BIOLOGY
OF
46,
REPRODUCTION
Testicular JON
Department
284-289
(1992)
Synchrony:
E. SIITERI,3
Evaluation
ALICE
of Biochemistry
and
F. KARL,
Biophysics,
and Analysis C. LINDER,
CAROL
Washington
of Different MICHAEL
and
State
University,
Protocols1
D. GRISWOLD2
Pullman,
Washington
99164-4660
ABSTRACT Using
the
vitamin
A depletion-replacement
degree
of synchronization
retinol
alone.
protocol
In
protocol
A smaller value
of animals
(point and
not
was
to calculate
in the different
a cycle
protocol
can
chronize
testes
reproducibly
of
by an adaptation
B demonstrated
protocol 50%
the
two
h for
provides
stages
treatments.
our
testicular
stages
the
of
strain
days
of
synchrony more
are
The
of vitamin
protocol experimental
advanced
and
material
50%
reproducibility achieved
high
the
are
less
results
from
of
the was
both that
molecular
animals.
a more
The and
use
used
of either
ability
cellular
of
constant
were
the
of
With
midpoint
protocols
while
of synchrony. the
use
Animals
Individual
contrast,
advanced)
indicate degree
study
synchrony.
among
In
and by
to retinoL
for quantifying was
obtained
Our
in a higher for
the was
of synchrony.
of synchrony rats.
examined synchrony
as a supplement
method
variability
degree
A results
(A),
A depletion
published
a lower
midpoints
we
protocol
although
of Sprague-Dawley
synchrony, sufficient
final
synchrony,
original
of a previously
with
300
provide
to selected
the
degree
between
duration
during
testicular
In the
a reproducible
at which
cycle
to obtain
protocols.
used
was
analyzed
treated
model
different
two
acid
A demonstrated
group
synchrony
B, retinoic
of 56 rats were
A, a total
by protocol
treated
rat
by
obtained
to synevents
of
spermatogenesis.
INTRODUCTION The
cycle
of the
seminiferous
the continual development been well characterized
cells
epithelium
that results in
and release of spermatozoa has by morphological, histochemical,
original
Clermont the cellular
morphological
[1], fourteen associations
testis specimens. The use of the
description
by Leblond
testis
synchronized
by means
and
complete
cessation
of spermatogenesis
of re-
When retinol, with or without retinoic acid supplementation during the final portion of vitamin A depletion, is provided within a specific time after depletion, spermatogenesis is reinitiated in a synchronous fashion such that the testis contains only three-to-four stages of the cycle at any point in time [6-8]. This is in contrast to the normal asynchronous rat testis in which all fourteen stages of the cycle are found in the seminiferous tubules at all times. The fredepend on testis, germ
October 7, 1991. February 18, 1991. ‘This work was supported by NIH Grants HD-10808 and HD-25846. 2Correspondence. 3Current address: Department of Cell Biology and Neuroanatomy, Received
Minnesota,
4-135
Jackson
Hall,
321
Church
Street,
Minneapolis,
MN
the
cycle
in a nor-
provides a model for the study in the control of the cycle of the
the length of the seminiferous epithelial cycle in our strain of Sprague-Dawley rats synchronized by our protocol of vitamin A depletion and replacement. Our results indicate that retinol alone provides a better degree of synchrony than retinol with retinoic acid supplementation and that the
Accepted
University
through
These authors presented a procedure for quantifying the degree of synchrony on the basis of the ratio of stage frequencies of synchronized testes over the stage frequencies of control rats. In our analysis, we used a simplified approach based on the method of Van Beek and Meistrich that also yields a quantitative assessment, but one that can be easily determined from a graphic representation of the data. The objectives of our report were 1) to evaluate testicular synchrony using only retinol in a large group of rats; 2) to compare these results with synchrony obtained with rats supplemented with retinoic acid; and 3) to determine
in mammals.
quencies of occurrence of the different stages the duration of each stage. In the synchronized
progress
Recently Van Beek and Meistrich [7] used a modification of the original method for obtaining synchrony. In their method, rats on a vitamin A (retinol)-deficient diet are maintained with supplemented retinoic acid. This treatment alleviates the symptoms of vitamin A deficiency in all tissues except for those involving vision and spermatogenesis. Retinol administered to the retinoic acid-treated rats successfully resulted in synchronized spermatogenesis [7].
tinol depletion and replacement has been recently described as a model for the study of stage-specific expression of Sertoli-cell-secreted proteins and their mRNA [3]. Vitamin A in the form of retinol is required for the maintenance of spermatogenesis [3-5]. Vitamin A deprivation leads to the
tubules
experimental manipulation of the elements involved seminiferous epithelium.
stages of the cycle are defined by observed in cross sections of fixed
rat
the
recent report indicates that synchronization persists for up to 18 wk from the time of vitamin A replacement [9]. This
and biochemical methods in the rat [1, 2]. The cycle is defined by the recurring appearance of specific germ cell types within given regions of the seminiferous tubule. According to the
within
mal fashion, resulting in spermiation approximately every 12-13 days. Synchronization of the seminiferous epithelial cycle continues for at least three spermiation events, and a
former method is a reliable technique for obtaining that contain seminiferous tubules at selected stages seminiferous epithelial cycle.
of
55455.
284
testes of the
SYNCHRONY:
TESTICUlAR
MATERIALS
AND
METHODS
EVALUATION
AND
ANALYSIS
285
99.999. I
SI..
II
III
V
IV
VI
viii
VII
Iqx
XII
XIII
XIV
99.99.
Preparation
of Synchronized
Vitamin
A-depleted
Testis
rats
were
99.9.
raised
as
previously
de-
ton State University, Pullman, WA) weaned at 20 days of age and weighing an average of 40 g were fed a vitamin A-deficient diet (U.S. Biochemical, Cleveland, OH) ad libitum for 9-11 wk. Testicular regression was determined pation, and rats received i.p. injections of 7.5 mg (Sigma, St. Louis, MO) in 0.4 ml 50% ethanol/sterile
diets
The mented
were with
Rats tration
changed
then 1 mg
retinol
per
epithelium.
in the retinol
The
for
chow
by palretinol water.
subsequent
reinitiation Vil-VIJI of the
stages
was
as described
The
use
of retinoic
the
systemic
exception
of the
accurately
72 post vitamin used retinoic
(Diet
supple-
80 70
a E (.5 a.’
a 0
10 5
a U-
B)
acid
in Van
injections
Beek
pronounced
effects
(7.5
ginning of the retinoic ferences were observed data from the remaining were pooled.
and
the
rats (20 days
mg
Meistrich
[7]. to re-
49 days. acid (10 of retiriol as described start of the the rats in
visual
the
system were
put
Their diet was mg/kg food) for and supplemenabove for proexperiment (rats this experiment
of retinoic
acid supplementation. in the data from 10 rats, the data
with
of age)
for
i.p.)
during on Days
deficiency, on
acid
at the
be-
Since no difthese rats and the for all 16 animals
The body weights of animals in both treatment groups increased linearly up to Week 7 or 8 of each treatment followed by a period of 1-2 wk during which there was no gain
or
loss
of weight
(data
not
killed
by carbon
vical dislocation. Testes were
dioxide removed
asphyxiation and
and
cleared
cer-
of the
epididymis and fat tissue, and a portion of a testis from each rat was fixed in Bouin’s fixative, embedded in paraffin, and stained with hematoxylin-eosin and counterstained with periodic acid Schiff’s reagent for histological examination. Stage frequencies in treated rats were determined by light microscopic classification (400X) of at least 200 tubules and 2 cross sections per testis according to the criteria estabby Leblond
and
Clermont
[1]. Control
testes
I
.1 .01 .
.001
#{149}
I -. 0.2
values
-
r
--.
.___
-
0.4
-
0.8
0.6
1
Fraction of a Cycle FIG. 1. Determination of SFs and MPs. The cumulative frequency of the seminiferous epithelial stages observed in a testis were plotted on a probability scale as shown. The points at which each curve crossed the 84.1 and 15.9 percentiles were noted, and the distances A to B and C to D were defined as the window widths. The SF was a ratio of the distances AB to CD where AB was the window width for normal controls, and CD was the window width for synchronized rats. The vertical lines denote the duration of stages in terms of a fraction of a cycle. The MPs were the points at which the curves crossed the 50% point. The open circles represent the stage frequency of control rats determined experimentally; the open triangles represent the expected stage frequency of control rats calculated from duration of stages 1181; and the closed circles represent the stage frequency of synchronized rat testis (59 days PVA).
were
determined
cross
sections
from in each
Quantflcation
ation factor determined mulative ability reedy
examination
of 500
tubules
and
4
of 4 animals.
of Synchrony
The data were Meistrich [7] with
points centiles.
shown).
Histology Rats were
:
after and
is reported
of retinol
H?
a
seen
the time the arrest occurs killed
were
supplementation
Sixteen
stages
-
--
.
30 20
0 =
0’
A administration (PVA). acid supplementation,
symptoms
spermatogenesis.
received
from that
is known
it
on a vitamin A-deficient diet then supplemented with retinoic an additional 29 days. Injection tation of the diet with retinol tocol A began 78 days after the were 98 days of age). Six of
lished
90
>
U)
epithelial
of spermatogenesis cycle [6]. Animals
54, 55, 59, 63, 64, and Protocol B, which
net
95
a
a
7 days.
seminiferous
testis can be predicted administration since
lieve
rat
rat
99.
0 a 0
were killed at selected times after retinol administo obtain specific stages of the cycle of the semi-
niferous
and
to normal
C
a
scribed [10]. In protocol A, Sprague-Dawley rats (obtained from the Laboratory Animal Resource Facility of Washing-
(SF) and graphically
frequencies scale, from
analyzed by the one modification; the
method that
midpoint of synchrony as illustrated in Figure
of each
stage
were
and the window widths the graph by noting the
at which These
of Van Beek and is, the synchroniz-
the curve percentiles
crossed define
the
plotted
(MP) were 1. The cuon
a prob-
were
determined distance between
dithe
the 15.9 and 84.1 perwindow width that is
equal to plus and minus one standard deviation. The ratio of window width of the control testes to the synchronized testis thus determined the SF. The SF, as previously defined,
is independent
of the
duration
and can be used to describe synchrony the cycle and in different experiments SF was
determined
by using
the
window
of any
width
for control rats, which is constant, i.e. 68.3%. width of the control rats was similar whether mental lished
data or calculated data on the duration
single
stage
in various parts of [7]. The value for the
values determined of each stage were
of the
data
The window the experifrom used.
pubThe
286 MP
SIITERI
is the 50th percentile
of the
point, or the median
ET AL.
I
stage
II
III
IV
V
VI
VII
VIII
IX
X
XI
54DAY
distribution, and is presented in terms cycles traversed. Thus, an MP 5.3 means
of the number of that 5.3 cycles have
18
Il
8
6
7
55DAY 14
been stages
traversed is 30%
teria follows: tained
we
and that of the way
the center throught
distribution cycle. The
of cri-
were part of a group observed comprised
number of tubules. meet these criteria
5
MPs
remain
the
ing
34
JI3
IS
software,
Abacus
Concepts,
A us-
obtained
from
72 PVA. These synchronized
in the majority of animals prbtocol B. The synchronized
I) stages of the cycle. The results of treatment A for 56 rats where synchrony was achieved rats that
Figure 2. Data from not included. experiments sentative
These
did not achieve synchrony
data were
conducted of animals
over for
with protocol are shown in
compiled
from
a 1 yr period
which
at least
a series of
and
three
were
are
repre-
samples
were
available for each PVA time point. In a total of 121 animals that were used in this and in other studies from this laboratory, the vitamin A depletion-replacement protocol produced synchrony in approximately 80% of the rats. The remaining animals were not synchronized (5%), exhibited
18 20
6
4
60
10
31
9
6
19
49
21
16
42
25 5 5 9 37 20 16 18140 ________22 5 ________ 27 40111 23 38 10 35 48 17 25 30 20 6 35 40 15 8 i8li8 5 12 12 7 21 26 10
24 29
13 10
10 16
10
34 40 41 40
28 24 32
10
40
32
10 15 5
23 9 IS 42
33 25 27 18
22 21
11 7
10 7 8
10
19
15
20
27
19
13
16
18
26
24
8.9
10
3.3
5.4
4.6
8.1
I5
17
3.3
by this
SF and
MP values
nized
tubules
able between rats, given PVA period, siderably. However, cycle, was Despite
93
± 8
ranging from 1.6 to 11.6. Even within a the synchronization values varied conthe MP, plus or minus one stage of the
reproducible within any given PVA time period. some variability in the stage frequencies within
each sample to treat each
and a large variation in the SFs, it was PVA time
period
as a group
and
possible determine
8.00
5.70
8
5.00 10.4
5.74 5.71
5
5.50
5.73
5.80 7.40 3.90 4.00 1.60 3.30
5.77 5.78 6.23 6.23 6.37 6.33
5.27
5.30
2.27
0.41
6.5
6
1.00
0.58
3.3
the mean stage frequencies. The mean stage frequencies (average of percentages for each stage) for each PVA period are shown in Table 1. Both the mean SF and mean MP val-
average
was
5.28 5.29 5.51 5.13 5.11 5.63 5.71 5.73 5.74 5.73
method.
for
56 animals
4.3
5.22
FIG. 2. Stage frequencies of individual rats on days PVA and calculated SFs and MPs (protocol A). The stages comprising the highest percentages of tubules are boxed; stages comprising less than 5% of tubules were not included. Dashes represent values that could not be determined
PVA group
with a range of 71 %-100%. 2 that the SF was highly vari-
4.6
11.6
3.50 4.30 2.90 5.00 4.30 1.70 6.90 5.80 5.20 6.50
16
±SD
1RL
NP 4.90 4.92 4.95 4.93 4.97 5.00 4.93 4.84 5.01 5.00 5.01 4.94 4.97 5.15 5.07 5.03 4.92 5.01 5.03 4.99 5.11 5.03 5.00 4.91 4.94 5.15 5.16 5.37 5.24 5.30 5.22
15
5
9
#{128}AN
each
of these
6
13
obtained
testes
7
7
values
deviation) from Figure
8
3
average
in the
21
5
ues
(mean ± standard It can be seen
13
39
12 22 14 17 20 22 19
tubules synchro-
than
64
13
86 68 78 78 37 59 45
segment
more
10
95
one
around
10
8
cycle; 8%) or had a majority of unrecovered The average percentage of recovered and
(synchrony
20
27
10
________
6 11
10 12
5
of the (7%).
parasynchrony
9
27
using both testes were
animals killed on days 54, 55, 59, 63, 64, and time points were selected to obtain testes in early (Il-VI), mid (VII-VIII) and late (XII-
8 1914
I98 22
DAY
720AY
33
24
RESULTS
of spermatogenesis protocol,A and
40
43
6
64
43
2816
61 30
Inc.,
18
34
3
5
of
15fl
9
19
by readministration in the synchronization
25
5
50
CA.
Depletion of vitamin A followed retinol to developing rats resulted
28
10
40
63 DAY
Berkeley,
16
7
17
62
Efli
same.
graphics
26
5
4
10 5
SE+
26 25
Ja
Analysis
StatView
31
26
stages, of the
from protocols signed-rank test
14
27
12
fl18
are considered, comparisons different treatments. Addition-
factors obtained by the Wilcoxon
56
10
18
The synchronization and B were compared
39
8
6
59DAY
Statistical
13 16
28
25
24 7
7
52 31
4.70 11.6 3.10 6.50 3.90 4.00 6.50 5.50 5.80 3.30 3.40 11.6 5.80 4.30 4.00 4.00 5.50 4.50 4.50 4.70 4.50 5.20 2.90 8.00 5.00 4.70 4.50 3.00 4.30 3.40 5.50
XIV
7
26
Although excluding tubules that do not tends to inflate SF values compared to
analysis in which all tubules may stillbe made between the
of related at least 5%
XIII 27 65
DII
used to determine testicular synchrony were as tubules were considered synchronized if they cona normal complement of germ cells, if they con-
tained stages that and if the stages
ally,
of the the next
XII 26 22
5.71 be
obtained
±
0.04
for
(mean
significantly
±
different
all animals,
5.27
When
synchronization
± 2.27
for
SD),
compare
favorably
all animals.
For
respectively,
from
the
±
and
63 PVA, 5.68
Day the
and
5.30
was
generated
do
to the
example, not
2.17
appear
average
SF and
0.41,
respectively.
±
by protocol
MP
B, which
utilized retinoic acid supplementation, the resulting frequencies (Fig. 3) were similar to those described ure 2. As in protocol A, the SFs were highly variable the
MPs were
testes from significantly
tightly
grouped
for each
PVA time
to for
stage in Figwhile
point.
the rats treated according to protocol lower SFs compared to those from rats
The
B had treated
TESTICUlAR
according
to protocol
the
MP was
A (p
SYNCHRONY:
0.02,
z = -2.483).
both
treatments
OF
46,
REPRODUCTION
Testicular JON
Department
284-289
(1992)
Synchrony:
E. SIITERI,3
Evaluation
ALICE
of Biochemistry
and
F. KARL,
Biophysics,
and Analysis C. LINDER,
CAROL
Washington
of Different MICHAEL
and
State
University,
Protocols1
D. GRISWOLD2
Pullman,
Washington
99164-4660
ABSTRACT Using
the
vitamin
A depletion-replacement
degree
of synchronization
retinol
alone.
protocol
In
protocol
A smaller value
of animals
(point and
not
was
to calculate
in the different
a cycle
protocol
can
chronize
testes
reproducibly
of
by an adaptation
B demonstrated
protocol 50%
the
two
h for
provides
stages
treatments.
our
testicular
stages
the
of
strain
days
of
synchrony more
are
The
of vitamin
protocol experimental
advanced
and
material
50%
reproducibility achieved
high
the
are
less
results
from
of
the was
both that
molecular
animals.
a more
The and
use
used
of either
ability
cellular
of
constant
were
the
of
With
midpoint
protocols
while
of synchrony. the
use
Animals
Individual
contrast,
advanced)
indicate degree
study
synchrony.
among
In
and by
to retinoL
for quantifying was
obtained
Our
in a higher for
the was
of synchrony.
of synchrony rats.
examined synchrony
as a supplement
method
variability
degree
A results
(A),
A depletion
published
a lower
midpoints
we
protocol
although
of Sprague-Dawley
synchrony, sufficient
final
synchrony,
original
of a previously
with
300
provide
to selected
the
degree
between
duration
during
testicular
In the
a reproducible
at which
cycle
to obtain
protocols.
used
was
analyzed
treated
model
different
two
acid
A demonstrated
group
synchrony
B, retinoic
of 56 rats were
A, a total
by protocol
treated
rat
by
obtained
to synevents
of
spermatogenesis.
INTRODUCTION The
cycle
of the
seminiferous
the continual development been well characterized
cells
epithelium
that results in
and release of spermatozoa has by morphological, histochemical,
original
Clermont the cellular
morphological
[1], fourteen associations
testis specimens. The use of the
description
by Leblond
testis
synchronized
by means
and
complete
cessation
of spermatogenesis
of re-
When retinol, with or without retinoic acid supplementation during the final portion of vitamin A depletion, is provided within a specific time after depletion, spermatogenesis is reinitiated in a synchronous fashion such that the testis contains only three-to-four stages of the cycle at any point in time [6-8]. This is in contrast to the normal asynchronous rat testis in which all fourteen stages of the cycle are found in the seminiferous tubules at all times. The fredepend on testis, germ
October 7, 1991. February 18, 1991. ‘This work was supported by NIH Grants HD-10808 and HD-25846. 2Correspondence. 3Current address: Department of Cell Biology and Neuroanatomy, Received
Minnesota,
4-135
Jackson
Hall,
321
Church
Street,
Minneapolis,
MN
the
cycle
in a nor-
provides a model for the study in the control of the cycle of the
the length of the seminiferous epithelial cycle in our strain of Sprague-Dawley rats synchronized by our protocol of vitamin A depletion and replacement. Our results indicate that retinol alone provides a better degree of synchrony than retinol with retinoic acid supplementation and that the
Accepted
University
through
These authors presented a procedure for quantifying the degree of synchrony on the basis of the ratio of stage frequencies of synchronized testes over the stage frequencies of control rats. In our analysis, we used a simplified approach based on the method of Van Beek and Meistrich that also yields a quantitative assessment, but one that can be easily determined from a graphic representation of the data. The objectives of our report were 1) to evaluate testicular synchrony using only retinol in a large group of rats; 2) to compare these results with synchrony obtained with rats supplemented with retinoic acid; and 3) to determine
in mammals.
quencies of occurrence of the different stages the duration of each stage. In the synchronized
progress
Recently Van Beek and Meistrich [7] used a modification of the original method for obtaining synchrony. In their method, rats on a vitamin A (retinol)-deficient diet are maintained with supplemented retinoic acid. This treatment alleviates the symptoms of vitamin A deficiency in all tissues except for those involving vision and spermatogenesis. Retinol administered to the retinoic acid-treated rats successfully resulted in synchronized spermatogenesis [7].
tinol depletion and replacement has been recently described as a model for the study of stage-specific expression of Sertoli-cell-secreted proteins and their mRNA [3]. Vitamin A in the form of retinol is required for the maintenance of spermatogenesis [3-5]. Vitamin A deprivation leads to the
tubules
experimental manipulation of the elements involved seminiferous epithelium.
stages of the cycle are defined by observed in cross sections of fixed
rat
the
recent report indicates that synchronization persists for up to 18 wk from the time of vitamin A replacement [9]. This
and biochemical methods in the rat [1, 2]. The cycle is defined by the recurring appearance of specific germ cell types within given regions of the seminiferous tubule. According to the
within
mal fashion, resulting in spermiation approximately every 12-13 days. Synchronization of the seminiferous epithelial cycle continues for at least three spermiation events, and a
former method is a reliable technique for obtaining that contain seminiferous tubules at selected stages seminiferous epithelial cycle.
of
55455.
284
testes of the
SYNCHRONY:
TESTICUlAR
MATERIALS
AND
METHODS
EVALUATION
AND
ANALYSIS
285
99.999. I
SI..
II
III
V
IV
VI
viii
VII
Iqx
XII
XIII
XIV
99.99.
Preparation
of Synchronized
Vitamin
A-depleted
Testis
rats
were
99.9.
raised
as
previously
de-
ton State University, Pullman, WA) weaned at 20 days of age and weighing an average of 40 g were fed a vitamin A-deficient diet (U.S. Biochemical, Cleveland, OH) ad libitum for 9-11 wk. Testicular regression was determined pation, and rats received i.p. injections of 7.5 mg (Sigma, St. Louis, MO) in 0.4 ml 50% ethanol/sterile
diets
The mented
were with
Rats tration
changed
then 1 mg
retinol
per
epithelium.
in the retinol
The
for
chow
by palretinol water.
subsequent
reinitiation Vil-VIJI of the
stages
was
as described
The
use
of retinoic
the
systemic
exception
of the
accurately
72 post vitamin used retinoic
(Diet
supple-
80 70
a E (.5 a.’
a 0
10 5
a U-
B)
acid
in Van
injections
Beek
pronounced
effects
(7.5
ginning of the retinoic ferences were observed data from the remaining were pooled.
and
the
rats (20 days
mg
Meistrich
[7]. to re-
49 days. acid (10 of retiriol as described start of the the rats in
visual
the
system were
put
Their diet was mg/kg food) for and supplemenabove for proexperiment (rats this experiment
of retinoic
acid supplementation. in the data from 10 rats, the data
with
of age)
for
i.p.)
during on Days
deficiency, on
acid
at the
be-
Since no difthese rats and the for all 16 animals
The body weights of animals in both treatment groups increased linearly up to Week 7 or 8 of each treatment followed by a period of 1-2 wk during which there was no gain
or
loss
of weight
(data
not
killed
by carbon
vical dislocation. Testes were
dioxide removed
asphyxiation and
and
cleared
cer-
of the
epididymis and fat tissue, and a portion of a testis from each rat was fixed in Bouin’s fixative, embedded in paraffin, and stained with hematoxylin-eosin and counterstained with periodic acid Schiff’s reagent for histological examination. Stage frequencies in treated rats were determined by light microscopic classification (400X) of at least 200 tubules and 2 cross sections per testis according to the criteria estabby Leblond
and
Clermont
[1]. Control
testes
I
.1 .01 .
.001
#{149}
I -. 0.2
values
-
r
--.
.___
-
0.4
-
0.8
0.6
1
Fraction of a Cycle FIG. 1. Determination of SFs and MPs. The cumulative frequency of the seminiferous epithelial stages observed in a testis were plotted on a probability scale as shown. The points at which each curve crossed the 84.1 and 15.9 percentiles were noted, and the distances A to B and C to D were defined as the window widths. The SF was a ratio of the distances AB to CD where AB was the window width for normal controls, and CD was the window width for synchronized rats. The vertical lines denote the duration of stages in terms of a fraction of a cycle. The MPs were the points at which the curves crossed the 50% point. The open circles represent the stage frequency of control rats determined experimentally; the open triangles represent the expected stage frequency of control rats calculated from duration of stages 1181; and the closed circles represent the stage frequency of synchronized rat testis (59 days PVA).
were
determined
cross
sections
from in each
Quantflcation
ation factor determined mulative ability reedy
examination
of 500
tubules
and
4
of 4 animals.
of Synchrony
The data were Meistrich [7] with
points centiles.
shown).
Histology Rats were
:
after and
is reported
of retinol
H?
a
seen
the time the arrest occurs killed
were
supplementation
Sixteen
stages
-
--
.
30 20
0 =
0’
A administration (PVA). acid supplementation,
symptoms
spermatogenesis.
received
from that
is known
it
on a vitamin A-deficient diet then supplemented with retinoic an additional 29 days. Injection tation of the diet with retinol tocol A began 78 days after the were 98 days of age). Six of
lished
90
>
U)
epithelial
of spermatogenesis cycle [6]. Animals
54, 55, 59, 63, 64, and Protocol B, which
net
95
a
a
7 days.
seminiferous
testis can be predicted administration since
lieve
rat
rat
99.
0 a 0
were killed at selected times after retinol administo obtain specific stages of the cycle of the semi-
niferous
and
to normal
C
a
scribed [10]. In protocol A, Sprague-Dawley rats (obtained from the Laboratory Animal Resource Facility of Washing-
(SF) and graphically
frequencies scale, from
analyzed by the one modification; the
method that
midpoint of synchrony as illustrated in Figure
of each
stage
were
and the window widths the graph by noting the
at which These
of Van Beek and is, the synchroniz-
the curve percentiles
crossed define
the
plotted
(MP) were 1. The cuon
a prob-
were
determined distance between
dithe
the 15.9 and 84.1 perwindow width that is
equal to plus and minus one standard deviation. The ratio of window width of the control testes to the synchronized testis thus determined the SF. The SF, as previously defined,
is independent
of the
duration
and can be used to describe synchrony the cycle and in different experiments SF was
determined
by using
the
window
of any
width
for control rats, which is constant, i.e. 68.3%. width of the control rats was similar whether mental lished
data or calculated data on the duration
single
stage
in various parts of [7]. The value for the
values determined of each stage were
of the
data
The window the experifrom used.
pubThe
286 MP
SIITERI
is the 50th percentile
of the
point, or the median
ET AL.
I
stage
II
III
IV
V
VI
VII
VIII
IX
X
XI
54DAY
distribution, and is presented in terms cycles traversed. Thus, an MP 5.3 means
of the number of that 5.3 cycles have
18
Il
8
6
7
55DAY 14
been stages
traversed is 30%
teria follows: tained
we
and that of the way
the center throught
distribution cycle. The
of cri-
were part of a group observed comprised
number of tubules. meet these criteria
5
MPs
remain
the
ing
34
JI3
IS
software,
Abacus
Concepts,
A us-
obtained
from
72 PVA. These synchronized
in the majority of animals prbtocol B. The synchronized
I) stages of the cycle. The results of treatment A for 56 rats where synchrony was achieved rats that
Figure 2. Data from not included. experiments sentative
These
did not achieve synchrony
data were
conducted of animals
over for
with protocol are shown in
compiled
from
a 1 yr period
which
at least
a series of
and
three
were
are
repre-
samples
were
available for each PVA time point. In a total of 121 animals that were used in this and in other studies from this laboratory, the vitamin A depletion-replacement protocol produced synchrony in approximately 80% of the rats. The remaining animals were not synchronized (5%), exhibited
18 20
6
4
60
10
31
9
6
19
49
21
16
42
25 5 5 9 37 20 16 18140 ________22 5 ________ 27 40111 23 38 10 35 48 17 25 30 20 6 35 40 15 8 i8li8 5 12 12 7 21 26 10
24 29
13 10
10 16
10
34 40 41 40
28 24 32
10
40
32
10 15 5
23 9 IS 42
33 25 27 18
22 21
11 7
10 7 8
10
19
15
20
27
19
13
16
18
26
24
8.9
10
3.3
5.4
4.6
8.1
I5
17
3.3
by this
SF and
MP values
nized
tubules
able between rats, given PVA period, siderably. However, cycle, was Despite
93
± 8
ranging from 1.6 to 11.6. Even within a the synchronization values varied conthe MP, plus or minus one stage of the
reproducible within any given PVA time period. some variability in the stage frequencies within
each sample to treat each
and a large variation in the SFs, it was PVA time
period
as a group
and
possible determine
8.00
5.70
8
5.00 10.4
5.74 5.71
5
5.50
5.73
5.80 7.40 3.90 4.00 1.60 3.30
5.77 5.78 6.23 6.23 6.37 6.33
5.27
5.30
2.27
0.41
6.5
6
1.00
0.58
3.3
the mean stage frequencies. The mean stage frequencies (average of percentages for each stage) for each PVA period are shown in Table 1. Both the mean SF and mean MP val-
average
was
5.28 5.29 5.51 5.13 5.11 5.63 5.71 5.73 5.74 5.73
method.
for
56 animals
4.3
5.22
FIG. 2. Stage frequencies of individual rats on days PVA and calculated SFs and MPs (protocol A). The stages comprising the highest percentages of tubules are boxed; stages comprising less than 5% of tubules were not included. Dashes represent values that could not be determined
PVA group
with a range of 71 %-100%. 2 that the SF was highly vari-
4.6
11.6
3.50 4.30 2.90 5.00 4.30 1.70 6.90 5.80 5.20 6.50
16
±SD
1RL
NP 4.90 4.92 4.95 4.93 4.97 5.00 4.93 4.84 5.01 5.00 5.01 4.94 4.97 5.15 5.07 5.03 4.92 5.01 5.03 4.99 5.11 5.03 5.00 4.91 4.94 5.15 5.16 5.37 5.24 5.30 5.22
15
5
9
#{128}AN
each
of these
6
13
obtained
testes
7
7
values
deviation) from Figure
8
3
average
in the
21
5
ues
(mean ± standard It can be seen
13
39
12 22 14 17 20 22 19
tubules synchro-
than
64
13
86 68 78 78 37 59 45
segment
more
10
95
one
around
10
8
cycle; 8%) or had a majority of unrecovered The average percentage of recovered and
(synchrony
20
27
10
________
6 11
10 12
5
of the (7%).
parasynchrony
9
27
using both testes were
animals killed on days 54, 55, 59, 63, 64, and time points were selected to obtain testes in early (Il-VI), mid (VII-VIII) and late (XII-
8 1914
I98 22
DAY
720AY
33
24
RESULTS
of spermatogenesis protocol,A and
40
43
6
64
43
2816
61 30
Inc.,
18
34
3
5
of
15fl
9
19
by readministration in the synchronization
25
5
50
CA.
Depletion of vitamin A followed retinol to developing rats resulted
28
10
40
63 DAY
Berkeley,
16
7
17
62
Efli
same.
graphics
26
5
4
10 5
SE+
26 25
Ja
Analysis
StatView
31
26
stages, of the
from protocols signed-rank test
14
27
12
fl18
are considered, comparisons different treatments. Addition-
factors obtained by the Wilcoxon
56
10
18
The synchronization and B were compared
39
8
6
59DAY
Statistical
13 16
28
25
24 7
7
52 31
4.70 11.6 3.10 6.50 3.90 4.00 6.50 5.50 5.80 3.30 3.40 11.6 5.80 4.30 4.00 4.00 5.50 4.50 4.50 4.70 4.50 5.20 2.90 8.00 5.00 4.70 4.50 3.00 4.30 3.40 5.50
XIV
7
26
Although excluding tubules that do not tends to inflate SF values compared to
analysis in which all tubules may stillbe made between the
of related at least 5%
XIII 27 65
DII
used to determine testicular synchrony were as tubules were considered synchronized if they cona normal complement of germ cells, if they con-
tained stages that and if the stages
ally,
of the the next
XII 26 22
5.71 be
obtained
±
0.04
for
(mean
significantly
±
different
all animals,
5.27
When
synchronization
± 2.27
for
SD),
compare
favorably
all animals.
For
respectively,
from
the
±
and
63 PVA, 5.68
Day the
and
5.30
was
generated
do
to the
example, not
2.17
appear
average
SF and
0.41,
respectively.
±
by protocol
MP
B, which
utilized retinoic acid supplementation, the resulting frequencies (Fig. 3) were similar to those described ure 2. As in protocol A, the SFs were highly variable the
MPs were
testes from significantly
tightly
grouped
for each
PVA time
to for
stage in Figwhile
point.
the rats treated according to protocol lower SFs compared to those from rats
The
B had treated
TESTICUlAR
according
to protocol
the
MP was
A (p
SYNCHRONY:
0.02,
z = -2.483).
both
treatments
