Restoration of spermatogenesis after scrotal replacement of experimentally cryptorchid rat testis: assessment of germ cell apoptosis and eNOS expression

Abstract

Objectives. Cryptorchidism has been shown to induce germ cell apoptosis. Nitric oxide (NO), a ubiquitous free radical produced by the nitric oxide synthases (NOSs), has been associated with apoptosis in a number of cell types. We examined the effect of experimental cryptorchidism and subsequent orchidopexy on germ cell apoptosis and endothelial NOS (eNOS) expression. Methods. Prepubertal rats were rendered unilaterally cryptorchid, and 14 days later, orchidopexy was performed on a subset of these rats. Forty days after the initial procedure, testes were harvested from experimental and sham-operated rats for immunohistochemical studies. Apoptosis was detected by in situ 3′-end-labeling of DNA with digoxigenin-ddUTP, and eNOS protein was detected using an eNOS monoclonal antibody. Results. Cryptorchid testes were characterized by diffuse hypospermatogenesis and had a 25-fold increase in apoptotic germ cells per cross-sectional area compared with sham-operated testes ( P < 0.05). By contrast, the number of apoptotic germ cells per cross-sectional area in orchidopexied testes was not significantly different from that of sham-operated testes. In addition to its known expression in Leydig, Sertoli, and vascular endothelial cells, eNOS was detected in the cytoplasm of degenerating germ cells. Consecutive testis sections stained for eNOS and cellular DNA fragmentation demonstrated co-localization of eNOS protein and germ cell apoptosis. Conclusions. In our experimental model, cryptorchidism induced germ cell apoptosis, and orchidopexy lowered the levels of germ cell apoptosis. Our data also support a role of eNOS in germ cell degeneration.