Testis and antler dysgenesis in sitka black-tailed deer on Kodiak Island, Alaska: Sequela of environmental endocrine disruption?

Abstract

It had been observed that many male Sitka black-tailed deer (Odocoileus hemionus sitkensis) on Kodiak Island, Alaska, had abnormal antlers, were cryptorchid, and presented no evidence of hypospadias. We sought to better understand the problem and investigated 171 male deer for phenotypic aberrations and 12 for detailed testicular histopathology. For the low-lying Aliulik Peninsula (AP), 61 of 94 deer were bilateral cryptorchids (BCOs); 70% of these had abnormal antlers. Elsewhere on the Kodiak Archipelago, only 5 of 65 deer were BCOs. All 11 abdominal testes examined had no spermatogenesis but contained abnormalities including carcinoma in situ-like cells, possible precursors of seminoma; Sertoli cell, Leydig cell, and stromal cell tumors; carcinoma and adenoma of rete testis; and microlithiasis or calcifications. Cysts also were evident within the excurrent ducts. Two of 10 scrotal testes contained similar abnormalities, although spermatogenesis was ongoing. We cannot rule out that these abnormalities are linked sequelae of a mutation(s) in a founder animal, followed by transmission over many years and causing high prevalence only on the AP. However, based on lesions observed, we hypothesize that it is more likely that this testis-antler dysgenesis resulted from continuing exposure of pregnant females to an estrogenic environmental agent(s), thereby transforming testicular cells, affecting development of primordial antler pedicles, and blocking transabdominal descent of fetal testes. A browse (e.g., kelp) favored by deer in this locale might carry the putative estrogenic agent(s).

Figure 1

Kodiak Archipelago, showing the AP of Kodiak Island and numbers of NCO, UCO, and BCO SBTD shot at sites above the dashed line, on the AP and on Sitkinak Island. Note predominance of BCOs on the AP.

Figure 2

Concentrations of testosterone (ng/mL) in serum prepared from blood collected shortly after death from NCO and BCO deer. Abbreviations: BCO-NA, BCO deer with normal antlers; BCO-AbA, BCO deer with abnormal antlers without velvet; and BCO-Velv, BCO deer whose antlers retained velvet. Data grouped as NCO-NA, NCO deer with normal antlers. Geometric means were 3.81, 1.73, 2.03, and 0.22 ng/mL, respectively.

Figure 3

Germ cell atypia. (A) Atypical germ cells (arrows), characterized by abnormal nuclei, pale cytoplasm, and perinuclear cytoplasmic inclusions, in the testis of NCO deer 03-10. These abnormal germ cells are CIS-like cells, also called intratubular germ cell neoplasia. Note the location of these cells in the basal compartment of a seminiferous tubule (where premeiotic germ cells are normally located). Primary spermatocytes and spherical spermatids are seen adluminally. PAS and hematoxylin staining. (B) Atypical germ cells in a seminiferous tubule lacking any normal germ cell. Arrow designates a mitotic figure. Note foamy, hypertrophic Leydig cells. Differential interference contrast microscopy. Scale bars = 25 μm.

Figure 4

Testicular CIS. (A) CIS-like cells (arrows) in the basal aspect of a seminiferous tubule from the testis of NCO deer 03-12. Compare the size and abnormal contours of the nuclei in these cells with those of spermatogonia in the lower tubule. Methylene blue and Safranin-O staining. (B) An intratubular tumorous nodule (designated by arrows) containing CIS-like cells in BCO deer 03-14. Note the foaminess and hypertrophy of Leydig cells and compare with those in A. Methylene blue and Safranin-O staining. (C) Transmission electron micrograph of an atypical germ cell from the testis of BCO deer 03-7. Note abnormal nuclear contours, chromatin clumps, and altered nucleolonema in large nucleolus, swollen mitochondria, and unusual membranous profiles. (D) Transmission electron micrograph of a cluster of CIS-like cells from the testis of BCO deer 03-6. Note meandering, marginated nucleoli typical of seminomatous lesions and swollen mitochondria in each of these cells. Scale bars: A and B = 25 μm; C and D = 2 μm.

Figure 5

Testicular tumors. (A) Intratubular seminoma-like cells in BCO deer 03-14. Note considerable variation in size and shape of nuclei; such tumors have been designated as anaplastic. PAS and hematoxylin staining; differential interference contrast microscopy. (B) Intratubular, solid Sertoli cell tumor in the testis of BCO deer 03-6. Note regressed Leydig cells in the interstitium and compare with foamy, hypertrophic Leydig cells in association with the seminoma in A. Methylene blue and Safranin-O staining. (C) Leydig cell tumor (LCT) causing distortion of seminiferous tubules (ST) containing only Sertoli cells in the testis of BCO deer 03-03. (D) Stromal cell tumor in the testis of BCO deer 03-6. H&E staining. Scale bars: A and B = 25 μm; C and D = 100 μm.

Figure 6

Neoplastic lesions and concretions in the rete testis. (A) Carcinoma, with proliferation of dysplastic epithelial cells (normally cuboidal) of the rete testis invading the stromal elements, in the testis of BCO deer 03-11. Methylene blue and Safranin-O staining. (B) Adenoma of rete tubules in the testis of BCO deer 03-6. Hypertrophic glandular epithelial cells with PAS-positive secretions are evident. PAS and hematoxylin staining. (C) Microlithiasis within rete testis tubules in the testis of BCO deer 03-13. PAS and hematoxylin staining. Scale bars: A = 25 μm; B and C = 50 μm.

Figure 7

Timing during ontogeny of the testes, antler pedicles, and limbs in SBTD and postulated actions of an endocrine disruptor to transform testicular cells, alter the antler pedicle primordia, and disrupt transabdominal descent of the testes. Testicular testo: testosterone concentration in total testicular tissue. At “S,” the future scrotum is visible externally as scrotal swellings. Timing of development in SBTD based on data for mule deer (Hudson and Browman 1959) and red deer (Li and Suttie 2001; Lincoln 1973) scaled to gestation length; duration of gestation assumed as 203 days in SBTD and mule deer and 233 days in red deer. In model animals, transabdominal descent of the testes is blocked by estrogens, whereas passage through the inguinal canal is blocked by antiandrogens (Klonisch et al. 2004). Insulin-like peptide-3 (Insl3) is involved in transabdominal testicular descent; hence, altered expression of Insl3 and or a Hoxa gene could cause cryptorchidism. Products of the fibroblast growth factor gene family (i.e., FGFs) promote early outgrowth of limb buds, with axis patterning controlled by the sonic hedgehog (Shh) gene. FGF genes as well as Shh have been detected in the growth region of regenerating antlers of red deer postnatally (Li and Suttie 2001). The primary agent causing abnormalities such as those seen in SBTD, if caused by an endocrine disruptor, might be a phytoestrogen, estrogenic mycotoxin, alkyl phenol, organochlorine, or polychlorinated biphenyl (see text). An antiandrogen might be a co-agent.