Time response of rat testicular alterations induced by cryptorchidism and orchiopexy

Abstract

Cryptorchidism is one of the main risk factors for infertility and testicular cancer. Orchiopexy surgery corrects cryptorchidism effects. Different models of cryptorchidism developed in the rat include surgery. We assessed testicular alterations in rats submitted to surgical cryptorchidism and examined their potential for reversibility at different time points in order to verify time dependency effect(s) on the recovery of the undescended testes. Cryptorchidism was induced in 3-week-old rats. Animals were euthanized 3, 6 or 11 weeks after surgery to evaluate the morphological progression of cryptorchidism-induced germinative epithelial alterations. Other groups underwent orchiopexy 3, 5 or 9 weeks after surgical cryptorchidism, before or after puberty. Animals were euthanized 3 or 8 weeks after orchiopexy. Controls underwent sham surgery at the same time points as the surgical groups. Cryptorchid testes showed decreased weight, germinative epithelial degeneration, apoptosis and vacuolation, corresponding to impairment of spermatogenesis and of Sertoli cells. Some tubules has a Sertoli cell-only pattern and atrophy. The intensity of damage was related to the duration of cryptorchidism. After orchiopexy, spermatogenesis completely recovered only when testicular relocation occurred before puberty and the interval for recovery was extended. These results indicate that age, sexual maturity and extension of germ cell damage were relevant for producing germ cell restoration and normal spermatogenesis. We provide original observations on the time dependency of testicular alterations induced by cryptorchidism and their restoration using morphologic, morphometric and immunohistochemical approaches. It may be useful to study germ cell impairment, progression and recovery in different experimental settings, including exposure to exogenous chemicals.

Keywords: cryptorchidism; germ cell damage; orchiopexy; testicular damage.

FIGURE 1

Experimental design. Sprague‐Dawley rats were surgically made cryptorchid at the weaning (3rd week of age or DPN 21st postnatal day). Some cryptorchid groups (CPT) were euthanized 3, 6 or 11 wks after the surgeries (CPT + 3, CPT + 6, CPT + 11, respectively). For evaluation of reversibility of testicular alterations induced by cryptorchidism, other cryptorchid groups were submitted to orchiopexy (ORC) 3, 5 or 9 wks after cryptorchidism (ORC3, ORC5 and ORC9, respectively) and euthanized after 3 or 8 wks. Corresponding sham‐operated controls (not shown) were euthanized simultaneously

FIGURE 2

Groups submitted to cryptorchidism (CPT) only or to recovery of cryptorchidism alterations through orchiopexy (ORC). A, Absolute testes weights (g). B, Seminiferous tubule diameter. C, Incidence of vacuoles within the seminiferous epithelium. D, Number of Sertoli cells per tubule. Cryptorchid groups euthanized 3, 6 or 11 wks after the surgery: CPT + 3, CTP + 6 and CPT + 11, respectively. Orchiopexy at the 3rd (ORC3), 5th (ORC5) or 9th (ORC9) week after the cryptorchidism procedure. These animals were euthanized either 3 (groups ORC3 + 3, ORC5 + 3 and ORC9 + 3) or 8 (groups ORC3 + 8, ORC5 + 8 and ORC9 + 8) weeks after orchiopexy. CTL: Control group. Comparisons were performed using Student's t test, (*P < .05). Values are presented as mean ± SD. Numbers in parentheses: number of animals per group

FIGURE 3

Histology of the testes from animals submitted to cryptorchidism (CPT) or to cryptorchidism followed by orchiopexy (ORC); testes from control animals are shown in the insets a, b and c. A, Seminiferous epithelium of a CPT + 3 animal. Several apoptotic germ cells are present (black narrow arrow), some located in the centre of the tubule, detached from the epithelium, characterizing exfoliation (black arrowhead). a, Seminiferous tubule in a control animal at the 6th week. B, Seminiferous epithelium of a CPT + 6 animal showing vacuoles (black wide arrows). b, Seminiferous tubule of a CPT + 6 control animal. C, Sertoli cell–only (SCO) tubule of a CPT + 3 animal. Sertoli cells show round nuclei; some of them appear to be fixed to the epithelium base, indicating possible dysfunction in the cell (black chevron arrows). c, SCO tubule showing immature Sertoli cells (black chevron arrows). D, Atrophic tubule in a CPT + 11 animal showing deformation of its normal conformation and severe impaired spermatogenesis (Sertoli cell–only pattern) tubule. E, Seminiferous tubule of an ORC3 + 8 animal. Spermatogenesis is complete, although vacuoles may be found in some tubules (black wide arrow). F, Atrophic seminiferous tubule of an ORC9 + 8 animal

FIGURE 4

Immunohistochemistry for cytokeratin 18 (CK18). A, Immature testis of a normal 5‐d‐old rat showing positive CK18 staining in the cytoplasm of Sertoli cells. B, Seminiferous tubules of a CPT + 11 animal. No staining was observed in Sertoli cells. b, Control CPT + 11 animal. C, Testis of an ORC3 + 8 animal showing no CK18‐positive Sertoli cells. c, Control ORC3 + 3 animal. D, Seminiferous tubules of an ORC9 + 8 animal showing no staining for CK18. d, Control ORC9 + 8 animal

FIGURE 5

Mitosis and caspase‐3 expression in the rat testis. CPT + 3 animal showing no mitosis (A) and high number of caspase‐3‐positive cells (B), 400×. ORC9 + 8 animal with cells undergoing division (C) and no caspase‐3 expression (D), 400×. a‐d, Inset for each figure shows its respective control. E, Mitotic Index (%). F, Caspase‐3 Labeling Index (%). Comparisons were performed using Wilcoxon rank‐sum test (*P < .05)