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UNIT 16.4Histopathology of the Male ReproductiveSystem II: Interpretation
Toxicants can affect the male reproductive
system at a broad range of target sites including
the testis, epididymis, secondary sex organs,
and the neuroendocrine/CNS regulatory sys-
tem. No single endpoint can adequately reflect
changes at such a diversity of sites, but it hasbeen repeatedly demonstrated that his-
topathologic evaluation represents the most
sensitive and reliable indicator of toxicologic
disturbance in the tract as a whole (Linder et
al., 1992; Ulbrich and Palmer, 1995). This is
only true if the appropriate tissues have been
taken and have been fixed and sampled in such
a way that subtle or localized changes are ade-
quately preserved and presented to the patholo-
gist for examination. These preparative proce-
dures are dealt with in UNIT 16.3. Even if material
has been appropriately fixed and sampled, it is
incumbent on the pathologist to have an ade-
quate understanding of the organization and
dynamics of spermatogenesis in the species
under investigation. Without this, lesions will
be missed or the significance of findings will
be misinterpreted. Although it is beyond the
scope of this unit to provide this knowledge, an
attempt is made to illustrate why and when such
knowledge is essential.
Once identified, interpretation of the signifi-
cance of the findings to overall reproductive
function and their implications for functional
or morphological changes in the rest of thereproductive tract relies on a basic knowledge
of reproductive physiology. Some examples of
common chemically induced findings in the
reproductive tract, their possible etiology, and
their likely functional significance are pro-
vided. The pattern of morphological change as
well as the specificity and severity of the find-
ings are significantly influenced by the duration
of dosing, such that the approach to examina-
tion is different in a short-term versus a long-
term study. This unit includes a section provid-
ing practical guidance on how to evaluate tes-
ticular and epididymal sections for evidence ofa chemically induced effect, including what to
look for and how hard to look for it. This
involves using knowledge of staging to identify
when cells are missing or when cells are inap-
propriately present.
Terminology of findings and how to grade
severity is a unique problem in the testis;
whether to grade on the basis of number of germ
cells affected, number of tubules affected, or
both; whether to use broad terminology such as
tubular atrophy and tubular degeneration, or to
use specific terminology that differentiates be-
tween effects on specific types of germ cells
and their stage localization. The decision isstrongly influenced by the type of study and the
specificity of the present findings.
As with the toxicological evaluation of any
tissue, the species, age, and history of sponta-
neous pathology need to be taken into account.
These factors are particularly important with
respect to the testis because, in some species,
such as dog, the relatively high incidence of
spontaneous degenerative lesions, confounded
by the small group size of animals used can
provide difficult data for interpretation. In ad-
dition, the use of immature or borderline mature
animals, which is the case for some regulatory
studies, can mask or be mistaken for toxicologi-
cally induced lesions. It is important that these
limitations are realized. Most of the discussion
in this unit relates to the rat because this is the
most commonly used species in toxicological
research. It is also the species for which most
information is available. Other species are spe-
cifically mentioned where appropriate.
ESSENTIAL KNOWLEDGE FOREVALUATION AND
INTERPRETATION OFTESTICULAR PATHOLOGY
Spermatogenesis and theSpermatogenic Cycle
The process and regulation of spermato-
genesis is essentially similar in all mammalian
species. Stem cell spermatogonia proliferate by
mitosis and produce a population of differenti-
ating or committed spermatogonia. These in
turn divide and produce spermatocytes, which
undergo DNA replication and divide by meiosis
to produce haploid spermatids. A complex se-
ries of morphological transformations of thesimple round spermatid finally produces a dif-
ferentiated sperm, which is released into the
tubule lumen and transported on into the
epididymis. In the rat, the whole process takes
56 days with spermatogonial divisions taking
2 weeks, spermatocyte meiosis lasting >3
weeks, and spermatid development also requir-
ing 3 weeks. The arrangement of the different
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populations of germ cells within the seminifer-
ous tubule is also similar between species, with
Sertoli cells providing a supporting structural
framework for discrete layers of the different
germ cell types. Spermatogonia are always at
the base of the tubule and progressively more
mature germ cells are found in layers moving
toward the lumen. In all species, 3 or 4 genera-
tions of germ cells are developing within a
tubule at any given time. The development ofeach of these generations occurs in synchrony
with each other, giving rise to specific and
predictable cellular associations (Fig. 16.4.1).
The complete sequence of cellular associations
is referred to as the cycle of spermatogenesis
while the individual cell associations form the
stages of the cycle. Each stage is therefore
defined by its germ cell complement and con-
sequently, identifying the stage defines what
cells should be in that tubule (and what cells
are missing). In order for a pathologist to detect
subtle changes in germ cell loss or, in the case
of spermatid retention, the inappropriate pres-ence of a population of germ cells, a thorough
understanding of the cellular makeup of the
spermatogenic cycle is essential.
Species-Specific Variations in theOrganization of Spermatogenesis
Although the fundamentals of the sperma-
togenic cycle are similar between species, there
are certain details that vary. These can have a
significant impact on histopathologic evalu-
ation.
Number of stagesThe number of stages and their cellular
makeup varies between species and depends on
the morphological criteria used by the classifi-
cation system. It is important that the patholo-
gist is familiar with the germ cell development
and the stage map of the species under investi-
gation. A highly recommended text that ex-
plains in detail how to stage tubules in a number
of common species and provides stage maps
for each species is provided by Russell et al.
(1990).
Duration
The duration of spermatogenesis (the time
taken for a spermatogonium to develop into
sperm) and the duration of the spermatogenic
cycle (the time taken to complete a cycle of cell
associations) varies between species. This in-
formation is important so that the pathologist
can predict how long it should take for any
particular cell to reach a later cell type (e.g., if
a toxicant affects leptotene spermatocytes, how
long will it take before the animal becomes
infertile?). Alternatively, if a particular cell type
is missing at some defined period after dosing,
knowing the dynamics of the spermatogenic
cycle will allow extrapolation as to what stage
of development that cell was in when dosing
began. Software programs have also been de-
veloped to calculate this type of information
(Hess and Chen, 1992).
Cell associations
The organization of cell associations
along the length of the tubule is linear for
most mammalian species, including most
species of monkey used in toxicological stud-
ies. This means that a tubular cross section
normally contains only one cell association
and that the adjoining length of tubule (which
is often the adjacent tubule in a cross section
of testis) will generally contain the consecu-
tive stage. This is not the case in humans
where cell associations are arranged in a heli-cal pattern resulting in a mosaic of cell asso-
ciations in a single cross section. In dogs,
although only one stage is present in a cross
section, adjoining lengths of tubule do not
necessarily contain consecutive stages.
Disturbances in SpermatogenesisAlmost regardless of the cellular target of
toxicity within the reproductive system, the
most common morphological consequence of
injury is a disturbance in spermatogenesis. This
is because spermatogenesis is dependent on, or
sensitive to, functional perturbations in mostother parts of the reproductive tract. Spermato-
genic disruption may reflect a direct effect on
the seminiferous epithelium, affecting either
the Sertoli cell or any one of the germ cell
populations, or it may occur as a secondary
response to altered hormone levels, altered vas-
cular supply or altered fluid balance, either
within the testis or within the epididymis. It is
therefore extremely important that distur-
bances in spermatogenesis are detected. The
pattern of disturbance can be very specific and
diagnostic of the mechanism of toxicity, but
generally, this is only seen during the early
development of the lesion. With longer periods
of dosing, the development of maturation de-
pletion (whereby death of a specific precursor
germ cell causes the progressive loss of its
descendant generations), reduces the specific-
ity of the pattern of spermatogenic disturbance
as the tubules become depleted of more and
more germ cells.
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1
15
15 19 15
1
EP
In
EP
In
8
19
MP
PL
14
D
Z
A
11
LP
LA
A
Z
D8
1
1411
LP
EP
In
I VIII XI
XI XIV
I VIII
XIV I
PL
MPL
A
Figure 16.4.1 Stages of the spermatogenic cycle of the rat. Cell associations for 4 of the 14 stages
of the spermatogenic cycle of the rat (stages I, VIII, XI, XIV). Spermatogonia: A, type A; In,
intermediate;Spermatocytes: PL, preleptotene; L, leptotene; Z, zygotene; EP, early pachytene; MP,
mid pachytene; LP, late pachytene; D, dividing. Spermatids:1, 8, 11, 14, 15, and 19 indicates steps
1 to 19 of spermatid development. The tubular cross sections (stages I, VIII, XI, and XIV) show the
arrangement of cells within the seminiferous epithelium. The columns of cells at the base of the
figure show the maturation of the cells during one spermatogenic cycle. Each generation of cells
develops sequentially. During stage VIII, the mature step 19 spermatids are shed into the lumen(arrows) while a new generation develops from stem cell spermatogonia. As spermatocytes
undergo meiotic division (D) in stage XIV, they produce step 1 spermatids and the cell association
returns to stage I to begin another cycle. (Reproduced from Creasy, 1997, with permission.)
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Regulatory Guidelines and the Role ofStaging in HistopathologicExamination of the Testis
Recently revised regulatory guidelines have
placed increased emphasis on the importance
of histopathology for detecting toxicological
effects in the male reproductive system. Rec-ommendations have been made, not only for
fixation and staining procedures but also for the
microscopic examination of tissues, providing
examples of findings that should be recorded
(Table 16.4.1). During the drafting of these
guidelines, there was much discussion relating
to the subject of staging of testes, and al-
though there is no mention of staging in the
final issued guidelines, the issue has become
surrounded by confusion. The ability to recog-
nize stages of the spermatogenic cycle is im-
portant in order for the pathologist to recognize
when cells are missing or are inappropriately
present. Due to the lack of understanding of this
concept, there has been a move to expect the
pathologist to produce a quantitative assess-
ment of stagese.g., a frequency distribution
of tubules for individual stages of the sperma-
togenic cycle. While this may be useful infor-
mation in an investigative study to determine
whether the dynamics of the spermatogenic
cycle have been disturbed (Hess, 1990), it is
inappropriate to carry out in a regulatory study,
which is designed as a screening study to detect
effects on spermatogenesis. Knowledge ofstaging should be used in a qualitative way to
evaluate the normality of the cellular makeup
of the seminiferous tubules. In other words, the
testis should be examined with an under-
standing of the normal progression of the stages
of the spermatogenic cycle. This approach is
explained below. For a more detailed discus-
sion of this issue see Creasy (1997) and Chapin
and Conner (1999).
COMMON TOXICOLOGICALLYINDUCED FINDINGS AND THEIRPOSSIBLE SIGNIFICANCE
As with any tissue, the cellular response to
injury is limited and at times, nonspecific.
However, certain aspects of the early patho-
genesis of toxicologically induced lesions inthe testis and accessory tissues can provide
important information on the mechanism of
injury. Additional information can be found in
Nolte et al. (1995), Creasy (2001), and Creasy
and Foster (2001).
Testes
Germ cell degeneration/multinucleate
aggregates
Whether spontaneous or induced, death of
germ cells appears to occur predominantly
through apoptosis, a process that is closely
regulated by the Sertoli cell (Lee et al., 1997,
1999). This is particularly true for spermatogo-
nia, which may be seen apoptosing in occa-
sional stage XII tubules. However, many of the
dying cells do not have the classic morphologi-
cal appearance of apoptotic cells. Dying sper-
matocytes generally develop cytoplasmic eos-
inophilia and nuclear pyknosis while round
spermatids show chromatin margination. If cell
death progresses rapidly, then the apoptotic cell
is rapidly phagocytized by the surrounding Ser-
toli cell cytoplasm and all evidence of cell deathis rapidly removed. Cell death and phagocy-
tosis of the remains can be complete within 24
hr, so if the process is not examined during this
time span, the only evidence of cell death will
be an absence of the cell (cell depletion). If the
degenerative process is slow, then adjacent
germ cells belonging to the same cohort, may
form a multinucleate syncitium (symplast,
multinucleate giant cells) probably due to the
Table 16.4.1 Specific EPA and OECD Recommendations for Histopathological Examination of the Testesand Epididymides in Studies to Detect Effects on Reproduction and Fertility
Testis Epididymis
Detailed histopathological examination of testes
should be conducted in order to identify
treatment-related effects such as:
Examination of the intact epididymis should include
the caput, corpus, and cauda. The epididymis should
be evaluated for:
Retained spermatids Leukocyte infiltration
Missing germ cell layers or types Sperm granulomas
Multinucleate giant cells Change in prevalence of cell types
Sloughing of spermatogenic cells into the lumen Absence of clear cells in the cauda epithelium
Aberrent cell types in the lumen
Phagocytosis of sperm
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breakdown of the cytoskeletal fibers supporting
the interconnecting cytoplasmic bridges. Mult-
inucleate aggregates are less readily phago-
cytized by Sertoli cells and are present for
longer periods and therefore more frequently
seen. They are most often composed of round
spermatids, but can also be formed by fusion
of neighboring spermatocytes or elongating
spermatids.
Germ cell depletion
This is the most common sequel to sperma-togenic disturbance and is generally a conse-
quence of germ cell death rather than exfolia-
tion. It may be seen as a generalized or partial
depletion of the germ cells or it may only affect
a specific cell type (e.g., spermatogonia).
Sometimes a specific cell type within specific
stages may be affected (e.g., pachytene sper-
matocytes in stages XII and XIII). Once the cell
has been phagocytized, the only way of recog-
nizing the lesion is by the abnormal cellular
association of individual stages of the sperma-
togenic cycle and the progressive development
of maturation depletion with time (Fig. 16.4.2).
The appearance of the testis, in terms of what
cells are missing, will depend largely on how
severe the initial effect was and how long after
dosing the testis is examined.
Instead of a specific cell type being killed,
a focal cohort of cells within a tubule may be
affected and result in a focal blow out of the
epithelium. This may be due to an effect on afew adjacent spermatogonia, which then fail to
produce their cohort of spermatocytes and sper-
matids, or on one or two Sertoli cells, which
are then unable to support spermatogenesis.
Partial or generalized germ cell depletion
may affect only a small number of tubular
profiles or a large proportion of the tubules.
When only a few scattered tubules are affected,
it is not possible to determine whether they
day of dosing
ES
Sg
ES
Sg
RSES
Sg
Sg
RSPS
1 week
2 weeks 4 weeks
Figure 16.4.2 Development of maturation depletion following daily dosing with a spermatocytetoxicant. Time-dependent progression of maturation depletion following cell-specific damage to
pachytene spermatocytes (PS). If the tubule is examined on the day of dosing, spermatocyte
degeneration and necrosis will be seen (top left). Phagocytosis of the necrotic cells by Sertoli cells
results in their rapid disappearance and because dosing continues, newly formed pachytene
spermatocytes will also be killed. Examination of the same stage tubule after 1 week of dosing (top
right) will reveal an absence of pachytene spermatocytes. After 2 weeks of dosing (one spermato-
genic cycle duration) pachytene spermatocytes will still be missing but round spermatids will also
be absent because their precursor cells were destroyed in the previous cycle (bottom left). Similarly,
after 4 weeks of dosing (bottom right), pachytene spermatocytes, round spermatids, and elongated
spermatids will be absent, leaving only spermatogonia. This progressive loss of subsequent germ
cells following injury to a previous cell type is termed maturation depletion. ES, elongated sperma-
tids; RS, round spermatids; PS, pachytene spermatocytes; Sg, spermatogonia. (Reproduced from
Creasy, 1997, with permission.)
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represent multiple convolutions of the same
tubule or focal segments of multiple affected
tubules. Prolonged dosing with a number of
testicular toxicants may cause generalized
germ cell depletion, affecting a large proportion
of the tubules. It generally represents an ad-
vanced or end-stage lesion, and in order to
elucidate the primary target cell, a time course
study needs to be carried out.
It is not possible to say whether spermato-genic depletion is or is not reversible without
carrying out an appropriate study. If spermato-
gonia are still present, then the lesion is poten-
tially reversible but if the Sertoli cells are func-
tionally compromised, spermatogenesis may
not be supportable. The chronic effects of 2,5-
hexanedione on the rat testis exemplify this.
Although spermatogonia remain and are seen
to divide, spermatogenesis does not recover.
This is thought to be due to the inhibition of a
critical Sertoli cell factor (Blanchard et al.,
1998). Conversely, spermatogonia may be sig-
nificantly depleted, but if the Sertoli cells arefunctionally intact and sufficient time is al-
lowed for stem cell renewal and repopulation
(and this may require several spermatogenic
periods), substantial recovery may be seen
(Meistrich, 1986).
Germ cell exfoliation
Loss of adhesion between Sertoli cell and
germ cell, or shearing of Sertoli cell cytoplasm
(as seen with cytoskeletal disrupting agents)
will result in exfoliation of germ cells into the
lumen of the seminiferous tubule and sub-
sequent transport of the cells to the rete testisand the epididymis. The exfoliated cells may
appear morphologically normal but are rapidly
removed from the testis. Once the cells have
been removed, cell depletion is the only recog-
nizable finding. Lumenal germ cells may also
be present as a result of handling trauma at
necropsy. Care must be taken to distinguish
between real and artifactual exfoliation (Foley,
2001). Abnormal residual bodies shed into the
lumen can sometimes be mistaken for exfoli-
ated germ cells. These generally occur as a
result of disturbances in spermiation (see be-
low).
Tubular vacuolation
Vacuolation within or between Sertoli cells
is a common early sign of Sertoli cell damage.
The vacuoles may be solitary and situated
amongst the germ cells at varying depths
throughout the epithelium. It is generally not
possible by light microscopy to determine
whether they are intra- or extra-cellular. In other
cases, intracellular microvacuolation or swel-
ling may be seen affecting the basal area of the
Sertoli cell cytoplasm and causing germ cell
displacement and disorganization. Such find-
ings are suggestive of disturbances within the
Sertoli cell and may represent alterations in the
smooth endoplasmic reticulum or in fluid ho-
meostasis. Vacuolation may also be seen in
end-stage lesions, associated with extensivegerm cell loss. In this situation, it should not be
regarded as a primary effect on the Sertoli cell.
Occasional solitary vacuoles are sometimes
seen in tubules from normal testes but these are
generally few in number. Vacuoles in the basal
compartment of the tubule, surrounding sper-
matogonia are generally fixation-induced arti-
facts due to osmotic shrinkage.
Tubular contraction
Reduction in the overall diameter of the
seminiferous tubule will occur as a result of
germ cell depletion and/or as a result of reducedsecretion of seminiferous tubule fluid. Seminif-
erous tubule fluid is secreted by the Sertoli cell
and maintains the lumenal size, which varies
with the stage of spermatogenesis. This is an
androgen-dependent function of the Sertoli cell
and will be affected by altered testosterone
secretion. Another major regulatory factor for
fluid secretion is the presence of elongating and
elongated spermatids. Therefore, if these cells
are depleted, fluid production and conse-
quently lumenal size are decreased. Germ cell
loss and decreased fluid will have a significant
effect on testis weight.
Tubular dilatation
Dilatation of the tubular lumen will occur as
a result of increased lumenal fluid volume. This
can occur through increased secretion by the
Sertoli cell or decreased expulsion of fluid from
the tubule, which is thought to be a function of
the contractile peritubular cells. Also decreased
resorption of fluid by the epithelial cells of the
rete and efferent ducts or obstruction of the
outflow (e.g., a sperm granuloma) can cause
increased tubular fluid. The increased fluid vol-
ume will generally be reflected by an increased
weight of the testis unless there is an accompa-
nying significant cell loss. The pathological
consequences of the finding depend on the
severity and duration of the effect. Prolonged
increased pressure on the seminiferous epithe-
lium will generally result in pressure atrophy
of varying degrees and may also lead to inspis-
sated sperm and granulomatous inflammation.
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Spermatid retention
This is a subtle but relatively common find-
ing that may be caused by a number of chemi-
cals as well as by hormonal disturbance. It is
characterized by the retention of step 19 sper-
matids (which should be released during stage
VIII) through stages VIII to XII. The position
of the retained spermatids varies with different
chemicals. In some cases, e.g., boric acid
(Chapin and Ku, 1994), the retained spermatidsremain in a predominantly lumenal position
through stages VIII to XI and are then pulled
down into the basal cytoplasm of stage XII
tubules where they are phagocytized. With
other chemicals the step 19 spermatids are
rapidly pulled down into the basal cytoplasm
and phagocytized during stages VIII to XI,
leaving very few in a lumenal position. The
formation and behavior of the residual bodies
is often also disturbed with residual bodies of
abnormal shape and size being seen in the
tubular or epididymal lumen. Descent and
phagocytosis of residual bodies normally occursduring stages IX to XI but in cases of spermatid
retention this may be delayed into stage XII. The
pathological significance of spermatid retention
can be varied. It is often associated with abnor-
mal sperm parameters (number, motility, or
morphology) and it may be associated with
alterations in fertility parameters. If homogeni-
zation resistant spermatids are measured, the
retained spermatids should be reflected by an
increase in this parameter. However, identifica-
tion by histopathology is a much more sensitive
endpoint since it can detect very small numbers
of phagocytized spermatids.
Tubular necrosis
While germ-cell necrosis proceeds by apop-
tosis, tubular necrosis is characterized by co-
agulative (oncotic) necrosis of Sertoli and germ
cells. Sertoli cells are normally highly resistant
to cell death even though they may be very
sensitive to functional perturbations. Conse-
quently, they are often the only cell left lining
severely damaged tubules (Sertoli cellonly
tubules). Ischemia is one of the few situations
where they are killed. The effects of this can beseen with cadmium toxicity, which damages
the testicular capillary endothelium. It can also
be seen following administration of vasoactive
compounds such as serotonin. Necrosis and
loss of the Sertoli cells from tubules is the major
characteristic of the lesion, and this is associ-
ated with gross disorganization and necrosis of
the germ cells as well as stasis of sperm in the
tubular lumen. Due to the loss of the Sertoli cell
blood-tubule barrier, the changes are also ac-
companied by an inflammatory infiltrate,
which is an otherwise rare accompaniment to
toxic injury. Tubular necrosis is a serious irre-
versible lesion because Sertoli cells are unable
to proliferate and the affected tubules are likely
to involute and be replaced by scar tissue.
Dilated rete
Both ends of the seminiferous tubules emptyinto the rete. Most of the tubule fluid is reab-
sorbed in the epithelium of the rete and efferent
ducts. If there is an obstruction in the efferent
ducts or in the epididymis, the fluid back-pres-
sure will cause the rete to dilate and if the
obstruction is severe, the back pressure will
progressively dilate the seminiferous tubules.
The tubules in the area of the rete also appear
to be a preferential location for some testicular
toxicants, but this should not be confused with
the transitional tubuli rectii that join the
seminiferous tubules to the rete and can be
mistaken for depleted tubules.
Leydig cell atrophy/hypertrophy/
hyperplasia/adenoma
Testosterone secretion is the major function
of the Leydig cell and the abundance of smooth
endoplasmic reticulum in the cell reflects this
activity. Increased stimulation by luteinizing
hormone results in functional hypertrophy and
hyperplasia. With prolonged gonadotropin
stimulation in the rat, Leydig cell hyperplasia
will usually progress to adenoma formation.
Many classes of compounds with diverse
chemical structures have been shown to pro-duce this effect in the rat but the significance to
man is considered limited (Clegg et al., 1997).
Decreased secretion of testosterone, whether
through inhibition of biosynthesis or decreased
gonadotropin stimulation, will lead to atrophic
changes in the Leydig cell.
Recognition of atrophy, hypertrophy, and
hyperplasia on a qualitative basis is not easy
unless the changes are marked. Contraction of
tubules due to cell loss will result in an apparent
increase in the volume of the interstitial space.
This may or may not be contributed to by a real
increase in size and number of Leydig cells, but
quantitative analysis may be necessary to sepa-
rate real from apparent effects.
Epididymis
Lumenal germ cells/debris
Cells and residual bodies exfoliated from the
testis will be transported into the epididymis.
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This can serve as useful confirmatory evidence
for changes seen in the testis. It can also alert
the pathologist to changes that may have been
overlooked in the testis. Occasional exfoliated
germ cells are sometimes seen in normal ani-
mals, and in immature and peripubertal animals
this number is significantly increased. Abnor-
mal residual bodies may also be detected in the
epididymis as a consequence of disturbed sper-
miation in the testis. The presence or absenceof germ cells in the luminal contents can also
aid the pathologist in evaluating whether appar-
ently exfoliated germ cells in the lumina of
seminiferous tubules are real effects or artifacts
of trimming; such artifacts will not be present
in epididymal lumena.
Epithelial vacuolation
Microvacuolation of the epididymal epithe-
lium can be seen as a specific chemically in-
duced finding. Macrovacuolation and cribri-
form change (infolding of the epithelium
within itself) is often seen accompanying con-traction of the atrophic aspermic epididymis.
This may represent a normal mechanism of
surface area reduction but has also been re-
ported as a toxicologic change (Foley, 2001).
Epithelial vacuoles are also sometimes seen as
a normal finding in some species at the junction
of the corpus and cauda. Since fluid absorption
and secretion are both major functions of the
epididymal epithelium, vacuolation is a likely
sequel to disturbance of either function.
Epithelial inflammation and sperm
granulomaThe antigenically foreign sperm in the
epididymal lumen and in the seminiferous tu-
bule are in an immunologically protected envi-
ronment. The protection is afforded by the
lumenal tight junctions between epithelial cells
in the excurrent ducts and by the basal occlusive
junctions between Sertoli cells in the testis. If
these barriers are damaged, then an inflamma-
tory response against the sperm develops and
generally progresses to form a sperm granu-
loma. This is a chronic, progressive lesion and
in the coiled epididymal duct has the added
complication of causing obstruction to the pas-
sage of sperm. Furthermore, the oxidative free
radicals produced by inflammatory cells in con-
tact with sperm can lead to genotoxic damage,
which may have implications for male-medi-
ated congenital defects and post-implantation
losses (Chellman et al., 1986). The efferent
ducts, which join the caput epididymis with the
rete testis, are a particular site for damage.
Certain chemicals, e.g., carbamates, cause
sperm stasis and inflammation of these ducts
resulting in partial or complete obstruction to
sperm transit and secondary dilatation of
seminiferous tubules. The mechanism may be
through increased fluid absorption resulting in
sperm stasis and inflammation (Hess, 1998).
The efferent ducts are also a frequent site for
the occurrence of spontaneous sperm granu-
lomas. In species such as the dog, they oftenform blind ending tubules that contain inspis-
sated sperm, which can develop inflammation
and progress to sperm granulomas.
Ductular dilatation/interstitial edema
This can occur as a result of fluid imbalance
mediated through the vasculature or inhibited
fluid reabsorption by the epithelial cells. In-
flammatory infiltrate and sperm granulomas
are a frequent consequence.
Prostate/Seminal Vesicles
Acinar atrophy
Secretory activity by the prostate and semi-
nal vesicles is a sensitive, androgen-dependent
function. Decreased circulating testosterone
levels, or interference with androgen receptors
in these two tissues will result in reduced se-
cretion leading to atrophic changes. These may
be detected by organ weight changes as well as
by microscopic changes
PRACTICAL APPROACH FOREXAMINATION OF THE TESTIS
AND EPIDIDYMIS FORTOXICOLOGICAL EFFECTS
The approach used is influenced by the du-
ration of the study. Cell- and stage-specific
disturbances in spermatogenesis are usually
only seen in short duration studies of
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decreased sperm and fluid content. An in-
creased weight in either tissue generally re-
flects increased fluid content, which is either
interstitial or tubule fluid. Increased interstitial
fluid will be seen as edema and suggests a
vascular-mediated lesion while increased tu-
bule fluid will be reflected by dilated tubular or
ductal lumen size. There are various possible
reasons for this (see Common Toxicologically
Induced Findings and Their Possible Signifi-cance).
2. If testicular homogenization-resistant
spermatids (HRS) and/or epididymal sperm
have been counted, review these data. A de-
crease in HRS indicates a reduced number of
elongated spermatids. This could be due to a
direct effect on these cells or due to maturation
depletion following effects on an earlier cell
type (the answer should be apparent by his-
topathology). HRS data are particularly useful
for confirming or alerting the pathologist to
slight reductions in sperm production which
may not be immediately obvious by qualitativehistopathology. A reduction in HRS should be
reflected by a decrease in epididymal sperm
count, but only if sufficient time has elapsed
between release of the reduced numbers of
sperm from the testis and their arrival in the
cauda epididymis or vas (2 weeks). If caudal
sperm are decreased in the absence of any effect
on HRS, a direct effect on epididymal sperm or
on sperm transit time is likely. An increase in
HRS numbers suggests retention of elongated
spermatids in the testis and should be confir-
mable by pathology.
3. Examine the testis at low power. Lookfor obvious depletion or disorganization of
germ cells within the epithelium or exfoliation
of germ cells into the lumen. Look for occa-
sional atrophic tubules (shrunken tubules lined
only by Sertoli cells) and determine whether
the number is increased over control levels.
Look for an increase in the number of vacuoles
within the tubular epithelium. Look for tubular
dilatation or tubular contraction.
4. At higher power, randomly scan tubules
and check that the appropriate cell layers are
present in their approximately normal num-
bersi.e., that stages I to VIII contain a layer
of spermatogonia, a layer of pachytene sperma-
tocytes, and several layers of round spermatids
interspersed with elongated spermatids. Stages
IX to XIV should contain a layer of pre-
pachytene spermatocytes, several layers of late
pachytene or dividing (stage XIV) spermato-
cytes, and several layers of elongating sperma-
tids. This does not require individual identifi-
cation of stages, just a familiarization with the
cellular make up of the two halves of the sper-
matogenic cycle. It will allow for the identifi-
cation of when a cell population is missing. This
is particularly important in studies of28-days
duration, where maturation depletion may not
have progressed to produce an obvious lesion.
If, for example, in a 28-day study in the rat,
spermatogonia are killed, the most obvious
finding in the terminal kill animals will be aloss of prepachytene spermatocytes in stages
IX to XIV. Otherwise the testes may appear
superficially normal. Check Leydig cells for
relative number and evidence of hypertrophy,
atrophy, or vacuolation. However, bear in mind
that the morphological appearance of the Ley-
dig cell is not a very sensitive indicator of
function.
5. Identify a few tubules between stages IX
to XI (there will be relatively few) and examine
these at high power for evidence of sperm
retention. There should be only one population
of elongating spermatids at the lumen. Alsoexamine a few stage XII tubules for evidence
of sperm head phagocytosis in the basal Sertoli
cell cytoplasm. These may occasionally be seen
in normal stage XII tubules but rarely exceed
more than 2 to 3 per tubule cross section.
Check a few stage VII tubules to ensure ap-
proximately normal numbers of step 19 (ma-
ture) sperm at the lumen and a normal appear-
ing layer of residual bodies at the lumen. Also
check stage VII tubules for any evidence of
degenerate pachytene spermatocytes and round
spermatids. Decreased testosterone levels will
lead to an increased rate of degeneration inthese cells in stages VII. The number of cells
affected at any one time can be small (2 to 3
cells per tubule cross-section) but this stage-
specific lesion is characteristic for and the most
sensitive marker of decreased testosterone lev-
els in the testis. Effects will become progres-
sively more obvious with time, due to matura-
tion depletion and direct effects on the elongat-
ing spermatids.
6. Examine the epididymis at low power for
evidence of reduced sperm content, sperm
granulomas, interstitial inflammation, or
edema.
7. At higher power, examine ductal con-
tents for evidence of testicular germ cells or
residual bodies (increased above control lev-
els). Examine epididymal epithelium for pres-
ence of vacuoles, inflammation, or altered cel-
lular characteristics or complement compared
with controls. If any alterations in sperm or
cellular content of the epididymis is seen, go
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Table 16.4.2 Rapid Reference Guide to Evaluation and Interpretation of Weight Changes and HistopathologicFindings in the Reproductive Tract
Finding/observation What to look for Possible causes
Increased testis weight Seminiferous tubular lumen dilatation Increased seminiferous tubule fluid that may
be due to obstruction of outflow, decreased
emptying of tubules, decreased resorption of
fluid by rete/epididymis, increased production
by Sertoli cell
Increased interstitial fluid (interstitialedema)
Altered hemodynamics, injury to vascularendothelium, reduced lymphatic drainage
Decreased testis weight Germ cell depletion Disruption of spermatogenesis through effects
on germ cells, Sertoli cells, hormonal
disturbance, or blood supply
Seminiferous tubule lumen contraction Decreased production of seminiferous tubule
fluid that may result from loss of elongating
spermatids and/or decreased testosterone
production
Increased epididymal weight Increased interstitial fluid Altered hemodynamics, injury to vascular
endothelium, reduced lymphatic drainage
Increased ductular fluid Decreased resorption of fluid by rete, efferent
ducts, and caput epitheliumSperm granulomas May be spontaneous but may be induced by
any agent causing inflammation or damage to
the epididymal epithelial lining
Decreased epididymal
weight
Reduced sperm content and contraction
of ductular lumen size
Disruption of spermatogenesis resulting in
reduced sperm production or release from the
testis
Decreased weight of
seminal vesicles and/or
prostate
Atrophic changes in the secretory
epithelium and decreased secretory
product
Reduced levels of circulating testosterone,
inhibition of 5- reductase, or disruption of
androgen receptor binding
Germ cell loss Is a specific cell type(s) affected? Does
the germ cell loss fit into a pattern of
maturation depletion or is it
nonspecific? Is it focal or diffuse, is itpartial or generalized?
The pattern of the germ cell loss will provide
valuable clues as to the likely mechanism of
injury, but this will also be very much
influenced by the duration of the study (seemain text for detail). The pathogenesis of
germ cell loss is best investigated in a short
time-course study.
Loss of elongate and
elongating spermatids
Degeneration of step 7 spermatids and
pachytene spermatocytes in stage VII
tubules
Disruption of testosterone secretion, which
may be caused by direct effects on the Leydig
cells or endocrine mediated effects.
Alternatively, direct effects on elongating
spermatids
Degeneration/apoptosis of
germ cells
Is a specific cell type affected? Are the
dying cells restricted to a specific
tubular stage? Are the affected cells
forming multinucleate aggregates?
The cause may be direct toxicity to the
affected germ cell but it may also be mediated
through a stage-specific disturbance to the
Sertoli cell. Apoptotic cells are rapidly
removed. Multinucleate aggregates suggest a
slow, nonspecific degenerative process
continued
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back to the testis and examine carefully, since
this probably reflects spermatogenic distur-
bance.
NOMENCLATURE AND SEVERITYGRADING OF SPERMATOGENICDISTURBANCE (GERM CELLDEPLETION/GERM CELLDEGENERATION)
The nomenclature used to describe deple-
tion and degeneration in the seminiferous epi-
thelium will depend on the specificity of the
findings seen, and this is most often related to
the duration of the study. In a 1- or 2-year
chronic study, any disturbance in spermato-
genesis is likely to show as generalized germ
cell depletion from some or all of the seminif-
erous tubules. Due to the duration of dosing and
the effects of maturation depletion, there is
unlikely to be any specificity in the germ cells
lost or in the stages of tubules affected. The
lesion seen is an end-stage lesion and therefore
nonspecific terminology and simple severity
grading based on proportion of tubules affected
can be used (Table 16.4.3). Regulatory studies
of28 days duration or investigational time-
course studies are much more likely to demon-
strate specific patterns of germ cell loss and
degeneration that may be restricted to specific
stages of the spermatogenic cycle. The termi-
nology used will depend on the specificity ofsuch findings. Examples of general and specific
findings are provided in Table 16.4.4.
The employment of a severity grading sys-
tem will also depend on the nature of the find-
ings. A general grading system based on the
proportion of tubules affected by a given find-
ing can be used for most nonspecific findings
(Table 16.4.2). Grading becomes difficult for
cell-specific and stage-specific findings. For
example, if 50% of the pachytene spermato-
cytes in 100% of stage VII tubules are degen-
erate, how should this be graded? Although
100% of stage VII tubules are affected, this only
constitutes 20% of the total number of tubules
in the testis cross section, and then only a
proportion of the spermatocytes within the tu-
bule are affected. Such situations have to be
dealt with on a case-by-case basis and the
terminology for each finding has to be made
sufficiently detailed to impart the necessary
information.
Germ cell exfoliation Presence of exfoliated germ cells in the
rete and epididymal lumens
Disruption of Sertoli/germ cell junctions
leading to loss of adhesion; disruption of
Sertoli cell cytoskeletal fibers leading to
sloughing of apical Sertoli cell cytoplasm and
attached germ cells
Macro/micro tubularvacuolation (in the absence
of severe germ cell
injury/loss)
Is this located in the basal Sertoli cellcytoplasm or scattered as large vacuoles
throughout tubule? Look for
accompanying or additional focal germ
cell loss (suggesting focal Sertoli cell
damage).
Disturbance of Sertoli cell function leading tovacuolation of organelles or disturbance of
fluid balance. Do not confuse with
osmotic-induced fixation artifact.
Necrosis and
disorganization of tubular
contents (including Sertoli
cells)
Evidence of acute inflammatory
infiltrate around affected tubules
Disturbance in hemodynamics or damage to
the vascular endothelium leading to ischemic
necrosis
Spermatid retention Alteration in epididymal sperm
parameters (morphology, motility, and
count) and possible increase in HRS
Disturbance in testosterone secretion, in
Sertoli cell function, or in spermatid
development leading to failure in spermiation
Dilated seminiferous tubulelumens
Blockage of efferent ducts orepididymal duct; evidence of
pressure-induced germ cell loss
Increased seminiferous tubule fluid that maybe due to obstruction of outflow, decreased
emptying of tubules, decreased resorption of
fluid by rete/epididymis, increased production
of fluid by Sertoli cell
Table 16.4.2 Rapid Reference Guide to Evaluation and Interpretation of Weight Changes and HistopathologicFindings in the Reproductive Tract, continued
Finding/observation What to look for Possible causes
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ARTIFACTS, SPONTANEOUSPATHOLOGY, AND IMMATURITY
Preparative and Fixation ArtifactsAs with any tissue, fixation and processingartifacts as well as spontaneous pathology need
to be distinguished from toxicologic changes.
Bouins fixation results in appreciable tubular
shrinkage, which is more marked in the center
than at the periphery of the testis. The enlarged
interstitial area surrounding these shrunken tu-
bules can be mistaken for edema. Formalin fixa-
tion, particularly in large animal testes can result
in sufficiently severe cellular shrinkage that the
cells appear pyknotic and the epithelium appears
extensively vacuolated. Pressure from forceps
during necropsy, or cutting into the testis beforeit is adequately fixed can result in displacement
of germ cells into the tubular lumen that can be
mistaken for exfoliation. For a review of some of
the more common artifacts see Foley (2001).
ImmaturityIn the testis, an additional factor that needs
to be considered is the age and maturity of the
animal. In the peripubertal animal, spermato-
genesis is incomplete and inefficient. This is
characterized by reduced numbers of elongat-
ing and elongated spermatids and a signifi-
cantly increased population of degenerating
germ cells of all types (spermatogonia, sperma-
tocytes, and spermatids). Significant numbers
of exfoliated germ cells and cell debris in the
epididymal ducts and an absence or reduction
of sperm usually accompany this. This appear-
ance can be indistinguishable from chemically
induced effects on spermatogenesis. In regula-
tory toxicity studies, this has proved a particular
problem with respect to dogs since the regula-
tory guidelines recommend starting studies
with dogs of an immature age (5 to 7 months).
In studies of 13 weeks duration, the dogs arethen on the borderline of maturity. Small group
sizes and significant variations in the age that
dogs attain full sexual maturity (8 to 12 months)
can lead to difficulties in separating the appear-
ance of varying levels of immaturity from
chemically induced changes.
Primates used in toxicity testing are fre-
quently immature and the variation between
Table 16.4.3 Semiquantitative Grading System toRecord the Severity of Germ Cell Degeneration orDepletion in Seminiferous Tubules
Severity gradeApproximate proportion oftubules affected
1 (minimal) 75% tubules affected
Table 16.4.4 Examples of NonSpecific and Specific Terminology to Categorize Germ Cell Loss andDegeneration in Seminiferous Tubulesa
Nonspecific Specific
Tubules with generalized germ cell depletion Depletion/degeneration spermatogonia
Tubules with partial germ cell depletion Depletion/degeneration prepachytene spermatocytes
Tubules with focal germ cell depletion Depletion/degeneration pachytene spermatocytes
Occasional Sertoli cellonly (atrophic) tubules Depletion/degeneration round spermatidsGerm cell degeneration/multinucleate aggregates Depletion/degeneration elongating spermatids
Depletion/degeneration elongated spermatids
aThe choice of whether to use nonspecific or specific terminology depends on the cell specificity of the changes seen. In longer duration
studies, germ cell loss is often patchy and nonspecific but shorter duration studies are more likely to show a cell-specific pattern of germ
cell loss. If necessary, the cell-specific changes can be further specified in terms of the individual spermatogenic stage or range of stages
affected. Each of the findings can then be graded using the approximate percentage of tubules affected (see Table 16.4.3).
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age and maturity of individuals within a study
can be marked. As a general guide, Cynomolo-
gous monkeys
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Rehm, S. 2001. Spontaneous testicular lesions inpurpose bred beagle dogs. Toxicol. Pathol.28:782-787.
Russell, L.D., Ettlin, R.A., Sinha-Hikim, A.P., andClegg, E.D. 1990. Histological and his-topathological evaluation of the testis. pp. 62-193. Cache River Press, Clearwater, Florida.
Ulbrich, B. and Palmer, A.K. 1995. Detection ofeffects on male reproductionA literature sur-vey.J. Am. Coll. Toxicol. 14:2293-3327.
KEY REFERENCESChapin and Conner, 1999. See above.
This chapter provides an overview on how to ap-
proach and carry out histopathological evaluationof the testis including the use of staging. It alsoreviews the inter-relationship of morphologicchanges in the testis with functional outcome anddiscusses the utility of sperm parameters.
Creasy, 1997. See above.
This key reference provides more in-depth consid-eration of the proper use of an understanding ofspermatogenesis in the histopathologic interpreta-tion of testis lesions, and will help the pathologist
understand the proper application of staging in theregulatory framework.
Creasy and Foster, 2001. See above.
This chapter provides a general overview of thestructure, function and physiology of the male repro-ductive system as well as responses of the system to
toxicologic disturbance.
Knobil, E., Neill, J., Greenwald, G., Markert, C., andPfaff, D. 1994. The Physiology of Reproduction,2nd Ed. Raven Press, New York.
This is an invaluable and comprehensive referencetext dealing with all aspects of reproductive physi-ology.
Russell et al., 1990. See above.
This is an essential reference text for beginners aswell as those experienced in testicular histopathol-ogy. It provides detailed instruction on how to iden-tify stages of the spermatogenic cycle in the rat,mouse, and dog. It also provides a wealth of infor-mation on testicular biology, histopathological andtoxicological evaluation, fixation, ultrastructureand much more.
Contributed by Dianne M. Creasy
Huntingdon Life SciencesEast Millstone, New Jersey
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