The Effects of Growth Factor on Testicular Germ Cell Apoptosis in the Stallion

Apoptosis is necessary for both initiation and control of spermatogenesis; however, an increase in apoptosis can lead to subfertility/infertility in stallions, causing substantial financial loss in the equine industry. The ability of stem cell factor (SCF), leukemia-inhibiting factor (LIF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and estradiol (E2), alone or in combination, to prevent apoptosis of germ cells in short-term equine testicular cultures was examined. Testicular tissue was sectioned into approximately 2-mm cubes and placed in media-filled culture chambers. Concentrations of SCF (100 ng/mL), LIF (10 ng/mL), GM-CSF (5 ng/mL), and E2 (10−9 mol/l) were added alone or in combination to each well. After 6 hours in culture, the tissue was fixed and immunohistochemically (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) stained for apoptosis detection. Apoptotic cells per 100 Sertoli cell nuclei within seminiferous tubules were counted until the 500th Sertoli cell nuclei was reached. This counting procedure was used for each slide. An analysis of variance (ANOVA) with a Tukey's test was used to compare apoptotic rates. In comparison with the control, GM-CSF alone lowered apoptosis by 34.77%. GM-CSF–treated tissue combined with SCF and LIF as well as GM-CSF combined with SCF, LIF, and E2 reduced apoptosis when compared with the control (37.45% and 44.40%, respectively) or other treatment combinations. GM-CSF alone reduced apoptosis; results suggest possible synergy for the combinations of SCF and LIF with GM-CSF and for E2 with SCF, LIF, and GM-CSF.