1979. A sequential analysis of meiosis in the mouse using a restricted spermatocyte population obtained by a hydroxyurea/triaziquone treatment. Chromosoma.
BIOLOGY
OF
35, 587-59
REPRODUCTION
Duration
1 (1986)
of the Cycle of the Seminiferous Epithelium in the Rhesus Monkey (Macaca mu/atta)’
D. G. de ROOIJ,
M.M.A.
van
ALPHEN,
Department State
and
of Cell
University Medical 3511 HG The
H. J. G. van
and Its Stages
de KANT
Biology
of Utrecht School Utrecht
Netherlands
ABSTRACT Doses of 1 Gy or more of X-irradiation killed all B spermatogonia present in the testis, and during the first 3 weeks after irradiation, virtually no new B spermatogonia were formed. The number of A pale spermatogonia decreased during the first cycle of the seminiferous epithelium while the number of A dark sperlnatogonia only began to decrease during the second cycle after irradiation. In this study, the duration of the cycle of the seminiferous epitbelium in the rhesus monkey was estimated to be 10.5 days (SE = 0.2 days). This was determined following the depletion of germinal cells in the seminiferous epitbelium during the first 3 weeks after irradiation. The duration of each of the 12 stages of the cycle was also determined.
Our observations of the progress of germinal ficient to kill all B spermatogonia, all spermatocytes sperm atids within about 31 days.
cell
depletion disappeared
INTRODUCTION
In
mammals,
tubules
undergoes
known This the one and
the
as the
a cycle
cycle has successive given area Clermont,
sociations are most
epithelium of
the
seminiferous
The
been called identified
Courot
in many et al.,
1970;
of
successive
the stages by the
mammalian Clermont,
the timing of spermatogenesis tion of the effects of irradiation
and
stages
species 1972).
series of in any (Lebbond cellular
of the various its
changes
epithelium.
complete appearing tubule”
as-
1973). Unfortunately, published for the important laboratory
been
(reviews Knowledge
and it forms kinetics. the duration 16.0 days in
sufand
for
all
the
base
studies
of the
the cycle has human (Heller
on been and
Clermont, 1963), 11.6 days in Macaca arctoides (Clermont and Antar, 1973), 9.3 days mM. fascicularis (Dang, 1970) and 10.2 days in Saimiri sciureus, Cercopithecus sabaeus and Papio cynocephalus (Barr,
cycle and steps of has
that after a dose of X-irradiation the testis within about 17 days,
togenic cells, spermatogonial In primates, found to be
seminiferous
series
been defined as “a cellular associations of the seminiferous
development of the spermatids. The duration of the cycle determined
the
repetitious
1952).
have often
of
revealed from
conflicting results have rhesus monkey, M. mulatta, animal. Barr (1973) found
been an the
duration of the cycle to be 9.5 days, and Arsenieva et al. (1961) estimated it to be 10.5 days. In these studies, the development of [3 HI thymidine-labeled
in of
is useful for the evaluaand drugs on sperma-
cells (Barr, 1973) or the depletion of the epithelium after X-irradiation (Arsenieva et ab., 1961) was followed. A possible cause for the varied results could be that in both studies, only one interval of more than one cycle’s duration after irradiation was studied. Clermont and Antar (1973), using the data of Arsenieva et al. (1961), calculated a cycle duration
Accepted March 12, 1986. Received November 29, 1985. This work was supported by the J. A. Cohen Institute for Radiopathology and Radiation Protection, Leiden, The Netherlands. 2Reprint requests: Dr. D. G. de Rooij, Department of Cell Biology, State University of Utrecht, Medical School, Nic. Beetsstraat 22, 3511 HG Utrecht, The Netherlands.
of of
11.8 days. the cycle
studying 587
the
We now have and its stages time
course
determined in the rhesus of
the
the duration monkey by
depletion
of
the
588
ROOIJ
germinal
cells
in
the
seminiferous
epithelium
after
stage had MATERIALS
Nineteen Primate
mature Center
were exposed doses varying kV, 10 mA, testicular
AND
rhesus TNO,
METHODS
monkeys Rijswijk,
(M.
at
mulatta)
The
Netherlands,
were
shielded
with
biopsies
different taken days, and days.
at
from each animal testes. After irradiation, the
following
at
were taken the biopsies
intervals:
2 biopsies
one The
4
biopsies
formol,
were
embedded
(JB4;
fixed
Polysciences
sections
were
Inc., The
made.
periodic acid-Schiff Epitheliab stages togonia were of Clermont described Adark
(Ad)
tively,
as
and done
fluid
or glycol
Warrington, were
sections
(PAS) reaction and the various
identified and Lebbond
as A1
in Bouin’s
in paraffin
and
according
by
PA), and 5-urn stained by the
and hematoxylin. types of sperma-
spermatogonia
Apale
(Ap) Clermont
or Zenker-
to the description The cells originally
(1959).
A2
were
spermatogonia and
irradiation,
the
same
progress
of
the
depletion
to calculate
during
Table
the thus
2 compares taken
the
time
between the
the
progress
at intervals
the
percentage
of
that
depletion
had
middle was
of
the
found
to
two
between
(1976).
after
with
time
stages.
duration
shortest 10.5
±
of the
two
intervals of
of the
depletion
the
4 days
one
apart.
cycle
passed,
proceeded
to
Furthermore,
when
it the
depletion
have proceeded to the transition zone stages, it was considered to have proto the border between these stages. a potential error that, depending on
the duration of the stages variation in the calculated especially when the depletion taken at short intervals after mean
(Table stages
duration
at least
assumed
and
interval
percentage
deriving
calculating
was
type
epithelium were seen
at
of the
cycle,
concerned,
would cause duration of the cycles, is compared in biopsies one another. Indeed the
as calculated
from
the
7
intervals between biopsies, was found to be 0.4; when calculated from the 7 longest
intervals, it was 10.5 ± 0.1 days. Knowing the duration of the cycle or 252 h, it was possible to calculate each of the 12 stages (Table 1).
named respec-
Hermo
passed
after
ceeded exactly This introduced
methacrylate
cell
taken
used
in all biopsies
3 after 7 days, 3 after 8 days, 2 after 10 days, 3 after each of the intervals of 11, 14 and 21 Also, biopsies were used from 4 control mon-
keys. The
cycle
In
to which
to calculate the duration of the cycle data on the relative duration of the 1), was
cycle.
from were after
biopsies
irradiation. The observed
(Table
lead.
determined
the depletion of the seminiferous proceeded (Fig. 1). No differences
was used 2). The
to local irradiation of the testes with between 0.5 and 4 Gy of x-rays at 300 HVL 3mm Cu, 0.3 Gy/min. The extra-
regions
it was
between
Testicular biopsies were taken before and interval during the first 3 weeks after irradiation.
two
AL. irradiation
X-irradiation.
the
ET
to be 10.5 the duration
days of
RESULTS
In biopsies of testes frequency and relative
from 4 control monkeys, duration of the stages
cycle
by
were
determined
total of about After doses togonia were first taken
3 wk from
numbers
new
cohorts
be produced. study. The considerable the numbers Day 7 and
identifying
the
the of the
stages
in a
3000 tubular cross-sections (Table 1). of 1 Gy or more, virtually no B spermafound at any of the intervals during the
after irradiation. In some of the biopsies testes that received 0.5 Gy, appreciable of B spermatogonia remained present, and of
primary
spermatocytes
continued
These testes were excluded from Ap spermatogonia were still present numbers at 4 days after irradiation, of these cells at longer intervals
were clearly diminished after irradiation. The
to
this in but at Ad
remained present in high numbers up Day 14, but were clearly less in number at Day 21. In the 19 biopsies taken during the first 3 wk after
TABLE 1. Frequency and duration of the stages of the cycle of the seminiferous epithelium of the rhesus monkey, Macaca mulatta.
Stage
No. tubular cross-sections
Frequency of stages
Duration of each stage (h)a
1 II III IV V VI VII
371 124 201 135 159 422 406
0.121 0.041 0.066 0.044 0.052
30.5
VIII IX X XI XII
365 271 140 241 224
0.119 0.089
spermatogonia
to
0.138 0.133
0.046
0.079 0.073
3059 aBased
on cycle length of 252
h.
10.3 16.6
11.1 13.1 34.8 33.5
30.0 22.4 11.6 19.9 18.4
TIMING
OF
SPERMATOGENESIS
IN THE
DISCUSSION At
doses
spermatogonia and during
of
1 Gy
or
more
no new B spermatogonia were tion, no abnormal degeneration spermatids
observed,
was
developed in a normal development of the present at the time gressing depletion took place. The determine
the
X-irradiation,
all B
formed. After of spermatocytes
and
these
cells
irradiaand
duration
of the
cycle.
the epithelium was used After
a dose
MONKEY
589
appreciable numbers formed spermatocytes
testes.
These
of B spermatogonia were seen in biopsies
testes
were
The type B spermatogonia from the testes while a first of
apparently
way. As a result of the further spermatocytes and spermatids of irradiation, a gradually pro-
of germinal cells from rate of this depletion
and
of several study.
present during irradiation were killed, first 3 weeks after irradiation, virtually
the
Gy, newly
0.5
of
RHESUS
A
spermatogonia
fmdings
are
spermatogonia
development togonia only
to
(Clermont
of
only
and
was
in
excluded
disappeared clearcut drop first
seen
accordance
Lebbond, in
numbers
1959).
The
during
this
quickly in numbers
at Day
with the proliferate continuously into sperm atocytes while the divide during a short period
decrease
from
7. These
fact that B during their A sperm aof the cycle
Ad spermatogonia the
second
cycle
ii
cells missing
0
C.-
CC-
a)
U) -D
cells
!Y1IIIXXIXflIfluI&21I
BpreL
L
present
s
XXIIUffl1!!
Z
P
DMrSpt
FIG. 1. Depletion of the seminiferous epithelium of the rhesus monkey, Macaca mulatta, at horizontal bars indicate the epithelial stage to which the depletion had proceeded. In case the between two stages, the bar has been drawn from the middle of the first stage to the middle of bars with a slope corresponding to a cycle duration of 10.5 days. On the abscissa, cell types have Significance of letters used: 8, B spermatogonia; preL, preleptotene spermatocytes; L, Z, P, D, and diplotene stage respectively; M, meiotic divisions; rSpt, round spermatids.
cell type various intervals of time after X-irradiation. The depletion front was found in the transition zone the next stage. A line has been drawn through the been indicated at the stage in which they arise. spermatocytes in leptotene, zygotene, pachytene
ROOIJ
590 TABLE
2. Calculation
the depletion
Progress depletiona
induced
of the duration
of the cycle of the seminiferous
ET
AL.
epithelium
in the rhesus
mulatta)
the progress
from
atDay4
From:
% of Cycle duration
10 11 14
9.9 9.5 10.0 10.5 10.4
To: PinIl/Ill P in VI Pin VI/VII Pin VIII/IX
atnay8 at Day at Day at Day
rSptinVl
atDay2l
40.4 63.5 70.4 95.6 163.5
P in VI/VIl Pin VIII/IX rSptinVl
at Day 11 at Day 14 atDay2l
40.1 65.3 133.2
10.0 10.7 10.5
To: at Day
7
From: Pin Il/Ill
at Day
8
To: P in VIII/IX rSpt in VI
at Day at Day
14 21
55.2 123.1
10.9 10.6
From: Pin VI
at Day
10
To: P in VIII/IX rSpt in VI
at Day at Day
14 21
32.1 100.0
11.0
11
To: rSpt
in VI
at Day
21
93.1
10.7
14
To: rSpt
in VI
at Day
21
67.9
10.3
Pin
of
Calculated duration of one epithelial cycle (days)
of the
From: ZinIX
(Macaca
monkey
by X-irradiation.
I
From: Pin VIIVII
at Day
From: Pin VIlI/IX
at Day
12.5
Mean aSignificance meant
of
letters
after irradiation. (1976), these action
only
are
decreased.
this
notion
become togonia
cells
Rooij, In
the
The as
zygotene
the
numbers
been
published
be
Ad
spermatogonia
the
duration
are
pachytene; between
and Hermo come into in
line
with
appear
to
numbers of A spermaquantitative study on
(Van of
of
but
is currently this study
Alphen the
in the rhesus days. This result
et al. (1961),
P, zone
spermatogonia
after irradiation initial results
elsewhere
epithelium 10.5 ± 0.2
of Arsenieva
of A results
numbers out. Some
1986). this study,
spermatocytes; the transition
present
active only after the decrease. A detailed
seminiferous found to that
Z,
had reached
According to Clermont are reserve cells that
when
spermatogonial being carried have
used:
that the front of depletion
cycle
and of
De the
monkey was is similar to
in contrast
to that
of Barr (1973) and to the result calculated by Clermont and Antar (1973) from the data of Arsenieva et al. (1961). The difference with the results of Barr (1973) probably lies in a different interpretation of epithelial Stages after
VII and VIII. administration
According to Barr of [3 H] thymidine,
(1973), the
2 h most
rSpt,
round
these two
spermatids.
When
two
stages
10.5 ± 0.2
are given
(e.g. Il/Ill), it is
stages.
advanced in Stage
labeled cells VII. However,
Antar (1973) found in Stage VIII to
are preleptotene in M. arctoides, 85% show
leptotene spermatocytes [3 H] thymidine. The the cycle are similar (Clermont monkey,
[3 H] thymidine.
present
duration
10.3-10.6
the data of Barr from the present Arsenieva process to
et take
duration
Clermont leptotene would be Stage VIII succeeding
in
al. 408
143.5% of one cycle.
therefore, in the spermatocytes
Stage In days
(1973), results.
which
VIII shortly that case,
can
be
stages
(1973) the end
calculated
is clearly
(1961) reported h. Using this
of the
and Antar stage to
and
of the tubular cross-sections labeled preleptotene and
and Antar, 1973); labeled (pre)leptotene
of
spermatocytes Clermont
by 3 h after injection of cellular associations composing in M. arctoides and M. mulatta
be similarly jection of
relative
(± SE)
found
not the value
by these
calculated that of the meiotic
rhesus may
after ina cycle from different meiotic and the authors, from the divisions
of one cycle duration, i.e. from cycle to the end of Stage XII of the Indeed, from this a cycle duration
OF SPERMATOGENESIS
TIMING
of
11.8
Table
days
can
2 from
be calculated.
Arsenieva
However,
et
al.
the
(1961)
data
suggests
cycle
10.8 10.5
duration.
days value
length To those that
From
can be calculated, lying within the
of Stage
the
of Arsenieva determining
etal. the
cycle
Figure
rat Oud
1, a line
the depletion through the
with by
the the
not
the
development of et al., 1982).
not affect on duration of the
of 10.5 days. it is clear that
B
used,
would
the duration of the cycle
the seminiferous epiinjection of cytostatic
the
stages
As the
killed.
Rooij
to which
has been corresponding
drawn to a
this line passes estimate of 10.5
line is extrapolated to the it intersects the abscissa at spermatogonia
spermatocytes. This indicates the B spermatogonia-but
the depletion indicates before all spermatocytes all spermatids have
(De
The
observed
divide
into
that with few if any progress
MONKEY
591
ACKNOWLEDGMENTS
and
tending
from
the
monkeys,
Radiobiological
and
Dr.
Institute
B. TNO,
Hogeweg
and
Rijswijk,
Dr.
J. Zoetelief
for the irradiation.
Arsenieva M, Dubinin NP, Orlova NN, Bakulina ED, 1961. Radiation analysis of duration of meiotic phases in spermatogenesis of monkey (Macaca mulatta). DokI Akad Nauk SSSR 141:1486-89 Barr AB, 1973. Timing of spermatogenesis in four nonhuman primate species. Fertil Steril 24:381-89 Clermont Y, 1972. Kinetics of spermatogenesis in mammals: the cycle of the seminiferous epithelium togonia. Physiol Rev 5 2:198-236
and
the mode
of renewal
of sperma-
Clermont Y, Antar M, 1973. Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides. Am J Anat 136:153-66 Clermont Y, Hermo L, 1976. Spermatogonial stem cells and their behaviour in the seminiferous epithelium of rats and monkeys. In: Cairnie AB, Lala PK, Osmond DG (eds.), Stem Cells of Renewing Cell Populations. New York Academy Press, pp. 273-87 Clermont Y, Leblond CP, 1955. Spermiogenesis and other mammals as shown by the technique. Am J Anat 96:229-5 3
of man, monkey, ram “periodic acid-Schiff”
Clermont Y, Leblond CP, 1959. Differentiation and renewal of spermatogonia in the monkey, Macacus rhesus. Am J Anat 104:237-74 Courot M, Hochereau MT, Ortavant R, 1970. Spermatogenesis. In: Johnson AD, Gomes WR, Van Denmark NL (eds.), The Testis, Vol. 1. New York Academic Press Inc., pp. 339-432 Dang DC, 1970. Le cycle de l’pithlium sminif#{234}redu singe crabier (Macaca fascicularis). Doctorate thesis. Facult des Sciences, Paris Dc Rooij DG, Kramer MF, 1970. The effect of three alkylating agents on the seminiferous epithelium of rodents. I. Depletory effect. Virchows Arch B Cell Pathol 4:267-7 5 Heller CG, Clermont Y, 1963. Spermatogenesis in man: an estimate of its duration. Science 140:184-85 Kluin PhM, Kramer MF, Dc Rooij DG, 1982. Spermatogenesis in the immature mouse proceeds faster than in the adult. mt J Androl Leblond
5:282-94 C?, Clermont
the seminiferous 548-73 Oakberg EF, 1956.
Oud
of
that it takes about 17 days and about 31 days before disappeared from the testis.
the
REFERENCES
and
suggested
irradiation
indicating
Stage
when
results
and golden hamster et al., 1979).
this
spermatocytes-are
(1973)
by
does effect
days is reliable. When moment of irradiation, preleptotene the doses
Barr
front had proceeded bars, with a slope
duration all bars,
VI
his
following the cells (Kluin
was seen when depletion thelium was followed after
cycle through
of
similar results were found in duration of the cycle was deterthe depletion after irradiation
irradiation Also, no
mouse, 1970;
between
(1961),
(Oakberg, 1956) or by [3 H] thymidine-labeled
drugs in the and Kramer,
duration
the difference variation induced
difference
However, when the following
Apparently, of the cycle.
a cycle
RHESUS
The authors are grateful to Mr. H. D. Wiersma and Mr. Tb. van Schie from the Primate Center TNO, Rijswijk, for performing the operations
that
VI.
explain
be accurate. the mouse mined by
In
this,
THE
in
these authors included the preleptotene phase in the meiotic process. In that case, 408 h represents the interval between Stage VI of one cycle to the end of Stage XII of the succeeding cycle, or about 15 7.5% of one
IN
Van
Y, 1952. Definition of the stages epithelium in the rat. Ann NY Duration
of
spermatogenesis
of the cycle of Acad Sci 55:
in the
mouse
and
timing of stages of the cycle of the seminiferous epithelium. Am J Anat 99: 507-16 JL, Dc Jong JH, Dc Rooij DG, 1979. A sequential analysis of meiosis in the mouse using a restricted spermatocyte population obtained by a hydroxyurea/triaziquone treatment. Chromosoma 71:237-48 Alphen MMA, epithelium radiation.
Dc of
Rooij
DG,
1986.
Depletion
the rhesus monkey, Macaca Br J Cancer (Suppl VII) 5 3:102-104
of the seminiferous mulatta,
after X-ir-
