Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation

doi:10.1016/S1671-2927(11)60197-1

Journal of Integrative Agriculture ›› 2011, Vol. 10 ›› Issue (12): 1958-1967.DOI: 10.1016/S1671-2927(11)60197-1

Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation

ZHANG Zhen, ZHOU Xuan, LI Hui-xia, CUI Qun-wei, YU Jing , WANG Gen-lin

1.College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R.China

收稿日期:2011-01-13 出版日期:2011-12-01 发布日期:2011-12-19
通讯作者: Correspondence WANG Gen-lin, Tel: +86-25-84395045, Fax: +86-25-84395314, E-mail: glwang@niau.edu.cn
作者简介:ZHANG Zhen, E-mail: zzgxu@163.com
基金资助:
This study was supported by the National Key Technology R&D Program of China (2006BAD04A12).

Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation

ZHANG Zhen, ZHOU Xuan, LI Hui-xia, CUI Qun-wei, YU Jing , WANG Gen-lin

1.College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R.China

Received:2011-01-13 Online:2011-12-01 Published:2011-12-19
Contact: Correspondence WANG Gen-lin, Tel: +86-25-84395045, Fax: +86-25-84395314, E-mail: glwang@niau.edu.cn
About author:ZHANG Zhen, E-mail: zzgxu@163.com
Supported by:
This study was supported by the National Key Technology R&D Program of China (2006BAD04A12).

摘要/Abstract

摘要： CatSper is a unique Ca2+ channel-like protein family exclusively expressed in the testis and sperm, and plays important roles in sperm motility, capacitation, acrosome reaction and sperm-egg interactions. Here we studied the mechanism of regulation of CatSper2-dependent Ca2+ influx, extracellular and intracellular Ca2+ on sperm hyperactivated motility. The siRNA duplexes were transfected into the sperm cells by electroporation (EP) to silence the expression of CatSper2. The results for targeted disruption of CatSper2 showed the highest silence efficiency 77.7% (P<0.05), the hyperactivated sperm rate calculated by computer-assisted semen analysis (CASA) analysis of interferenced sperm was significantly lower 11.1% than the control 99.2%. Flow cytometry (FCM) detection of the intracellular Ca2+ concentration of interferenced sperm was 50 nmol L-1 higher than the normal. It was remarkably lower than hyperactivated sperm with 200-1 000 nmol L-1 (P<0.05). It was not sufficient to evoke hyperactivation. To trigger hyperactivation, the sperm were incubated in 50 μmol L-1 thimerosal or 5 mmol L-1 procaine, it sharply increased the intracellular Ca2+ via two different channels. CASA and FCM detection indicated that intracellular Ca2+ is required for initiating hyperactivation while extracellular Ca2+ is essential to maintain the process. We concluded that to mediate sperm hyperactivation, it is essential to inhibit Ca2+ peak present with targeted disruption of CatSper2 expression. This provides important prospective for further exploration of signal channel of sperm hyperactivated motility, potential factors for male infertility and provide further reference to the exploration of male contraceptive drug targets of male reproduction.

关键词: sperm hyperactivation motility, CatSper2, Ca2+ channel, RNAi, electroporation (EP)

Abstract: CatSper is a unique Ca2+ channel-like protein family exclusively expressed in the testis and sperm, and plays important roles in sperm motility, capacitation, acrosome reaction and sperm-egg interactions. Here we studied the mechanism of regulation of CatSper2-dependent Ca2+ influx, extracellular and intracellular Ca2+ on sperm hyperactivated motility. The siRNA duplexes were transfected into the sperm cells by electroporation (EP) to silence the expression of CatSper2. The results for targeted disruption of CatSper2 showed the highest silence efficiency 77.7% (P<0.05), the hyperactivated sperm rate calculated by computer-assisted semen analysis (CASA) analysis of interferenced sperm was significantly lower 11.1% than the control 99.2%. Flow cytometry (FCM) detection of the intracellular Ca2+ concentration of interferenced sperm was 50 nmol L-1 higher than the normal. It was remarkably lower than hyperactivated sperm with 200-1 000 nmol L-1 (P<0.05). It was not sufficient to evoke hyperactivation. To trigger hyperactivation, the sperm were incubated in 50 μmol L-1 thimerosal or 5 mmol L-1 procaine, it sharply increased the intracellular Ca2+ via two different channels. CASA and FCM detection indicated that intracellular Ca2+ is required for initiating hyperactivation while extracellular Ca2+ is essential to maintain the process. We concluded that to mediate sperm hyperactivation, it is essential to inhibit Ca2+ peak present with targeted disruption of CatSper2 expression. This provides important prospective for further exploration of signal channel of sperm hyperactivated motility, potential factors for male infertility and provide further reference to the exploration of male contraceptive drug targets of male reproduction.

Key words: sperm hyperactivation motility, CatSper2, Ca2+ channel, RNAi, electroporation (EP)

ZHANG Zhen, ZHOU Xuan, LI Hui-xia, CUI Qun-wei, YU Jing , WANG Gen-lin . Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation[J]. Journal of Integrative Agriculture, 2011, 10(12): 1958-1967.

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Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation

Delivery of CatSper2 siRNA into Rat Sperms by Electroporation Repressed Ca2+ Influx During Sperm Hyperactivation

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