Recent studies have demonstrated apoptotic DNA fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis apoptotic DNA fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two injections at 2-day intervals). Pretreatment with the GnRH antagonist increased apoptotic DNA fragmentation in rats between 16-32 days of age but not in younger and adult animals demonstrating an age-related change in gonadotropin dependence. To identify the exact testis cell type undergoing apoptosis, in situ analysis of DNA fragmentation was performed. In rats at 16-24 days of age, spermatocytes in selected tubules were found to have increased DNA fragmentation. In contrast, neither Leydig cells nor Sertoli cells were affected. In 32-day-old and adult animals, increased DNA fragmentation was seen in early primary spermatocytes of some tubules. Treatment with GnRH antagonist increased the number of cells with DNA fragmentation as well as percentage of tubules affected. In animals between 16-32 days of age, meiotic spermatocytes were labeled, whereas early spermatids were also labeled in 24- and 32-day-old animals. In adult animals, the level of apoptotic DNA fragmentation was not affected by GnRH antagonist treatment. However, DNA isolated from specific stages of the seminiferous tubules of adult animals showed stage-specific changes of apoptotic DNA fragmentation with 2-fold higher levels found in stages I and XII-XIV compared with stage VIII. In situ analysis of adult testis demonstrated that spermatocytes were the major cell type affected. In conclusion, the present study demonstrated that at least three factors determine the onset of apoptosis of the male germ cells: 1) the developmental stage of the animal; 2) serum levels of gonadotropins, especially FSH; and 3) specific stage of the seminiferous epithelial cycle. The present approach provides the basis for future analysis of the role of gonadotropins and other factors in the regulation of testis cell degeneration in normal and pathological states.