Testicular alterations in cryptorchid/orchiopexic rats chronically exposed to acrylamide or di-butyl-phthalate

Abstract

Exposure of Sprague-Dawley (SD) rats to acrylamide (AA) or di-butyl-phthalate (DBP) from the 12th gestational day to the 16th postnatal week (PNW) has been shown to reduce the effectiveness of orchiopexy in recovering the testicular alterations associated with experimental cryptorchidism established at weaning. Herein, we provide information about the long-term effects of AA or DBP on the testes of cryptorchid/orchiopexic rats. Male offspring exposed in utero to 10 mg/kg/day AA or 500 mg/kg/day DBP underwent bilateral surgical cryptorchidism at the 3rd PNW and orchiopexy at the 6th week, with continuous exposure to the chemicals through diet until the 58th week. Regardless of the test chemical, there were severe qualitative/quantitative alterations in the seminiferous tubules and increased numbers of Leydig cells. There was an increase and decrease in the number of tubules with c-Kit- and placental alkaline phosphatase-labeled germ cells, respectively, as compared to those in the control group, suggesting an imbalance between apoptosis and cell proliferation processes. The histological scores of the testicular lesions at the end of this one-year study were higher than those in the previous 16-week study, indicating that exposure of rats to the toxicants AA or DBP enhanced the testicular alterations induced by the chemicals beginning at the intra-uterine life, and impaired the effectiveness of orchiopexy in restoring the testes to normal morphology. Although the present experimental protocol does not completely replicate the natural human undescended testes, our findings may contribute to understanding the alterations occurring in cryptorchid/orchiopexic testes potentially exposed to exogenous chemicals for extended periods.

Keywords: acrylamide; chronic toxicity; di-butyl-phthalate; orchiopexy; rat surgical cryptorchidism; testicular germ cells.

Fig. 1.

Experimental design. Dams were exposed by gavage to a dose of 10 mg/kg/day AA or 500 mg/kg/day DBP during the gestational days 12 to 21. At the 3rd postnatal week (weaning), the male offspring underwent surgery for cryptorchidism, which was reversed by orchiopexy 3 weeks later. After weaning, the animals were exposed to the chemicals through diet. Euthanasia was performed when the rats were 58 weeks old. The number of animals in each group represents the number of animals that survived to the end of the study.

Fig. 2.

Body weights of Sprague-Dawley rats. Generalized linear mixed-effects model with gamma distribution. ^aAA/CPT-R and ^bDBP/CPT-R significantly differed from control (week 2: AA/CPT-R p=0.000 and DBP/CPT-R p=0.008; week 12: AA/CPT-R p=0.002 and DBP/CPT-R p=0.394); ^cSignificant differences between the chemically-exposed groups (week 2: p=0.000; week 12: p=0.002).

Fig. 3.

Testes of Sprague-Dawley rats (H&E-stained) at 58 weeks of age. (A, 100 µm) Control - normal seminiferous tubules with complete spermatogenesis. Treated groups with several histopathological changes: (B, 100 µm) vacuolization (dotted arrow), multi-nucleated germ cells (asterisk and b, inset - 20 µm), germ cells exfoliation (black arrow); (C, 100 µm) hyperchromatic nuclei and pleomorphic cytoplasm (dotted arrow and c, inset - 20 µm), and interstitial edema (triangle); (D, E, 50 µm) tubules with “Sertoli cells only” (black arrow), “apparent” Leydig cell hyperplasia (black arrowhead); (d, inset - 50 µm) intratubular calcification of debris within an atrophic tubule; (F, 100 µm) tubular atrophy with spermatogenesis arrest occurring among apparently normal tubules.

Fig. 4.

Testes of Sprague-Dawley rats at 58 weeks of age. Immunohistochemistry staining for c-Kit and PLAP (50 µm, solid red arrows). (a and d, inset) - Human classical seminoma, positive control for immunohistochemistry; c-Kit: (A) Control – Positive staining scattered among basal spermatogonia up to the level of primary spermatocytes, (B) AA/CPT-R and (C) DBP/CPT-R - Spermatogonia occurred diffusely in the germinative epithelium, with weak positive cytoplasmic staining. PLAP: (D) Control – Positive staining scattered among basal spermatogonia up to the level of primary spermatocytes, (E) AA/CPT-R and (F) DBP/CPT-R - Weak positive cytoplasmic staining in germ cells, with a decrease in the number of labeled tubules, especially in the AA/CPT-R group. Non-stained cells in the dotted red arrows.