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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

Supplement 13

Contributed by Dianne M. CreasyCurrent Protocols in Toxicology (2002) 16.4.1-16.4.14

Copyright 2002 by John Wiley & Sons, Inc.

16.4.1

MaleReproductiveToxicology


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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.

Supplement 13 Current Protocols in Toxicology

16.4.2

Histopathology ofthe Male

ReproductiveSystem II:

Interpretation


3/14

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.)

Current Protocols in Toxicology Supplement 13

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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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Interpretation


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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.)


16.4.5



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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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Interpretation


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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.


16.4.7



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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


9/14

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


16.4.9



10/14

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


16.4.10



Interpretation


11/14

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


16.4.11



12/14

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).


16.4.12



Interpretation


13/14

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


16.4.14



Interpretation

current protocols in toxicology ii

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