Variability in Weight and Histological Appearance of the Prostate of Beagle Dogs Used in Toxicology Studies

Animals

These dogs were provided either by Harlan France (Gannat, France) or by Marshall Farms, Inc. (North Rose, NY, USA). Marshall Farms USA dogs were in the range of twenty-five to eighty-nine weeks of age, whereas dogs from Harlan France were from 23 to 108 weeks of age. No data were available for Harlan dogs from fifty-one to ninety weeks of age. For statistical analysis, groups of ten-week intervals were arbitrarily formed.

Histological data from 125 male beagle dogs, from 23 to 108 weeks of age (birth date provided by supplier) from the control groups of twenty-seven acute and chronic toxicity studies were examined. In addition, terminal body weight and prostate weight were also available from another thirty-two male beagle dogs from eight other studies. The studies were conducted in an Association for Accreditation and Assessment of Laboratory Animal Care (AAALAC)-accredited facility, in compliance with European regulations governing the housing and use of animals. During the course of all studies, there were no differences of diet, housing, and exercise between the dogs from different commercial sources.

Necropsy

At the end of the experimental period, the dogs were euthanized with an overdose of barbiturates, and a full macroscopic postmortem examination was performed. Animals were weighed, and all major organs—including prostate, testes, and epididymides—were dissected free of fat and the weights recorded. Prostatic sections were prepared according to MDS standard operating procedures and were standard across studies. Prostate samples were fixed in 10% buffered formalin, whereas testis and epididymides samples were fixed in modified Davidson’s fixative. They were then processed into paraffin wax, sectioned at 3–4 μm, and routinely stained with hematoxylin-eosin (HE).

Microscopic Examination

The prostatic sections were re-examined and the histological appearance recorded in a consistent fashion, using the following criteria:

Testis and epididymide maturity was also evaluated to allow an animal-by-animal correlation with prostate morphology. As described by Foley (2001), the immature testis was characterized by small-diameter, immature tubules, often without a lumen, lined by Sertoli cells and immature germ cells; the presence of multinucleated giant cells; and the absence of spermatids and spermatozoa. Immature animals may have few or no spermatozoa within the epididymal duct. By contrast, the mature testis was characterized by visible lumina in seminiferous tubules, the presence of spermatids and spermatozoa, and infrequent multinucleated giant cells.

Statistical analysis was performed using R Development Core Team (R Foundation for Statistical Computing, Vienna, Austria, 2007) A Student test was used to compare changes in terminal body weight and prostate weight between animals originating from the two different suppliers. Correlations between different parameters (age, terminal body weight, and absolute prostate weight) were determined by the linear regression method.

Terminal Body Weight and Prostate Weight

Terminal body weight and absolute and relative prostate weight were tabulated by week of age and supplier. The mean terminal body weight was higher and statistically significant in dogs from Harlan France than in dogs from Marshall Farms USA (Figure 1 and Table 1). The difference in mean relative prostate weight was not statistically significant between the two suppliers for the period between twenty-three and fifty weeks of age (Figure 2 and Table 2).

There was a positive correlation between terminal body weight and age in dogs from Marshall Farms USA and from Harlan France (p <.01) (Figure 3). In dogs from both suppliers, there was a positive correlation (p <.01) between absolute prostate weight and age (Figure 4). The correlation between terminal body weight and absolute prostate weight was positive in dogs from the two suppliers (p <.01). Relative prostate weight also increased with age between twenty-three and fifty weeks, however, implying that the prostate increased in weight relatively more than the rest of the body during this period (Figure 5).

Microscopic Findings

Owing to the absence of animals from Harlan France of fifty-one to ninety weeks of age, most histological data were derived from Marshall Farms dogs. There were no differences in histological appearance between dogs from the two sources at the same stage of maturity.

The microscopic findings were consistent with what was described by Evans (1993). Prostates from sexually immature animals were characterized by a branching ductular system. In contrast to the normal mature gland, the acinar portions of the gland were not developed and had little lumen, and there was no evidence of secretory activity (eosinophilic material) in the acinar cell cytoplasm or acinar lumen. Immature prostatic epithelium was characterized by a cuboidal to flattened basophilic epithelium with a high nucleocytoplasmic ratio (Figure 6). The amount of connective tissue was relatively higher in the immature gland than in the fully developed gland. This stroma was composed predominantly of prominent fibrous connective tissue, and smooth muscle was seen in the outer capsule and in major trabeculae radiating into the gland (Evans 1993). The percentage of grade 1 or 2 acini (totally or partially immature) decreased with age, and the percentage of grade 3 acini (mature) increased with age (Figure 7).

By contrast, mature prostates in these studies were characterized by compound tubuloalveolar glands, which radiated from their duct openings into the urethra. The acinar portion of the gland was dilated to form an alveolar structure and contained primary and secondary infoldings of secretory epithelium, which projected into the alveolar lumina. Mature prostatic epithelium was characterized by columnar epithelial cells with basal nuclei and abundant eosinophilic apical cytoplasm (Figure 8). The lumina were slightly dilated, although in the majority of cases there was no secretory material. When present, prostatic secretion was characterized by a deeply eosinophilic secretion dilating the acini but without compression of adjacent acini (Figures 9 and 10). Secretion increased with age between twenty-three and eighty-nine weeks of age. Focal areas of acini with eosinophilic intraluminal content were sometimes found in prostates showing features of immaturity.

In mature prostates, acinar dilation presented two patterns. Simple dilation was defined as many dilated acini with or without luminal eosinophilic secretion, which did not compress the adjacent acini. Acini were lined by a flattened basophilic epithelium with a high nucleocytoplasmic ratio (Figure 11a). Focal glandular ectasia was defined as focal dilation of a few acini with eosinophilic content, sharply demarcated, with compression of the adjacent prostatic parenchyma. Acini were lined by a columnar epithelium with basal nuclei and abundant eosinophilic apical cytoplasm (Figure 11b). The alveoli were separated by a dense stroma that also contained smooth muscle cells. These smooth muscle septa were continuous with the smooth muscle and fibrous tissue located in the periurethral area (Evans 1993).

The patterns of inflammation were classified as:

The incidences of dilation and inflammation are presented in Table 3. No relation could be established between inflammatory lesions and dilation pattern. With the available data, there is no clear relation between the incidence of these changes (dilation and inflammation) and the age of animals.

Testis Maturity

The percentage of immature and mature testes according to age is summarized in Figure 13. There was a strong correlation between maturity of prostate and maturity of testes and epididymides in each dog. Moreover, 90% of thirty-one- to forty-week-old dogs from Harlan France were mature, whereas only 10% of dogs from Marshall Farms USA were mature at the same age. Sexual maturity appeared to occur earlier in dogs from Harlan France than in dogs from Marshall Farms USA.

In summary, the sources of variation in prostate weight between individual animals were (1) the sexual maturity of the animals (sexual maturity being reached between forty-one and fifty weeks in Marshall Farm dogs but at thirty-one to forty weeks in Harlan France dogs); (2) the source of the animals was very important in this result, as Harlan France dogs were heavier at a given age and sexually mature earlier than Marshall’s dogs, but there were no differences in histological appearance between dogs from the two sources at the same age of maturity; and (3) individual variation between dogs of the same sexual maturity.