Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer.

Phosphorylated histone H2A.x (H2AX139ph) is a key factor for the repair of DNA double-strand breaks (DSBs) and the presence of H2AX139ph foci indicates the sites of DSBs. In this study, we characterized the presence of H2AX139ph during in vitro development of porcine embryos produced by IVF and somatic cell nuclear transfer (SCNT). Pronuclear stage embryos produced by IVF had, on average, 9.2 H2AX139ph foci per pronucleus. The number of H2AX139ph foci was higher in the 2-cell-stage embryos than in the 4-cell-stage embryos fixed at 48 h post-fertilization. The percentage of H2AX139ph-positive nuclei was higher in SCNT embryos that were activated with ionomycin (ION) alone than in those activated with ION and strontium chloride (ION+Sr(2+)). A negative correlation was found between the percentage of H2AX139ph-positive cells and the total number of cells per embryo in day 7 blastocysts produced by IVF or SCNT. Based on the detection of H2AX139ph foci, the findings of this study indicate that DSBs occur in a high proportion of porcine embryos produced by either IVF or SCNT; fast-cleaving embryos have fewer DSBs than slow-cleaving embryos; the oocyte activation protocol can affect DNA integrity in SCNT embryos; and better-quality blastocysts have fewer DSBs. We propose that the presence of H2AX139ph foci can be a useful marker of embryo quality.

Adiponectin enhances in vitro development of swine embryos.

Recent studies have shown that factors from adipose tissue influence and regulate the reproductive system. Hormones such as leptin and resistin are now known to regulate several reproductive processes. Adiponectin is the most abundant protein secreted by adipose tissue, and its circulating concentration is inversely related to adiposity and body mass index. Little is known about the involvement of adiponectin in reproduction. In the present study, the effect of recombinant adiponectin on the meiotic maturation and early embryo development in vitro was investigated, using porcine oocytes. Adiponectin receptors, AdipoR1 and AdipoR2, were found to be expressed in porcine oocytes and cumulus cells of both small and large follicles. Both AdipoR1 and AdipoR2 were immunolocalized to cumulus-oocyte complexes (COCs), oocytes, and early developing embryos. When included in oocyte maturation medium for 46 h, adiponectin significantly decreased the frequency of meiotic immature oocytes derived from large follicles (3-6 mm) but not from small follicles (<3mm). From studies of oocytes matured in the presence of adiponectin and mitogen-activated protein kinase (MAPK) pathway inhibitors MEK1 (PD98059), MEK1/2 (U0126), and p38MAPK (SB203580) it was concluded that adiponectin enhances oocyte maturation thought the p38MAPK pathway. Finally, a superior rate of embryo development to the blastocyst stage was achieved by embryos cultured in the presence of adiponectin. These results indicate that adiponectin has a positive effect on the meiotic maturation and in vitro embryo development of porcine oocytes and suggests a physiological role for this adipokine in early development in mammals.

Intelligent Graph Layout Using Many Users' Input.

In this paper, we propose a new strategy for graph drawing utilizing layouts of many sub-graphs supplied by a large group of people in a crowd sourcing manner. We developed an algorithm based on Laplacian constrained distance embedding to merge subgraphs submitted by different users, while attempting to maintain the topological information of the individual input layouts. To facilitate collection of layouts from many people, a light-weight interactive system has been designed to enable convenient dynamic viewing, modification and traversing between layouts. Compared with other existing graph layout algorithms, our approach can achieve more aesthetic and meaningful layouts with high user preference.

Pre- and postimplantation development of swine-cloned embryos derived from fibroblasts and bone marrow cells after inhibition of histone deacetylases.

The present study assessed changes in epigenetic markers and pre- and postimplantation development in somatic cell nuclear transfer (SCNT) porcine embryos after treatment with the inhibitor of histone deacetylases (HDACi), Trichostatin A (TSA). Embryos were generated using in vitro matured oocytes and nuclei from either a male fetal fibroblast (FF) cell line or bone marrow cells (BMC) from three adult sows. After nuclear transfer, oocytes were either exposed or not to 10 ng/mL TSA for 10 h starting 1 h after cell fusion. Samples of one-cell stage and cleaved (two- to four-cell stage) embryos were fixed at 15 to 18 h or 46 to 48 h after cell fusion and immunocytochemically processed to detect histone H3 acetylation at lysine 14 (H3K14ac) or histone H3 dimethylation at lysine 9 (H3K9m2) using specific primary antibodies. TSA treatment increased the immunofluorescent signal for H3K14ac in cleaved embryos derived from both FF and BMC but did not affect H3K9m2. Development to the blastocyst stage was increased by TSA treatment (45.2 vs. 23.9%) in embryos produced from FF cells but not in those produced from BMC (30.6 vs. 27.4%). Cloned piglets were produced from both treatments when day 5 to 6 blastocyst-stage embryos derived from FF cells were transferred into the uterus of recipient females. Cloned piglets were also produced after the transfer of TSA-treated blastocysts derived from BMC of adult sows but not from control embryos. These findings suggest that the inhibition of histone deacetylases have similar effects on enhancing H3K14ac in SCNT embryos reconstructed from different cell types but the effect on in vitro and in vivo development seems to differ according to the nuclear donor cell type.

Parthenogenetic activation and subsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone I treatment.

This study was designed to evaluate the parthenogenetic activation of porcine oocytes matured in vitro for a varied period after combined electric pulse (EP; 1500 V/cm, 100 microsec) and Butyrolactone I (BL I). After 36 h of maturation culture, the rates of activated oocytes and oocytes with two pronuclei were significantly lower than those of oocytes cultured for 42 and 48 h after EP. However, when treated by a combined EP and BL I (150 microM), these rates increased to the same level as 42 and 48 h oocytes. When oocytes cultured for 48 h and activated by a combined EP and BL I treatment were subsequently cultured in mNCSU37 medium, the rates of embryos cleaved and developed to the blastocyst stage were significantly higher than those in Whitten's medium. In contrast, when activated oocytes were cultured in mNCSU37 medium under two oxygen environments (5% vs 20% O(2)), there was no difference in the rates of cleavage, blastocyst formation and nuclear numbers per blastocyst. Our results demonstrated that the combined EP and BL I treatment of porcine oocytes matured in vitro is capable of producing high rates of good quality blastocysts when cultured in a suitable in vitro condition.