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

Hur, Chang-Gi,Kim, Eun-Jin,Cho, Seong-Keun,Cho, Young-Woo,Yoon, Sook-Young,Tak, Hyun-Min,Kim, Chang-Woon,Choe, Changyong,Han, Jaehee,Kang, Dawon Journals of Reproduction and Fertility 2012 Reproduction Vol.143 No.5

<P>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.</P>

The histone deacetylase inhibitor scriptaid enhances nascent mRNA production and rescues full-term development in cloned inbred mice.

Van Thuan, Nguyen,Bui, Hong-Thuy,Kim, Jin-Hoi,Hikichi, Takafusa,Wakayama, Sayaka,Kishigami, Satoshi,Mizutani, Eiji,Wakayama, Teruhiko Journals of Reproduction and Fertility 2009 Reproduction Vol.138 No.2

<P>Since the birth of Cumulina, the first mouse clone produced by somatic cell nuclear transfer (SCNT), the success rate of cloning in mice has been extremely low compared with other species and most of the inbred mouse strains have never been cloned. Recently, our laboratory has found that treatment of SCNT mouse embryos with trichostatin A, a histone deacetylase inhibitor (HDACi), improved the full-term development of B6D2F1 mouse clones significantly. However, this was not effective for the inbred strains. Here, we show for the first time that by treating SCNT embryos with another HDACi, scriptaid, all the important inbred mouse strains can be cloned, such as C57BL/6, C3H/He, DBA/2, and 129/Sv. Moreover, the success of somatic nuclear reprogramming and cloning efficiency via nuclear transfer technique is clearly linked to the competent de novo synthesis of nascent mRNA in cloned mouse embryos.</P>

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

Hur, Chang-Gi,Choe, Changyong,Kim, Gyu-Tae,Cho, Seong-Keun,Park, Jae-Yong,Hong, Seong-Geun,Han, Jaehee,Kang, Dawon Journals of Reproduction and Fertility 2009 Reproduction Vol.137 No.2

<P>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.</P>

Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos.

Kwon, D J,Park, C K,Yang, B K,Cheong, H T Journals of Reproduction and Fertility 2008 Reproduction Vol.135 No.5

<P>We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.</P>

Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos.

Wee, Gabbine,Shim, Jung-Jae,Koo, Deog-Bon,Chae, Jung-Il,Lee, Kyung-Kwang,Han, Yong-Mahn Journals of Reproduction and Fertility 2007 Reproduction Vol.134 No.6

<P>Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the alpha-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the alpha-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.</P>