Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to inf...Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/ or openers of these ion channels could interfere with sperm function. In this study, in vivo electmporation （EP） and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower （P〈0.05） hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca2＋ peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hvoeractivation.展开更多
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 stu...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 Ca〉 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-t 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 pmol L-t thimerosal or 5 mmol L-1 procaine, it sharply increased the intracellular Ca2＋ via two different channels. CASA and FCM detection indicated that intracellular Ca〉 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 Ca〉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.展开更多
文摘Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/ or openers of these ion channels could interfere with sperm function. In this study, in vivo electmporation （EP） and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower （P〈0.05） hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca2＋ peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hvoeractivation.
基金supported by the National Key Technology R&D Program of China (2006BAD04A12)
文摘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 Ca〉 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-t 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 pmol L-t thimerosal or 5 mmol L-1 procaine, it sharply increased the intracellular Ca2＋ via two different channels. CASA and FCM detection indicated that intracellular Ca〉 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 Ca〉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.
