정자의 수정능 획득에서의 Glycogen Synthase Kinase-3 α/β 기능

Abstract

생쥐의 부정소 정자와 사람의 정자에서 GSK-3α/β의 발현과 기능을 분석하였다. 정자의 수정능 획득에 주요한 PKA, PI-3 kinase, CaMKKα/β와 같은 조절효소들과 GSK-3α/β의 억제성 인산화, phospho-tyrosine proteome, 정자 운동성 간의 상관관계를 분석하였다. 생쥐와 사람 정자에서 GSK-3α의 발현이 우세하였다. 생쥐와 사람 정자에서 GSK-3α/β의 억제는 수정능 획득과 운동성을 촉진하였다. 생쥐 정자에서 GSK-3α/β는 머리의 post acrosomal region과 꼬리에서 발현되었다. 부정소 두부 보다 미부 정자가 GSK-3α/β의 양과 serine (9/21) 억제성 인산화 정도가 높았다. 생쥐와 사람 정자의 GSK-3α/β는 protein kinase A에 의존적으로 억제성 인산화가 증가하였다. PI-3 kinase 억제제 (LY294002)는 사람 정자에서 GSK-3α/β의 억제성 인산화 및 phospho-tyrosine proteome을 증가시킨 반면 생쥐 정자에서는 GSK-3α/β의 억제성 인산화와 phospho-tyrosine proteome을 감소시켰다. 사람 정자에서 CaMKKα/β의 억제제 STO-609는 phospho-tyrosine proteome는 크게 증가시킨 반면 GSK-3α/β억제성 인산화에는 영향이 없었다. 생쥐 정자에서 GSK-3α/β 억제제 LiCl는 GSK-3α/β의 억제성 인산화를 증가시켰지만 phospho-tyrosine proteome는 감소시켰다. 생쥐 정자와 사람 정자에서 GSK-3α/β 특이적 억제제인 BIO (6-bromoindirubin-3-oxime)는 GSK-3α/β 억제성 인산화와 phospho-tyrosine proteome를 증가시켰다. 사람과 생쥐 정자에서 calcium ionophore A23187은 GSK-3α/β의 억제성 인산화와 정자 운동성을 가역적으로 감소시켰다. 사람과 생쥐의 정자에서 BIO는 A23187에 의한 GSK-3α/β의 억제성 인산화, phospho-tyrosine proteome, 정자 운동성 억제효과를 상쇄하였다. 정자의 GSK-3α/β 활성은 PI-3 kinase, PKA등의 kinase 활성과 calcium에 의해 밀접하게 조절되며, 이러한 정보는 남성불임 환자의 정자 수정능력 증진 치료법 개발에 적용 가능할 것으로 사료된다.|In an effort to verify the function of GSK-3α/β in spermatozoa we examined the expression of GSK-3α/β in mouse epididymal sperm and human ejaculated spermatozoa together with the regulatory role of kinases such as PKA, PI-3 kinase and CaMKKα/β crucial for sperm capacitation on the inhibitory phosphorylation of GSK-3α/β, total phospho-tyrosine proteome and motility in vitro. In mouse and human sperm, both GSK-3α and GSK-β isoforms are present and the expression of GSK-3α is dominant. Inhibition of GSK-3α/β promoted the capacitation and motility of mouse and human spermatozoa. In mouse epididymal spermatozoa GSK-3α/β was expressed in the post-acrosomal region of head and tail. The amount of GSK-3α/β and serine (9/21) inhibitory phosphorylation were much greater in cauda epididymal sperm compared to that of caput. Inhibitory phosphorylation of GSK-3α/β of human and mouse sperm was largely dependent on protein kinase A activation. Inhibition of PI-3 kinase increased inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome in human sperm. In mouse sperm, however, inhibition of PI-3 kinase resulted in decrease in inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome. In human sperm, STO-609, a CaMKKα/β inhibitor did not change the inhibitory phosphorylation of GSK-3α/β but protein tyrosine phosphorylation was increased. In mouse sperm, LiCl which inhibits GSK-3α/β increased inhibitory phosphorylation of GSK-3α/β but decreased phospho-tyrosine proteome. BIO (6-bromoindirubin-3-oxime), a specific inhibitor for GSK-3α/β increased inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome in mouse as well as human sperm. A23187, Ca2+ ionophore reversibly decreased inhibitory phosphorylation of GSK-3α/β and motility in mouse and human spermatozoa. In A23187-trated mouse and human sperm motility was recovered by BIO together with increase in inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome. In sperm, GSK-3α/β activity is tightly regulated by several kinases directly and indirectly, and which could be applicable for development of therapy potentiating the sperm fertility.; In an effort to verify the function of GSK-3α/β in spermatozoa we examined the expression of GSK-3α/β in mouse epididymal sperm and human ejaculated spermatozoa together with the regulatory role of kinases such as PKA, PI-3 kinase and CaMKKα/β crucial for sperm capacitation on the inhibitory phosphorylation of GSK-3α/β, total phospho-tyrosine proteome and motility in vitro. In mouse and human sperm, both GSK-3α and GSK-β isoforms are present and the expression of GSK-3α is dominant. Inhibition of GSK-3α/β promoted the capacitation and motility of mouse and human spermatozoa. In mouse epididymal spermatozoa GSK-3α/β was expressed in the post-acrosomal region of head and tail. The amount of GSK-3α/β and serine (9/21) inhibitory phosphorylation were much greater in cauda epididymal sperm compared to that of caput. Inhibitory phosphorylation of GSK-3α/β of human and mouse sperm was largely dependent on protein kinase A activation. Inhibition of PI-3 kinase increased inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome in human sperm. In mouse sperm, however, inhibition of PI-3 kinase resulted in decrease in inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome. In human sperm, STO-609, a CaMKKα/β inhibitor did not change the inhibitory phosphorylation of GSK-3α/β but protein tyrosine phosphorylation was increased. In mouse sperm, LiCl which inhibits GSK-3α/β increased inhibitory phosphorylation of GSK-3α/β but decreased phospho-tyrosine proteome. BIO (6-bromoindirubin-3-oxime), a specific inhibitor for GSK-3α/β increased inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome in mouse as well as human sperm. A23187, Ca2+ ionophore reversibly decreased inhibitory phosphorylation of GSK-3α/β and motility in mouse and human spermatozoa. In A23187-trated mouse and human sperm motility was recovered by BIO together with increase in inhibitory phosphorylation of GSK-3α/β and phospho-tyrosine proteome. In sperm, GSK-3α/β activity is tightly regulated by several kinases directly and indirectly, and which could be applicable for development of therapy potentiating the sperm fertility.