| Boar sperm is very sensitive to cold shock and oxidative damage during freezing-thawing, due to high content of unsaturated fatty acids in the phospholipids of the plasma membrane and its low resistance to oxidation. Frozen-thawed boar spermatozoa are less fertile than fresh or cooled semen and the reasons are not yet completely understood. Despite of that motility, structure and function of sperm are affected to some degree under cryopreservation, the physiological indices, i.e. motility, could still reach the required values; However, the pregnancy rate and farrowing rate of these sperms are relatively lower than those of normal ones. The mechanisms is still unclear. Especially, little attention has been paid on the relationship among the quality characteristics and activity of mitochondria, tyrosine phosphorylation and freezing-thawing. Six Landrace boars semen samples were chose in the present study. The straw frozen semen samples were created by diluent BTS and supplemented with OEP and a-tocopherol (VE) as cryoprotection and antioxidant. The phosphatidylserine in plasma membrane, mitochondrial membrane potential and sperm viability were determined by Annexin-V/PI, JC-1 fluorescent antibody kit and flow cytometry (FCM) correspondingly. We systematically analyzed the influence of different cryoprotection on frozen-thawed sperm function and tyrosine phosphorylation, and thus could get a relatively clear relationship among plasma membrane integrity, mitochondrial activity, elivision and antiprotection. This is crucial for assessment of fertilization ability in cryobiology of frozen-thawed boar sperm. Based on the obtained data, we found the following results,1. The mitochondrial membrane potential had a significant enhancement (p<0.05) after equilibrated and cryopreservated. The ratios of low mitochondrial membrane potential sperms in fresh, microthermal samples and thawing semen samples are 17.10%,37.89% and 94.44% respectively. The ratios of low mitochondrial membrane potential sperms increased 20.80% and 77.34% corresponding, after equilibrated and cryopreservated. It testifies that cryopreservation could increase depolarization of mitochondrial membrane potential, leading to significantly decrease in activity of sperm mitochondrial.2. Proportion of active sperm after cryopreservated (50.89%) was significantly lower than the fresh (86.87%) (p<0.05), including apoptosis-like sperm (15.93%) increased by about 5 percentage points camparing with the fresh (5.62%). It revealed the cryopreservation can not only decrease the mitochondrial membrane potential and membrane integrity of boar sperm, but also induce active sperm to go into a apoptosis-like trance3. Supplement of cryoprotection (OEP) and antioxidant (VE) improve membrane integrity, viability and mitochondrial activity (High mitochondrial membrane potential) of frozen-thawed sperm strongly, especially the combination of OEP and VE has a greater efficiency on improving the quality of frozen-thawed sperm.4. Sixteen boar sperm proteins are phosphorylated on tyrosine residues after being incubated in capacitating medium in vitro, their mollecular weight are approximately 126kDa, 108kDa,79kDa,69kDa,58kDa,50-42kDa,37kDa,34kDa,32kDa,26kDa,23kDa,20kDa, 15kDa,14kDa and 13kDa. Among these, the proteins of 50kDa to 42kDa have a higher level of expression and are constitutively phosphorylated on tyrosine during various capacitating or tempreture conditions. The tyrosine phosphorylated proteins with mollecular weight of approximately 23kDa,14kDa and 13kDa has not been reported so far.5. The tyrosine phosphorylation of frozen-thawed sperm is significantly lower than that in the fresh. Among them, of high mollecular weight proteins of 126kDa,108kDa,79kDa,69kDa, 58kDa and 32kDa have a obviously decrease in the tyrosine phosphorylation.6. After being supplemented with OEP and VE, the tyrosine phosphorylations of small mollecular weight proteins are greater than that control group. The tyrosine phosphorylation of proteins with mollecular weight about 13kDa and 37kDa are the greatest. Camparing with OEP, VE improves the tyrosine phosphorylation of proteins with mollecular weight 19kDa and 32kDa are more efficiency. Moreover, tyrosine phosphorylation of sperm proteins of mollecular weight 79kDa,58kDa,34kDa,32kDa,15kDa and 13kDa with treatment using the combination of OEP and VE are higer than the control.In frozen-thawed boar sperm, the correlation between the plasma membrane integrity and motility and tyrosine phosphorylation of protein with mollecular weight 32kDa is high.It reveals that cryopreservation could not only destruct mitochondrial plasma, but also increase the ratio of low mitochondrial membrane potential sperms. at the same time, tyrosine phosphorylation is significantly weakened, which might also be one of reasons for low natality for cryopreservated semen.
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