Gossypol-Induced Damage to Mitochondria of Transformed Sertoli Cells JOHN M. ROBINSON, PhD, NONGNUJ TANPHAICHITR, PhD, and ANTHONY R. BELLVE, PhD Studies on gossypol-induced morphologic changes in transformed Sertoli cells (TM4) were performed at both light- and electron-microscopic levels. Exposure ofTM4 cells to 5 gg gossypol/ml for >1 hour has severe, delete- rious effects on the structure and function of mitochon- dria. Mitochondrial function in TM4 cells was monitored by employing a fluorochrome, Rhodamine 123, which accumulates rapidly in mitochondria having a high trans- membrane potential. In gossypol-treated TM4 cells, Rhodamine 123 mitochondrial staining was reduced significantly 1 hour after the drug addition and reached a minimal level at 3 hour. Concomitantly, cytoplasmic GOSSYPOL, a plant disesquiterpene found in cotton- seed, has been used as a male contraceptive in China.1-4 The compound inhibits spermatogenesis as well as sperm motility in man. The antifertility action of gos- sypol has also been demonstrated to occur in rats, mice, hamsters, and cynomolgus monkeys.2 Mature male rats force-fed with gossypol (20 mg/kg body weight) daily for 3 weeks exhibit epididymal spermatozoa that were malformed, having degenerated mitochondria and dis- placed outer dense fibers.5 Alterations in mitochondria were observed in advanced spermatids of Steps 18 and 19. Spermatogenesis halts after 4 weeks of this gossypol administration67'; damage was evident in several or- ganelles, such as nuclear vacuolation of pachytene sper- matocytes and detachment of sperm acrosomes.' The initial impairment of sperm mitochondria ob- served in gossypol-treated animals suggests that these organelles may be the primary targets of the drug's ac- tion. Other in vitro studies, performed mostly at gos- sypol concentrations >50 jM (25 jg/ml) also support this hypothesis.8-'7 Exposure of ejaculated or epididy- mal sperm to gossypol causes a decrease in the activities of mitochondrial-related enzymes, including sperm- specific LDH-C4,8'9 malate dehydrogenase, glutathione- From the Department of Pathology, Physiology and Biophysics, and Obstetrics and Gynecology and the Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, and Dana Biomedical Institute, Beth Israel Hospital, Boston, Massachusetts vacuoles were detected even at the light-microscopic level. Electron-microscopic studies revealed that these vacuoles were distended mitochondria. The morphology of these damaged organelles changed gradually, starting with the transformation of the tubular mitochondria into the rounded forms. Cristae concurrently collapsed onto the organelles' periphery. In addition, the ground matrixes disappeared, and the mitochondria appeared as empty vacuoles. Further evidence that these vacuoles were dis- tended mitochondria was derived from the cytochemi- cal localization of cytochrome c oxidase in these vacuole- like structures. (Am J Pathol 1986, 125:484-492) S-transferase, fumarase, and pyruvate dehydrogenase.8 Human spermatozoa exposed to gossypol convert fruc- tose to CO2 at a reduced rate, suggesting a defect in glycolysis and/or mitochondrial function in the Krebs' cycle.10,11 The drug also perturbs sperm mitochondrial transmembrane potential,"2 possibly by interacting with the mitochondrial membrane. In accordance with this observation, oxygen consumption of gossypol-treated sperm is altered from the control levels.'3 In addition, gossypol may interact with the lipid bilayer of the sperm plasma membrane. " Any of these adverse effects may contribute to the marked decrease in sperm motility'5-'- and capacitation.'4 "8 Sertoli cells play important roles in promoting sper- Supported in part by Rockefeller Foundation Grant RF82014, NIH Grant AL 17945, and Center Grant HD06645. Accepted for publication July 11, 1986. Dr. Bellve's address is Center of Cellular and Molecular Urology, College of Physicians and Surgeons of Columbia University, 630 W. 168th Street, New York, NY 10032. Address reprint requests to John M. Robinson, PhD, Department of Pathology, Harvard Medical School, 25 Shat- tuck Street, Boston, MA 02115. 484