Experimental Research Of Influence Of K~+ Channel During Human Sperm Volume Regulation In Vitro

Object:Mammalian and human sperm spermatozoa passing through the epididymis experience increasing osmolality in the luminal environment and mature cells are stored in fluids hyper-osmotic to serum. When ejaculated into the female tract, they encounter a hypo-osmotic challenge which initiates the process of regulatory volume decrease (RVD).The K+ channel take part in this process. In the present study,we used the K+ channel blocker quinine blocking the K+ channel to observe involvement and mechanisms of separate K+ channel in sperm RVD.Methods:First, we select human sperm samples which parameter is normal and every sperm was optimized by gradient centrifugation. After optimization,each sperm was divided into two parts, the first part of the optimized sperm was divided into five groups, respectively, were added to the osmotic pressure of 200,275,290,310,330 mmol/kg BWW solution,of which the 200,275,290 mmol/kg groups were each divided into three groups again:as quinine concentrations were 0.1mmol/L,0.3mmol /L and control group,11 groups of sperm were incubated in water bath at 37℃,after 30 minutes,these sperms were analysed by CASA. Then these eleven samples of sperm suspension were smeared and stained with Diff-Quik method.Then the rate of tail coiling of sperm sample was counted by microscope. The second part of the sperm after optimization was divided into two groups:the first group was made a final concentration of quinine by 0.3mmol/L, the second group was the control group. Two groups of sperm were incubated in water bath at 37℃,after 30 minutes, both groups take Flow Cytometry to reflect the sperm volume by the meaning of FSC.Results:1) The experimental results showed that as osmolarity increased from 200mmol/Kg to 290mmol/Kg, VCL of sperm showing a rising trend, but the difference was not significant (P> 0.05), but with the increasing of concentration of quinine,VCL of sperm had significant changed (P< 0.05); with the osmolarity increased from 200mmol/Kg to 290mmol/Kg, LIN of sperm had no significant changed (P> 0.05). With the increasing of concentration of quinine, LIN of sperm went on declining, there are significant differences (P<0.05);AS osmolarity increased from 200mmol/Kg to 275mmol/Kg, there are significant differences in sperm VSL (P <0.05), although as the osmolarity rose from 275mmol/Kg to 290mmol/Kg, the in VSL of sperm increase, the difference was not significant (P> 0.05). In the 275 mmol/Kg group,with quinine concentration increased, sperm VLS downward trend had significant difference (P<0.05); With the rise of osmolarity and quinine concentration, sperm ALH had no significant changes (P> 0.05).2) Of the 200,275,290 mmol/Kg groups,as the concentration of quinine increased, the sperm tail coiling rate increased, the difference is significant (P<0.05), In the 290mmol/Kg group, with quinine concentration increased, the rate of sperm tail coiling significantly different (P<0.01).Of the quinine blank groups, with the osmotic pressure increased from 200mmol/Kg to 290mmol/Kg, the rate of sperm tail coiling decreased gradually, which had significant differences (P<0.05); Of all the quinine 0.1 mM quinine groups, with the osmolarity increased, the rate of sperm tail coiling decreased gradually, whicn had significant differences (P<0.05); q Of all the quinine 0.1 mM quinine groups, with the osmolarity increased, the rate of sperm tail coiling decreased gradually, whicn had significant differences (P<0.05).3)measure FSC values of control group, Triton, and 0.3 mM quinine treatment groups by flow cytometry,then, calculate the difference of FSC between Triton treatment and control group and the difference of FSC between Triton treatment and 0.3mM quinine group, statistical results showed that there was significant difference (P <0.01).Conclusion:After quinine blocking potassium channels of sperm,RVD regulation of sperm function was disordered,if these sperm came into the hypotonic environment where was similar to the female reproductive tract, sperm volume increased, the sperm tail became coiling, momentum from flagellum swing decreased and result in Sperm VCL, VSL and LIN reduction, these change directly affected sperm motility,。reduced exercise capacity directly affected the sperm penetration of cervical mucus barrier and the migration of sperm in the fallopian tubes, which will result in the occurrence of male infertility. RVD regulation of sperm dysfunction may be one of the important reasons for male infertility.