Vizel Ruth, Hillman Pnina, Ickowicz Debby, Breitbart Haim
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel.
Biochim Biophys Acta. 2015 Sep;1850(9):1912-20. doi: 10.1016/j.bbagen.2015.06.005. Epub 2015 Jun 17.
The A-kinase anchoring protein (AKAP) family is essential for sperm motility, capacitation and the acrosome reaction. PKA-dependent protein tyrosine phosphorylation occurs in mammalian sperm capacitation including AKAP3. In a recent study, we showed that AKAP3 undergoes degradation under capacitation conditions. Thus, we tested here whether AKAP3 degradation might be regulated by its tyrosine phosphorylation.
The intracellular levels of AKAP3 were determined by western blot (WB) analysis using specific anti-AKAP3 antibodies. Tyrosine phosphorylation of AKAP3 was tested by immunoprecipitation and WB analysis. Acrosome reaction was examined using FITC-pisum sativum agglutinin.
AKAP3 is degraded and undergoes tyrosine-dephosphorylation during sperm capacitation and the degradation was reduced by inhibition of tyrosine phosphatase and enhanced by inhibition of tyrosine kinase. Sperm starvation or inhibition of mitochondrial respiration, which reduce cellular ATP levels, significantly accelerated AKAP3 degradation. Treatment with vanadate, or Na(+) or bicarbonate depletion, reduced AKAP3-degradation and the AR rate, while antimycin A or NH4Cl elevated both AKAP3-degradation and the AR degree. Treatment of sperm with NH4Cl enhanced PKA-dependent phosphorylation of four proteins, further supporting the involvement of AKAP3-degradation in capacitation. To demonstrate more specifically that sperm capacitation requires AKAP3-degradation, we inhibited AKAP3-degradation using anti-AKAP3 antibody in permeabilized cells. The anti-AKAP3-antibody induced significant inhibition of AKAP3-degradation and of the AR rate.
Sperm capacitation process requires AKAP3-degradation, and the degradation degree is regulated by the level of AKAP3 tyrosine phosphorylation.
Better understanding of the molecular mechanisms that mediate sperm capacitation can be used for infertility diagnosis, treatment and the developing of male contraceptives.
A激酶锚定蛋白（AKAP）家族对精子活力、获能及顶体反应至关重要。PKA依赖的蛋白酪氨酸磷酸化发生在包括AKAP3在内的哺乳动物精子获能过程中。在最近的一项研究中，我们发现AKAP3在获能条件下会发生降解。因此，我们在此测试AKAP3降解是否受其酪氨酸磷酸化的调控。
使用特异性抗AKAP3抗体，通过蛋白质印迹（WB）分析测定细胞内AKAP3水平。通过免疫沉淀和WB分析检测AKAP3的酪氨酸磷酸化。使用异硫氰酸荧光素（FITC）标记的豌豆凝集素检测顶体反应。
在精子获能过程中，AKAP3发生降解并经历酪氨酸去磷酸化，酪氨酸磷酸酶抑制可减少降解，酪氨酸激酶抑制则增强降解。精子饥饿或线粒体呼吸抑制会降低细胞ATP水平，显著加速AKAP3降解。用钒酸盐处理、或Na⁺或碳酸氢盐缺失会降低AKAP3降解及顶体反应率，而抗霉素A或氯化铵则会提高AKAP3降解及顶体反应程度。用氯化铵处理精子会增强PKA依赖的四种蛋白的磷酸化，进一步支持AKAP3降解参与获能过程。为更具体地证明精子获能需要AKAP3降解，我们在通透细胞中使用抗AKAP3抗体抑制AKAP3降解。抗AKAP3抗体可显著抑制AKAP3降解及顶体反应率。
精子获能过程需要AKAP3降解，且降解程度受AKAP3酪氨酸磷酸化水平调控。
更好地理解介导精子获能的分子机制可用于不孕症诊断、治疗及男性避孕药开发。
