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Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.

In the mouse, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1o) enzyme. The objectives of this study were to identify the alternative splicing variants of Dnmt1 in porcine oocytes and determine the gene expression pattern of the different Dnmt1 isoforms during embryo development. A rapid amplification of cDNA ends (RACE ) system was used to amplify the 5' cDNA end of Dnmt1 isoforms in porcine oocytes. RNA levels of the Dnmt1 isoforms were analyzed in porcine oocytes and embryos. DNMT1 protein expression of oocytes and somatic cells were analyzed by western blot and immunostaining. Two new Dnmt1o RNA isoforms were identified--Dnmt1o1 and Dnmt1o2. The previously reported somatic Dnmt1 isoform (Dnmt1s) was expressed at low but constant levels in oocytes and embryos from the two-cell to the blastocyst stage. Abundant RNA levels of Dnmt1o1 and Dnmt1o2 were detected in oocytes and embryos from the two- to the eight- to 16-cell stage. Levels of these Dnmt1o transcripts were low at the morula and blastocyst stages. Although Dnmt1s was present in all the somatic cell types analyzed, Dnmt1o1 and Dnmt1o2 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by western blot analysis, Dnmt1o1 and Dnmt1o2 RNAs translate one DNMT1o enzyme. Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, whereas somatic cells produce only DNMT1s protein. DNMT1o is localized mainly in the nuclei of oocytes and early embryos, whereas DNMT1s is expressed in the ooplasm cortex of oocytes and cytoplasm of early embryos.

In this study, the effect of the steps involved in zona-free somatic cell nuclear transfer (SCNT) on oocyte transcripts was investigated in sheep. To establish the reliable combined electrical-chemical activation for zona-free oocytes, oocytes were first exposed to an electrical pulse and then treated with 18 chemical activation regimens designed through modifying duration and concentration of ionomycin and 6-dimethyl aminopurine (6-DMAP), which is routinely used for SCNT. Electrofusion-mediated nuclear transfer significantly reduced transcript abundances of CCNB1, POU5F1, NPM2, GMMN, and CX43 compared to intact oocytes. Maximum parthenogenetic blastocyst development was obtained when oocytes were submitted to electric pulse and then to (1) 5 μM ionomycin for 5 or 2.5 min, both followed by 2 h of incubation with 6-DMAP (41.7±1.1, and 42.4±1.4%, respectively), (2) 5 μM ionomycin for 1 min+6-DMAP for 4 h (43.1±1.4%), and (3) 2.5 μM ionomycin for 1 min+6-DMAP for 2 h (42.4±1.4%), with significant differences compared to all the other groups. Statistical assessment of interactions between duration and concentration of ionomycin and duration of 6-DMAP exposure revealed that (1) concentration of ionomycin may be a more important factor than its duration, (2) both a long exposure period and a low concentration of ionomycin had marked decreasing effects on parthenogenetic development of zona-free oocytes, and (3) high duration of exposure to 6-DMAP can reduce parthenogenetic development. Despite an activation preference of parthenogenetic oocytes, a significantly higher rate of cloned blastocyst development was observed when reconstructed oocytes were activated with 5 μM ionomycin for 5 min rather than 2.5 μM ionomycin for 1 min (8.8±2.5 vs. 1.25±2.2%). These results suggested that SCNT steps have determining effects on oocyte transcripts and activation preferences of the reconstituted oocytes compared to intact counterparts. In this sense, reconstituted oocytes may need a higher concentration of ionomycin for a longer period than intact oocytes.

In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity.

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

In our previous study (Tsuji et al., 2010 ), administration of hCG to recipients around the timing of implantation significantly increased the in vivo development of mouse embryos after somatic cell nuclear transfer (SCNT) until day 10.5, but did not increase the development to full term. The present study was undertaken to examine whether cotransfer of fertilized embryos or parthenogenetic embryos prevents the embryonic loss of SCNT embryos after day 10.5, allowing them to develop to full term. We found that compared with SCNT embryo transfer alone, full-term development of SCNT embryos slightly, but not significantly, increased by cotransfer of mouse hybrid blastocysts derived from BDF1 (C57BL/6×DBA) female×ICR male into the oviducts of recipients administered hCG (2.0% vs. 5.5%). This was not the case with the cotransfer of blastocysts from an ICR female×ICR male (2.5% vs. 2.2%) or parthenogenetic blastocysts from BDF1 female (3.0% vs. 2.0%). Furthernore, when SCNT blastocysts were transferred into the uteri of recipients, full-term development did not increase even with the cotransfer of hybrid blastocysts. The mechanisms of the effect of cotransfer of fertilized and parthenogenetic embryos on the full-term development of SCNT mouse embryos are discussed.

We investigated the individual and combined effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor 2 (bFGF2) on the derivation and maintenance of rabbit embryonic stem cell lines isolated from parthenogenetic activated embryos (p-rES). First, we demonstrated that p-rES cell lines can be prevented from differentiation via LIF (STAT3) and bFGF2 (MEK-ERK1/2 and PI3K-AKT) signaling on MEF feeders. High levels of ERK1/2 and AKT activities were crucial for maintaining p-rES cells in an undifferentiated state. Although the p-rES cells under the influence of LIF (500, 1000, and 2000 U/mL) or bFGF2 (5, 10, and 20 ng/mL) alone showed enhanced expression in the pluripotency markers, the highest levels of marker expressions coincided with the simultaneous presence of LIF (1000 U/mL) and bFGF2 (10 ng/mL). The phosphorylation status of LIF and bFGF2 downstream signaling molecules including STAT3, ERK, and AKT was also intensively involved in the maintenance of p-rES cell proliferation and self-renewal. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors caused remarkable losses of self-renewal capacity of p-rES cells. We conclude that bFGF2 and LIF by itself are self-sufficient in maintaining the state of undifferentiation and self-renewal of rabbit p-ES cells, yet are most effective when acting concomitantly.

An innovative technique called high hydrostatic pressure (HHP) treatment has recently been reported to improve the cryosurvival of gametes and embryos in certain mammalian species, including the mouse, pig, and cattle. In the present study the parthenogenetic activation (PA) of pig oocytes caused by HHP treatment was investigated in different holding media with or without Ca(2+). The efficiency of activation was tested at different pressure levels and media including T2 (HEPES-buffered TCM-199 containing 2% cattle serum), and mannitol-PVA fusion medium with (MPVA + Ca(2+)) or without Ca(2+) and Mg(2+)(MPVA). The results showed that HHP cannot induce PA in T2, but only in MPVA + Ca(2+) with low Ca(2+) concentration and MPVA without Ca(2+). The highest activation efficiency was achieved with 10 min HHP treatment using 100 or 200 bars for oocytes in MPVA + Ca(2+) or MPVA, respectively. In the light of these results, the possible source of Ca(2+) during activation was investigated. It was found that even after a total of 30-min wash with TL-HEPES-PVA buffer without Ca(2+) before HHP treatment in MPVA, the oocytes could still be activated, indicating the possibility of an intracellular Ca(2+) source caused cytoplasmic free Ca(2+) elevation. In conclusion, parthenogenetic activation could be induced by HHP in certain holding media with low or zero Ca(2+) content. Further experiments are needed to identify the exact mechanisms of activation.

Currently, a major hurdle in the progress of human embryonic stem (hES) cell research is the lack of human oocytes with which to perform experiments. The collection process is a logistical and ethical challenge and usually involves the use of excess oocytes donated after assisted reproduction procedures. We collected resected human ovarian tissue after routine surgical procedures. Oocytes were isolated from the tissue and matured in vitro to the meiosis II (MII) stage, when the first polar body is extruded. With the large antral and smaller preantral follicles, the efficiencies of the maturation were nearly 50% and 25%, respectively. The quality of the matured oocytes was assessed by inducing parthenogenesis and >50% of the in vitro matured oocytes were competent enough to develop pronuclei and 33% developed at least to the two-cell stage 48 h after activation. Parthenotes continued to develop by 72 h but with significant blastomere fragmentation. These results provide evidence that resected ovarian tissue, which is normally discarded, may be a suitable alternative source for oocytes in hES cell research.

Somatic cell nuclear transfer (SCNT) efficiency is still low in rabbit. Previous studies indicated that trichostatin A (TSA) treatment could improve cloning efficiency and term development in the mouse, and cotransfer of parthenogenetic (PA) embryos benefited the pregnancy of cloned embryos in porcine and the mouse. In this study we investigated the effect of TSA treatment on the term development of the SCNT rabbit embryos, and the possibility of the pregnancy maintenance of clones by cotransfer of PA embryos. The SCNT embryos were produced by fusing cumulus cells with enucleated cytoplasts before activation by electrical stimulation, and Dimethylaminopurine (6-DMAP) and Cyclohexamide (CHX) treatments. They were cultured in EBSS-complete medium regardless of their treatment with or without TSA. In vitro developmental data showed no differences in the cleavage and the blastocyst rates, and the blastocyst cell number between the TSA-treated and the untreated SCNT embryos. Two of the six recipients became pregnant after the embryo transfer (ET) in the TSA-treated group, and one pregnant female delivered seven live and three stillborn pups. The death of all live pups occurred within an hour to 19 days. Four of the seven recipients became pregnant in the TSA-untreated group. Three of them gave birth to six live and eight stillborn pups. Four pups of the TSA-untreated group have grown into adulthood, and three of them produced progeny. Cotransfer of three to four PA embryos with 26-32 SCNT embryos to the same recipient resulted in pregnancy and birth rates statistically no different compared to the control SCNT ET group. In conclusion, our results indicate that TSA treatment has a limited effect on the in vitro development of the SCNT embryos; furthermore, both the TSA-treated and the untreated clones can develop to term in rabbits, but none of the offspring from TSA-treated embryos survived to adulthood in our experiment.

Little is known about parthenogenesis in the human ovary. What is known is related to patients with teratoma in their medical history. Ovarian surface epithelium (OSE) was often proposed as a source of ovarian stem cells with an embryonic character in the past, and was also termed "germinal epithelium." The aim of this study was to isolate putative stem cells from OSE scrapings, to set up an OSE cell culture, to follow the in vitro oogenesis and possible formation of parthenogenetic embryos in 21 postmenopausal women with no naturally present follicles and oocytes. Small round cells with a bubble-like structure and with a diameter from 2 to 4 microm were isolated from the material obtained by OSE scrapings in all women. They expressed early embryonic developmental markers such as stage-specific embryonic antigen-4 (SSEA-4) surface antigen and Oct-4, Nanog, Sox-2, and c-kit transcription factors. These cells were separated by density gradient centrifugation and grown in vitro, where they proliferated and formed embryoid body-like structures. Their markers of pluripotency such as telomerase activity were decreased during in vitro culture and they did not form teratoma after the injection into SCID mice. Some of them grew intensively and reached a diameter of approximately 20 microm after 5-7 days of culture. In the OSE cell culture, oocyte-like cells developed among them, which reached a diameter up to 95 mum, and expressed Oct-4, c-kit, VASA, and ZP2 transcription markers after 20 days of culture. Some of them expressed a zona pellucida-like structure and rarely germinal vesicle- and polar body-like structures. At the same time, parthenogenetic blastocyst-like structures developed, which expressed transcription markers Oct-4, Sox-2, and Nanog and were normal for chromosomes X, Y, 13, 16, 18, 21, and 22. In conclusion, the discovered cells expressed embryonic stem cell markers, gave rise to embryoid body-, oocyte-, and blastocyst-like structures, and might be involved in the human reproduction and formation of ovarian tumors.

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S. population, and is shared by different racial groups. These hpSC-Hhom lines demonstrate typical human embryonic stem cell morphology, expressing appropriate stem cell markers and possessing high levels of alkaline phosphatase and telomerase activity. Additionally, injection of these cell lines into immunodeficient animals leads to teratoma formation. G-banded karyotyping demonstrates a normal 46,XX karyotype in lines hpSC-Hhom-1 and hpSC-Hhom-4, and chromosomal anomalies in lines hpSC-Hhom-2 and hpSC-Hhom-3, both derived from the same donor. HLA genotyping of all four hpSC-Hhom lines demonstrates that they are HLA homozygous. Furthermore, in the case of HLA heterozygous donors, the hpSC-Hhom lines inherit the haplotype from only one of the donor's parents. Single-nucleotide polymorphism (SNP) data analysis suggests that hpSC-Hhom lines derived from HLA heterozygous oocyte donors are homozygous throughout the genome as assessed by SNP analysis. The protocol used for deriving these HLA homozygous stem cell lines minimizes the use of animal-derived components, which makes them more appealing for potential clinical application.

Several studies report that meiotic maturation of porcine oocytes can be reversibly preserved. The present study examined how long meiotic maturation can be suppressed. The first experiment determined the preservation medium suitable for reversibly suppressing meiotic maturation of porcine oocytes. The second experiment examined the in vitro developmental potential of oocytes maintained in meiotic arrest after parthenogenetic activation and nuclear transfer of somatic cells. Preservation of cumulus-oocyte complexes with NCSU-37 medium containing 10% follicular fluid, 1 mM dibutyryl cyclic AMP, and follicular shell pieces for 24-96 h at 39 degrees C did not affect oocyte maturation compared with controls (94-98% vs. 98%). The potential of parthenogenetically activated and nuclear-transferred oocytes maintained in meiotic arrest for 24-48 h to develop into blastocysts was not significantly different from that of controls (20-25% vs. 18% and 8-11% vs. 9%, respectively). The present study demonstrated that meiotic maturation of porcine oocytes can be suppressed after preservation for 48 h at 39 degrees C without decreasing oocyte maturation competence or the ability of oocytes to develop to at least the blastocyst stage.

The objective of this study was to evaluate the in vitro and in vivo developmental competence of parthenogenetic (parthenote) pig embryos derived from ovulated and in vitro matured (IVM) oocytes. A total of four experiments were carried out. These demonstrated that the mean blastocyst rates from stimulated ovulated and IVM pig oocytes were not significantly different (61% vs. 46%, p > 0.05) following in vitro culture. Both ovulated and IVM pig parthenotes were able to develop in vivo for 30 days. Parthenote fetuses collected 21 and 30 days post estrus were morphologically normal but significantly smaller and lighter than fertilized controls (p < 0.01). IVM pig parthenotes stopped development around 31 days post estrus.