Study of sperm fertilizing ability and intracellular calcium of sperm

1) Ca_v2.3 (α_<1E>) Ca^<2+>通道缺失小鼠精母细胞Ca^<2+>电流分析。目的精子内钙流入是成功受精的先决条件。电压依赖性Ca^<2+>通道（VDCC）被认为是Ca^<2+>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明确。在哺乳动物雄性生殖系细胞中发现了低电压激活（LVA） Ca^<2+>电流，并对其电生理特性进行了研究。虽然最初建议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^<2+>通道的α_12.3 （α_<1E>）亚基是否编码LVA电流，本文进行了研究。设计采用全细胞膜片钳和逆转录聚合酶链反应（RT-PCR）对Ca_v2.3+/+和Ca_v2.3-/-m厚线精母细胞进行实验。材料与方法采用8 ~ 20周龄C57BL/6和129/Sv杂交背景的野生型（Ca_v2.3+/+）或纯合突变型（Ca_v2.3-/-）小鼠，均采用盲法。在室温（23.26℃）下，采用膜片钳全细胞模式记录Ca^<2+>电流，放大器（EPC-8， HEKA，德国）。同时，对Ca_v2.3+/+和Ca_v2.3-/-小鼠粗线精母细胞进行逆转录聚合酶链反应（RT-PCR）实验。结果Ca_v2.3+/+和Ca_v2.3-/-小鼠急性分离粗线精母细胞的全细胞电流表现为典型的LVA Ca^<2+>电流和动力学，无显著差异。单细胞RT-PCR结果显示Ca_v2.3+/+和Ca_v2.3-/-小鼠粗线精母细胞中Cacnalgin的表达，并记录了LVA Ca^<2+>电流。结论Ca_v2.3通道对粗线精母细胞LVA Ca^<2+>电流无明显影响。相反，Ca_v3家族，如Ca_v3.1可能是粗线精母细胞中LVA电流的可能候选人。背景：据报道，孕酮通过与质膜结合，调节多种精子功能。过度激活被认为是获能过程的一部分。为了探讨黄体酮引起的细胞内钙（[Ca^<2+>]_i）升高与细胞过度活化的关系，本实验进行。方法：在人精子中加入黄体酮，根据伯克曼标准，测定细胞内钙反应的变化和顶体反应精子的百分比。结果：孕酮给药后[Ca^<2+>]_i从基线至峰值升高与高活精子百分比呈正相关（r=0.685, p<0.0001）。孕酮给药后[Ca^<2+>]_i从基线到平台增高（r=0.668, p<0.0001），过夜孵育后[Ca^<2+>]_i的基线浓度也与高激活精子百分比呈正相关（r=0.500, n=60, p<0.0001）。孵育过夜后，高活化精子的比例和顶体反应精子的比例均有所提高。黄体酮诱导顶体反应和[Ca^<2+>]_i升高也呈剂量依赖性[Ca^<2+>]_i从基线到峰值升高与VAP（平均路径速度）、VSL（直线速度）、VCL（曲线速度）、ALH（侧头位移幅度）呈正相关，与BCF（心跳交叉频率）负相关。从基线到平台的[Ca^<2+>]_i升高与VAP、VSL、VCL、ALH、BCF之间也存在这种关系。结论：这些数据提示，过度活化的发生与细胞内钙的升高密切相关。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