Gradual Regeneration of Mouse Testicular Stem Cells after Exposure to Ionizing Radiation

Author(s): Marvin L. Meistrich, Nancy R. Hunter, Norio Suzuki, Patricia K. Trostle and H.
Rodney Withers
Source: Radiation Research, Vol. 74, No. 2 (May, 1978), pp. 349-362
Published by: Radiation Research Society
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RADIATION RESEARCH 74, 349-362 (1978)

Gradual Regenerationof Mouse Testicular Stem Cells

after Exposureto Ionizing Radiation'

MARVIN L. MEISTRICH, NANCY R. HUNTER, NORIO SUZUKI,

PATRICIA K. TROSTLE, AND H. RODNEY WITHERS

Section of ExperimentalRadiotherapy,The Universityof Texas System CancerCenter,

M. D. AndersonHospital and TumorInstitute, 6723 BertnerAvenue,
Houston, Texas 77030

MEISTRICH,M. L., HUNTER, N. R., SUZUKI, N., TROSTLE,P. K., AND WITHERS,
H. R. Gradual Regeneration of Mouse Testicular Stem Cells after Exposure to Ion-
izing Radiation. Radiat. Res. 74, 349-362 (1978).
The regeneration of mouse testicular stem cells during 60 weeks after exposure
to 600 or 1200 rad of -y-radiationwas examined. Restoration of spermatogenesis de-
pended on stem cell survival, regeneration, and differentiation. Several assays were
employed to measure the number of stem cells and their ability to repopulate the
seminiferous epithelium as follows. Assay 1: The percentage of repopulated tubular
cross sections was determined histologically at various times after irradiation.Assay 2:
Mice were irradiated and, after given time intervals to allow for regenerationof stem
cell numbers, a second dose was given. The percentage of repopulated tubular cross
sections was determined5 weeks later. Assay 3: The ability of the stem cells to produce
spermatocytes and spermatids was assayed by the levels of the germ cell specific
isoenzyme, LDH-X. Assay 4: The ability of the stem cells to produce sperm was
assayed by the number of sperm heads in the testes. In addition, the ability of the
stem cells to produce functional spermatozoa was measured by the fertility of the
animals. The results obtained were as follows. All assays demonstrated that gradual
regeneration of stem cell number occurred simultaneously with repopulation of the
seminiferous epithelium by differentiating cells derived from stem cells. The regenera-
tion kinetics of stem cells followed an exponential increase approaching a dose-de-
pendent plateau below the level prior to irradiation. The doubling time for stem cells
during the exponential portion was about 2 weeks. After 600 rad of irradiation, the
number of stem cells assayed by sperm head counts or by LDH-X activity increased
from 5% of the unirradiatedlevel to a plateau of 80%. A greater proportionalincrease
in stem cell number, from 0.1 to 10% of control, was observed after 1200 rad, but
the plateau level was lower than after 600 rad. The regenerationof stem cell number
after depletion by irradiation was gradual and incomplete, and only partially restored
spermatogenesis. Correlation of regeneration with fertility data demonstrated that
fertility was reestablished when sperm production returned to about 15% of control
levels.
1This investigation was supported by Grants CA 17364, CA 06294, CA 11138, and CA 11430
awarded by the National Cancer Institute, DHEW, and by research Grant PCM 76-08836
awarded by the National Science Foundation.

349

0033-7587/78/0742-0349$02.00/0
Copyright ? 1978 by Academic Press, Inc.
All rights of reproduction in any form reserved.

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350 MEISTRICH ET AL.

INTRODUCTION
Radiation produces sterility in male mammals which may be either tem-
porary or permanent (1). After irradiation, fertility is maintained for about
3 weeks because of the radioresistance of the spermatocytes, spermatids, and
spermatozoa (2). Then sterility occurs first because of production of dominant
lethal mutations in spermatids and spermatocytes (3, 4) and subsequently
because of the radiosensitivity of the differentiating spermatogonia (5) which
results in a depletion of sperm during the time interval when these sperma-
togonia would have become sperm. The spermatogonial stem cell is more radio-
resistant (6, 7). Surviving stem cells will differentiate to produce spermatozoa
which will, if produced in sufficient numbers, result in restoration of fertility.
The length of time prior to restoration of fertility (sterile period) has been
shown to be dose dependent (8, 9), and measurement of this time has been
proposed as an assay of stem cell survival (9). Although the length of the sterile
period is certainly related to stem cell survival, other factors such as the re-
generation of the numbers of stem cells and the kinetics of their differentiation
into functional spermatozoa must also be involved. The purpose of this study
was to measure the kinetics of regeneration of the numbers of stem cells after
irradiation and the repopulation of differentiated germinal cells from the sur-
viving stem cells and then to relate these data to the recovery of fertility.
The terms "regeneration" and "repopulation" are often used interchangeably.
However, in the context of these studies it will be useful to make a distinction
to describe two different processes. The term "regeneration" will be used to
describe the increase in the numbers of stem cells and the term "repopulation"
to describe the differentiation of the stem cells and the refilling of the tubule
by their progeny.
Until recently, methods have not been available for quantitative measure-
ment of stem cell survival. Conflicting models for the identity of the stem cell
have been proposed (7, 10-14), complicating any histological measurement of
stem cell survival. Using one model for stem cell survival, Erickson has ob-
tained a survival curve for stem spermatogonia in the rat (15).
An alternative approach has been to measure the survival of the stem cell
by its functional ability, i.e., the ability to sustain spermatogenesis. This func-
tional criterion has been used to assay stem cell survival by counting the frac-
tion of seminiferous tubular cross sections which are repopulated by colonies
of spermatogenic cells (6, 16, 17). In this study we have employed the colony
assay in two ways. In the first case, we irradiated mice with 1200 rad and
counted the fraction of repopulated tubular cross sections at various post-
irradiation times. One difficulty with this assay is that the proximity of several
stem cells in one region of the tubule, as happens during regeneration (18),
would result in an underestimation of stem cell numbers. In the second method,
we irradiated mice with 600 rad, waited a given time after irradiation for re-
generation of stem cell number to occur, gave a second "knock-down" dose
of 600 rad, and then counted the fraction of repopulated tubular cross sections
5 weeks later. The latter method has the advantages that most clusters of stem
cells are removed by the second dose and that by using a fixed time between

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 REGENERATIONOF TESTIS STEM CELLS 351

the final dose and the assay, the colony size is constant. The limitation is that
synchronization and redistribution of cells within their cycle continues for
a long time after the first dose, altering the radiosensitivity of the cells to the
second dose.
Because of the disadvantages of the colony assay, we have developed two
other assays for the survival of the stem cells based on their ability to produce
spermatocytes, spermatids, and spermatozoa. These assays consist of measuring
the levels of the enzyme, LDH-X, and the numbers of sperm heads in testic-
ular homogenates.
LDH-X, the X isozyme of lactate dehydrogenase, is found only in sperma-
tocytes, beginning at the mid-pachytene stage, spermatids, and spermatozoa
(19, 20). Since this isozyme can be specifically assayed using the substrate,
a-ketovalerate, the level of LDH-X can be readily determined in testicular
homogenates (21). This level is a measure of the numbers of spermatocytes
and spermatids which have developed from the surviving stem cells.
Sperm heads, identified in testicular homogenates after sonication, are nuclei
of steps 12 to 16 spermatids (22). Such preparations contain only distinctively
shaped sperm heads and quantitation is relatively easy and rapid by microscopic
counting. The numbers of sperm heads provide a measure of the number of
surviving stem cells and their ability to differentiate to produce sperm.
The results obtained using the various assays described indicate that re-
generation of stem cell numbers occurs very slowly after depletion by 600 or
1200 rad of y-radiation, and the restoration of spermatogenesis is incomplete.

MATERIALS AND METHODS

Animals.2 C3Hf/Bu mice maintained in a specific pathogen free colony were

used throughout. Unless otherwise indicated, animals received their first dose
of irradiation between 8 and 9 weeks of age.
Irradiation procedure. Irradiation was carried out using a pair of 37Cs sources,
with a 3-cm diameter field. Mice, awake at the time of irradiation, were re-
strained in Lucite boxes and were positioned such that only the testes and
surrounding organs received radiation. The dose rate was 1040 rad/min for
all doses of 600 rad or above, and 200 rad/min for doses of 300 rad. When mice
were given 2400 rad, in order to avoid excessive mortality from bowel injury,
the irradiation was fractionated into two doses of 1200 rad each, given 1 week
apart.
Histological procedures. After the mice were sacrificed, testes were removed
and fixed in Bouin's solution for 2 hr, stored in 70% ethanol overnight, and
then stored in 10% buffered formalin. Testes were cut transversely to the long
axis at the equator, subjected to routine histological processing and sectioning,
and stained with hematoxylin and eosin.

2
Animals used in this study were maintained in facilities approved by the American Asso-
ciation for Accreditation of Laboratory Animal Care, and in accordance with current United
States Department of Agricultureand Department of Health, Education, and Welfare, National
Institutes of Health regulations and standards.

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352 MEISTRICHET AL.

Counts of repopulated tubules. In each testicular cross section counts of re-

populated and total tubular cross sections were made. The ratio of these two
counts was designated the repopulation index, RI (16). From the RI the number
of surviving stem cells per tubular cross section, referred to as the stem cell
survival index, SSI, was calculated by applying the Poisson distribution
rules (6)
SSI = -loge (1 - RI).
This correction becomes appreciable when RI ? 0.3.
Sample preparation for sperm head counts and LDH-X assay. Immediately
after sacrifice, testes were placed, with the tunica albuginea intact, into dis-
tilled water (approximately 1 testis/ml). Each sample was then homogenized
with a Polytron homogenizer (Model PT10, Brinkmann Instruments) for 5 sec
(90% maximum) and sonicated with a Branson 20 KHz sonifier fitted with
a microprobe (model W185, Heat Systems, Inc.) for 30 sec at a setting of 3.
An aliquot was removed for sperm head counts. The remainder of the sample
was heated to 55?C for 5 min, chilled on ice, and centrifuged at 24,000gmax
for 15 min (19). The supernatant was used for LDH-X assay.
Sperm head counts. Sperm heads, i.e., sonication resistant spermatid nuclei
in steps 12 to 16 of development, were counted in a hemacytometer using a
microscope with phase contrast optics. Either a volume of 9 X 10-4 ml or
200 sperm heads were counted, whichever was reached first.
LDH-X assay. The LDH-X activity was assayed at room temperature by
spectrophotometric measurement of the oxidation of 1.5 X 10-4 M NADH in
the presence of 3 X 10-5 M a-ketovalerate in 0.05 M sodium phosphate buffer
(pH 7.4) (19). The time required for a reduction of optical density at 340 nm
of 0.05 was recorded and the activity was calculated. The activity was a nearly
linear function of the amount of enzyme present over a range of 3 orders of
magnitude. When the enzymatic activity was extremely low, duplicate assays
were run with and without the a-ketovalerate substrate. The apparent activity
in the absence of a-ketovalerate was attributed to nonspecific enzymatic loss
of NADH absorbance and was subtracted from the activity measured in the
presence of a-ketovalerate.
Fertility tests. The fertility of male mice was tested beginning 59 days after
irradiation. At that point, each mouse was placed with three virgin females.
The mice were checked daily for production of litters. The return to fertility
for each male was assumed to be the date of conception of the first litter,
i.e., 20 days prior to birth. Every several months the females were removed
and replaced by younger ones.
Fitting of regeneration curves. The regeneration of stem cells was assumed
to display exponential growth kinetics with an approach to a saturation value.
This type of growth may be characterized by a Gompertzian (23) function
of the form
y = ae-be-k'
where ae-" is the initial value of y, a is the final asymptotic value of y, k is
a coefficient describing the rate of growth during the exponential part of the

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 REGENERATION OF TESTIS STEM CELLS 353

__ 08- o 8 \
=006
-
E 046
0.4

/200rods

1 0?2 - H1

.- 0.1
cn -- 0.08-

0.06 -
10 20 30 40 50 60
TimeAfterIrradiation
(weeks)
FIG. 1. Stem cell survival index (SSI) as a function of time after irradiation. Each point
represents data obtained from 10 mice, irradiated with 1200 rad of y-radiationat 0 weeks and
sacrificedat the indicated times. Values are presented as means and standard errors.

curve, and t is the time. The increase factor (asymptotic value divided by
initial value) is given by eb. The doubling time during the exponential part
is given by 0.693/bk. The curves were fit by computer. Initial estimates of
the parameters were first obtained using linear regression to fit the initial part
of the rise. Subsequently, a nonlinear least-squares fitting routine was used
to obtain the best values of the parameters fit to the entire data set.

RESULTS

Assay 1: Fraction of Repopulated Tubular Cross Sections at Various Times

The numbers of testicular stem cells at various times after irradiation were
assessed by counts of repopulated tubular cross sections (Fig. 1). Mice, given
1200 rads of y-radiation, were sacrificed at specified times beginning 4 weeks
after irradiation. The SSI at 4 weeks was 0.08. With longer intervals before
sacrifice, the SSI increases gradually, reaching a plateau value of 1.19. Resto-
ration of spermatogenesis is far from complete since the SSI of 1.19 corresponds
to an RI of only 0.70.
The estimated doubling time along the exponential part of the curve is
5.0 weeks. However, several opposing modifying factors must be noted. First
of all, a colony derived from a single stem cell becomes larger with time, result-
ing in a greater probability of detection in a given cross section. On the other
hand, when a stem cell divides to produce two stem cells, the two cells will
most likely remain in the same region of the tubule. Assuming they produce
a colony of the same shape but twice the volume of a colony produced by one
cell, the probability that it will be detected in a random testicular cross sec-
tion is only 2*, or 1.26, instead of 2. With the latter correction, the doubling
time for stem cell number becomes 1.7 weeks.

Assay 2: Fraction of Repopulated Tubular Cross Sections After a Second Dose

of 600 Rad
Stem cell regeneration, after a dose of 600 rad, was measured by the following
method: After various time intervals, a second dose of 600 rad was given to
lower the SSI to a level which is convenient for counting. Repopulated tubular

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354 MEISTRICH ET AL.

Repair Synchronizatibn Regenertion Decline

1.0 nd Redistribution2 o o

0.5-

.? - 600*600reds

CO
CO y ^/ :.\"A?
n
C ._c- 00 * V- ,/\

*
- (singledosecontro/)

i '- 0.01

0.005 \

0 2 4 6 8 10 13 17 21 30 44 58
TimeBetween
Radiation
Doses(weeks)
FIG. 2. Stem cell survival index as a function of time between 2 doses of 600 rad each. At
week 0, mice were irradiated with 600 rad, then at the indicated time, irradiated again with
another 600 rad and sacrificed 5 weeks later for determination of the SSI. For comparison,
separate groups of mice were irradiated with 1200 rad at the time of the second dose and those
points are plotted at that time along the abscissa. Each point represents the mean of data from
5 mice. The average standard errors are indicated by error bars. Duplicate points taken at each
time represent two separate experiments. The lines are drawn through the means of these
two points.

cross sections were counted 5 weeks later. As a control for an effect of the age
of mice or alteration in irradiation conditions, additional mice from the same
group were also irradiated with a single dose of 1200 rad at each time of irra-
diation and sacrificed 5 weeks later.
The data shown in Fig. 2 demonstrate that the killing of stem cells by
1200 rad is essentially independent of age from 9 to 32 weeks of age. At 39 weeks
_ ' - - a a----.- o
- 300
rods ' 600 rods

2 Ia
rod
a 120 d
lo-, ^
A

.4_
-T
2400 rods
(:F4 2i..--

105
0( 5 ' , ' , ' ,A ',, ' A ' ', ,& ', ,
10 20 30 40 50 60
TimeAfterIrradiation
(weeks)
FIG. 3. LDH-X activity per testis of irradiated mice. Mice were locally irradiated with 300,
600, 900, 1200, or 2400 rad (the latter given as two doses of 1200 rad spaced 1 week apart).
All values are expressed as a fraction of the activity in unirradiatedmice sacrificed at the same
time. Each point represents the activity of a homogenate prepared from 3 mice (control 100,
300, 900, and 2400 rad), 5 mice (600 rad), or 10 mice (1200 rad). Duplicate points taken at
each time represent two separate experiments.

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 REGENERATION OF TESTIS STEM CELLS 355

and older, either the stem cell number decreases, the radiation sensitivity in-
creases, or their proliferative ability after radiation declines.
Fluctuations are observed in the numbers of stem cells surviving split doses
of radiation. The rapid rise in the SSI when the doses are separated by 4 to
24 hr is primarily due to repair of sublethal radiation injury. The fluctuations
in the SSI between 1 and 28 days are likely a result of synchronization of the
cells by the first dose into more resistant phases of their cycle and subsequent
progression and redistribution. If we assume that by 28 days these fluctuations
are over and cells are redistributed in the same state as prior to irradiation,
we can then attribute any increase which occurs after this time to regeneration
of stem cell number. Since the SSI rises from 0.049 at 4 weeks to 0.64 by 23
weeks, the stem cell number must undergo a 13-fold increase in this period.
Again it must be noted that the regeneration is still incomplete at 23 weeks
since the plateau level (SSI = 0.64) is lower than that which would be ob-
served with a single dose of 600 rad. A slight decline is also observed in this
curve with the older mice.

Assay 3: LDH-X Activity

After irradiation there is a marked drop in the activity of LDH-X with a
minimum at about 4 weeks after irradiation. This minimum reflects the killing
of the radiosensitive spermatogonia. A time of at least 6 weeks is required for
the differentiating progeny of a stem cell to reach the end of spermatogenesis.
Hence, only data on LDH-X levels, taken at times longer than 6 weeks, will
be used as a measure of survival and regeneration of stem cells (Fig. 3). For
doses of 600, 900, and 1200 rad the levels of LDH-X rise steadily after 7 weeks
and reach a plateau, significantly below unirradiated control levels. For doses
of 300 rad, the control levels are restored.
LDH-X activity in mouse testes given 2400 rad, which should be sufficient
to kill practically all of the spermatogenic stem cells, was measured as a control.

i a
,- ---_____-,---8 _---- ^
300 rods * ,/"-' 600 rods

10'0rt
7o 0_ - / / -A -d
900 rods d

0 o f
/
o
-2 r. 0
E 1? 1o

'-" .' ". 2400 rods

, , , I ,,
105
10 20 30 40 50 60
TimeAfterIrradiation
(weeks)
FIG. 4. Number of sperm heads per testis of irradiated mice. All values are expressed as a
fraction of the number of sperm heads in the unirradiated mice sacrificed at the same time.

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356 MEISTRICH ET AL.

There appears to be a very small amount of activity in these homogenates

which results in oxidation of NADH in the presence of a-ketovalerate. It is
likely that this is not due to LDH-X, but rather the presence of another enzyme
with a slight reactivity with this substrate. No regeneration of testicular stem
cells was detected after 2400 rad.

Assay 4: Numbers of Sperm Heads

After irradiation there is a marked drop in the numbers of sperm heads
(nuclei of elongated spermatids) reaching a minimum at about 5 weeks post-
irradiation. Subsequently, there is an increase in the numbers of sperm heads
until a plateau level is reached as in the case of LDH-X (Fig. 4). After 50 weeks
there appears to be a further decline in the numbers of sperm heads in irradiated
mice relative to unirradiated controls.

Analysis of Regeneration Curves

Additional information on the kinetics and extent of regeneration can be
obtained by mathematical fitting of the curves. The Gompertzian function
provided a reasonable fit to the experimental data. The parameters obtained
are expressed in a more useful form in Table I. The following conclusions can
be drawn from these data. The plateau levels of stem cell regeneration from
Assays 3 and 4 are 0.8 and 0.1 for 600 and 1200 rad, respectively. The amount

TABLE I
Parameters of Stem Cell Regeneration

Assay method Dose Range of fit Asymptotic Increase Doubling

(rad) (weeks) value factor time
(a) (ae-b) (weeks)
(0.693/bk)

Repopulated tubular
cross sections at
various times after
single dose (Assay 1) 1200 4-51 1.19" 14 5.0
Repopulated tubular
cross sections 5 weeks
after second dose
(Assay 2) 600 4-23 0.64a 13 0.6
LDH-X activity
(Assay 3) 600 7-51 0.76b 11 2.0
LDH-X activity
(Assay 3) 1200 7-51 0.10b 32 3.0
Sperm head counts
(Assay 4) 600 7-51 0.81b 21 1.6
Sperm head counts
(Assay 4) 1200 7-51 0.12b 185 1.9
a SSI.
b Fraction of unirradiatedcontrol.

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 REGENERATION OF TESTIS STEM CELLS 357

TABLE II
Relationship between Radiation Dose, Duration of Sterility, and
Sperm Production in C3H Mice

Dose Lengthof sterileperiod (days) Sperm numberat

(rad) median (range) time of conceptiona
(fractionof
unirradiatedcontrol)
(%)
600 85 (79-99) 19
900 195 (127-256) 13b
1200 >400 (None fertile) <10
a Calculated on the basis of number of testicular
sperm heads 9 days prior to restoration of
fertility. The 9-day period is the time required for sperm leaving the testes to pass through the
epididymis and vas deferens (24).
b Calculated by interpolation between sperm counts at 600 rad and 1200 rad.

of stem cell regeneration is between 10- and 20-fold after 600 rad (Assays 2, 3,
and 4) and between 30- and 200-fold after 1200 rad (Assays 3 and 4). The
lower amount of regeneration, 14-fold after 1200 rad measured in Assay 1,
is expected because of stem cell clustering. The time required for a doubling
of stem cell numbers is between 1.6 and 3.0 weeks (Assays 3 and 4). The shorter
time of 0.6 weeks measured by Assay 2 may be a result of the lack of complete
redistribution of stem cells 4 weeks after the first dose.

Fertility Tests
The time of return to fertility of the irradiated males was taken as the length
of the sterile period. The values for radiation doses of 600, 900, and 1200 rad
are given in Table II. It is seen that the length of the sterile period increases
with increasing doses; after 1200 rad the mice remained sterile throughout
the course of the experiment (i.e., 400 days).

DISCUSSION
In this study we have measured the regeneration of testicular stem cells
after doses of ionizing radiation which kill most of these cells. In the assays
used, we are counting the differentiated progeny of the stem cells. Hence, we
must consider the two processes acting; i.e., the regeneration of the depleted
stem cells, and the ability of the stem cells to differentiate to produce sperm.
We shall first review what is known about the normal pattern of stem cell
renewal and differentiation in untreated animals for comparison with what
happens after depletion. This is a steady-state process involving no change
in stem cell numbers and a continuous repopulation of the tubules with dif-
ferentiating cells. According to one recent model (13, 14), the stem cell is an
isolated undifferentiated type A spermatogonia, designated Ais. These cells
appear to be heterogeneous; some have a short cell cycle (about 40 hr) and

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358 MEISTRICH ET AL.

200-300rads i

<100ratds A
Ais Aa--
StemCell -5.3doys
Spermatozoa 16
> 60,000 rods \
i/4.' a d _ D,otiplofene
Secondorry
Step,jl'"-
4 2 k1,
I_d _q_e Y!7-6-
-\,
V"50 . .... Spermheads
I1 s~~,
SeOProQs I _LDH-X

FIG. 5. Kinetics and radiation sensitivities of spermatogenic cells in the mouse. Drawings
of the various cell types, modified from Clermont's work on the rat (11) are shown. The mode
of stem cell renewal is taken from Oakberg'smodel (13). The kinetics of spermatogenesisis also
taken from the work of Oakberg (13, 25, 26). Radiation sensitivities are given as LD50values.
The LD50 values for the differentiated mouse spermatogenic cells are taken from Refs. (2, 5,
and 27), those for A,a are estimated from Refs. (7, 28), and those for the Ais stem cell are esti-
mated from Refs. (7, 15) and our own unpublished data. The cell types during spermatogenesis
which contain LDH-X (19) are indicated by the solid bar. The stages of spermatogenesisduring
which the spermatid nucleus has the properties of a sperm head (22) are indicated.

others, probably the true stem cells, a much longer cycle (about 8.6 days)
(12-14).
The first apparent step in stem cell differentiation is division to produce
a pair of cells and then a syncytium of cells, designated Aai (14). These cells
yield A1 spermatogonia which continue through the differentiation sequence
(25) according to a rigid schedule as outlined in Fig. 5. An alternative model
of stem cell renewal proposes the existence of a resting radioresistant Ao sper-
matogonia, which only divides to repopulate the seminiferous epithelium after
depletion by a cytotoxic agent (10, 11). In the ensuing discussion, we shall
only utilize the first model discussed (13, 14).
The radiation sensitivities of the cells are also presented in Fig. 5. Practically
all of the differentiated spermatogonia, A1 through B, are killed by the radia-
tion doses employed, as are many of the Aai cells. The more mature spermato-
genic cells survive and progress through the developmental sequence with the
normal kinetics (29). Thus, 28 days after irradiation the only germinal cells
remaining in the testes must represent the progeny of the surviving stem cells.
The normal kinetics of differentiation also holds during repopulation. If all
the Ais spermatogonia that survived started differentiating immediately, it
would require 40.5 days to produce sperm. The repopulation of the seminiferous
epithelium after 100 rad occurs more quickly than this, indicating that Aai
spermatogonia also survive and begin the repopulation process (26). However,
higher doses, for example 300 rad, appear to kill these Aai spermatogonia (28).

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 REGENERATION OF TESTIS STEM CELLS 359

Therefore, it requires 8 days more before the initiation of repopulation after

300 rad, than after 100 rad (30). In addition to the fact that higher doses of
radiation result in cell killing further back in the spermatogonial differentia-
tion sequence, another factor to be considered is that at higher doses, regenera-
tion of the numbers of stem cells may occur for a longer time before differentia-
tion and repopulation of the seminiferous epithelium begins.
Regeneration of the numbers of stem cells and repopulation of the semi-
niferous epithelium by differentiation of stem cells may be viewed as competing
processes. At each stem cell division there is a probability, P, that each daughter
will remain a stem cell. In order to maintain steady-state levels of stem cell
numbers and spermatogenic cell differentiation, P must be 0.5. On the other
hand, in order to achieve regeneration of stem cells, P must be greater than 0.5.
During the first 10 days of recovery from a partial depletion of the seminiferous
epithelium by the alkylating agent, Myleran, P appears to approach 1 (18, 31).
This result indicates that stem cells first begin regenerating their numbers
and then start producing differentiating cells which repopulate the seminiferous
epithelium. A similar interpretation may be drawn from observations on rat
spermatogonia after X irradiation (15). This phenomenon would result in a
delay before repopulation of the seminiferous epithelium is observed. This
delay appears to be greater with higher doses (15, 30). However, the dose
response curves of the RI measured at various times after irradiation appear
to show little change in slope with time allowed for repopulation (17). This
result implies that variation of the delay in repopulation as a function of dose
is not a large effect.
In this study we propose the use of two parameters which reflect the number
and composition of differentiated spermatogenic cells to assay the survival
and regeneration of stem cells. The several criteria which are listed below,
appear to be satisfied, indicating validity of these new assays. First of all, the
amount of LDH-X per cell, the cell types which synthesize LDH-X, and the
formation of the sperm head are not directly affected by radiation (19); these
parameters decline only as a result of cell depletion. These parameters are
also unaffected by changes in hormonal levels (32). A second criterion must
be that the number of differentiating cells produced by a stem cell must be
constant during all phases of regeneration and equivalent to that which ocurs
during steady-state conditions prior to depletion. After partial depletion of
the seminiferous epithelium with Myleran, despite overshoot in the numbers
of A spermatogonia, the number of spermatocytes steadily recovers, and is
then maintained at control values (31, 33). Thus, some factor which maintains
normal production of differentiated cells from a stem cell must be present.
In addition, we can ascertain that no appreciable degeneration occurs during
repopulation since the plateau values for amount of regeneration obtained
with LDH-X are very similar to those obtained with sperm head counts.
It is important to choose proper times for measurement of LDH-X activity
and sperm head counts in order to use these assays to measure the number
of stem cells. After treatment (18), differentiating spermatogonia (Aai) first
reappear between 10 and 15 days later. Since an additional 40.5 days are re-

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 360 MEISTRICH ET AL.

quired for these differentiating spermatogonia to become spermatozoa, the use

of LDH-X and sperm head data to assay stem cell survival only becomes valid
at 7 to 8 weeks after irradiation.
There are differences in the times after irradiation when the progeny of the
stem cells can be detected by the different assays. For example, by Assay 1,
repopulating colonies can be detected when they reach B spermatogonia.
LDH-X first appears at the mid-pachytene stage and is also present in sper-
matids. Taking the midpoint of the time period when LDH-X is present, we
estimate that regenerating stem cells are detected 2.7 weeks later by the
LDH-X assay than by Assay 1. Likewise, the time difference between Assay 1
and sperm head counting is 3.6 weeks. On the other hand, the time scale used
in Assay 2 (Fig. 2) is taken to the time of the second dose. Thus, stem cell
regeneration should be more directly measured by this assay since the time
scale does not include any additional time of repopulation.
The slowness of the regeneration process, compared with other tissues, is
surprising (6). However, in the testes we might expect only one division of
stem cells during each 8.6 day cycle of the seminiferous epithelium. Other
workers have reported that 10 days after treatment with 30 mg/kg Myleran
the regenerating colonies contained an average of 2.3 stem cells, but by Day 15,
they contained an average of 7.2 stem cells (18). Their results indicate that
during recovery, stem cells can divide 3 times during one cycle (31, 33). In the
present experiments (Assay 2), as well as a previous study (6), no regeneration
of stem cell numbers was observed for 2 weeks after 600 rad of radiation. It is,
however, possible that some regeneration is occurring but is masked by syn-
chronous passage and redistribution of surviving stem cells into a sensitive
compartment. Nevertheless, regeneration of stem cell numbers is not great
during this time interval. The data from Assays 3 and 4 indicate only one
doubling of stem cell numbers each 2 weeks (1.6 cycles). However, since we
are only observing stem cells which produce differentiated cells, a change in P
would somewhat alter the apparent kinetics of stem cell regeneration. If P
decreases as regeneration progresses, the actual doubling time would be longer
than that calculated from the data. Assuming that the stem cells divide once
every 8.6 days, that P is constant, and that the number of cells doubles every
14 days, we calculate P to be 0.76. This value is intermediate between pure
regeneration and the steady-state condition.
The incompleteness of the regeneration is also surprising. The factors limiting
the regeneration are not known. One hypothesis, that regeneration is limited
because some of the approximately 20 to 30 seminiferous tubules in the testes
(34) are absolutely depleted of stem cells so that no regeneration or repopula-
tion can occur in these tubules, was considered. However, regeneration of stem
cells approaches only 10% of control after 1200 rad, while the percentage of
repopulated tubular cross sections is 70%. Thus, repopulated tubules must
show incomplete regeneration of stem cell numbers and the above hypothesis
is rejected. Some feedback mechanism might exist which shuts off regeneration
before it is complete, but it is not apparent how this could be dose-dependent.
Another possibility is that radiation damage to the testes limits the ability

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 REGENERATION OF TESTIS STEM CELLS 361

of the seminiferous tubules to support the level of spermatogenesis prior to

irradiation. Finally, we have observed a decline in sperm production even in
unirradiated mice about 1 yr old. This aging phenomenon could limit the re-
generation and produce an apparent plateau.
The relationship between stem cell survival and regeneration, repopulation
of spermatogenic cells, and restoration of fertility has been further investigated.
After doses of 600 and 900 rad, fertility is restored when the number of sperm
reach 15% of control values. A similar estimate of 10% was obtained using
only 200 rad (4). This sterility results from a reduction in egg fertilization
by the reduced number of sperm (4), and not dominant lethal mutations car-
ried by the sperm (8). Thus, restoration of fertility occurs at a fixed value of
repopulation of spermatogenic cells. The time required to reach this level is
a function of stem cell survival and the kinetics of regeneration and repopulation.

ACKNOWLEDGMENTS
We thank W. Larry Wilborn and his staff for the supply and care of the mice used in these
experiments. We also thank Marsha L. Frapart for excellent technical assistance and Dr. Howard
Thames for assisting with the analysis of the growth curves.

RECEIVED: September 24, 1977

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