seen in the air-dried preparations on day 18. The fact that Oud et
al. (1979) and Dietrich & Mulder (1981) recorded a 2 to 3-day duration for diplotene
remains, however, unexplained, but it may be that some delay in spermatogenic
development had occurred in their experiments because of the use of drugs to restrict
the spermatocyte population.
From the point of view of morphological progression, we were interested particularly in two questions. One was the nature of the XY pairing sequence at
pachytene, the other was the possible existence of a diffuse stage at diplotene.
On the former question, the early studies of Solari (1970) on sectioned spermatocytes, and later studies of Tres (1977) and Moses (1980) using spreading
techniques, have shown that the X and Y chromosomes of the mouse are the last to
pair at zygotene and the first to commence desynapsis in mid-pachytene. These
observations we were able to confirm in our study. Spreads of late zygotene nuclei
(first seen on day 10 post-partum (p.p.)) showed a few cells in which the X and Y
could be recognized as darker-staining single structures. (In the majority of cells at
this time, however, recognition of the sex elements is extremely difficult, if not
impossible.) The autosomes, meanwhile, were almost fully paired along their length,
only occasional ends remaining unpaired. By early pachytene (also first seen on day
10 p.p.), pairing had occurred in the XY pair in all cells examined and it was at this
stage that the longest XY SC was observed, occupying, on average, about two-thirds
of the total length of the Y axis. By late pachytene (day 14p.p.), end-to-end pairing
characterized the XY bivalent in the majority of cells and this was the configuration
that remained also through diplotene (first seen on day 17 p.p.). Thus, the trend from
maximum length XY SC at early pachytene to end-to-end pairing by late pachytene,
Meiosis in pubertal male mice
261
described originally by Solari (1970) was confirmed in our study. We were also able
to confirm two other interesting aspects of XY morphology, described by Tres (1977),
in spread spermatocyte preparations of the adult male mouse. One was the frequent
splitting of the axes into two or sometimes more filaments at the mid/late pachytene
stages (see Fig. 4c, D) ; the other was the appearance of fusiform swellings on the sex
chromosome axes, beginning at late pachytene and becoming even more pronounced
at diplotene (see Fig. 4E, F). We were, however, unable to confirm a third observation
made by Tres (1977). She has claimed that a gradual shortening of the X and Y axes
takes place through zygotene, pachytene and diplotene. When measurements on the
whole genome were made in our mice, however, no change in length, for either the
XY or the autosomal axes, was detectable between early and late pachytene (zygotene
cells rarely allowed XY identification to be made), but from late pachytene onwards,
a gradual lengthening occurred. This was an observation consistent with the findings
of Moses (19776) for the XY bivalent of the Chinese hamster.
The concept of a diffuse stage in late meiotic prophase in the male mouse, comparable to that reported in many plants, has been discussed at length by Kldgterska",
Natarajan & Ramel (1976) and Kla'Sterska' (1977). Such a stage in the male, having
an interphase-like appearance, has been postulated to correspond to the diffuse
diplotene or dictyate stage of the female (Wilson, 1925). KliSterskd et al. (1976)
demonstrated the existence of a diffuse-like stage in mouse and rhesus monkey spermatocytes prepared by squashing in combination with a modified C-banding
technique. She believes that an equivalent stage may exist in most male mammals,
including man. The later studies of Oud et al. (1979) provided additional proof of a
stage when extreme despiralization of the chromosomes occurred in late prophase.
These authors introduced a new nomenclature, subdividing the diplotene stage into
'pre-diffuse', 'diffuse' and 'post-diffuse'. In the diffuse stage, which they estimated to
be short in duration, the nucleus was seen in their air-dried preparations to be filled
with a network of fine threads, only the sex vesicle and heterochromatic regions
staining darkly. No description of this stage was given, however, in later studies made
by Dietrich & Mulder (1981) employing the use of spreading techniques.
Our own air-dried preparations from pubertal males, made by a technique different
from those used by Oud et al. (1979), provide no evidence however, for a diffuse stage
as described by these authors. What we have referred to throughout our paper as
'early' diplotene does, however, correspond well to their description of pre-diffuse
diplotene and our 'late' diplotene stage corresponds well to their post-diffuse
diplotene. We could not, however, find an intermediate stage between the two. When
silver-stained spreads were examined, a marked lengthening of SCs was obvious
throughout diplotene, as mentioned earlier in the Discussion, but at no time were we
able to find a stage at which the silver-stained axial elements virtually disappeared
altogether into an interphase-like state, comparable to that seen in silver-stained
spread preparations of mouse dictyate oocytes (Speed, 1982). We were unable
therefore to provide evidence for a truly diffuse stage in the male mouse at diplotene,
using the techniques described. The possibility still remains, however, that the diffuse stage in the male mouse is of such a transient nature that it has been missed.
262
P. Goetz, A. C. Chandley and R. M. Speed
The authors are grateful to Mr Andrew Ross and Miss Liz Peffers for assistance in connection with
the preparation of EM specimens and to Mr Douglas Stuart for help in the preparation of the plates.
The senior author, Dr P. Goetz, carried out the work while the recipient of a Wellcome Research
Followship, 1982-83.
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(Received 6 June 1983-Accepted 19 August 1983)