Dy. Gao et al., HYPEROSMOTIC TOLERANCE OF HUMAN SPERMATOZOA - SEPARATE EFFECTS OF GLYCEROL, SODIUM-CHLORIDE, AND SUCROSE ON SPERMOLYSIS, Biology of reproduction, 49(1), 1993, pp. 112-123
Hyperosmotic stress, which cells experience during the freezing proces
s, and its release during the warming process are both related to cryo
injury. 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 ) t
ype of solute, and 4) solute concentration. Human sperm samples were d
ivided into three aliquots. The aliquots were equilibrated at 0, 8, an
d 22-degrees-C, respectively. Different hyperosmotic solutions were pr
epared by addition of either a permeating cryoprotective agent (glycer
ol) or nonpermeating solutes (sucrose, non-ionic; or NaCl, ionic) to i
sotonic Mann's Ringer solution. Aliquots of the prepared solutions wer
e equilibrated at 0, 8, and 22-degrees-C, respectively. A small volume
(2.5 mul) of each sperm aliquot was quickly mixed with 50 mul of each
hyperosmotic solution at the corresponding temperature. After times r
anging from 5 s to 5 min, 10 mul of each hyperosmotic cell suspension
was abruptly returned to an isosmotic environment by mixing with 500 m
ul of Mann's Ringer solution at the corresponding temperature. The pla
sma membrane integrity of cells after exposure to hyperosmotic stress
and after return to isosmotic conditions was measured by a dual staini
ng (carboxyfluoroscein diacetate and propidium iodide) technique and f
low cytometry. The morphology of the treated cells was observed by sca
nning electron microscopy of freeze-substituted sperm. The results ind
icate that human spermatozoa exhibited a significant posthypertonic ly
sis/injury, i.e., loss of membrane integrity, when returned to isosmot
ic conditions after exposure to hyperosmotic solutions of NaCl or sucr
ose. The higher die hyperosmolality, the more serious the cell injury.
The majority of the cells (> 50% ) lost membrane integrity when die o
smolality was a 2000 mOsm. In contrast, if the sperm were not returned
to isosmotic conditions, the majority of the sperm in the hyperosmoti
c solutions appeared to maintain membrane integrity. For a given highe
r hyperosmolality (> 1000 mOsm), posthypertonic spermolysis was reduce
d with a decrease of temperature. Cell survival was also affected by t
ime of cell exposure to hyperosmotic environments before cells were re
turned to the isotonic condition. The shorter the time, the higher the
cell survival. When exposed to hyperosmotic glycerol solutions that w
ere isotonic with respect to electrolytes, few cells lost their membra
ne integrity if the osmolality of glycerol was < 3000 mOsm. For a fixe
d 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-degrees-C, 8-degrees-C, and 22-degrees-C, respectively. Sp
ermolysis 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 s
permolysis. The scanning electron micrographs showed the distinct morp
hology of the spermatozoa experiencing the different osmotic condition
s. The abnormality of spermatozoa that underwent posthypertonic treatm
ent was demonstrated especially clearly.