Study of sperm fertilizing ability and intracellular calcium of sperm

1)Ca_v2.3（α_）Ca ^&lt;2 +&gt;通道缺失小鼠精母细胞Ca ^&lt;2 +&gt;电流分析精子内钙内流是受精成功的先决条件。<1E>电压依赖性Ca ^&lt;2 +&gt;通道（VDCC）被认为是Ca ^&lt;2 +&gt;内流的重要途径之一，尽管其在精子功能中的生理作用尚未阐明。在哺乳动物雄性生殖系细胞中，低电压激活（LVA）的Ca ^2+电流已被发现，并对其电生理特性进行了研究。虽然最初提出Ca_v2.3通道产生LVA电流，但Ca_v2.3通道的身份尚未完全澄清。为了研究电压依赖性Ca ^&lt;2 +&gt;通道α_12.3（α_）亚基是否编码LVA电流，本研究采用全细胞膜片钳和逆转录聚合酶链反应（RT-PCR）技术，对Ca_v2.3 +/+和Ca_v2.3-/-小鼠粗线期精母细胞进行了研究。<1E> ...更多信息 材料和方法在所有实验中以盲法使用具有C57 BL/6和129/Sv杂交背景的雄性野生型（Ca_v2.3 +/+）或纯合突变型（Ca_v2.3-/-）小鼠（8 - 20周龄）。在室温（23.26 ℃）下，用放大器（EPC-8，HEKA，德国）以膜片钳全细胞模式记录Ca ^&lt;2 +&gt;电流。结果急性分离的Ca_v2.3 +/+和Ca_v2.3-/-小鼠粗线期精母细胞的全细胞电流显示出典型的LVA Ca ^&lt;2 +&gt;电流和动力学特征，但无显著性差异。单细胞RT-PCR结果显示，Ca_v2.3 +/+和Ca_v2.3-/-小鼠粗线期精母细胞中有Canalgin的表达，并记录到LVA Ca ^&lt;2 +&gt;电流。相反，Ca_v3家族如Ca_v3.1可能是负责粗线期精母细胞LVA电流的候选者。2）人类精子的超激活与细胞内钙的关系背景：孕酮通过与质膜结合来调节精子的许多功能。超活化被认为是获能过程的一部分。为探讨孕酮引起的精子胞内钙（[Ca ^&lt;2 +&gt;] i）升高与精子超活化的关系，本研究采用Burkman标准，测定孕酮对精子胞内钙反应的影响，同时测定精子顶体反应率.结果：孕酮引起的精子胞内钙升高与精子超活化率呈显著正相关（r = 0.685，p &lt;0.0001）. [Ca[Ca ^&lt;2 +&gt;]_i、孕酮给药后[Ca ^&lt;2 +&gt;]_i从基线到平台期的增加（r = 0.668，p &lt;0.0001）和过夜孵育后[Ca ^&lt;2 +&gt;]_i的基线浓度也与高活化精子的百分比呈正相关（r = 0.500，n = 60，p &lt;0.0001）。孵育过夜后，超活化精子的百分率和顶体反应精子的百分率增加。[Ca ^2 +] i从基线到峰值的增加与VAP（平均路径速度）、VSL（直线速度）、VCL（曲线速度）、ALH（头侧位移幅度）呈正相关，与BCF（搏动交叉频率）呈负相关。[Ca ^&lt;2 +&gt;]_i升高与VAP、VSL、VCL、ALH、BCF之间也存在相关性。少

1)Analysis of Ca^<2+> currents in spermatocytes from mice lacking Ca_v2.3 (α_<1E>) Ca^<2+> channel.ObjectiveCalcium influx into sperm is a prerequisite for successful fertilization. The voltage-dependent Ca^<2+> channel (VDCC) is thought to be important as one of the pathways of this Ca^<2+> influx, although its physiological role in sperm function has not been clarified. In mammalian male germ-line cells, low-voltage-activated (LVA) Ca^<2+> current has been identified and its electrophysiological properties have been studied. Although Ca_v2.3 channel was initially suggested to generate an LVA current, the identity of the Ca_v2.3 channel has not been completely clarified yet. To investigate whether α_12.3 (α_<1E>) subunit of the voltage-dependent Ca^<2+> channel codes for the LVA current, this study was conducted.DesignWhole-cell patch clamp and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed in pachytene spermatocytes from Ca_v2.3+/+ and Ca_v2.3-/-m … More ice.Materials and MethodsMale wild-type (Ca_v2.3+/+) or homozygous mutant (Ca_v2.3-/-) mice with a hybrid background of C57BL/6 and 129/Sv (aged 8 to 20 weeks) were used in all the experiments in a blind manner. The Ca^<2+> currents were recorded at room temperature (23.26℃) by means of a whole-cell mode of the patch clamp recording with an amplifier (EPC-8, HEKA, Germany). Also, reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed in pachytene spermatocytes from Ca_v2.3+/+ and Ca_v2.3-/-mice.ResultsWhole-cell current in acutely dissociated pachytene spermatocytes from Ca_v2.3+/+ and Ca_v2.3-/-mice displayed a typical profile of LVA Ca^<2+> currents and kinetics with no significant differences. Single-cell RT-PCR revealed the expression of Cacnalgin the pachytene spermatocytes from Ca_v2.3+/+ and Ca_v2.3-/- mice in which LVA Ca^<2+> currents were actually recorded.ConclusionThese results suggest that the Ca_v2.3 channel makes no detectable contribution to the LVA Ca^<2+> current in the pachytene spermatocyte. Instead, Ca_v3 family such as Ca_v3.1 may be the likely candidates responsible for the LVA currents in pachytene spermatocytes.2)Relationship between hyperactivation and intracellular calcium of human spermBACKGROUND :Progesterone has been reported to modulate numerous sperm functions through the binding of progesterone to plasma membrane. Hyperactivation is recognized as a part of the process of capacitation. In order to investigate the relationship between progesterone evoked intracellular calcium ([Ca^<2+>]_i) increase and hyperactivation, this study was conducted.METHODS :Progesterone was added to human sperm and the percentages of hyperactivated sperm, based on the Burkman's criteria, the changes of intracellular calcium response, and the percentages of acrosome reacted sperm were measured.RESULTS :There was a significant positive correlation between [Ca^<2+>]_i increase from baseline to peak after the administration of progesterone and the percentage of hyperactivated sperm (r=0.685, p<0.0001). [Ca^<2+>]_i, increase from baseline to plateau after the administration of progesterone (r=0.668, p<0.0001) and the baseline concentration of [Ca^<2+>]_i after overnight incubation was also positively correlated with the percentage of hyperactivated sperm (r=0.500, n=60, p<0.0001). The percentage of hyperactivated sperm and the rate of acrosome reacted sperm were enhanced after overnight incubation. Progesterone induced acrosome reaction and [Ca^<2+>]_i increase were also dose dependent [Ca^<2+>]_i increase from baseline to peak was positively correlated with VAP (average path velocity), VSL (straight line velocity), VCL (curvilinear velocity), ALH (amplitude of lateral head displacement), and negatively correlated with BCF (beat cross frequency). This relationship was also observed between [Ca^<2+>]_i increase from baseline to plateau and VAP, VSL, VCL, ALH, BCF.CONCLUSIONS :These data suggested that the occurrence of hyperactivation is closely related with the elevation of intracellular calcium. Less