Histopathology of the Male Reproductive System 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 system. No single endpoint can adequately reflect changes at such a diversity of sites, but it has been repeatedly demonstrated that histopathologic 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 adequately preserved and presented to the pathologist for examination. These preparative procedures 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 adequate 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 significance of the findings to overall reproductive function and their implications for functional or morphological changes in the rest of the reproductive 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 provided. The pattern of morphological change as well as the specificity and severity of the findings are significantly influenced by the duration of dosing, such that the approach to examination is different in a short-term versus a longterm study. This unit includes a section providing practical guidance on how to evaluate testicular and epididymal sections for evidence of a 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 inappropriately 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
Contributed by Dianne M. Creasy
Current Protocols in Toxicology (2002) 16.4.1-16.4.14 Copyright 2002 by John Wiley & Sons, Inc.

UNIT 16.4

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 between effects on specific types of germ cells and their stage localization. The decision is strongly 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 spontaneous 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 addition, the use of immature or borderline mature animals, which is the case for some regulatory studies, can mask or be mistaken for toxicologically 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 specifically mentioned where appropriate.

ESSENTIAL KNOWLEDGE FOR EVALUATION AND INTERPRETATION OF TESTICULAR PATHOLOGY Spermatogenesis and the Spermatogenic Cycle
The process and regulation of spermatogenesis is essentially similar in all mammalian species. Stem cell spermatogonia proliferate by mitosis and produce a population of differentiating or committed spermatogonia. These in turn divide and produce spermatocytes, which undergo DNA replication and divide by meiosis to produce haploid spermatids. A complex series of morphological transformations of the simple round spermatid finally produces a differentiated 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 requiring 3 weeks. The arrangement of the different

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populations of germ cells within the seminiferous 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 generations of germ cells are developing within a tubule at any given time. The development of each 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 consequently, 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 presence of a population of germ cells, a thorough understanding of the cellular makeup of the spermatogenic cycle is essential.

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 developed 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 studies. 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 consecutive stage. This is not the case in humans where cell associations are arranged in a helical pattern resulting in a mosaic of cell associations 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.

Species-Specific Variations in the Organization of Spermatogenesis

Although the fundamentals of the spermatogenic cycle are similar between species, there are certain details that vary. These can have a significant impact on histopathologic evaluation. Number of stages The number of stages and their cellular makeup varies between species and depends on the morphological criteria used by the classification system. It is important that the pathologist is familiar with the germ cell development and the stage map of the species under investigation. A highly recommended text that explains 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 information 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

Disturbances in Spermatogenesis
Almost 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 most other parts of the reproductive tract. Spermatogenic 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 vascular supply or altered fluid balance, either within the testis or within the epididymis. It is therefore extremely important that disturbances 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 depletion (whereby death of a specific precursor germ cell causes the progressive loss of its descendant generations), reduces the specificity of the pattern of spermatogenic disturbance as the tubules become depleted of more and more germ cells.

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I
15 1 EP In

VIII
19 8 MP PL

XI
11

XIV
14

LP L A

D Z A

15

19

11

14

15

LP

EP

MP L PL A Z A

EP In

In

VIII

XI

XIV

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, , early pachytene; MP , intermediate; Spermatocytes: PL, preleptotene; L, leptotene; Z, zygotene; EP 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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Table 16.4.1 Specific EPA and OECD Recommendations for Histopathological Examination of the Testes and Epididymides in Studies to Detect Effects on Reproduction and Fertility

Testis Detailed histopathological examination of testes should be conducted in order to identify treatment-related effects such as: Retained spermatids Missing germ cell layers or types Multinucleate giant cells Sloughing of spermatogenic cells into the lumen

Epididymis Examination of the intact epididymis should include the caput, corpus, and cauda. The epididymis should be evaluated for: Leukocyte infiltration Sperm granulomas Change in prevalence of cell types Absence of clear cells in the cauda epithelium Aberrent cell types in the lumen Phagocytosis of sperm

Regulatory Guidelines and the Role of Staging in Histopathologic Examination 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. Recommendations 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 although there is no mention of staging in the final issued guidelines, the issue has become surrounded by confusion. The ability to recognize stages of the spermatogenic cycle is important 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 assessment of stagese.g., a frequency distribution of tubules for individual stages of the spermatogenic cycle. While this may be useful information 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 of staging 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 understanding of the normal progression of the stages of the spermatogenic cycle. This approach is explained below. For a more detailed discussion of this issue see Creasy (1997) and Chapin and Conner (1999).

COMMON TOXICOLOGICALLY INDUCED FINDINGS AND THEIR POSSIBLE SIGNIFICANCE

As with any tissue, the cellular response to injury is limited and at times, nonspecific. However, certain aspects of the early pathogenesis of toxicologically induced lesions in the 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 spermatogonia, which may be seen apoptosing in occasional stage XII tubules. However, many of the dying cells do not have the classic morphological appearance of apoptotic cells. Dying spermatocytes generally develop cytoplasmic eosinophilia and nuclear pyknosis while round spermatids show chromatin margination. If cell death progresses rapidly, then the apoptotic cell is rapidly phagocytized by the surrounding Sertoli cell cytoplasm and all evidence of cell death is rapidly removed. Cell death and phagocytosis 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

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ES RS PS Sg day of dosing 1 week ES RS Sg

ES Sg 2 weeks 4 weeks Sg

Figure 16.4.2 Development of maturation depletion following daily dosing with a spermatocyte toxicant. 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 spermatogenic 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 spermatids; RS, round spermatids; PS, pachytene spermatocytes; Sg, spermatogonia. (Reproduced from Creasy, 1997, with permission.)

breakdown of the cytoskeletal fibers supporting the interconnecting cytoplasmic bridges. Multinucleate aggregates are less readily phagocytized 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 spermatogenic disturbance and is generally a consequence of germ cell death rather than exfoliation. 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 spermatocytes 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 spermatogenic 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 a few adjacent spermatogonia, which then fail to produce their cohort of spermatocytes and spermatids, 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

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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 advanced 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 spermatogenic depletion is or is not reversible without carrying out an appropriate study. If spermatogonia are still present, then the lesion is potentially reversible but if the Sertoli cells are functionally compromised, spermatogenesis may not be supportable. The chronic effects of 2,5hexanedione 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 significantly depleted, but if the Sertoli cells are functionally intact and sufficient time is allowed 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 subsequent transport of the cells to the rete testis and 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 recognizable 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 exfoliated germ cells. These generally occur as a result of disturbances in spermiation (see below). 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 swelling may be seen affecting the basal area of the Sertoli cell cytoplasm and causing germ cell displacement and disorganization. Such findings are suggestive of disturbances within the Sertoli cell and may represent alterations in the smooth endoplasmic reticulum or in fluid homeostasis. Vacuolation may also be seen in end-stage lesions, associated with extensive germ 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 spermatogonia are generally fixation-induced artifacts 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 reduced secretion of seminiferous tubule fluid. Seminiferous 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 consequently 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 volume will generally be reflected by an increased weight of the testis unless there is an accompanying significant cell loss. The pathological consequences of the finding depend on the severity and duration of the effect. Prolonged increased pressure on the seminiferous epithelium will generally result in pressure atrophy of varying degrees and may also lead to inspissated sperm and granulomatous inflammation.

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Spermatid retention This is a subtle but relatively common finding that may be caused by a number of chemicals as well as by hormonal disturbance. It is characterized by the retention of step 19 spermatids (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 spermatids remain 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 occurs during 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 abnormal sperm parameters (number, motility, or morphology) and it may be associated with alterations in fertility parameters. If homogenization resistant spermatids are measured, the retained spermatids should be reflected by an increase in this parameter. However, identification 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 apoptosis, tubular necrosis is characterized by coagulative (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. Consequently, 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 be seen 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 associated 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 accompanied by an inflammatory infiltrate, which is an otherwise rare accompaniment to toxic injury. Tubular necrosis is a serious irreversible 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 empty into the rete. Most of the tubule fluid is reabsorbed 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-pressure 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 produce 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 separate 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 animals, and in immature and peripubertal animals this number is significantly increased. Abnormal residual bodies may also be detected in the epididymis as a consequence of disturbed spermiation in the testis. The presence or absence of germ cells in the luminal contents can also aid the pathologist in evaluating whether apparently 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 epithelium can be seen as a specific chemically induced finding. Macrovacuolation and cribriform change (infolding of the epithelium within itself) is often seen accompanying contraction of the atrophic aspermic epididymis. This may represent a normal mechanism of surface area reduction but has also been reported 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 granuloma The antigenically foreign sperm in the epididymal lumen and in the seminiferous tubule are in an immunologically protected environment. 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 inflammatory response against the sperm develops and generally progresses to form a sperm granuloma. This is a chronic, progressive lesion and in the coiled epididymal duct has the added complication of causing obstruction to the passage of sperm. Furthermore, the oxidative free radicals produced by inflammatory cells in contact with sperm can lead to genotoxic damage, which may have implications for male-mediated 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 granulomas. In species such as the dog, they often form blind ending tubules that contain inspissated 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. Inflammatory infiltrate and sperm granulomas are a frequent consequence.

Prostate/Seminal Vesicles
Acinar atrophy Secretory activity by the prostate and seminal vesicles is a sensitive, androgen-dependent function. Decreased circulating testosterone levels, or interference with androgen receptors in these two tissues will result in reduced secretion leading to atrophic changes. These may be detected by organ weight changes as well as by microscopic changes

PRACTICAL APPROACH FOR EXAMINATION OF THE TESTIS AND EPIDIDYMIS FOR TOXICOLOGICAL EFFECTS
The approach used is influenced by the duration of the study. Cell- and stage-specific disturbances in spermatogenesis are usually only seen in short duration studies of <28 days. As discussed above, the kinetics of spermatogenesis combined with the process of maturation depletion, result in a progressive, germ cell loss, which becomes more generalized and affects more stages of spermatogenic cycle the longer dosing continues. Table 16.4.2 provides a rapid reference guide to common changes and their possible etiologic significance. 1. Review the testis and epididymis weights. Absolute weights are generally more useful than relative values because testis, like brain weight, is less influenced by body weight than most other tissues. A decrease in testis weight generally reflects germ cell loss and decreased tubule fluid production, while a decreased epididymal weight is likely to reflect

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decreased sperm and fluid content. An increased weight in either tissue generally reflects 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 tubule 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 Significance). 2. If testicular homogenization-resistant spermatids (HRS) and/or epididymal sperm have been counted, review these data. A decrease 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 histopathology). HRS data are particularly useful for confirming or alerting the pathologist to slight reductions in sperm production which may not be immediately obvious by qualitative histopathology. 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 confirmable by pathology. 3. Examine the testis at low power. Look for obvious depletion or disorganization of germ cells within the epithelium or exfoliation of germ cells into the lumen. Look for occasional 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 numbersi.e., that stages I to VIII contain a layer of spermatogonia, a layer of pachytene spermatocytes, and several layers of round spermatids interspersed with elongated spermatids. Stages IX to XIV should contain a layer of prepachytene spermatocytes, several layers of late pachytene or dividing (stage XIV) spermatocytes, and several layers of elongating spermatids. This does not require individual identifi-

cation of stages, just a familiarization with the cellular make up of the two halves of the spermatogenic cycle. It will allow for the identification of when a cell population is missing. This is particularly important in studies of 28-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 a loss 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 Leydig 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. Also examine 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 approximately normal numbers of step 19 (mature) sperm at the lumen and a normal appearing 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 in these 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 stagespecific lesion is characteristic for and the most sensitive marker of decreased testosterone levels in the testis. Effects will become progressively more obvious with time, due to maturation depletion and direct effects on the elongating 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 contents for evidence of testicular germ cells or residual bodies (increased above control levels). Examine epididymal epithelium for presence of vacuoles, inflammation, or altered cellular 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 Histopathologic Findings in the Reproductive Tract

Finding/observation Increased testis weight

What to look for Seminiferous tubular lumen dilatation

Possible causes 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 Altered hemodynamics, injury to vascular endothelium, reduced lymphatic drainage Disruption of spermatogenesis through effects on germ cells, Sertoli cells, hormonal disturbance, or blood supply Decreased production of seminiferous tubule fluid that may result from loss of elongating spermatids and/or decreased testosterone production Altered hemodynamics, injury to vascular endothelium, reduced lymphatic drainage Decreased resorption of fluid by rete, efferent ducts, and caput epithelium May be spontaneous but may be induced by any agent causing inflammation or damage to the epididymal epithelial lining Disruption of spermatogenesis resulting in reduced sperm production or release from the testis Reduced levels of circulating testosterone, inhibition of 5- reductase, or disruption of androgen receptor binding 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 (see main text for detail). The pathogenesis of germ cell loss is best investigated in a short time-course study. 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 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

Decreased testis weight

Increased interstitial fluid (interstitial edema) Germ cell depletion

Seminiferous tubule lumen contraction

Increased epididymal weight Increased interstitial fluid Increased ductular fluid Sperm granulomas

Decreased epididymal weight Decreased weight of seminal vesicles and/or prostate Germ cell loss

Reduced sperm content and contraction of ductular lumen size Atrophic changes in the secretory epithelium and decreased secretory product 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 it partial or generalized?

Loss of elongate and elongating spermatids

Degeneration of step 7 spermatids and pachytene spermatocytes in stage VII tubules

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?

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Table 16.4.2 Rapid Reference Guide to Evaluation and Interpretation of Weight Changes and Histopathologic Findings in the Reproductive Tract, continued

Finding/observation Germ cell exfoliation

What to look for Presence of exfoliated germ cells in the rete and epididymal lumens

Possible causes

Macro/micro tubular vacuolation (in the absence of severe germ cell injury/loss)

Necrosis and disorganization of tubular contents (including Sertoli cells) Spermatid retention

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 Disturbance of Sertoli cell function leading to Is this located in the basal Sertoli cell cytoplasm or scattered as large vacuoles vacuolation of organelles or disturbance of fluid balance. Do not confuse with throughout tubule? Look for accompanying or additional focal germ osmotic-induced fixation artifact. cell loss (suggesting focal Sertoli cell damage). Evidence of acute inflammatory Disturbance in hemodynamics or damage to infiltrate around affected tubules the vascular endothelium leading to ischemic necrosis Disturbance in testosterone secretion, in Sertoli cell function, or in spermatid development leading to failure in spermiation 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 of fluid by Sertoli cell

Alteration in epididymal sperm parameters (morphology, motility, and count) and possible increase in HRS Dilated seminiferous tubule Blockage of efferent ducts or lumens epididymal duct; evidence of pressure-induced germ cell loss

back to the testis and examine carefully, since this probably reflects spermatogenic disturbance.

NOMENCLATURE AND SEVERITY GRADING OF SPERMATOGENIC DISTURBANCE (GERM CELL DEPLETION/GERM CELL DEGENERATION)
The nomenclature used to describe depletion and degeneration in the seminiferous epithelium 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 spermatogenesis is likely to show as generalized germ cell depletion from some or all of the seminiferous 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 of 28 days duration or investigational time-

course studies are much more likely to demonstrate specific patterns of germ cell loss and degeneration that may be restricted to specific stages of the spermatogenic cycle. The terminology used will depend on the specificity of such findings. Examples of general and specific findings are provided in Table 16.4.4. The employment of a severity grading system will also depend on the nature of the findings. A general grading system based on the proportion of tubules affected by a given finding 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 spermatocytes in 100% of stage VII tubules are degenerate, 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 tubule 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.

Male Reproductive Toxicology

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Table 16.4.3 Semiquantitative Grading System to Record the Severity of Germ Cell Degeneration or Depletion in Seminiferous Tubules

Severity grade 1 (minimal) 2 (slight) 3 (moderate) 4 (marked) 5 (severe)

Approximate proportion of tubules affected <5% of tubules affected 5% to 25% tubules affected 25% to 50% tubules affected 50% to 75% tubules affected >75% tubules affected

Table 16.4.4 Examples of NonSpecific and Specific Terminology to Categorize Germ Cell Loss and Degeneration in Seminiferous Tubulesa

Nonspecific Tubules with generalized germ cell depletion Tubules with partial germ cell depletion Tubules with focal germ cell depletion Occasional Sertoli cellonly (atrophic) tubules Germ cell degeneration/multinucleate aggregates

Specific Depletion/degeneration spermatogonia Depletion/degeneration prepachytene spermatocytes Depletion/degeneration pachytene spermatocytes Depletion/degeneration round spermatids 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).

ARTIFACTS, SPONTANEOUS PATHOLOGY, AND IMMATURITY Preparative and Fixation Artifacts

As with any tissue, fixation and processing artifacts 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 tubules can be mistaken for edema. Formalin fixation, 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 before it 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).
Histopathology of the Male Reproductive System II: Interpretation

Immaturity
In the testis, an additional factor that needs to be considered is the age and maturity of the

animal. In the peripubertal animal, spermatogenesis is incomplete and inefficient. This is characterized by reduced numbers of elongating and elongated spermatids and a significantly increased population of degenerating germ cells of all types (spermatogonia, spermatocytes, 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 appearance can be indistinguishable from chemically induced effects on spermatogenesis. In regulatory toxicity studies, this has proved a particular problem with respect to dogs since the regulatory guidelines recommend starting studies with dogs of an immature age (5 to 7 months). In studies of 13 weeks duration, the dogs are then 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 appearance of varying levels of immaturity from chemically induced changes. Primates used in toxicity testing are frequently immature and the variation between

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age and maturity of individuals within a study can be marked. As a general guide, Cynomologous monkeys <4 years of age are likely to show varying levels of immaturity. An additional problem with primates is the effect of hierarchical status on testosterone levels and testicular development and regression. In most routine regulatory studies using rodents, the guidelines recommend using animals of an age that are sexually mature by the termination of the study. In addition, there is much less variation in the age at which rodents attain sexual maturity. However, if a study design calls for the use of prepubertal animals, the increased rate of germ cell degeneration and the relative disorganization of the epithelium in this age of animal should be appreciated.

points for Human Health Risk Assessment. (G. Daston and C. Kimmel, eds). pp. 28-41. ILSI Press, Washington, D.C. Chapin, R.E. and Ku, W.W. 1994. The reproductive toxicity of boric acid. Environ. Health Persp. 7:87-91. Chellman, G.J., Bus, J.S., and Working, P.K. 1986. Role of epididymal inflammation in the induction of dominant lethal mutations in Fisher 344 rat sperm by methyl chloride. Proc. Natl. Acad. Sci. U.S.A. 83:8087-8091. Clegg, E.D., Cook, J.C., Chapin, R.E., Foster, P.M.D., and Daston, G.P. 1997. Leydig cell hyperplasia and adenoma formation: Mechanisms and relevance to humans. Reprod. Toxicol. 11:107-121. Creasy, D.M. 1997. Evaluation of testicular toxicity in safety evaluation studies: The appropriate use of spermatogenic staging. Toxicol. Pathol. 25:119-131. Creasy, D.M. 2001. Pathogenesis of male reproductive toxicity. Toxicol. Pathol. 29:64-76. Creasy, D.M. and Foster, P.M.D. 2001. Male reproductive system, Chapter 10. In Handbook of Toxicologic Pathology, 2nd Edition (W. Haschek, C. Rousseaux, and M. Wallig, eds.) Academic Press, San Diego. Foley, G.M. 2001. Overview of male reproductive pathology Toxicol. Pathol. 29:49-63. Hess, R.A. 1990. Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat seminiferous epithelium: Light microscopic observation of perfusion-fixed and plastic embedded testes. Biol. Reprod. 43:525-542. Hess, R.A. 1998. Effects of environmental toxicants on the efferent ducts epididymis and fertility. J. Reprod. Fertil. Suppl. 53:247-259. Hess, R.A. and Chen, P. 1992. Computer tracking of germ cells in the cycle of the seminiferous epithelium and prediction of changes in cycle duration in animals commonly used in reproductive biology and toxicology. J. Androl. 13:185-190. Lee, J., Richburg, J.H., Younkin, S.C., and Boekelheide, K. 1997. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology 138:2081-2088. Lee, J., Richburg, J.H., Shipp, E., Meistrich, M.L., and Boekelheide, K. 1999. The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis. Endocrinology 140:852-858. Linder, R.E., Strader, L.F., Slott, V.L., and Suarez, J.D. 1992. Endpoints of spermatotoxicity in the rat after short duration exposures to 14 reproductive toxicants. Reprod. Toxicol. 6:491-505. Meistrich, M.L. 1986. Critical components of testicular function and sensitivity to disruption. Biol. Reprod. 34:17-28. Nolte, T., Harleman, J.H., and Jahn, W. 1995. Histopathology of chemically induced testicular atrophy in rats. Exp. Toxicol. Pathol. 47:267-286. Male Reproductive Toxicology

Spontaneous Pathology
Dog testes present a particular problem in terms of the level of spontaneous degenerative lesions and the prevalence of incomplete spermatogenesis in many tubules. Rehm (2001) has reported and quantified the incidence of various degenerative lesions in the testes of pure bred beagle dogs at various ages. Dogs 8 to 11 months of age showed a 27% incidence of testes with hypospermatogenesis (tubules with partial depletion of germ cells), a 100% incidence of testes with degenerating germ cells, and a 45% incidence of testes with segmental atrophy/hypoplasia. Since these changes are often indistinguishable from the nonspecific spermatogenic changes induced by chemical toxicants and because regulatory studies generally employ small group sizes, interpretation of slight increases in incidence can be a significant problem. Rodent testes generally demonstrate a far lower incidence of spontaneous lesions. Unilateral or bilateral generalized tubular atrophy (Sertoli cellonly tubules) are not uncommon findings and occasional animals with partial or focal depletion of germ cells are also seen. However, in comparison with large animals, the use of larger group sizes generally allows ready distinction between spontaneous and treatment-related effects.

LITERATURE CITED
Blanchard, K.T., Lee, J., and Boekelheide, K. 1998. Leuprolide, a gonadotropin-releasing hormone agonist reestablishes spermatogenesis after 2,5hexanedione-induced irreversible testicular injury in the rat resulting in normalized stem cell factor expression. Endocrinology 139:236-244. Chapin, R.E. and Conner, M.W. 1999. Testicular histology and sperm parameters. In An Evaluation and Interpretation of Reproductive End-

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Rehm, S. 2001. Spontaneous testicular lesions in purpose bred beagle dogs. Toxicol. Pathol. 28:782-787. Russell, L.D., Ettlin, R.A., Sinha-Hikim, A.P., and Clegg, E.D. 1990. Histological and histopathological evaluation of the testis. pp. 62193. Cache River Press, Clearwater, Florida. Ulbrich, B. and Palmer, A.K. 1995. Detection of effects on male reproductionA literature survey. J. Am. Coll. Toxicol. 14:2293-3327.

Creasy and Foster, 2001. See above. This chapter provides a general overview of the structure, function and physiology of the male reproductive system as well as responses of the system to toxicologic disturbance. Knobil, E., Neill, J., Greenwald, G., Markert, C., and Pfaff, D. 1994. The Physiology of Reproduction, 2nd Ed. Raven Press, New York. This is an invaluable and comprehensive reference text dealing with all aspects of reproductive physiology. Russell et al., 1990. See above. This is an essential reference text for beginners as well as those experienced in testicular histopathology. It provides detailed instruction on how to identify stages of the spermatogenic cycle in the rat, mouse, and dog. It also provides a wealth of information on testicular biology, histopathological and toxicological evaluation, fixation, ultrastructure and much more.

KEY REFERENCES
Chapin and Conner, 1999. See above. This chapter provides an overview on how to approach and carry out histopathological evaluation of the testis including the use of staging. It also reviews the inter-relationship of morphologic changes in the testis with functional outcome and discusses the utility of sperm parameters. Creasy, 1997. See above. This key reference provides more in-depth consideration of the proper use of an understanding of spermatogenesis in the histopathologic interpretation of testis lesions, and will help the pathologist understand the proper application of staging in the regulatory framework.

Contributed by Dianne M. Creasy Huntingdon Life Sciences East Millstone, New Jersey

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