Exogenous stem cell factor (SCF) compensates for altered endogenous SCF expression in 2,5-hexanedione-induced testicular atrophy in rats.

2,5-Hexanedione (2,5-HD) is a Sertoli cell toxicant that causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few committed type A spermatogonia remain in the seminiferous epithelium. A majority of the stem cell progeny differentiate into type A spermatogonia, but then, rather than continuing to differentiate, undergo apoptosis. We hypothesized that the cause for germ cell apoptosis was, at least in part, a deficiency in the function of stem cell factor (SCF), a paracrine growth factor normally made by Sertoli cells. To test this hypothesis, rats were exposed to 1% 2,5-HD for 5 wk and killed at various times after toxicant exposure. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine that, unlike what was observed in control testes, the majority of SCF was expressed in the soluble form after 2,5-HD injury. In vitro co-culture experiments were used to establish the appropriate dose of SCF to administer in vivo. A continuous intratesticular delivery system was established and used to expose 2,5-HD-treated rats to SCF for 2 wk. Animals were exposed to bromodeoxycytidine (BrdCyd) for 2 days before being killed in order to assess the effect of SCF on germ cell proliferation. SCF caused a statistically significant increase in the number of germ cells positive for bromodeoxyuridine (BrdUrd), indicating that SCF promoted survival and/or stimulated proliferation of the remaining germ cells. We conclude that SCF expression is disrupted after 2,5-HD-induced testicular atrophy and that exogenous administration of SCF promotes recovery of spermatogenesis.

Fate of germ cells in 2,5-hexanedione-induced testicular injury. II. Atrophy persists due to a reduced stem cell mass and ongoing apoptosis.

The Sertoli cell toxicant 2,5-hexanedione (2,5-HD) causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few spermatogonia remain in the seminiferous epithelium. In this study, the number, type, and fate of the remaining germ cells were determined. Male Sprague-Dawley rats were exposed to 1% 2,5-HD in drinking water for 5 weeks and then sacrificed 12 or 40 weeks after the start of exposure. Cell counts determined that the stem cell population was diminished in size, but made up a significant portion of the remaining germ cells. The remaining germ cells were primarily type A spermatogonia. Modeling of spermatogonial divisions suggested that most spermatogonia undergo degeneration at the level of type A3 spermatogonia after 2,5-HD-induced atrophy. Apoptosis was demonstrated to occur in the remaining germ cells by nuclear morphology and in situ analysis of DNA fragmentation. Quantitation indicated that apoptosis occurred in a majority of stem cell progeny. We conclude that the irreversibility of 2,5-HD-induced testicular injury results from the reduced size of the stem cell population as well as a block in germ cell development at the level of type A spermatogonia.

Fate of germ cells in 2,5-hexanedione-induced testicular injury. I. Apoptosis is the mechanism of germ cell death.

2,5-hexanedione (2,5-HD) is a Sertoli cell toxicant which causes germ cell loss and testicular atrophy in the rat. The mechanism of germ cell death over the course of 2,5-HD treatment is not known nor is the reason why residual germ cells do not repopulate the seminiferous epithelium following toxicant withdrawal. In the current study, the role of apoptosis in germ cell loss was studied. Male Fischer rats were treated for up to 5 weeks with 1% 2,5-HD in the drinking water and killed between 0 and 12 weeks after the start of toxicant exposure. Apoptosis was assessed in control and treated animals by (1) DNA fragmentation detected by gel electrophoresis, (2) cellular morphology on plastic sections, and (3) DNA fragmentation in situ by terminal deoxy-nucleotidyl transferase-mediated digoxigenin-UTP nick end label (TUNEL) staining of testis cross sections. All three indices demonstrated a substantial increase in apoptosis which peaked at 5 weeks of 2,5-HD treatment. Morphological analysis determined that apoptosis occurred in germ cells of the seminiferous epithelium. DNA fragmentation determined by gel electrophoresis was barely detectable until 5-6 weeks of toxicant exposure. However, TUNEL staining of testis cross sections indicated that germ cell apoptosis increased after as early as 2 weeks of toxicant exposure, providing a highly sensitive biological marker of toxicant-induced testicular injury. These data also suggested a differential sensitivity of germ cells to toxicant exposure with spermatid apoptosis occurring first at 4-5 weeks of treatment followed by apoptosis of spermatocytes and spermatogonia between 6 and 12 weeks. Together, these data demonstrate that apoptosis is the mechanism of germ cell loss in 2,5-HD-induced testicular injury.

Stem cell kinetics in rat testis after irreversible injury induced by 2,5-hexanedione.

Stem cells provide a continuous supply of committed progenitor cells for the process of spermatogenesis. In rodents, stem cells have been identified as single, undifferentiated type A spermatogonia. The rate of stem cell division has not been definitively determined because of difficulty in locating stem cells among a normal compliment of germ cells. The testicular toxicant 2,5-hexanedione (2,5-HD) induces irreversible testicular atrophy with only Sertoli cells and spermatogonia remaining after injury. Stem cell kinetics could be assessed in this toxicant model because of the absence of most mature germ cells. It is also not known if 2,5-HD-exposed rats possess an active stem spermatogonia population. Charles River CD rats were exposed to 1% 2,5-HD in drinking water for 5 wk. At 7 or 35 wk following toxicant exposure, rats were exposed to bromodeoxycytidine continuously via Alzet mini-pumps for 1-28 days. Serial cross sections of testis were used to identify single stem spermatogonia and to determine whether the cells were positive or negative for bromodeoxyuridine incorporation. We obtained a continuous labeling index for stem cells from rats 7 and 35 wk after 2,5-HD exposure and found that stem cells had a cell cycle time of approximately 8-14 days at both time points after toxicant exposure. In conclusion, we have developed a method for the assessment of stem cell kinetics and verified the presence of an actively dividing stem cell population in irreversibly injured testes.

Colchicine disrupts the cytoskeleton of rat testis seminiferous epithelium in a stage-dependent manner.

Sertoli cell microtubules play an important role in the process of spermatogenesis. We investigated the effects of colchicine, a microtubule-disrupting agent, on the seminiferous epithelium. Rats were injected intratesticularly with 0.004-40 micrograms colchicine/testis. Colchicine had a dose-related effect on seminiferous tubule fluid secretion and completely blocked secretion at a dose of 40 micrograms colchicine/testis. Colchicine also resulted in a dose-related decrease in testes weight 2 and 8 wk after injection. When 40 micrograms colchicine/testis was used, testis morphology showed a time-dependent increase in the incidence of sloughing over a time course of 1, 3, 6, and 16 h. Quantitative analysis demonstrated that stage IX-XIV seminiferous tubules were most sensitive to sloughing. Changes in the distribution of tubulin immunostaining within Sertoli cells occurred preferentially in stage VII-VIII seminiferous tubules, which were most resistant to sloughing. In addition, colchicine resulted in disruption of vimentin filaments in stage IX-XIV seminiferous tubules, which correlated with the stage-dependent sensitivity of sloughing. We propose that the stage dependence of colchicine-induced effects reflects the dynamic and stage-dependent role of microtubules in spermatogenesis. Furthermore, cellular structures other than microtubules, such as vimentin filaments, may be important for maintaining the structural integrity of the seminiferous epithelium.