/ . Embryol. exp. Morph. Vol. 38, pp. 115-186, 1977
Printed in Great Britain
175
Preleptotene chromosome condensation
stage in human foetal and neonatal testes
By J. M. LUCIANI, 1 MONIQUE DEVICTOR1 AND A. STAHL1
From the Laboratory of Histology and Embryology, Marseille
SUMMARY
A preleptotene stage of chromosome condensation analogous to that already described
in various plants and in the oocytes of several animal species has been observed in the human
foetal testis. Contrary to what has been previously described, this stage in the testis is not
followed by decondensation leading to leptotene filaments. This observation underlines the
problem of the precise significance of this stage and its relation to initiation of meiosis. It is
suggested that meiosis may be initiated during this condensation phase and that the male
germ cell, despite its XY chromosome constitution, tends to evolve towards meiosis. This
proposal pleads in favour of both the role of somatic cells in the inhibition of meiosis in the
male foetus and the role of environmental factors rather than genetic constitution of the
germ cell in meiotic induction.
INTRODUCTION
The existence of a chromosomal condensation period, followed by decondensation, occurring between the last premeiotic interphase and leptotene, has
been described in several plant species (for review see Walters, 1972; Bennett &
Stern, 1975). Such a period of chromosomal condensation also exists in animals
as demonstrated by Davis in 1908 and Wilson in 1928 who described in several
invertebrates the presence of compact and irregular masses of chromatin,' the
prochromosomes'. We have observed a preleptotene stage with prochromosomes in the human foetal oocyte (Stahl & Luciani, 1971), the rabbit oocyte
(Devictor-Vuillet, Luciani & Stahl, 1973) and the sheep oocyte (Mauleon,
Devictor-Vuillet & Luciani, 1976). Despite certain morphological differences,
an homology exists between the prochromosome condensation stage in
animals and the preleptotene contraction described in plants.
Such a preleptotene chromosome condensation stage has never been observed
at the onset of human male meiosis (Luciani, 1970; Ferguson-Smith, 1972;
Hulten & Lindsten, 1973).
Analysis of histological sections and cytological preparations in the human
foetal testis allowed us to observe images of prochromosomes (Figs. 7, 9)
analogous to those already described in the human foetal oocyte.
1
Authors' address: Laboratoire d'Histologie et Embryologie II, Faculte de Medecine, 27,
bd Jean-Moulin, 13385 Marseille Cedex 4, France.
176
J. M. LUCIANI, M. DEVICTOR AND A. STAHL
As a result, there exists in the human foetal testis a preleptotene stage of
chromosome condensation which is not followed by a decondensation stage
normally leading to leptotene filaments.
MATERIAL AND METHODS
The testes from 13 foetuses and two 2-day-old neonates were examined. Foetal
age was established according to both the date of the last menstruation and
foetal measurements (crown-rump; crown-heel) leading to the following results:
8 weeks (1), 12 weeks (2), 13 weeks (2), 15 weeks (1), 16 weeks (1), 18 weeks (1),
19 weeks (2), 22 weeks (l), 7 months (i), i\ months (l). We employed classical
histological techniques, and for chromosomal study the same cytological
technique used in treating foetal ovaries (Luciani, Devictor-Vuillet, Gagne &
Stahl, 1974). Cytological preparations were stained using Giemsa solution and
for three foetuses (14 weeks, 19 weeks and 2 days post-partum) using the G 11
technique of Gagne & Laberge (1972).
RESULTS
1. Morphology of preleptotene condensation stage
As in the human oocyte, the preleptotene chromosomal condensation stage in
the male may be divided into four phases: early, middle, advanced and complete
condensation. This condensation stage itself is preceded by one of pre-condensation. The only missing stage in the foetal testis is that of decondensation
leading to leptotene. Morphological aspects of this condensation stage are
identical regardless of foetal age.
During the precondensation stage, the chromatin of premeiotic interphase
nuclei consists of numerous, small and dark staining masses connected to one
another by barely visible fine threads (Figs. 1, 2). These masses correspond to
centromeric heterochromatin.
The onset of the condensation stage is marked by an increase in length of the
chromosome threads which then surround the chromatin masses (Fig. 3).
As condensation continues the filaments become thicker and more irregular
in appearance (Fig. 4). The chromosomal masses become more voluminous
and are composed of the loops and folds originating from the filaments, whereas
the not yet incorporated filaments connect the masses to each other (Fig. 5).
At a later stage of spiralization, there are 46 well individualized chromosome
masses (Figs. 6, 7). Each mass is clearly seen to be constituted by the tangled
filaments (Figs. 6-7 and 9C). One or more nucleoli are visible in relation with
the chromosome masses, yet the exact nature of this relationship is difficult to
establish. Quinacrine mustard staining followed by examination under u.v. light
demonstrates that the satellites are in contact with the nucleoli, whereas specific
staining of the C9 heterochromatin by the technique of Gagne & Laberge (1972)
Chromosome condensation in human testes
Scales indicate 10 {im.
Fig. 1. Late premeiotic interphase nucleus. Foetus at 13 weeks.
Fig. 2. Nucleus at precondensation stage. Note the individualization of darkstained chromatin segments corresponding to centromeric heterochromatin.
Foetus at 13 weeks.
Fig. 3. Nucleus at very early preleptotene chromosome condensation stage showing
chromosomal strands surrounding each dark segment. Foetus at 13 weeks.
Fig. 4. Early condensation: chromosomal filaments become longer and thicker.
Foetus at 12 weeks.
111
178
J. M. LUCIANI, M. DEVICTOR AND A. STAHL
10/tm
7
Fig. 5. Mid-condensation: loops and folds originating from thefilamentsconstitute
irregulai masses. Foetus at 19 weeks.
Figs. 6, 7. Late condensation: coiled and tangled filaments constitute 46 separate
chromosomal masses. In Fig. 6, note the presence of two nucleoli (/?) in close contact with several chromosomes. Foetus at 13 weeks.
Fig. 8. Nucleus at full condensation: 46 separate chromosomes, each presenting
two chromatids. Foetus at 13 weeks.
Chromosome condensation in human testes
179
Fig. 9. (A) Histological section of a seminiferous tubule showing several germ cells,
one of which can be clearly identified as a nucleus at late condensation stage
(arrow). Foetus at 18 weeks. (B) Detail of a late condensation-stage nucleus from
histological section showing the 'prochromosomes' in close contact with the nuclear
membrane. Foetus at 19 weeks. (C) Detail of condensation from cytological preparations: loops and folds of tangled filaments constitute each chromosome.
Foetus at 13 weeks.
does not demonstrate any particular nucleolar association at this stage in contrast to its usual localization as pointed out by Gagne, Laberge & Tanguay
(1973).
Morphology at this stage corresponds to that of the prochromosome nuclei
previously observed in human oocytes. These nuclei are also visible on the
histological sections (Fig. 9 A, B) permitting observation of most chromosome
masses pressed against the nuclear membrane. Increased condensation is
accompanied by continual increase in thickness of the chromosome threads.
At maximal condensation, individual chromosomes, composed of two distinctly separate chromatids exhibit both thicker and thinner regions (Fig. 8).
This irregular outline permits their distinction with prometaphase mitotic
chromosomes. In the foetal testis, this stage is rarely observed.
180
J. M. LUCIANI, M. DEVICTOR AND A. STAHL
(A) OVARY
Full
condensation
Early
decondensation
Late
decondensation
\
. Leptotene
Precondensation
(B) TESTIS
Full
condensation
4 , .<
' g ^
Late
c? $•
condensation
Lack of
decondensation.
Mid
condensation
By-pass
Early
condensation
Reversion to
mitosis (?)
Precondensation
Fig. 10. Comparative behaviour between foetal ovary and testis at the moment of
meiotic initiation. (A) In the ovary, initiation of meiosis may occur during the preleptotene chromosome condensation stage. According to the timing at which
meiotic initiation occurs, varying degrees of condensation are observed. Condensation is followed by decondensation leading to leptotene. (B) In the testis, initiation
of meiosis does not take place during preleptotene chromosome condensation.
With lack of initiation, decondensation does not occur and the germinal cells probably revert to a mitotic state.
Chromosome
condensation in human
181
testes
Table 1. Distribution of the number of preleptotene condensation
stages according to foetal age
Reference
TEF 12(8 weeks p.c.)
TEF 16(12 weeks p.c.)
TEF 17 (12 weeks p.c.)
TEF 13 (13 weeks p.c.)
TEF 5 (13 weeks p.c.)
TEF 18(15 weeks p.c,)
TEF 4 (16 weeks p.c.)
TEF 8 (18 weeks p.c.)
TEF 1 (19 weeks p.c.)
TEF 11 (19 weeks p.c.)
TEF 19(22 weeks p.c.)
TEF 15 (7± months p.c.)
TEF 3 (2 days p.p.)
Number of
cells
Number of
cells at
gonia stage
Number of
cells at early
condensation
stage
Number of
cells at midcondensation
stage
50
50
50
50
50
50
50
50
50
50
50
50
50
40
42
46
46
38
46
42
40
45
35
40
41
31
6
6
1
1
7
3
6
7
4
7
8
9
13
4
2
3
3
5
1
2
3
1
8
2
2
6
p.c, Post-conception ;p.p., post-partum.
2. Quantitative study of preleptotene condensation according to foetal age
We looked to see if a difference in distribution of the various stages with
respect to foetal age could be detected. Table 1 shows that no significant difference exists. This preleptotene condensation stage is present very early since
it can be seen in the 8-week-old testis whose seminiferous tubules are already
well formed. This study demonstrated the existence of individual variation and
in particular showed that the condensation process is present at the same time
in no more than 20 % of male germinal cells. In addition, this process continues,
as in the ovary, throughout the foetal period.
These results seem to indicate the existence of an incessant renewal of those
germinal cells which have reached condensation: whereas a certain number of
cells (no more than 20 %) evolve towards the condensation stage, those having
already attained this stage become uncondensed and either degenerate or revert
to a mitotic state.
3. Comparative study with the ovary
Morphological aspects of the preleptotene condensation stage are identical in both the 8-week-old testis and ovary. The differences are quantitative
as the number of cells in the 'gonia' stage and in the condensation stage is
higher in the ovary. At such an early phase of gonadal differentiation the despiralization stage is not observed in the ovary and only becomes apparent as
do the first leptotene stages in the ovary at 10 weeks. Differences between
182
J. M. LUCIANI, M. DEVICTOR AND A. STAHL
testis and ovary are rapidly accentuated with the apparition of the first pachytene nuclei at 11 weeks and the first diplotene nuclei at 13 weeks (unpublished
data).
Regardless the age, full condensation is rarely attained in the foetal testis
and the degree of contraction always remains inferior to that observed in
the human oocyte. This variation in degree of contraction is actually well
established: Walters (1972) demonstrated it in various species of Lilium and we
have observed it in comparing human and rabbit oocytes (Devictor-Vuillet et al
1973).
4. Absence of chromosome pairing during preleptotene condensation
The use of morphological criteria for chromosome identification and in
particular the use of a specific staining technique for the heterochromatic
segments of the C9 chromosome (Gagne & Laberge, 1972) showed that no
evidence exists for the pairing of homologous chromosomes. On the contrary,
chromosomes appear to be randomly distributed, except the nucleolar chromosomes which nevertheless do not pair with one another.
5. What becomes of the cell ?
The following point is certain: the nucleus does not despiralize to yield
leptotene filaments. In addition, we were not able to observe under light microscopy signs of cytoplasmic or nuclear degeneration. Thus it is probable that a
process of reversion to a mitotic behaviour takes place leading to formation of
interphase nuclei.
DISCUSSION
Observation of a chromosomal stage of condensation common to both ovary
and testis may shed new light on this still poorly understood phenomenon and
on the mechanisms of meiotic initiation.
For Hilscher et al (1974) this process of chromosomal condensation characterizes the postmitotic oocyte nuclei resulting from the last mitotic division,
whereas for most authors (Walters, 1970, 1972; Stahl & Luciani, 1971; Burns,
1972; Devictor-Vuillet et al 1973; Bennett & Stern, 1975; Mauleon et al
1976) such condensation, followed by decondensation, is considered to be a
true preleptotene stage constituting in various species a regular form of onset
in meiotic prophase. Recently, Bennett & Stern (1975), studying a Lilium
hybrid Black Beauty, situated the onset of this process after the G2 phase of the
last premeiotic interphase. A preliminary study in the sheep embryo after
injection of tritiated thymidine has led Mauleon et al (1976) to similar conclusions.
This preleptotene stage of chromosome condensation is situated at a transition phase between the mitotic and meiotic states. In certain organisms the
transition is precise as the germinal cells evolve directly from premeiotic inter-
Chromosome condensation in human testes
183
phase to leptotene. In other organisms, transition is less clearly defined: meiosis
begins, yet some fluctuations can take place and a reversion to mitosis may
occur, the chromosomes becoming condensed as in mitotic prophase. However,
reversion is only partial and before reaching metaphase, the chromosomes
despiralize and form the elongated filaments of the leptotene nucleus. This
hypothesis formulated in 1970 led Walters to suggest in 1972 that the preleptotene stage of chromosomal condensation results from a temporary absence of
certain substances necessary for the initiation and development of meiosis.
Certain experimental results support this hypothesis. Mauleon (1973),
studying cultures of sheep ovaries demonstrated the existence of a critical
moment before which the oogonia are unable to develop beyond the stage of
chromosome condensation and enter into meiosis. However, if the ovary is
secondarily transplanted the oogonia are able to undergo meiosis. The same
results were obtained by Wolff in the mouse (1952) and Challoner in the hamster
(1975). Nevertheless, acceptance of those results must be reserved as Byskov
(personal communication), under different culture conditions in the hamster,
did not find this critical period.
Recent studies on meiotic initiation emphasize the existence of meiotic
determinants to which are added the effects of a favourable local or general
environment (reviewed by Mauleon, 1975). In the absence of one or more of
these factors, chromosome decondensation might not take place.
Foetal testes of both man and sheep are examples where the condensation
stage is not followed by decondensation.
A certain number of foetal spermatogonia seem to evolve as if their mitotic
behaviour would change to a meiotic one but without development of meiosis.
This failure of germ cells to undergo meiosis may be a result of the presence of
'a meiosis-preventing substance' secreted by the male gonad (Byskov & Saxen,
1976) and the inhibitory action of the Sertoli cells. The latter, as demonstrated
by Jost (1972) in the rat, surround the germinal cells from the beginning of
testicular differentiation, thus completely isolating them. Such a pattern is
present in the 10-week-old human foetal testis (Gondos & Hobel, 1971), and
even from the 8th week on as we have observed in the present study. The
inhibitory role of the Sertoli cells is certainly of importance, even though a few
cases of early meiotic initiation have been reported in the cat (Ohno et al. 1962)
and in the mouse (Ozdzenski, 1972). The frequency of this latter phenomenon is
augmented by disturbing the relation between germinal and somatic cells, for
example by transplantation (Ozdzenski, 1972).
Results from the present study suggest that initiation of meiosis may take
place during the course of the chromosome condensation stage. Thus, in agreement with Bennett & Stern (1975), we believe that in those species without a
condensation process meiosis is initiated at G2 of premeiotic interphase. In
species presenting a condensation stage, the initiation of meiosis occurs later on,
taking place during the chromosome condensation stage (Fig. 10). Decondensa-
184
J. M. LUCIANI, M. DEVICTOR AND A. STAHL
tion which leads to leptotene is consequently initiated and the cell enters
meiosis; such is the case in the oocyte. In addition, variations in the timing of
initiation may explain both the variable degree of condensation observed among
different species (Walters, 1972; Devictor-Vuillet et ah 1973) and that a stage of
full spiralization is rarely reached (Fig. 10). With the foetal testis, in the absence
of initiation, decondensation does not occur and the germinal cell probably
returns to an interphase state yielding the foetal spermatogonium (Fig. 10).
This study also implies that the male germinal cell, despite its chromosomal
constitution, tends to evolve towards meiosis, as suggested by the presence of
a condensation stage. Decondensation does not appear and meiosis does not
develop since this cell does not receive any initiation, and is the object of
inhibition by somatic cells. This hypothesis of equal evolutive potential in male
and female germ cells finds support in the recent studies of Byskov & Saxen
(1976): the female gonad secretes a 'meiosis-inducing-substance'. Male germ
cells are triggered into entering meiosis when a not yet differentiated testis is
cultured together with older ovaries. Seemingly contradictory data are presented
by the results of studies on experimental XXjXY chimeras in the mouse
(McLaren, Chandley & Kofman-Alfaro, 1972): foetal testicular germ cells
observed to be in meiosis present an XX constitution. There are no XY cells.
Yet, the authors themselves state that the XX cells alone enter meiotic prophase
under the influence of neighbouring XX somatic cells.
The results of the present study seem to support the view that environmental
factors rather than genetic programming take part in the priming of meiosis.
We are greatly indebted to Professor Ruf and his colleagues (Department of Obstetrics and
Gynaecology) for their kind cooperation in providing human foetal testes.
We wish to thank Miss Noelle Leguillou for photographic assistance.
This investigation was supported by I.N.S.E.R.M. ATP no. 25-78-48, and C.N.R.S. ERA
no. 397.
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