Abstract
This study was conducted to characterize spontaneous testicular and epididymal microscopic findings in eighty control beagle dogs from toxicity studies. Hypospermatogenesis, characterized by randomly scattered missing spermatids and/or spermatocytes within seminiferous tubules, was observed in 75% of dogs six to seven months of age and declined to fewer than 10% in dogs over eleven months of age. Atrophy/hypoplasia of seminiferous tubules, characterized by subcapsular triangular clusters of tubules containing no germ cells, was observed in 25 to 40% of dogs under twelve months old, decreasing with age to 14 to 17% in dogs twelve to thirty-six months old. Retained spermatids, multinucleate giant cells, intracytoplasmic vacuoles (presumably in Sertoli cells), and swollen spermatocytes were common findings of minimal severity. Six- and seven-month-old dogs had lower testicular weights, less filling of the epididymal tails with sperm, and a two-fold higher incidence of abnormal epididymal content compared to dogs more than eight months of age. Most male beagles were histologically sexually mature by eight to nine months of age. This study confirms published reports that dogs at least ten months of age at necropsy usually are adequate for routine microscopic evaluation of the testes. If evaluation of spermatogenesis is critical, the incidental findings can be minimized by using males over twelve months of age.
Introduction
Histopathology is a sensitive method for evaluating adverse effects of chemicals on spermatogenesis (Rehm 2000; Takayama et al. 1995; Ulbrich and Palmer 1995). Knowledge of spontaneous microscopic findings in normal dog testes is required before drug-related lesions can be distinguished from spontaneous or age-related testicular findings. Although beagle dogs are commonly used as a nonrodent model for evaluating chemical toxicity, there are limited data on spontaneous testicular findings in these animals (Hottendorf and Hirth 1974; James and Heywood 1979; Rehm 2000). Rehm described the features and incidences of common spontaneous findings in the testes of fifty beagle dogs from eight to twenty months of age with a relatively small sample of dogs in each age group, specifically excluding dogs considered immature from her report (Rehm 2000). Our report extends Rehm’s observations by describing microscopic findings in testes from eighty additional control beagles ranging from six months to three years of age. Some of the beagles in our report were younger and older than those described by Rehm, and we did not selectively exclude any animals based on age. This manuscript demonstrates novel relationships regarding testicular weight and spermatogenic changes in control beagle dogs six to seven months of age. This information will assist toxicologic pathologists in distinguishing spontaneous and treatment-related findings of the testes and epididymides of laboratory beagles.
Materials and Methods
Animals
Testes from eighty control beagle dogs (aged six to thirty-six months) were obtained from toxicology studies conducted between 2000 and 2005 at Pfizer Global Research and Development, Groton, Connecticut. All dogs were obtained from Marshall Farms (North Rose, NY). Animals were housed singly in stainless steel cages in environmentally stable rooms (18°C –22°C, 40 to 60% humidity, and twelve-hour light cycle). They were fed Advanced Protocol High Density Diet 5L66 (PMI Nutrition International) once daily and had ad libitum tap water. Procedures were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International, and were approved by the Institutional Animal Care and Use Committee.
Histologic Evaluation
Testes were dissected free of epididymides and adjacent tissue at necropsy, weighed, and fixed by immersion in either 10% neutral buffered formalin (eight of eighty total dogs) or modified Davidson’s fluid (seventy-two of eighty total dogs). Following fixation, cross sections through the middle of the testes and longitudinal sections of the epididymides were embedded in paraffin, sectioned at 5 μm, stained with hematoxylin and eosin, and evaluated by a single pathologist using light microscopy. In almost all cases, the full testicular cross section was available for examination. In a few cases with very large testes, one edge of the testis was trimmed away so that the specimen would fit into a standard cassette. Most epididymal sections contained head, body, and tail. Histologic examination was conducted using nomenclature and semiquantitative severity grades as previously published (Rehm 2000; Lanning et al. 2002). This semiquantitative scoring system categorized severity as minimal (score 1) for fewer than 5% tubules affected, slight (score 2) for 5%–25%, moderate (score 3) for 25%–50%, marked (score 4) for 50%–75%, and severe (score 5) for more than 75% tubules affected. An additional category (score 0) representing absence of the finding was included. The spermatogenic stages of tubules containing findings were qualitatively assessed when possible (Creasy 1997; Russell et al. 1990).
Each testis was evaluated independently, and findings were categorized as either bilateral or unilateral. Hypospermatogenesis was further characterized according to the germ cell layer (spermatocyte, round spermatid, and/or elongate spermatid) that exhibited the greatest cell loss. Assessment of epididymides included a semiquantitative estimate of the percentage of all ductular lumens filled with sperm and qualitative assessment of abnormal intraluminal cell types, epithelial changes, and interstitial changes.
Imaging
Slides were examined with an Olympus BX50 light microscope, and selected images were captured using a high-resolution camera (Qimaging 5.3RTV). Images were captured using QCapture Pro 5.0.1.25 software (QImaging, Surrey, BC, Canada) at 2048 x 1536 dpi resolution. Grayscale conversion, brightness/contrast adjustments, and plate production were performed in Adobe Photoshop 6.0 for Windows (Adobe Systems, Inc., San Jose, CA, 2007).
Statistical Analysis
Combined testes weights and incidences of hypospermatogenesis and atrophy/hypoplasia were analyzed independently by age group. Data were analyzed using one-way analysis of variance followed by Newman-Keul’s multiple range post hoc test (p < .05). Histopathologic grades were rank-ordered prior to statistical analysis. Quantitative results were expressed as means ± standard deviations.
Results
Testicular Weight
Significantly lower paired testicular weights were seen in six- and seven-month-old dogs (8.1 ± 1.2 g) compared to testicular weights from dogs aged eight to thirty-six months (range: 11.0 ± 3.9 g to 14.8 ± 2.3 g) (Figure 1). Testicular weights appeared to plateau at eight months of age, with ranges as follows: six and seven months (6.5–10.4 g), eight months (10.6–18.3 g), nine months (5.5–16.2 g), ten months (9.3–12.7 g), eleven months (9.3–17 g), twelve to twenty-four months (8.3–19.1 g), and twenty-four to thirty-six months (11.5–17.7 g).
Testicular Microscopic Findings
Hypospermatogenesis (absence of some or all of the germ cells within individual tubules) was a common finding, occurring in 20% of all dogs (sixteen of eighty dogs). Of these sixteen animals, five dogs (31.3%) had bilateral changes (Table 1). Testicular and epididymal changes in control dogs are shown in Figures 2–9. Testes with hypospermatogenesis contained randomly scattered groups of one to fifteen tubules with partial to complete absence of germ cells. Multifocal absence of spermatocytes was observed in six dogs, whereas lower numbers of round spermatids were seen in twelve dogs. All sixteen dogs demonstrated multifocal absence of elongate spermatids (Figures 2 and 3). Hypospermatogenic tubules often contained small amounts of intraluminal debris, giant cells, and/or swollen spermatocytes. The severity of hypospermatogenesis was minimal (score 1) in 5% (four of eighty dogs), slight (score 2) in 10% (eight of eighty dogs), moderate (score 3) in 13.8% (eleven of eighty dogs), and marked (score 4) in 3.8% (three of eighty dogs) (Table 2). In addition, there was an age distribution to the incidence of hypospermatogenesis, which was observed in 75% of dogs six to seven months of age, declining in incidence with age to less than 10% in dogs over eleven months of age.
Tubules with atrophy/hypoplasia were typically arranged in well-demarcated, subcapsular, triangular clusters of three to fifty seminiferous tubules lacking all germ cells, as described by Rehm (2000) (Figures 4 and 5). These clusters were readily observed at low magnification and immediately juxtaposed with normal tubules at the borders of the clusters. The tubules in these clusters were lined only by elongate Sertoli cells with round nuclei, were smaller in diameter with smaller tubular lumina than normal tubules, contained no luminal content, and lacked evidence of interstitial inflammation or basement membrane thickening. Atrophic/hypoplastic tubules were seen in 26.3% of all dogs (twenty-one of eighty dogs); four of these (19%) exhibited bilateral lesions. The incidence was clearly affected by age, with 25%–40% of dogs under twelve months old having this finding, decreasing with age to 14%–17% in dogs twelve to thirty-six months old. Eleven dogs exhibited both atrophy/hypoplasia and hypospermatogenesis.
All other histopathological changes were of minimal severity. These changes included retained spermatids, multinucleate giant cells, swollen spermatocytes, apoptotic cells, intracytoplasmic vacuoles within tubules (presumably within Sertoli cell), and dilated tubules.
Retained spermatids had elongated hyperchromatic nuclei and were located either basally (in or between Sertoli cells) or apically (adhered to the surface). These retained cells had condensed cytoplasm and were found in later stages of spermatogenesis (six and seven, rarely eight). Spermatid retention consisting of two to seven spermatids in an average of six tubules per testis was seen in 47.5% of dogs (thirty-eight of eighty). Of the thirty-eight dogs with retained spermatids, 79% (thirty of eighty) had bilateral findings.
Multinucleated giant cells were seen in 73.8% of dogs (fifty-nine of eighty) (Figure 6), and 85% of these dogs (fifty of fifty-nine) had a bilateral distribution. In each case, there were from one to six multinucleate cells per affected tubule with fewer than ten affected tubules per testis and no predilection for specific stages.
Tubules with swollen spermatocytes were observed in seventy-four of eighty dogs (93%), and in most cases were observed bilaterally (fifty-two of seventy-four). These swollen spermatocytes were found singly or in loose aggregates of two to eight cells per tubule, and there were typically fewer than ten affected tubules per testis (Figure 7). Swollen cells had abundant pale cytoplasm and marginated nuclei. Some swollen spermatocytes had condensed chromatin (pyknosis).
Apoptotic germ cells within the seminiferous epithelium were found in 10% of dogs (eight of eighty), with no apparent age correlation. Of the eight dogs with apoptotic cells, only two animals had this lesion bilaterally.
Rare intracytoplasmic vacuoles, presumed to be in Sertoli cells based on cellular morphology, were seen in 55% of dogs (forty-four of eighty).
Five percent (four of eighty) of dogs had dilated seminiferous tubules. Some dilated tubules in three of the four affected dogs contained aggregates of desquamated immature intraluminal germinal cells.
One dog had unilateral subacute coagulative necrosis of one fourth of the parenchyma, suggesting a spontaneous infarct or focal trauma, although the inciting cause was not determined.
Epididymal Changes
Epididymal ducts from most dogs contained sperm admixed with rare multinucleate giant cells and a few immature spermatids. Older dogs (eight to thirty months) had between 40 and 90% of all epididymal duct profiles filled with sperm with consistent filling of the tails. Dogs from six to seven months of age had 0%–10% of epididymal duct profiles filled with a mixture of sperm, round spermatids, spermatocytes, multinucleated giant cells, and cell debris (Figure 8). Most dogs six to seven months of age had little or no sperm in their epididymal tails, and ducts of the epididymal head and body were incompletely expanded with sperm. Interestingly, incomplete filling of tubules in the head and body also was seen in three of the nine-month-old dogs. Filling of the tail of the epididymis with normal sperm was determined to be the most appropriate epididymal finding for evaluating maturity. One to four small (less than one tubule diameter), unilateral, interstitial lymphoid aggregates were present in four dogs. In four other dogs, the epididymal epithelium bilaterally contained rare small intracytoplasmic vacuoles. Lastly, two dogs had bilateral cribriform change characterized by 50–200 μm intraepithelial spaces rimmed by compressed columnar cells, primarily in the tail of the epididymis (Figure 9).
Discussion
Focal hypospermatogenesis and/or tubular atrophy/hypoplasia occurred in all age groups, but they were more common in younger animals. Eighty-seven percent of dogs six to seven months old and 26.4% of dogs eight months through three years old had either hypospermatogenesis and/or atrophy/hypoplasia.
Although the interactions of Sertoli cells and germ cells in normal and abnormal spermatogenesis are not completely characterized, recent information from rodents and nonhuman primates suggests that interaction between Sertoli cells and germinal cells is bidirectional. Immature Sertoli cells are identified by positive staining for anti-Müllerian hormone, neural cell adhesion molecule, and cytokeratin 18, whereas mature Sertoli cells are labeled with inhibin B (Rajpert-De Meyts et al. 1999; Rey et al. 1994; Rey et al. 1999; Stosiek et al. 1990). Positive staining for immature Sertoli cell markers is found in tubules with hypospermatogenesis, suggesting that incomplete maturation of Sertoli cells is associated with partial or complete absence of germ cells (Cortes et al. 1987; Mann et al. 1997; Marshall and Plant 1996; Sharpe et al. 2003; Sharpe et al. 2000; Wang et al. 1989). On the other hand, it is known that germ cell loss triggers mature Sertoli cells to develop characteristics of immature cells (Foley 2001; Sharpe et al. 2003; Sharpe et al. 1993; Steger et al. 1999). If future studies in beagle dogs are consistent with information from rodents and primates, the terms atrophy/hypoplasia and hypospermatogenesis may not adequately reflect the mechanisms of action responsible for these findings. Abnormal tubules with similar appearances could develop through multiple mechanisms, and a single mechanism could produce morphologic findings compatible with both hypospermatogenesis and atrophy/hypoplasia. More descriptive terminology, such as “Sertoli-cell-only tubules” for atrophy/hypoplasia and “missing germ cells” for hypospermatogenesis, may be more appropriate.
Multinucleate giant cells, swollen germ cells, vacuolation of Sertoli cells, and retained spermatids of mild severity were common findings in the testes of normal beagles. Apoptosis and intraluminal cell debris were relatively uncommon, and when present they were quite mild in severity. Multinucleate giant cells are a common finding in clinically normal male dogs, rats, rabbits, and humans, but they also may be linked to starvation, thermal stress, surgical procedures, local injury, and toxicity (Russell et al. 1990). Swollen and degenerate spermatocytes near the basal cell layer of seminiferous tubules were common spontaneous findings that may be confused with toxicity.
The major epididymal change was high variability in filling of the epididymal tail with sperm. Reduced luminal filling with increased numbers of immature germ cells and cell debris may be seen in healthy, peripubertal dogs, but such findings may also result from prior testicular toxicity of two to fourteen days in duration (Abbott 1993; Foley 2001). In our study, six- to seven-month-old dogs consistently had a much lower percentage of the overall epididymal lumen filled with spermatozoa (0%–10%) compared with animals at least eight months of age (40%–90%), and the epididymal tails of most six- to seven-month-old dogs contained little or no sperm compared to older dogs. Filling of the tail of the epididymides with normal sperm was one indicator of sexual maturity. Dogs six to seven months of age had proportionately more epididymal intraluminal eosinophilic debris, intraluminal spermatocytes, multinucleate giant cells, and degenerate immature spermatids than observed in the older dogs. These findings correlated well with lower testicular weights and the increased incidence of hypospermatogenesis in the younger group. Although there was at least four-fold greater epididymal filling in older dogs (eight to thirty-six months), the high variability (40%–90%) may have been partly a result of artifactual loss of sperm during processing. Epithelial cell cytoplasmic vacuoles, cribriform change, and interstitial mononuclear infiltrates were considered spontaneous findings. The cause of spontaneous interstitial lymphocytic aggregates is unknown and may be difficult to distinguish from a drug effect if there was increased incidence or severity in treated animals compared with controls. However, small testicular or epididymal lymphocytic aggregates in the absence of other epididymal findings likely would be of negligible toxicological significance.
Testicular histology and testicular weights are routinely used to detect drug toxicity in male reproductive organs (Ulbrich and Palmer 1995). Knowledge of the timing of sexual maturation is important to the design of preclinical toxicity studies, since sexually immature beagle dogs exhibit histologic findings similar to drug toxicity end points such as low testicular weight, hypospermatogenesis, multinucleate giant cells, reduced epididymal filling, intraluminal cell debris, and swollen, retained, and apoptotic germ cells (James and Heywood 1979). Sexual maturity in male beagle dogs has been estimated to occur between thirty-five and forty-one weeks of age based on hormone levels, sperm analysis, histology, and testes weight (James et al. 1979; James and Heywood 1979; Kawakami et al. 1991).
Our data indicated that 75% of six- and seven-month-old dogs had microscopically evident hypospermatogenesis which sharply declined in incidence after eleven months of age. Similarly, 40% of six- to seven-month-old dogs had focal atrophy/hypoplasia, declining to about half this level in dogs older than twelve months of age. Interestingly, the testes weight range for nine-month-old dogs was quite variable, ranging from 5.5 to 16.2 g (Table 1). The three dogs (20% of nine-month-old dogs) that had microscopic findings of both hypospermatogenesis and atrophy/hypoplasia accounted for the lowest testes weights (5.5, 6.6, and 7.6 g) in this age group. The remaining dogs that did not exhibit both lesions had a testes weight range of 8 to 16.2 g (mean 12.1 ± 0.6 g), which is similar to the remaining age groups. These findings, along with evidence of reduced testis weights and microscopically reduced epididymal sperm content in the six- to seven-month age group compared with older dogs, are in line with data reporting that the greatest testicular development and maturation in dogs occurs between five and eight months of age (James and Heywood 1979; Kawakami et al. 1991).
The age-related differences in the incidences of atrophy/hypoplasia vary from a previous report, perhaps because our study included younger and older dogs than the previous study (Rehm 2000). Taken together, more frequent hypospermatogenesis, reduced testicular weights, and reduced epididymal filling with sperm in six- and seven-month-old dogs suggested that histologic sexual maturity occurred in most dogs by eight months of age. Occasional dogs nine months of age may still have features of immaturity, including decreased testes weights and decreased filling of the epididymal lumen with sperm. Our results support previously published recommendations that male beagles should be at least ten months of age at necropsy for routine microscopic evaluation of the mature testis (Lanning et al. 2002). If critical evaluation of spermatogenesis and adult reproductive organs is imperative, use of dogs at least twelve months of age will reduce the incidence of spontaneous background findings that may be confused with toxicity.
Every study must be evaluated on a case-by-case basis to distinguish primary treatment-related effects, effects secondary to nonspecific stress or morbidity, and spontaneous findings. In some cases, a finding cannot be easily classified into a specific category. Testicular and/or epididymal findings in one or more treated dogs are more likely to be spontaneous and incidental if the findings: are known to occur as spontaneous findings; are unilateral rather than bilateral; occur in tubules intermixed with many normal tubules of the same stage; occur in the absence of other clearly treatment-related findings in the reproductive tract; are found in control animals as well as treated animals; demonstrate no dose response in incidence or severity; and/or are found inconsistently in a minority of the treated animals. When a dose response exists in incidence and/or severity of a finding that has been reported to occur spontaneously, the pathologist must weigh knowledge of spontaneous findings, mechanism(s) of intended and secondary actions of the test article, and all other study data (gross and microscopic findings, testicular and body weights, food consumption, etc.) with a thorough examination of treated and control animal tissues to determine if a treatment-related effect exists and is toxicologically meaningful.
In summary, hypospermatogenesis and atrophy/hypoplasia in seminiferous tubules are more common in young dogs than in older dogs. Dogs less than eight months of age have high incidences of hypospermatogenesis, lower testicular weights, and incomplete filling of epididymal tails with sperm, all compatible with immaturity. If evaluation of spermatogenesis is a critical end point in a toxicity study, males should be necropsied at ages no younger than ten months so that low testicular weights and microscopic findings found commonly in immature dogs do not complicate study interpretation.