Incomplete DNA methylation reprogramming in cloned embryos leads to poor cloning efficiency. Epigenetic modification agents can improve genomic methylation reprogramming and the development of cloned embryos, however, the effect of epigenetic modification agents on gene-specific methylation reprogramming remains poorly studied. Here, we investigated DNA methylation reprogramming of pluripotency (Oct4) and tissue specific (Thy1) genes during early embryo development in pigs. In this study, we found that compared with in vitro fertilized counterparts, cloned embryos displayed the disrupted patterns of Oct4 demethylation and Thy1 remethylation. When 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of cloned embryos, the transcripts of DNA methyltransferases (Dnmt1 and Dnmt3a), histone acetyltransferase 1 (Hat1) and histone deacetylase 1 (Hdac1) and the methylation and expression patterns of Oct4 and Thy1 became similar to those detected in in vitro fertilized counterparts. Further studies showed that Dnmt1 knockdown in cloned embryos enhanced the methylation reprogramming of Oct4 and Thy1 and promoted the activation of Oct4 and the silence of Thy1. In conclusion, our results demonstrated that cloned embryos displayed incomplete gene-specific methylation reprogramming and disrupted expression patterns of pluripotency and tissue specific genes, and epigenetic modification agents improved gene-specific methylation reprogramming and expression pattern by regulating epigenetic modification related genes. This work would have important implications in improving cloning efficiency.

We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 +/- 3.5 kDa, 79.0 +/- 3.0 kDa, and 113.5 +/- 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 +/- 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 +/- 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 +/- 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.

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Immature pig oocytes cultured for 36 h in a modified tissue culture medium 199B were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescence illumination, Ricinus communis agglutinin (RCA-I) and Lens culinaris agglutinin (LCA) bound to all oocytes, with the strongest fluorescence in either the outer region of the ZP (RCA-I) or throughout the ZP (LCA). However, Bandeiraea simplicifolia lectin-I (BS-I) and Ulex europaeus agglutinin-I (UEA-I) bound with either strong or weak intensity mainly to the outer and inner regions of the ZP, respectively, in ~70-90% of oocytes examined. Bandeiraea simplicifolia lectin-II (BS-II) bound to various regions of the ZP with different intensities in only 60% of oocytes examined. At 2 h after insemination in vitro, significantly fewer spermatozoa were bound to the ZP of lectin-treated oocytes than untrearted control oocytes. Of the lectins, RCA-I and LCA most inhibited sperm binding. At 12 h after insemination, penetration rates were similar in control oocytes and those treated with BS-I or BS-II, but penetration rates were lower in oocytes treated with UEA-I than those in untreated controls, and an almost complete block of sperm penetration was observed in oocytes treated with RCA-I or LCA. The incidence of monospermy was similar in untreated oocytes and those treated with BS-I or UEA-I, but it was higher in oocytes treated with BS-II. These results suggest that β-D-galactose and α-D-mannose residues in the pig ZP may act as primary sperm receptors and/or inducers of the sperm acrosome reaction and β-D-N-acetylglucosamine residues may be involved in the block of polyspermy. The variability in binding of BS-II to the ZP, which correlated with monospermy/polyspermy may reflect differences in the maturation state of individual oocytes. In future, this lectin might provide a means of monitoring maturation in vitro, leading to the development of improved culture systems. Key words: Pig, Oocyte, Zona pellucida, Lectin-binding, Gamete interaction.The zona pellucida (ZP), a relatively thick extracellular coat that surrounds mammalian oocytes, is known to mediate critical steps in fertilization including speciesspecific binding of spermatozoa, inducing the sperm acrosome reaction and preventing penetration of excess spermatozoa (reviewed in [1,2]). The mammalian ZP typically consists of a few glycoproteins which have different polypeptide chains and oligosaccharides [3][4][5][6][7][8] and evidence indicates that these oligosaccharides play an important role in sperm-oocyte recognition in all mammalian species (reviewed in [9][10][11]). Lectins are cell-agglutinating and sugar specific proteins or glycoproteins of non-immune origin that precipitate glycoconjugates having saccharides of appropriate complementarity [12]. Because of these properties, plant lectins have been used to help characterize the carbohydrate moieties of glycoproteins in the ZP of several mammalian species [13][14][15][16][17] including...

We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues.