BIOLOGY OF REPRODUCTION 53, 186-192 (1995)
Stem Cell Kinetics in Rat Testis after Irreversible Injury Induced by 2,5-Hexanedione'
Elizabeth K. Allard, Susan J. Hall, and Kim Boekelheide 2
Departmentof Pathology and LaboratoryMedicine, Brown University, Providence, Rhode Island
ABSTRACT
Stem cells provide a continuous supply of committed progenitor cells for the process of spermatogenesis. In rodents, stem cells have
been identified as single, undifferentiated type A spermatogonia. The rate of stem cell division has not been definitively determined
because of difficulty in locating stem cells among a normal compliment of germ cells. The testicular toxicant 2,5-hexanedione (2,5-HD)
induces irreversible testicular atrophy with only Sertoli cells and spermatogonia remaining after injury. Stem cell kinetics could be assessed
in this toxicant model because of the absence of most mature germ cells. It is also not known if 2,5-HD-exposed rats possess an active
stem spermatogonia population. Charles River CD rats were exposed to 1%2,5-HD in drinking water for 5 wk. At 7 or 35 wk following
toxicant exposure, rats were exposed to bromodeoxycytidine continuously via Alzet mini-pumps for 1-28 days. Serial cross sections of
testis were used to identify single stem spermatogonia and to determine whether the cells were positive or negative for bromodeoxyuridine
incorporation. We obtained a continuous labeling index for stem cells from rats 7 and 35 wk after 2,5-HD exposure and found that stem
cells had a cell cycle time of approximately 8-14 days at both time points after toxicant exposure. In conclusion, we have developed a
method forthe assessment of stem cell kinetics and verified the presence of an actively dividing stem cell population in irreversibly injured
testes.
INTRODUCTION
[1]. Furthermore, A4 cells had a stage-specific peak labeling
index and cell number that preceded a stage-specific increase in the number of A, and In spermatogonia [2].
In contrast, work carried out by Oakberg in mouse [3]
and Huckins in rat [4-7] suggested that there were additional classes of type A spermatogonia with type A cells
functioning as the true stem cell population. In this model,
isolated type A cells (termed type A~) divided about once
per cycle to form pairs (Apr) and eventually chains of precursor cells (Aai), which then differentiated into A, cells.
Stem cell renewal occurred when Apr cells separated by
more than 25 gtm to form new Ais cells.
Germ cell kinetics have been difficult to discern in testes.
Huckins [4, 6, 7] determined the cell cycle time of subclasses
of type A spermatogonia in rat testis. Cells could only be
followed through certain stages of the cycle by "continuous
labeling," which was achieved by repeated injections of 3HTdR. Type A,-A4 spermatogonia had a cell cycle time of 42
h, while type A, cells were found to have a longer cell cycle
time of 114-169 h [4]. Of the undifferentiated type A cells,
types Apr and A had a cell cycle time of 55 h, while type
Ai cells had a division time of greater than 60 h [6]. These
data added further support to the Huckins model, which
has become the more accepted model of stem cell division
Spermatogenesis occurs throughout the lifetime of the
male animal. This process takes place in the testis within
the seminiferous epithelium, which consists of Sertoli cells
and concentric layers of germ cells. As the germ cells mature, they are pushed towards the apical portion of the tubule. Spermatogonia, the most immature germ cells, line the
basement membrane of seminiferous tubules. In the rat,
there are three types of spermatogonia-A, intermediate
(In), and B. Stem cells, a subclass of type A spermatogonia,
provide a continuous supply of committed progenitor cells
that differentiate and divide into mature spermatozoa.
Two opposing models have been proposed for the differentiation process of stem cells in spermatogenesis. Studies have been based primarily on cell morphology, the
stage-specific appearance of cells, and cell kinetics
parameters. Using rat seminiferous tubule whole mounts,
Clermont and coworkers morphologically differentiated
five distinct classes of type A spermatogonia-Ao4 [1]. Type
A0 cells were termed the reserve stem cell population because they divided infrequently [1], had a low mitotic index,
and had a low labeling index after tritiated thymidine (3HTdR) exposure [2]. Therefore, the number of type A0 cells
was insufficient to function as the sole source of spermatogenic precursors. Type A4 spermatogonia were proposed
to be the true stem spermatogonia because they divided at
a specific stage to become both In and Al spermatogonia
[8].
n-Hexane exposure in the rat has been shown to induce
peripheral polyneuropathy and testicular germ cell loss
through the action of the toxic metabolite 2,5-hexanedione
(2,5-HD) [9]. The proposed target of 2,5-HD toxicity in the
testes is the Sertoli cell, the supportive cell of the seminiferous epithelium. Sertoli cell microtubules are altered after
exposure to 2,5-HD [10-13], followed by a decrease in seminiferous tubule fluid secretion and subsequent germ cell
loss [14, 15]. After exposure to 1% 2,5-HD for 5 wk, peak
Accepted March 7, 1995.
Received May 10, 1994.
'This publication was supported by grant numbers R01 E505033 and K04 E500193 from
the National Institute of Environmental Health Sciences, NIH.
2Correspondence: Kim Boekelheide, M.D., Ph.D., Department of Pathology and Laboratory Medicine, Division of Biology and Medicine, Brown University, Box G-B518, Providence, RI 02912. FAX: (401) 863-1971.
186
STEM CELL KINETICS AND RAT TESTIS
germ cell loss occurred 7 wk later [16], suggesting a time
delay between exposure and testicular injury.
Germ cell loss persisted up to 75 wk after 2,5-HD exposure [17], indicating that the injury was irreversible. Some
germ cells, principally type A spermatogonia, remained after 2,5-HD exposure. Based upon the frequency and isolated position of the type A cells, it was suggested that the
majority of the remaining germ cell population were stem
spermatogonia [17]. In addition, it was implied that the type
A cells were dividing because a few In and type B spermatogonia were also present in the atrophic seminiferous
tubules [171. Thus, the 2,5-HD-treated rat provides an intriguing model of testicular injury because of the presence
of persistent spermatogonia in the face of nearly lifelong
testicular atrophy.
This manuscript describes a novel method for the assessment of stem cell kinetics in 2,5-HD-treated rats. The
2,5-HD model of irreversible injury provides a facile system
by which to assess stem cell kinetics. Since the seminiferous
epithelium is devoid of mature germ cells after irreversible
injury, it is relatively easy to identify single germ cells. Moreover, it is of interest to determine whether or not stem spermatogonia are dividing after 2,5-HD exposure. Using the
definition that stem cells are single, undifferentiated type A
spermatogonia, these cells were identified in serial testis
cross sections. The labeling index of stem spermatogonia
was determined using continuous bromodeoxycytidine
(BrdCyd) exposure. BrdCyd is converted to bromodeoxyuridine (BrdUrd) in cells, which is then detected by immunohistochemistry.
MATERIALS AND METHODS
General
All chemicals were purchased from Sigma Chemical
Company (St. Louis, MO) unless mentioned otherwise. Photomicrographs were taken using a Zeiss Axiovert 35 microscope (Carl Zeiss, Inc., Thornwood, NJ) and Kodak Technical Pan 4125 film (Eastman Kodak Co., Rochester, NY).
Animals and Toxicant Exposure
Young adult (150-175 g, 42-46 days old) male Charles
River (Wilmington, MA) CD rats were housed in wire cages
and provided with food (Pro-Lab Rat, Mouse, Hamster chow
No. 3000; Farmer's Exchange, Framingham, MA) and water
ad libitum. The rats were maintained at 22 + 1IC under a
12-h light-dark cycle with 35% to 70% humidity. All rats
were acclimated for 1 wk prior to the onset of 2,5-HD exposure. Rats were exposed to 1% 2,5-HD (Aldrich Chemical
Co., Milwaukee, WI) in drinking water for 5 wk followed
by return to normal drinking water for the recovery period.
187
BrdCyd Exposure
Beginning 7 and 35 wk following 2,5-HD exposure, rats
were exposed to BrdCyd via Alzet minipumps (Alza Corp.,
Palo Alto, CA). The minipumps were used for short (up to
7 days, 1 l/h, Model 2001) and long-term (up to 28 days,
2.5 il/h, Model 2ML4) exposures. Short-term pumps were
filled with 0.3 M BrdCyd, while long-term pumps were filled
with 0.12 M BrdCyd; both nucleotide concentrations were
diluted in sterile phosphate-buffered saline (PBS). For shortterm exposures, the pumps were filled and then incubated
in sterile saline at 37°C for 4 h to begin the continuous release of BrdCyd. Rats were anesthetized with methoxyflurane (Pitman-Moore, Mundelien, IL), and the pumps were
implanted s.c. into the subscapular region. The incisions
were closed with surgical wound clips. Groups of rats were
exposed to BrdCyd 7 wk after 2,5-HD exposure for the following numbers of days: 1 (n = 3), 2 (n = 3), 4 (n = 3),
7 (n = 6), 14 (n = 3), 21 (n = 3), and 28 (n = 3). Other
groups of rats were exposed to BrdCyd 35 wk after 2,5-HD
exposure for the following numbers of days: 7 (n = 4), 14
(n = 4), and 21 (n = 3). Following BrdCyd exposure, the
rats were killed.
Serial Section Preparationand Staining
Both testes were removed, and the tunica albuginea was
punctured several times with a 26-gauge needle (Becton
Dickinson and Co., Franklin Lakes, NJ); the testes were
fixed in 10% neutral buffered formalin and then embedded
in glycol methacrylate (Reichart-Jung). The tissue was serially sectioned, with use of a Histocut microtome (ReichartJung), into one of two combinations (m): 10-10-3-3-310-10 or 10-6--3-3-3--6-10. Sections were placed on
poly-L-lysine-coated slides.
The middle 3-gm section was immunohistochemically
stained to detect BrdUrd. Sections were hydrolyzed in 1 N
HCI at 60°C for 20 min followed by etching in xylene. Next,
the sections were digested with 0.25 mg/ml protease XIV
for 90 min at 37 0C. PBS containing 5% normal goat serum
and 1% BSA was used as a nonspecific blocking agent.
Mouse monoclonal anti-BrdUrd (Dako, Glostrup, Denmark)
was used as a primary antibody; biotintylated goat antimouse IgG (Calbiochem, San Diego, CA) was used as a secondary antibody; and an ABC elite kit (Vector, Burlingame,
CA) was used to label the complex. All antibody incubations
were carried out at 37 0C. A peroxidase substrate consisting
of 0.25 mg/ml diaminobenzidine, 1% imidazole, and 0.033%
H2 02 was used to detect the complex. These sections were
counter-stained with periodic acid-Schiffs (PAS). The remaining serial sections were stained with PAS and hematoxylin (PAS/H).
Data Quantitation
Each serial section was divided into 10 quadrants using
a fine-tipped pen. Up to 2 stem spermatogonia were iden-
188
ALLARD ET AL.
FIG. 1. Testicular atrophy induced by 2,5-HD. Light micrograph of seminiferous
tubule cross sections, stained with PAS/H, from a rat 35 wk after a 5-wk exposure
to 2,5-HD. Seminiferous tubules contained Sertoli cells (arrows) and a few spermatogonia (arrowheads). The interstitial space (I)and seminiferous tubule lumens
(L)are indicated. x 500.
tified per quadrant with a range of 9 to 20 stem cells identified per animal. Labeling indices were determined in each
animal and averaged for each time point, and the standard
error was calculated.
StatisticalAnalysis
All statistical analyses were performed using StatViewSE + Software (Abacus Concepts, Inc., Berkeley, CA).
Cell kinetics parameters were derived, with modifications,
from the continuous labeling curve as described previously
[18].
RESULTS
Method to Assess Stem Cell Kinetics
Rats were exposed to 2,5-HD for 5 wk and then observed
postexposure for 7 or 35 wk. Testes from these rats showed
variable atrophy, as previously described [17], with most
seminiferous tubules containing only Sertoli cells and oc-
casional spermatogonia (Fig. 1). In order to determine the
cell cycle kinetics of stem spermatogonia in atrophic testes,
rats were continuously exposed to BrdCyd. In cells, BrdCyd
is converted into BrdUrd, which is incorporated into newly
synthesized DNA during S phase. Actively dividing cells
could be identified immunohistochemically using an antiBrdUrd antibody.
To determine if stem spermatogonia had incorporated
BrdUrd, testes were serially sectioned into the following
thicknesses or a slight modification thereof (pm): 10-10-33-3-10-10. The middle 3-pm section was immunostained
to detect BrdUrd, while the remaining sections were stained
with PAS/H. Analysis of the serial sections provided the
means to identify stem cells as true isolated type A spermatogonia in all three dimensions.
A flow chart of the decision-making process for the identification of stem spermatogonia is shown in Figure 2. A
potential stem cell was identified in one of the outer 3-pm
sections, which appeared single on the horizontal plane of
the section. The same cell had to be identified in the other
outer 3-pm section so that, by default, the cell was known
to traverse the middle BrdUrd immunolabeled 3-pm section.
Next, the four 10-pm sections were used to determine if the
cell was actually single. The thickness of the serial sections
permitted the identification of any neighboring germ cells
in the vertical plane. If no other germ cells were seen within
25 pm in all directions, the BrdUrd immunostained section
was used to determine if the identified stem spermatogonia
had incorporated BrdUrd (Fig. 3). Using this procedure, up
to 20 stem cells were identified per animal.
Labeling Index 7 and 35 Weeks after 2,5-HD Exposure
Two time points, 7 and 35 wk after 2,5-HD exposure,
were chosen for the assessment of stem cell kinetics. The
first time point was chosen based on the observation that
percent germ cell loss is greatest at 7 wk after a 5-wk exposure to 2,5-HD [16]. A second, later time point was chosen
in order to determine whether or not stem cell kinetics
changed over the course of irreversible atrophy.
Rats were exposed continuously to BrdCyd via Alzet
minipumps for 1 through 28 days. The stem cell labeling
index for each time point was determined using the serial
section method. Stem cells were actively dividing at 7 wk
after 2,5-HD exposure as indicated by the labeling index
curve (Fig. 4). Labeling index increased rapidly up to 7 days
and then slowed to a plateau phase, which is commonly
observed in continuous labeling index curves. The labeling
index curve was the same for stem spermatogonia in rats
35 wk after 2,5-HD exposure.
Multiple regression analysis showed that the data from 7
and 35 wk were not significantly different from each other
and could be pooled for further calculations. A linear regression analysis of the pooled data was performed to test
189
STEM CELL KINETICS AND RAT TESTIS
the fit to a linear model. The resulting quadratic coefficient
was negative and significantly different from zero (p <
0.05), indicating that the curve did not fit a linear model.
Growth fraction (GF), the percentage of cells actively dividing, as well as cell cycle time minus cell synthesis time
(Tc-T s) were calculated using the point where the plateau
phase of the curve begins. This point was estimated using
the intersection of two linearly fitted lines to the rapidly
rising (Days 1-7) and plateau portions (Days 14-28) of the
curve (Fig. 5). The slopes of each portion of the curve (rapidly rising portion, m = 0.049 days-1 ; plateau portion, m
= 0.014 days- 1) were shown to be significantly different
from each other within 85% confidence intervals (rapidly
rising portion, 2.9-6.8; plateau portion, 0.3-2.5). Using the
intersection point yielded a GF of 0.42 and a (Tc-Ts) value
of 8.4 days. Cell cycle time was also estimated using the
following equation: slope of the rapidly rising portion of
the curve = 2[11n(1 + GF)]/Tc [19]. Using this approach, Tc
was calculated to be 14.3 days.
DISCUSSION
Using rat testes serial cross sections, a method was developed for the identification of stem spermatogonia after
testicular injury. Further, continuous BrdCyd exposure was
used to assess the continuous labeling index of stem spermatogonia. This method verified the existence of a viable
stem spermatogonia population in the face of irreversible
testicular atrophy. The implications of this result may be relevant 1) to normal stem cell kinetics, 2) to characterizing the
2,5-HD model, and 3) to understanding human infertility.
After exposure to 2,5-HD, approximately 40 percent of
stem spermatogonia were actively dividing. The remaining
60 percent of the stem cells were either quiescent or slowly
cycling. Huckins provided evidence for the existence of a
slowly cycling stem cell population in normal testis, since a
small subset of As cells did not become labeled with 3 H-TdR
after a 48-h exposure [7] and some A,, cells were observed to
divide only once every 13 days [7]. The data presented here
support the observation of a quiescent or slowly cycling population of stem spermatogonia and suggest that a higher proportion of stem cells are quiescent. From this data, it cannot
be discerned if there is a higher proportion of slowly cycling
cells because of the 2,5-HD-induced injury. In addition, the
current data indicate that stem spermatogonia have a cell
cycle time of approximately 8-14 days after 2,5-HD exposure, compatible with a cell cycle time of greater than 60 h
as demonstrated by Huckins for normal rat testes [6].
Rats exposed to 2,5-HD for 5 wk do not recover up to
75 wk after injury [171, yet a few spermatogonia persist in
the seminiferous tubules. This investigation demonstrated
that a subset of these cells, the stem cells, are actively dividing, and that they have a similar Tc and GF at 7 or 35 wk
after 2,5-HD exposure. However, this finding does not ex-
f
~~~ ~ ~ ~
Find a single undifferentiated type
A spermatogonia ina 3 um PAS/H
section not within 25 urn of any
other spermatogonia, adjacent to
the peritubular cells, having a
visible cytoplasm.
No
I
Check to make sure that the same
cell
I isin the other 3 um PAS/H
section.
Yes
Check to make sure that no other
unairrerenuatea spermatogonia
are located inthe four 10 urn thick
sections within a 25 urn radius
around where the original cell was
located.
1
es
Check the 3 urn BrdU
immunostained section to
determine whether or not the
single cell ispositive or negative
Ifor BrdU immunostaining.
.
._
j
FIG. 2. Flow chart for identification of single stem spermatogonia and determination of BrdUrd uptake. The algorithm is depicted by which germ cells in seminiferous tubule cross sections were first identified, verified as single cells, and then
assessed for BrdUrd uptake. Up to 20 stem cells were assessed per animal.
plain the failure of spermatogenic recovery after 2,5-HD exposure and instead suggests that there is an impediment to
successful spermatogenesis. Since stem cells are dividing,
their progeny must be dying in order for atrophy to persist.
The death of differentiating spermatogonia suggests a failure of normal paracrine factors needed to support this population. The normal compliment of paracrine growth factors, especially those made by Sertoli cells, could be absent
from the seminiferous tubules due to the effect of the toxicant and subsequent injury to Sertoli cells. Spermatocytes
and spermatids have been shown to be important modulators of Sertoli cell function [20], raising the possibility that
the atrophy persists because of the absence of these cells.
190
ALLARD ET AL.
STEM CELL KINETICS AND RAT TESTIS
However, after irradiation or experimental cryptorchidism,
spermatids and spermatocytes are eliminated from the seminiferous epithelium, but full recovery can occur.
Assessment of stem cell kinetics is important to the field
of male infertility because stem spermatogonia are a crucial
target of cytotoxic injury. The number of stem cells remaining after cytotoxic injury may determine the possibility for,
and rate of, recovery [211. If there are no stem spermatogonia remaining after injury, obviously, the azoospermia
will be irreversible [21]. Likewise, it has been suggested that
if there are stem spermatogonia present after injury, recovery will occur at a rate proportional to the percentage of
stem spermatogonia remaining after injury [21]. However,
testicular germ cell recovery is also species-specific [22].
In mice, exposure to chemotherapeutic agents [231 or radiation [24-271 resulted in temporary azoospermia and a
relatively rapid recovery of spermatogenesis. These observations fit well with the model that stem cells remaining
after toxic exposure will lead to recovery. In contrast, in
humans, exposure to chemotherapeutic agents [28, 29] or
radiation [30, 31] have resulted in an extended period of
azoospermia before recovery. Clearly, stem cell recovery in
humans differs from mice because of the extended window
of azoospermia.
In rats, exposure to various toxicants [17, 32, 33] has resulted in irreversible testicular atrophy in the presence of
stem spermatogonia. These observations argue strongly
against the model that the presence of surviving stem cells
predicts recovery in this species. Moreover, the data presented here have shown that there are stem cells in testes
irreversibly injured by 2,5-HD, and that a portion of these
stem cells are actively dividing. It will be of interest to assess
stem cell kinetics in other models of irreversible atrophy in
order to determine if this state of atrophy can result from
more than one toxicant.
In summary, we have developed a method for the assessment of male stem cell kinetics in atrophic rat testes.
This method can be applied to models of testicular atrophy
in order to characterize the irreversibility of injury. In rats
exposed to 2,5-HD, the stem cells had a cell cycle time of
8 through 14 days at both 7 and 35 wk after 2,5-HD exposure. The existence of an active stem cell compartment sug-
FIG. 3. Serial testis cross sections were used to identify a single stem spermatogonium and to assess BrdUrd uptake. These light micrographs depict a portion of
a seminiferous tubule cross section containing an isolated stem spermatogonium
positive for BrdUrd immunostaining. A) 10-,pm section stained with PAS/H; B) 10pm section stained with PAS/H; C)3-pm section stained with PAS/H; D)3-pm section
immunostained to detect BrdUrd and counterstained with PAS; E)3-pm section
stained with PAS/H; F) 10-pm section stained with PAS/H; and G) 10-pm section
stained with PAS/H. The seminiferous tubule lumen (L)and interstitial space (I)are
marked. An arrow identifies the isolated stem spermatogonium, which appears in
C, D,and E. x 589.
100
191
/I I
-- ----
35 weeks
75-
x
a,
'O
Q
C
50-
-J
250
-·
E
g
0
E
I
7
E
I
m
I
14
21
Days
i
I
28
FIG. 4. Labeling index of stem spermatogonia after 2,5-HD exposure. Graph of
labeling index versus time for stem spermatogonia from animals 7wk (solid circles)
and 35 wk (open circles) after 2,5-HD exposure. Each point represents the mean +
SEM of 3-6 individual animals.
1
X
0)
Z
C
Q
_J
0
7
14
21
28
Days
FIG. 5. Estimation of intersect of rapidly rising and plateau portion of the continuous labeling index curve used to determine (T,-T,) and GF. Plot of labeling index
versus time for the mean of the pooled 7- and 35-wk 2,5-HD data showing linear
curve fits for rapidly rising (Days 1-7, open circles) and plateau portion (Days 1428, solid circles) of the curve. (T-T,) and GF are indicated on the graph.
192
ALLARD ET AL.
gests that it may be possible to induce recovery by maintaining the population of committed progenitor cells.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Eric Hall, Dr. John Richburg, and Ruth Rusch for their
time-saving technical assistance, and Drs. R. Allen White (University of Texas, M.D. Anderson
Cancer Center) and Bernard Cole for their expert assistance with data analysis.
REFERENCES
1. Clermont Y,Bustos-Obregon E. Re-examination of spermatogonial renewal in the rat by
means of seminiferous tubules mounted "in toro". AmJ Anat 1968; 122:237-248.
2. Bartmanska J, Clermont Y. Renewal of type A spermatogonia in adult rats. Cell Tissue
Kinet 1983; 16:135-143.
3. Oakberg EF. Spermatogonia stem-cell renewal in the mouse. Anat Rec 1971; 169:515532.
4. Huckins C. Cell cycle properties of differentiating spermatogonia in adult Sprague-Dawley rats. Cell Tissue Kinet 1971; 4:139-154.
5. Huckins C. The spermatogonial stem cell population in adult rats. I. Their morphology,
proliferation and maturation. Anat Rec 1971; 169:533-558.
6. Huckins C. The spermatogonial stem cell population in adult rats. II1.A radioautographic
analysis of their cell cycle properties. Cell Tissue Kinet 1971; 4:313-334.
7. Huckins C. The spermatogonial stem cell population in adult rats. III. Evidence for a
long-cycling population. Cell Tissue Kinet 1971; 4:335-349.
8. Meistrich ML, van Beek MEAB. Spermatogonia stem cells. In: Desjardins C, Ewing LL
(eds.), Cell and Molecular Biology of the Testis. New York: Oxford University Press;
1993:266-295.
9. Krasavage WJ, O'Donoghue JL, DiVincenzo GD, Terhaar CJ. The relative neurotoxicity
of methyl-n-butyl ketone, n-hexane and their metabolites. Toxicol Appl Pharmacol 1980;
52:433-441.
10. Boekelheide K. 2,5-Hexanedione alters microtubule assembly. I. Testicular atrophy, not
nervous system toxicity, correlates with enhanced tubulin polymerization. Toxicol Appl
Pharmacol 1987; 88:370-382.
11. Boekelheide K. Rat testis during 2,5-hexanedione intoxication and recovery. II. Dynamics of pyrrole reactivity, tubulin content, and microtubule assembly. Toxicol Appl Pharmacol 1988; 92:28-33.
12. Sioussat TM, Boekelheide K. Selection of a nucleation-promoting element following
chemical modification of tubulin. Biochemistry 1989; 28:4435-4443.
13. Boekelheide K, Eveleth J, Neely MD, Sioussat TM. Microtubule assembly is altered following covalent modification by the n-hexane metabolite 2,5-hexanedione. In: Witmer
CM (ed.), Biological Reactive Intermediates IV. New York: Plenum Press; 1990: 433-442.
14. Johnson KJ, Hall ES, Boekelheide K. 2,5-Hexanedione exposure alters the rat Sertoli cell
cytoskeleton. I. Microtubules and seminiferous tubule fluid secretion. Toxicol Appl Pharmacol 1991; 111:432-442.
15. Richburg JR, Redenbach DM, Boekelheide K. Seminiferous tubule fluid secretion is a
Sertoli cell microtubule-dependent process inhibited by 2,5-hexanedione exposure. Toxicol Appl Pharmacol 1994; 128:302-309.
16. Boekelheide K. Rat testis during 2,5-hexanedione intoxication and recovery. I. Dose
response and the reversibility of germ cell loss. Toxicol Appl Pharmacol 1988; 92:1827.
17. Boekelheide K, Hall SJ.2,5-Hexanedione exposure in the rat results in long-term testicular atrophy despite the presence of residual spermatogonia. J Androl 1991; 12:18-26.
18. Wright N, Alison M. Methodology in epithelial cell kinetics. In: The Biology of Epithelial
Cell Populations, vol. 1. Oxford: Clarendon Press; 1984: 97-202.
19. White RA, Fallon JF, Savage MP. On the measurement of cytokinetics by continuous
labeling with bromodeoxyuridine with applications to chick wing buds. Cytometry 1992;
13:553-556.
20. Sharpe R. Experimental evidence for Sertoli-germ cell and Sertoli-Leydig cell interaction.
In: Russell L, Griswold M (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993:
391-418.
21. Meistrich ML. Critical components of testicular function and sensitivity to disruption. Biol
Reprod 1986; 34:17-28.
22. Meistrich ML. Effects of chemotherapy and radiotherapy on spermatogenesis. Eur Urol
1993; 23:136-142.
23. van Keulen CJG, de Rooij DG. Spermatogenic clones developing from repopulating stem
cell surviving a high dose of an alkylating agent. Cell Tissue Kinet 1975; 8:543-551.
24. Meistrich ML, Hunter NR, Suzuki N, Trostle PJ, Withers HR. Gradual regeneration of
mouse testicular stem cell after exposure to ionizing radiation. Radiat Res 1978; 74:349362.
25. van den Aardweg GJMJ, de Ruiter-Bootsma AL, Kramer MF, Davids JAG. Growth and
differentiation of spermatogenic colonies in the mouse testis after irradiation with fission
neutrons. Radiat Res 1983; 94:447-463.
0
26. Erickson BH. Survival and renewal of murine stem spermatogonia following Co y radiation. Radiat Res 1981; 86:34-51.
27. Huckins C, Oakberg EF. Morphological and quantitative analysis of spermatogonia in
mouse testes using whole mouse seminiferous tubules. Anat Rec 1978; 192:529-542.
28. Meistrich ML, Wilson G, Brown BW, de Cuhna MF, Lipshultz LI. Impact of cyclophosphamide on long-term reduction in sperm count in men treated with combination chemotherapy for Ewing and soft tissue sarcomas. Cancer 1992; 70:2703-2712.
29. Schilsky RL, Lewis BJ, Sherins RJ,Young RC. Gonadal dysfunction in patients receiving
chemotherapy for cancer. Ann Intern Med 1980; 93:109-114.
30. Rowley MJ, Leach DR, Warner GA, Heller CG. Effect of graded doses of ionizing radiation
on the human testis. Radiat Res 1974; 59:665-678.
31. Meistrich ML, van Beek MEAB. Radiation sensitivity of the human testis. Adv Radiat Biol
1990; 14:227-268.
32. Ku WW, Chapin RE, Wine RN, Gladen BC. Testicular toxicity of boric acid (BA): relationship of dose to lesion development and recovery in the F344 rat. Reprod Toxicol
1993; 7:305-319.
33. Boekelheide K. Sertoli cell toxicants. In: Russell LD, Griswold MD (eds.), The Sertoli
Cell. Clearwater, FL: Cache River Press; 1993: 551-575.