Laser-induced immobilization and plasma membrane permeabilization in humanspermatozoa

We evaluated the potential use of a non-contact, 1.48 mu m wavelength diode laser for immobilization of human spermatozoa and permeabilization of the sperm membrane in different culture media. When we applied a single laser s hot near to the middle region of the sperm tail, spermatozoa could be immob ilized either temporarily or permanently, depending on the energy used. Abo ve an energy of 2 mJ in polyvinylpyrrolidone and 2-3 mJ in culture medium, a reliable permanent immobilization was achieved by permeabilization of the sperm tail membrane. We then explored the use of a double laser shot techn ique. Spermatozoa were temporarily immobilized by a first laser shot applie d near to the sperm tail followed by permeabilization with a second laser s hot aimed directly at the sperm tail. This sequential approach yielded perm anent immobilization at much lo ver energy values compared with the single shot technique. Following the injection of laser-treated spermatozoa, mouse oocytes underwent normal activation and pronuclear formation. We conclude that a non-contact 1.48 mu m diode laser system can be used for immobilizat ion of spermatozoa and for permeabilization of the sperm tail membrane. Thi s laser procedure may offer an alternative to currently used sperm pretreat ment prior to intracytoplasmic sperm injection.