Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.

The aim of the present study was to assess the effects of L-carnitine, an enhancer of lipid metabolism and mitochondrial activity, during in vitro maturation (IVM) on nuclear maturation and in vitro fertilisation of porcine follicular oocytes and subsequent embryo development. Mitochondrial functions, intracellular lipid content and reactive oxygen species (ROS) levels in oocytes were also investigated. L-carnitine supplementation in 0.6-5mgmL(-1) concentration during IVM significantly improved (P<0.05) the rates of metaphase-II (MII) stage oocytes compared with the control; however, fertilisation rates and monospermy were not improved. Although supplementation of IVM medium with L-carnitine significantly increased oocyte cleavage (P<0.05), further development to the blastocyst stage was not improved. The density of active mitochondria was significantly higher and the density of lipid droplets was significantly lower (P<0.05) in L-carnitine-treated oocytes compared with the control. Furthermore, the ROS levels in L-carnitine-treated oocytes were significantly lower than those in the control. In conclusion, enhancing mitochondrial functions by L-carnitine improved oocyte maturation and cleavage underlining the importance of lipid metabolism for nuclear and cytoplasmic maturation of porcine oocytes.

Abstract. We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines. Key words: Bovine, Nuclear transfer, Histone deacetylase inhibitor (J. Reprod. Dev. 57: [120][121][122][123][124][125][126] 2011) ive cloned offspring have been produced in many species by somatic cell nuclear transfer (SCNT), although the cloning efficiency is still very low. It has been reported that, in SCNT, embryo losses occur at various stages, and the anomalies in fetal and perinatal stages have been observed [1][2][3][4][5][6]. Because such abnormalities of clones are not transmitted to their progenies [7][8][9][10][11][12], most of the developmental problems of clones are believed to be the results of epigenetic defects [13]. In fact, several studies have revealed abnormal epigenetic modifications such as DNA methylation and histone modifications in SCNT embryos [14][15][16][17].A wide variety of histone deacetylase inhibitors (HDACi) of both natural and synthetic origin has been reported [18]. Trichostatin A (TSA), which is a natural product isolated from Streptomyces hygroscopicus [19], is one of the most frequently used drugs for various studies as a selective HDACi. Recently, it has been reported from two la...

To investigate the physiological importance of inhibin and estradiol in the regulation of FSH secretion during the follicular phase of the estrous-cycle in cows, animals were passively immunized against the two hormones. Sixteen cows were divided into four equal groups and given injections of prostaglandin F2 alpha (PG) i.m. twice at 8-h intervals on Day 10 of the estrous cycle (Day 0 = day of estrus) to induce luteal regression. At 48 h after the first PG injection, each group of four cows received injections of one of the following: 100 ml castrated goat serum (control serum), 100 ml antiserum against inhibin, 100 ml antiserum against estradiol, or a combination of 100 ml inhibin antiserum and 100 ml estradiol antiserum. The LH surge occurred within 2 days after injection of the control serum or inhibin antiserum, whereas it was not detected in either of the groups passively immunized against estradiol, indicating that passive immunization against estradiol blocks a positive feedback effect of estradiol on LH secretion. There was no clear difference in basal concentrations of LH among the four groups. Injection of the inhibin antiserum resulted in a marked increase (p < 0.01) in plasma concentrations of FSH compared with values in the control group, while there were no significant changes in concentrations of plasma FSH after injection of the estradiol antiserum. Combined administration of the inhibin and estradiol antisera also produced a marked increase (p < 0.01) in plasma concentrations of FSH. The FSH response to the combined immunization was longer in duration than the FSH response to immunization with inhibin alone.(ABSTRACT TRUNCATED AT 250 WORDS)