Age related changes in apoptosis of germ cells in normal and dibutyl phthalate-treated testes of Japanese quail (Coturnix coturnix japonica)

Abstract

Excess germ cells in normal and in pathological conditions are removed from testicular tissue by the mechanism of apoptosis. Studies on germ cell apoptosis in avian species are grossly lacking, and there are only a few reports of induced germ cell degenerations in the testicular tissue of birds. This study was designed to evaluate selected pro-apoptotic proteins, their mechanism of action and their involvement in germ cell apoptosis. Seminiferous tubule parameters such as seminiferous tubule diameter, seminiferous tubule epithelial height and seminiferous tubule lumen diameter were also investigated. In addition, the process of apoptosis of germ cells under normal conditions, and in pathological conditions induced by an endocrine disruptor, di-n-butyl phthalate (DBP) in the testes of Japanese quail (Coturnix coturnix japonica) was evaluated. Features of germ cell apoptosis were observed in birds of all age groups, namely pre-pubertal, pubertal, adult and aged under normal conditions. The morphological features of these apoptotic germ cells under normal conditions ranged from irregular nuclear membrane, ruptured nuclear membrane, condensed nuclear material and apoptotic bodies. The treated birds were administered a daily dosage of 10, 50, 200 or 400 mg/kg/body weight of DBP (dissolved in corn oil) for 30 days intra-gastrically by lavage. Apoptosis of germ cells in the quail under normal conditions and those of the DBP treated groups was evaluated using various methods. These included haematoxylin and eosin (H&E) staining, TdT dUTP Nick End Labeling (TUNEL) assay and electron microscopy, and results were similar to those observed in previous studies of germ cells and somatic cells of mammalian species. The observed morphological features of these apoptotic cells under pathological (DBP-treated testis) conditions were also similar to those under normal conditions, which ranged from irregular plasma and nuclear membranes in the early stage of apoptosis to rupture of the nuclear membrane, condensation of nuclear material, as well as fragments of apoptotic bodies, in later stages of apoptosis. These apoptotic germ cell features were similar to those observed in mammalian species, although the DBP doses were higher in reported data on mammalian species. Therefore, it is logical to propose that spermatogenic cells of Japanese quail seem to be more sensitive to DBP-induced germ cell degenerations compared to mammalian species. Therefore, more studies need to be conducted on the effects of DBP exposure on avian spermatogenesis.