. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. CELL MOTILITY 227 Reference: Biol. Bull 193: 227-22:1 (October, 1997) Effect of Gossypol on Spisula Sperm Observed with Real-time Confocal Microscopy, Polarized Light Microscopy, and Video Microscopy Phong T. Tran\ Ana DePina2, Sliinva Inoiie (Marine Biological Laboratory, Woods Hole, Massachusetts 02543), Rudolf Oldenbourg, Sheldon J. Segal, and Mario H. Burgos* Ingestion or injection of gossypol suppresses sperm produc- tion and sperm motility in vivo (reviewed in ref. 1). We report here an in vitro assay for observing gos
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. CELL MOTILITY 227 Reference: Biol. Bull 193: 227-22:1 (October, 1997) Effect of Gossypol on Spisula Sperm Observed with Real-time Confocal Microscopy, Polarized Light Microscopy, and Video Microscopy Phong T. Tran\ Ana DePina2, Sliinva Inoiie (Marine Biological Laboratory, Woods Hole, Massachusetts 02543), Rudolf Oldenbourg, Sheldon J. Segal, and Mario H. Burgos* Ingestion or injection of gossypol suppresses sperm produc- tion and sperm motility in vivo (reviewed in ref. 1). We report here an in vitro assay for observing gossypol action on individ- ual sperm. A sample of the sperm suspension was added to a perfusion chamber consisting of a polylysine-coated coverslip and a glass slide separated by two strips of double-sticky tape. Sperm from the bivalve mollusc, Spisula sulidisxima, were sus- pended in a solution of tillered seawater and Eosin-B. Eosin-B stains the nucleus of only dead sperm red (2). and it therefore serves as a marker for dead sperm. The sperm heads adhered tightly to the coverslip, often leaving the flagella free to move in solution. Filtered seawater containing 0-100/jM gossypol was perfused through the chamber. Observations were performed either with a real-time confocal microscope (3), the ' University of North Carolina. Chapel Hill. NC 27599. : Dartmouth College. Hanover. NH 03755. 3 Institute de Histologia y Embriologia. Universidad Nacional de Cuyo. Mendoza, Argentina. Pol-Scope polarized light microscope (4). or a bright field video microscope. Each of the above microscopy techniques allow for observa- tion of different aspects of sperm morphology, motility, and vitality. The real-time confocal, by imaging the fluorescence of Eosin-B-stained sperm, allows comparison of morphological changes between control and gossypol-treated sperm. The Pol- Scope allows observation of cellular fine-structures. Finally, bright field video microscopy has high temporal resolution (30
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