MORPHOLOGICAL AND TEMPORAL SEQUENCE OFMEIOTIC PROPHASE DEVELOPMENT AT PUBERTYIN THE MALE MOUSE
P. GOETZ', ANN C. CHANDLEY AND R. M. SPEEDMRC Clinical and Population Cytogenetics Unit, Western General Hospital, EdinburghEH4 2XU, U.K.
SUMMARY
The correct sequence of meiotic prophase development in the male mouse has been establishedby the use of pubertal males. The first wave of spermatogenesis at this time provides a uniqueopportunity to study progressive meiotic development in a direct way. Air-dried and micro-spreadanalyses have been carried out. Temporal and morphological progression at this time is entirelyconsistent with that occurring in the later waves of meiosis of the adult male. Morphological detailshows delayed pairing of the X and Y chromosomes relative to the autosomes. The longest XYsynaptonemal complex is seen in early pachytene cells, occupying up to 72% of the length of theY and 22% of the length of the X axis. By late pachytene, end-to-end pairing in the XY bivalentis established, the autosomal axes remaining fully paired. Desynapsis of the autosomes commencesat early diplotene. A 'diffuse' diplotene stage in the male, comparable to the dictyate stage of thefemale, could not be found. Marked lengthening of the XY and autosomal axes did, however, occurthrough the diplotene stage.
INTRODUCTION
The simple and rapid technique by which primary spermatocytes of mammals canbe prepared by micro-spreading (Counce & Meyer, 1973; Moses, 1977a,b) has led,in recent years, to a great expansion of knowledge concerning cellular morphology andchromosome pairing at meiotic prophase in a variety of species (see Moses, 1980, fora review). Complemented by serial reconstructions of whole nuclei (see Gillies, 1975,for a review), for accurate identification of individual stages in meiosis, much has beenlearned of the behaviour of chromosomes during normal and abnormal synapsis anddesynapsis, and the role of the synaptonemal complex (SC) in meiosis. Sequentialmeiotic development has to be inferred, however, from relationships between the XYpair, nucleolar development and autosome behaviour (Moses, 1980; Solari, 1980)since, among spermatocytes from mixed testicular suspensions of adult males, thereis 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 adultmale mice by the creation of two large gaps in the spermatogenic line usinghydroxyurea and triaziquone to kill spermatogonia. The two gaps enclosed a small,
•Permanent address: Department of Medical Genetics, 151 12 Prague 5, V uvalu 84, Czecho-slovakia.
CEL65
250 P. Goetz, A. C. Chandley and R. M. Speed
well-defined cohort of surviving spermatocytes in pre-meiotic interphase. Thedevelopment of this restricted spermatocyte population was then followed day by dayas 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, wouldsimply exploit the natural sequence of development at puberty in the male. Bysystematic sampling around the peri-pubertal period the morphological and temporalsequence 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 andspread preparations have been analysed, and descriptions of cell stages seen at bothlight- (LM) and electron-microscope (EM) levels are given. The advantages of cor-related light and EM studies have been emphasized by Dresser & Moses (1980).
Particular attention has been focussed on the behaviour and pairing sequence in theXY bivalent and on the possible existence of a 'diffuse' stage at diplotene.
MATERIALS AND METHODS
Testicular samplingMice 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 tenby destruction of excess females in order to allow vigorous development and to minimize growthdifferences between males. Over days 8—20 (the peri-pubertal period) males were killed daily andtestes removed. To obtain a sufficient number of cells in suspension, it was found necessary overdays 8-13, to pool the testes of 4-6 males from one litter. From day 13 today 20, preparations weremade from three individual males on each day. Because it was found that critical events in XYpairing were occurring over days 11 to 13 of development, additional males were killed at half-dailyintervals over this period. A check on inter- and intra-litter variation in male meiotic developmentwas also carried out. Testicular suspensions prepared from all ten males in two different litters onday 14 post-partum were analysed. Six males were obtained from one litter and four from the other.
Cell preparationFor analysis at the light-microscope (LM) level. Air-dried preparations were made according toEvans, Breckon & Ford (1964), and stained with carbol fuchsin (Carr & Walker, 1961). In ourexperience, this is the best possible combination for spermatogenic stage identification in air-driedpreparations 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 cellsuspension, drop for drop, with 0-15 M-sucrose on the slide. After 1 h of drying on the bench, theslide was fixed with 4% formaldehyde and washed with 0-4% buffered Photoflo. Staining wascarried out overnight using 50 % AgNCh (aqueous) at 60 °C, or by the rapid colloidal silver methodof 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 ofFelluga & Martinucci (1976) with minor modification. Spermatocytes were spread on Parlodion-coated slides, stained with AgNC>3 (Howell & Black, 1980), and examined at LM level. The bestspreads were selected, and cut from the film using a Leitz diamond slide marker, the small roundfilm 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 EM300at60kV.
Meiosis in pubertal male mice 251
Analytical methodsFor 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 werefollowed in development from preleptotene to diakinesis/metaphase I. In spreads, it is only possibleto examine cells up to late diplotene as the SCs (synaptonemal complexes) after that stage are brokendown and no longer visible by silver staining. Spread preparations provide a considerably greaterdegree 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 mostadvanced cell type observed at each sampling time was recorded. For the inter- and intra-littercomparisons, carried out on day 14, differential analyses were made on 100 cells from each male ofthe two litters sampled again using both air-dried and spread preparations.
To complement the LM analysis, morphological detail was also studied in photographic enlarge-ments 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 therelative degrees of contraction and elongation of the XY and autosomal bivalents throughoutpachytene and diplotene. Measurements were made using the cursor of a Digiplan electronicmeasurer (Reichert-Jung) to trace the silver-stained axes on the prints. Each measurement was madethree times and a mean calculated. Prints made from cells on EM grids were also examined formorphological detail and, in particular, were studied critically for detail of XY behaviour betweenzygotene and late diplotene.
RESULTS
Morphological analysis
Air-dried preparations. The principal prophase stages identified in air-driedpreparations as progressive sampling took place, are shown in Fig. 1.
At preleptotene, the nucleus is small and round (approx. 6/im in diameter), thechromatin being generally homogeneous (Fig. 1A). Leptotene and zygotene cannotreadily be distinguished from each other, but cells in these early prophase stages areslightly 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 ofchromatin condensation. Early pachytene cells are small with dense chromatin andoccasionally show the beginnings of the sex vesicle (Fig. lc); mid-pachytenes aremuch larger and show a prominent sex vesicle against a paler background of autosomalelements (Fig. ID).
Cells in late pachytene may easily be confused with those in mid-pachytene andearly diplotene. Nuclei are only slightly larger than those in mid-pachytene but theautosomal bivalents are more clearly delineated (Fig. 1E).
By early diplotene (Fig. IF), the nucleus is very large and pale-staining. Carefulexamination shows a double structure to the autosomal bivalents and a very prominentsex vesicle. Several clusters of darker staining bodies are present. In the past, thisstage 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 theearly diplotene stage, the autosomal bivalents having already commenced separation,at least in the interstitial regions. They named this stage 'pre-diffuse' diplotene. Bylate 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, latepachytene; 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 isreferred to by Oud et al. (1979) as 'post-diffuse' diplotene. Between pre-diffuse andpost-diffuse diplotene, these authors also identified a 'diffuse' diplotene stage in whichthe autosomal bivalents were completely despiralized and the nucleus was filled witha network of fine threads. Only the condensed sex vesicle and heterochromatic regionsof 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 (LMlevel). A. Preleptotene; B, leptotene; c, early zygotene; D, late zygotene; E, mid-pachytene; F, late pachytene; G, early diplotene; H, late diplotene. The sex bivalent isarrowed. 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 thatquite 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. Thecritical stages in development, as seen in progressive samplings over days 8 to 19 areshown 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 zygoteneshowing 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 withmeiotic 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 malemouse has not, as far as we can ascertain, been given before.
Fig. 2A shows the spread nucleus at preleptotene (LM level). The chromatin isdispersed, but scattered in it are many small silver-positive bodies and a number oflarger such bodies. At leptotene (Fig. 2B), these can still be seen, but now the firstfine silver-stained threads are discerned. In early zygotene (Fig. 2c), paired elementsare 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 Xand Y, although recognizable as long single dark-staining elements in a few latezygotene nuclei (see Figs 3A, 4A), cannot generally be identified at this stage butwhere they can be recognized, they appear to be delayed in pairing relative to theautosomes. 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 anyother time (see later). The individual lateral elements of the autosomal and XY SCscan be clearly seen at the EM level, while the unpaired segments of the X and Y axesare more darkly stained than the paired autosomal lateral elements. This darkerstaining 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 thinnerthan 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 andY 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 segmentsof autosomal SCs is seen, while differential thickening appears in the XY bivalent forthe first time. Such thickenings become ever more pronounced as fusiform swellingsthat 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 placefirst in the sub-terminal regions (see also Fig. 3c). The dark-staining ends of bivalentsbecome ever more prominent. By late diplotene (Figs 2H, 3D), autosomal desynapsisis extensive in the interstitial regions as the silver axes of separating homologuesballoon out. Terminal segments are still associated for the most part, either end-to-end 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-staininground body (Fletcher, 1979) with the XY bivalent. (Fig. 3c, D; Fig. 4E, F). This wasseen in 95 % of cells examined at this time. Earlier stages also showed it but withdeclining frequency (85% at late pachytene, 65% at mid-pachytene, 50% at earlypachytene). Prior to early pachytene, it could not be found. Its function remainsunknown.
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 stillunpaired (composite photograph from cell in Fig. 3A); B, early pachytene, XY SCoccupies more than one-third the length of the Y axis; c, mid-pachytene, XY SC occupiesless than one-third the length of the Y axis; D, late pachytene, X and Y now in end-to-endassociation; 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 XYbivalent. Bar, 1 jim.
Meiosis in pubertal male mice 257
Table 1. Meiotic progression in air-dried preparations. Differential analysis ofprophase cells observed on each day sampled
of cells analysed (n) = 1000; on all other days, n
Diplotene
13-722-021-020-0
= 300. MI,
Diakinesis/MI
3-04-01-6
metaphase I.
Table 2. Meiotic progression in spread preparations. Differential analysis of prophasecells observed on each day sampled
Days post-partum
89
1011121314151617181920
r
Prelep-totene
Sporadic98-064-334-024-020-714-112-313-09-08-3
10-710-7
Leptotene
1-721-739-330-027-329-624-018-016-711-012-715-7
Prophase stage
Zygotene, 'Early
0-310-016-01709-7
15-39-77-07-07-35-09-7
k ^Late
3-78-0
14-014-013-49-38-36-33-73-76-3
distributorA
tEarly
0-32-6
12-020-08-8
15-010-07-34 03-76-3
i(%)
PachyteneA
Mid
3-07-6
18-526-631-313-75-07-0
12-0
Late
0-60-33-0
12-318-320-031-727-7
Diplotene
21-740-725-411-7
Meiotic progression
The daily progression of cells, as seen from air-dried preparations, over days 8-20post-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 ofprecision in sub-staging could be obtained using spread preparations, which accountsprincipally for the conspicuous differences in the frequencies of different stages forthe two sets of data. Overall timings were, however, the same: early zygotene wasreached on day 9, early pachytene on day 10 and late pachytene on day 13. Diplotenefirst appeared on day 17, and diakinesis/metaphase I (only seen in air-dried prepara-tions) on day 18.
For the inter- and intra-litter variations between males, it was found that no sig-nificant timing differences existed. Thus, all the ten males analysed in two differentlitters 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, describedby 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 (whenpachytenes in sufficient number first appeared on the slides) to day 20. The propor-tions of pachytenes showing either unpaired XY axes, more than one-third of the Ypaired with the X, less than one-third of the Y paired with the X, or XY end-to-endpairing, were recorded. The result of the analysis is given in Table 3 and the morpho-logical detail is shown in Fig. 4B-D. Whilst at days 11 and 11-5, 64-5% of allpachytene cells observed showed an XY SC occupying more than one-third the totallength of the Y axis, by day 14 only 31-3% did so. Over days 16-20, the figuredeclined further as these early pachytene cells became diluted in the total pachytenepopulation. 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 mostextensive pairing between X and Y was indeed the earliest pachytene stage, as statedpreviously by Tres (1977) and Moses (1980).
Measurements made of the actual length of XY SC found over the early, mid andlate pachytene stages, and early and late diplotene stages showed that at earlypachytene, 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 insuccessive daily samples (expressed as percentages)
Days post-partum
11-11-512-12-5
14161820
• Numbers of analysedf Unpaired.
312732252625
cells in
X + Yf
19-33-76-20-07-70-0
photographic prints
SO1/3Y
64-563031-31607-780
(LM spread
SC<1 /3Y
16133-356-340-015-436-0
preparations).
End-to-end
0-00 06-2
44-069-256-0
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 nowoccupied only 24 % of the length of the Y and 9-6 % of the X. Between late pachyteneand 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 earlypachytene 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, range144-7-217-9/im), to late diplotene (mean 200-9/im, range 182-6-213-2/im). Therelative degree of elongation observed in the XY axes was the same as that found inthe autosomal axes throughout these stages.
DISCUSSION
The use of pubertal males to define the correct sequence of meiotic prophasedevelopment has two distinct advantages over other methods. The use of noxiousdrugs 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 ofmaturing 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 initialwave of meiosis at puberty might not be typical of later waves occurring in the adultmale. Our findings, however, indicate that in both timing and morphologicalprogression, this first wave of spermatogenesis is entirely representative. Cells atpuberty 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 laboratoryon adult males of the random-bred Q-strain (Kofman-Alfaro & Chandley, 1970) usingtritium autoradiography to trace the advancing front of cells labelled at the pre-meiotic 5-phase (preleptotene). Also within this 10-day period of development atpuberty, the time taken for cellular progression through individual prophase stageswas consistent with Oakberg's (1957) early estimations for the adult male, based onhistological sections. Thus, leptotene and zygotene each lasted approximately 1 day,pachytene approximately 6 days and diplotene approximately 1 day. These timingscould not, however, be established with any greater precision in the present studysince only 24-h sampling intervals were used.
The overall timing of prophase development found for our Swiss pubertal maleswas also consistent with that recorded previously by Nebel, Amarose & Hackett(1961) for pubertal males of the CF strain. Furthermore, there was a remarkableconstancy in temporal progression in the Swiss strain for males of the same age, bothwithin and between litters. Spermatogenic timing differences between strains, be-tween 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 encounteredin 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 radio-active tracers in adults are used, it is the faster rate of development of certain sper-matogenic cells that is recorded. In cytological studies on testicular sections, on theother hand, it is average timings that are given (see discussion by Sirlin & Edwards(1957), following Oakberg (1957)). Also, differences in the duration of stages withinprophase are to be found, the diplotene stage being notable in this respect. Accordingto 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, lastingonly 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. Thesedisparate results have arisen mainly out of the problem of recognition of the diplotenestage 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 onlyvery late diplotene cells, i.e. those approaching diakinesis, were recorded as such.WhenOude/a/. (1979) published their findings on air-dried material, however, manyof 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 ofknowledge gained from the studies of Oud et al. (1979), it would now seem that thediplotene stage in our pubertal males is of approximately 24h duration: earlydiplotenes first appeared in spread preparations on day 17 post-partum, whilediakinesis was first seen in the air-dried preparations on day 18. The fact that Oud etal. (1979) and Dietrich & Mulder (1981) recorded a 2 to 3-day duration for diploteneremains, however, unexplained, but it may be that some delay in spermatogenicdevelopment had occurred in their experiments because of the use of drugs to restrictthe spermatocyte population.
From the point of view of morphological progression, we were interested par-ticularly in two questions. One was the nature of the XY pairing sequence atpachytene, the other was the possible existence of a diffuse stage at diplotene.
On the former question, the early studies of Solari (1970) on sectioned sper-matocytes, and later studies of Tres (1977) and Moses (1980) using spreadingtechniques, have shown that the X and Y chromosomes of the mouse are the last topair at zygotene and the first to commence desynapsis in mid-pachytene. Theseobservations 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 Ycould be recognized as darker-staining single structures. (In the majority of cells atthis time, however, recognition of the sex elements is extremely difficult, if notimpossible.) The autosomes, meanwhile, were almost fully paired along their length,only occasional ends remaining unpaired. By early pachytene (also first seen on day10 p.p.), pairing had occurred in the XY pair in all cells examined and it was at thisstage that the longest XY SC was observed, occupying, on average, about two-thirdsof the total length of the Y axis. By late pachytene (day 14p.p.), end-to-end pairingcharacterized the XY bivalent in the majority of cells and this was the configurationthat remained also through diplotene (first seen on day 17 p.p.). Thus, the trend frommaximum 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 ableto confirm two other interesting aspects of XY morphology, described by Tres (1977),in spread spermatocyte preparations of the adult male mouse. One was the frequentsplitting of the axes into two or sometimes more filaments at the mid/late pachytenestages (see Fig. 4c, D) ; the other was the appearance of fusiform swellings on the sexchromosome axes, beginning at late pachytene and becoming even more pronouncedat diplotene (see Fig. 4E, F). We were, however, unable to confirm a third observationmade by Tres (1977). She has claimed that a gradual shortening of the X and Y axestakes place through zygotene, pachytene and diplotene. When measurements on thewhole genome were made in our mice, however, no change in length, for either theXY or the autosomal axes, was detectable between early and late pachytene (zygotenecells rarely allowed XY identification to be made), but from late pachytene onwards,a gradual lengthening occurred. This was an observation consistent with the findingsof Moses (19776) for the XY bivalent of the Chinese hamster.
The concept of a diffuse stage in late meiotic prophase in the male mouse, compar-able 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, havingan interphase-like appearance, has been postulated to correspond to the diffusediplotene 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 sper-matocytes prepared by squashing in combination with a modified C-bandingtechnique. 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 astage 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 tobe short in duration, the nucleus was seen in their air-dried preparations to be filledwith a network of fine threads, only the sex vesicle and heterochromatic regionsstaining darkly. No description of this stage was given, however, in later studies madeby Dietrich & Mulder (1981) employing the use of spreading techniques.
Our own air-dried preparations from pubertal males, made by a technique differentfrom those used by Oud et al. (1979), provide no evidence however, for a diffuse stageas described by these authors. What we have referred to throughout our paper as'early' diplotene does, however, correspond well to their description of pre-diffusediplotene and our 'late' diplotene stage corresponds well to their post-diffusediplotene. We could not, however, find an intermediate stage between the two. Whensilver-stained spreads were examined, a marked lengthening of SCs was obviousthroughout diplotene, as mentioned earlier in the Discussion, but at no time were weable to find a stage at which the silver-stained axial elements virtually disappearedaltogether into an interphase-like state, comparable to that seen in silver-stainedspread preparations of mouse dictyate oocytes (Speed, 1982). We were unabletherefore 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 dif-fuse 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 withthe 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 ResearchFollowship, 1982-83.
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COUNCE, S. J. & MEYER, G. F. (1973). Differentiation of the synaptonemal complex and thekinetochore in Locusta spermatocytes studied by whole mount electron microscopy. Chromosoma44, 231-253.
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