Chang-Gi Hur

Expression and localization of two-pore domain K+ channels in bovine germ cells

Two-pore domain K(+) (K(2P)) channels that help set the resting membrane potential of excitable and nonexcitable cells are expressed in many kinds of cells and tissues. However, the expression of K(2P) channels has not yet been reported in bovine germ cells. In this study, we demonstrate for the first time that K(2P) channels are expressed in the reproductive organs and germ cells of Korean cattle. RT-PCR data showed that members of the K(2P) channel family, specifically KCNK3, KCNK9, KCNK2, KCNK10, and KCNK4, were expressed in the ovary, testis, oocytes, embryo, and sperm. Out of these channels, KCNK2 and KCNK4 mRNAs were abundantly expressed in the mature oocytes, eight-cell stage embryos, and blastocysts compared with immature oocytes. KCNK4 and KCNK3 were significantly increased in eight-cell stage embryos. Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed at the membrane of oocytes and blastocysts. KCNK10 and KCNK4 were strongly expressed and distributed in oocyte membranes. These channel proteins were also localized to the acrosome sperm cap. In particular, KCNK3 and KCNK4 were strongly localized to the post-acrosomal region of the sperm head and the equatorial band within the sperm head respectively. These results suggest that K(2P) channels might contribute to the background K(+) conductance of germ cells and regulate various physiological processes, such as maturation, fertilization, and development.

K(+) efflux through two-pore domain K(+) channels is required for mouse embryonic development.

Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.

Acetylcholine rescues two-cell block through activation of IP3 receptors and Ca2+/calmodulin-dependent kinase II in an ICR mouse strain.

Acetylcholine (ACh) causes early activation events in mouse oocytes, but little is known about its precise role in the early embryonic development of mice. We aimed to determine whether and how ACh is capable of rescuing two-cell block in an in vitro culture system. ACh evoked different transient Ca2+ patterns showing a higher Ca2+ peak in the two-cell stage embryos (two-cells) than observed in mature oocytes. In early two-cells subjected to an in vitro two-cell block, xestospongin C (Xes-C), an IP3 receptor antagonist, significantly decreased the level of the ACh-induced Ca2+ increase. The reduction in the ACh-induced Ca2+ increase by Xes-C in late two-cells was lower than that in early two-cells. Furthermore, KN62 and KN93, both CaMKII inhibitors, were found to reduce the magnitude of the ACh-induced Ca2+ increase in early two-cells. The addition of ACh to the culture medium showed an ability to rescue in vitro two-cell block. However, the addition of ACh together with both Xes-C and CaMKII inhibitors or with either inhibitor separately had no effect on the rescue of two-cell block. Long-term exposure of late two-cells to ACh decreased morula and early blastocyst development and ACh had a differential effect on early and late two-cells. These results indicate that ACh likely rescues the in vitro two-cell block through activation of IP3R- and/or CaMKII-dependent signal transduction pathways.