| Co-Investigator(Kenkyū-buntansha) |
ASO Takeshi Tokyo Medical and Dental University, Department of Comprehensive Reproductive Medicine, Professor, 大学院医歯学総合研究科, 教授 (60093176)
KUBOTA Toshiro Tokyo Medical and Dental University, Department of Comprehensive Reproductive Medicine, Assistant Professor, 大学院医歯学総合研究科, 助教授 (50126223)
KOI Hideki Tokyo Medical and Dental University, Department of Comprehensive Reproductive Medicine, Research Associate, 大学院医歯学総合研究科, 助手 (20280969)
坂田 優 東京医科歯科大学, 大学院・医歯学総合研究科, 助手 (10334442)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2003: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2002: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2001: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
1)Analysis of Ca^<2+> currents in spermatocytes from mice lacking Ca_v2.3 (α_<1E>) Ca^<2+> channel.
Objective
Calcium 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.
Design
Whole-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 Methods
Male 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.
Results
Whole-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.
Conclusion
These 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 sperm
BACKGROUND :
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
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