Hyperosmotic tolerance of human spermatozoa: Separate effects of glycerol, sodium chloride, and sucrose on spermolysis

Abstract

Hyperosmotic stress, which cells experience during the freezing process, and its release during the warming process are both related to cryoinjury. To define optimal cooling or warming rates and prevent osmotic injury to human sperm, information is required regarding the osmotic tolerance of the cells as a function of 1) time, 2) temperature, 3) type of solute, and 4) solute concentration. Human sperm samples were divided into three aliquots. The aliquots were equilibrated at 0, 8, and 22°C, respectively. Different hyperosmotic solutions were prepared by addition of either a permeating cryoprotective agent (glycerol) or nonpermeating solutes (sucrose, non- ionic; or NaCl, ionic) to isotonic Mann's Ringer solution. Aliquots of the prepared solutions were equilibrated at 0, 8, and 22°C, respectively. A small volume (2.5 μl) of each sperm aliquot was quickly mixed with 50 μl of each hyperosmotic solution at the corresponding temperature. After times ranging from 5 s to 5 min, 10 μl of each hyperosmotic cell suspension was abruptly returned to an isosmotic environment by mixing with 500 μl of Mann's Ringer solution at the corresponding temperature. The plasma membrane integrity of cells after exposure to hyperosmotic stress and after return to isosmotic conditions was measured by a dual staining (carboxyfluoroscein diacetate and propidium iodide) technique and flow cytometry. The morphology of the treated cells was observed by scanning electron microscopy of freeze- substituted sperm. The results indicate that human spermatozoa exhibited a significant posthypertonic lysis/injury, i.e., loss of membrane integrity, when returned to isosmotic conditions after exposure to hyperosmotic solutions of NaCl or sucrose. The higher the hyperosmolality, the more serious the cell injury. The majority of the cells (> 50%) lost membrane integrity when the osmolality was ≥ 2000 mOsm. In contrast, if the sperm were not returned to isosmotic conditions, the majority of the sperm in the hyperosmotic solutions appeared to maintain membrane integrity. For a given higher hyperosmolality (> 1000 mOsm), posthypertonic spermolysis was reduced with a decrease of temperature. Cell survival was also affected by time of cell exposure to hyperosmotic environments before cells were returned to the isotonic condition. The shorter the time, the higher the cell survival. When exposed to hyperosmotic glycerol solutions that were isotonic with respect to electrolytes, few cells lost their membrane integrity if the osmolality of glycerol was < 3000 mOsm. For a fixed high osmolality (> 3000 mOsm), the lower the temperature, the higher the percentage spermolysis. At the highest glycerol concentration in this study, i.e., 4694 mOsm, the percentage spermolysis was 17%, 10%, and 2% at 0°C, 8°C and 22°C, respectively. Spermolysis caused by the removal of glycerol from the cells depended on the means by which the cells were returned to isotonic conditions. A one-step return to isotonic conditions resulted in serious spermolysis, while a multi- step (nine-step) procedure significantly reduced the spermolysis. The scanning electron micrographs showed the distinct morphology of the spermatozoa experiencing the different osmotic conditions. The abnormality of spermatozoa that underwent posthypertonic treatment was demonstrated especially clearly.