摘要
第一部分 钾离子通道在小鼠精子体外获能中的作用
以金霉素(chlortetracycline,CTC)荧光染色法评价小鼠精子的体外获能，并观察K~+离子载体缬氨霉素(Val)对小鼠精子体外获能的影响，CTC荧光染色后涂片观察，以B型精子的发生率来评价精子获能，B型精子发生率越高，获能越完全。
钾离子载体缬氨霉素能促使体细胞钾离子外流，实验以终浓度为50nmol·L-1的缬氨霉素处理小鼠精子，其B型精子发生率(％)与对照组相比显著增加(n=10，p＜0.05)，表明缬氨霉素能促进小鼠精子的体外获能，由于缬氨霉素能促使体细胞内钾离子外流，提示在精子体外获能过程中，钾离子外流是重要的一环。
进一步采用全细胞膜片箝技术记录小鼠粗线期精母细胞延迟整流钾电流(I_(DRK))，并分析其电流特性，同时观察缬氨霉素对I_(DRK)的影响。结果显示：在小鼠粗线期精母细胞上能记录到一个被缓慢激活
南京医科大学硕十学位论文
的外向钾电流，其幅度随去极化电压的升高而增大，该电流的上升幅
度具有明显的电压依赖性，但通道的开放没有明显的时间依赖性失
活。本实验使用的电极内液含有10mmol.L一’的caZ+鳌合剂EGTA，可
有效地防止胞内游离Ca2+浓度升高，因此该外向钾电流中基本不包含
Ca2+激活的钾通道电流，而且该电流对4一AP不敏感，但TEACI可显
著抑制该电流，因此可初步判断该电流为延迟整流钾电流(ID二)。
进一步观察到加入50nmol.L一’颧氨霉素后，小鼠生精细胞上延
迟整流钾电流显著增大，I一V曲线上调。分析实验结果:颧氛霉素通
过激活延迟整流钾通道，增加胞内钾离子的外流，从而使精子膜电位
发生超极化，进而促发小鼠精子体外获能。
第二部分氛戊菊醋对小鼠精子获能的影响及机制探讨
200川小鼠精子细胞悬液加入不同终浓度氰戊菊醋(Fen)染毒后，
eo:孵箱37℃培育2，J、时，结果显示:以终浓度(林mol·L一，)为。、
0.625、1 .25、2.5、5.0、10.0、20.0的Fen处理小鼠精子，其B
型精子发生率(%)分别为55.05士0.43、52.02士1 .29、47.13士0.87、
40.08士0.32、35.79士0.88、28.66土0.96、27.89士1 .56，半数有效剂量
(E DS。)为5尽mol·L一’。表明氰戊菊醋抑制小鼠精子体外获能，其作
用呈剂量一反应关系(P<0.05，n一12)，同时具有时间依赖性。
我们进一步探讨了Fen抑制小鼠精子体外获能机制，发现氰戊菊
醋可轻微抑制小鼠粗线期精母细胞延迟整流钾电流，提示氰戊菊醋主
要不是通过延迟整流钾离子通道途径影响精子体外获能。同时实验还
观察到，氰戊菊醋能显著抑制精子细胞膜的脂质流动性。我们选择l，
6一二苯已三烯(DPH)为荧光探针结合细胞染毒，测定小鼠精子细胞
膜脂质的荧光偏振度(P值)，来观察氰戊菊醋对精子细胞膜脂质流
动性的影响。结果表明，以O、0.625、1 .25、2.5、5.0、10.0、20.0
南京医科大学硕l学位论文
。molL一’的Fen处理小鼠精子，从2.5 0 mol·L一’起，氰戊菊醋即显著
增高小鼠精子细胞膜脂质DPH探针的荧光偏振度(P值)、降低膜脂
质流动度LFu(P<0.05，n=15):10抖mol·L一，一20林mol·L一，浓度的
氰戊菊醋组，其P值与LFU到达平台期。提示氰戊菊醋可增高小鼠
精子细胞膜脂质的荧光偏振度，降低细胞膜脂质流动性，并具有浓度
依赖关系。经相关分析发现，氰戊菊醋抑制小鼠精子体外获能及降低
精子细胞膜脂质流动性，这两者的作用剂量高度相关(:2=0 .968)，提
示氰戊菊醋可通过降低精子细胞膜的脂质流动性而抑制小鼠精子体
外获能。
Part one: Role of potassium channel on sperm in vitro capaictaion in mice
The primary aim of this study was to observe the correlation between the mouse sperm capacitaion state and K+ channel, and then to observe the effect of valinomycin (Val), a potassium ionophore, in sperm in vitro capacitation. Mouse in vitro sperm capacitation was assessed by the B pattern of staining by chlortetracycline (CTC), a Ca2+-dependent fluorescence microscopictechnique, and its is currently the assay of choice because it distinguishes five different stages of sperm activation: noncapacitated, capacitated acrosome-intact , capacitated acrosome-reacted, in an early stage of the AR and nonidentified sperm.
When spermatozoa were cultured in valinomycin (Val) at the
concentration of 50nmol.L-1, the capacitation was increased significantly (n=10,p<0.05).As we all know, valinomycin induces potassium efflux in cell, suggesting that potassium efflux is important in capatitation.
Then we recorded the delayed rectifier K+ currents(IDRK) in mouse pachytene spermatocyte using whole-cell patch clamp technique so as to determine their characteristics. Meanwhile we observed the effects of valinomycin (Val) on delayed rectified K+ currents presents in mouse pachytene spermatocyte. A outward current activated at -40mV was obtained when inward Ca2+ current and outward Ca2+-dependent potassium current were blocked by Cd2+and EGTA separated. The current increased dramatically when depolarization in creased from -40mV to +70 mV, without any deactivation in 400MS. And it had an outwardly rectifying current-voltage relationship. According to the characteristics of the current, we primarily inferred that it was delayed rectifier K+ current. Furthermore , 4-AP, a know transient inactivation K+ current blocker, did not significantly affect these K+ currents. The current were blocked by 20mmol.L-1 TEAC1, a know voltage-dependent K+ currents. These observations suggest that the outward K+ currents are generated by activate relayed rectifier K+ channels.
We found that 50nmol.L-1 Val increased the amplitude of IDRK currents significantly, up-regulated the I-V curve of IDRK current. These results suggest that valinomy promotes potassium ion efflux by activation of delayed rectifier potassium channels in mouse pachytene spermatocyte, and then mediated hyperpolarization itself appears capable of inducing sperm capacitation.
Part two: Effect of fenvalerate on sperm in vitro capaciation in mice and its mechanism
When spermatozoa were cultured in various fenvalerate
concentrations(umol .L-1, 0, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0) for 2 h in an atmosphere of 5%co2 in air at 37C, the percentage of spermatozoa showing pattern B was 55.05 + 0.43, 52.02+1.29, 47.13 + 0.87, 40.08+0.32, 35.79 + 0.88,28.66 + 0.96,27.89+1.56 separately, the ED50 is 5 u mol.L/-1.This suggest that fenvalerate can inhibit capacitation was in a concentration-dependent manner. In the same time we also observed that fenvalerate can inhibit capacitation in a time-dependent manner.
To study the mechanism of fenvalerate's action, we observe the effect of fenvalerate on IDRK channels in moue pachytene spermatocyte, was observed using patch clamp technique. The results showed that fenvalerate had a slightly inhibitory effect on the currents presented in moue pachytene spermatocyte, even though at higher fenvalerate concentration. These results indicated that 1DRK channel was not the primary regulation for fenvalerate to inhibit sperm capacitation.
Insecticides have been shown to partition into membrane and cause change in membrane fluidity. So at the same time we monitored the fluidity parameters to reveal the dynamic changes in sperm membrane due to fenvalerate interaction by measuring the fluorescence polarization of l,6-diphlnyl-1,3,5-hextriene DPH incorporated into the membrane. Fenvalerate was found to increase the DPH fluorescence polarization value in a concentration-dependent manner, implication that it makes the membrane less fluid. These results suggest that
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