embryonic development following somatic cell nuclear transfer

Epigenetic modification and expression of key pluripotent factors are critical for development, cell fate determination, and differentiation in early embryos. In this study, we systematically examined the dynamic patterns of histone modifications (H3K4me3 and H3K27me3) and Nanog expression during the development of preimplantation rabbit embryos. Rabbit oocytes, 1-, 2-, 4-, 8-, and 16-cell embryos, morulae, and blastocysts were collected at specific time points following superovulation and assessed for nuclear H3K4me3, H3K27me3, and Nanog expression by immunofluorescence microscopy...

Xist is the master regulator of X chromosome inactivation. In order to further understand the Xist locus in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-activator-like effectors-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which caused higher H3K9me3 followed by X chromosome opening and repression of X-linked genes in mouse embryonic fibroblasts, rather than affecting Xist expression, substantially improved the iPSC generation and the SCNT preimplantation embryo development...

With artificial insemination (AI) and other precision dependent assisted reproductive technologies (ART) being followed in large scale in human and animal reproduction, assessing semen quality and fertilizability is under continuous scrutiny. Various tests have been developed to predict semen quality, but so far no single, highly reliable test is available. In this regard, transcriptomic profiling of spermatozoa assumes significance as it carries the information about spermatogenesis, sperm function, and paternal roles in post-fertilization events...

The derivation of gametes from patient-specific pluripotent stem cells may provide new perspectives for genetic parenthood for patients currently facing sterility. We use current data to assess the gamete differentiation potential of patient-specific pluripotent stem cells and to determine which reprogramming strategy holds the greatest promise for future clinical applications. First, we compare the two best established somatic cell reprogramming strategies: the production of induced pluripotent stem cells (iPSC) and somatic cell nuclear transfer followed by embryonic stem cell derivation (SCNT-ESC)...

Reproductive technology revolutionized dairy production during the past century. Artificial insemination was first successfully applied to cattle in the early 1900s. The next major developments involved semen extenders, invention of the electroejaculator, progeny testing, addition of antibiotics to semen during the 1930s and 1940s, and the major discovery of sperm cryopreservation with glycerol in 1949. The 1950s and 1960s were particularly productive with the development of protocols for the superovulation of cattle with both pregnant mare serum gonadotrophin/equine chorionic gonadotrophin and FSH, the first successful bovine embryo transfer, the discovery of sperm capacitation, the birth of rabbits after in vitro fertilization, and the development of insulated liquid nitrogen tanks...

Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), 10 μM MG132 (MG), and 10 mM caffeine +10 μM MG132 (CA+MG)...

The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein ( Bos taurus ) fibroblasts depleted of their mtDNA, and oocytes derived from Angus ( Bos taurus ) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells...

BACKGROUND: Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young...

This study was conducted to evaluate the effects of cyclic AMP (cAMP) modulator cilostamide (CIL) and forskolin (FSK) treatment during in vitro growth (IVG) on growth, maturation, and embryonic development of cumulus-oocyte complexes (COCs) derived from small antral follicles < 3 mm in diameter (SAFCOCs). SAFCOCs were untreated (control) or treated with 20 μM CIL and/or 50 μM FSK for 2 days for IVG. Next, IVG oocytes were cultured for maturation and then induced for parthenogenesis (PA) or used as recipient ooplasts for somatic cell nuclear transfer (SCNT)...

Recent accomplishments in the field of somatic cell nuclear transfer (SCNT) hold tremendous promise to prevent rapid loss of animal genetic resources using ex situ conservation technology. Most of SCNT studies use viable cells for nuclear transfer into recipient oocytes. However, preparation of live cells in extreme circumstances, in which post-mortem material of endangered/rare animals is improperly retained frozen, is difficult, if not impossible. This study investigated the possibility of interspecies-SCNT (iSCNT) in Asiatic cheetah (Acinonyx jubatus venaticus), a critically endangered subspecies, using nuclei derived from frozen tissue in absence of cryo-protectant at -20 °C and in vitro matured domestic cat oocytes...

Buffalo are characteristic livestock of the Guangxi Zhuang Autonomous Region of China, but their low reproductive capacity necessitates the use of somatic cell nuclear transfer (SCNT). We investigated the effects of RG108 on DNA methylation in buffalo adult fibroblasts, and on subsequent SCNT embryo development. RG108 treatment (0, 5, 10, 20, and 100 mM) had no effect on cell morphology, viability, or karyotype (2n = 48), and cell growth followed a typical "S" curve. Immunohistochemistry showed that relative DNA methylation gradually decreased as RG108 concentration increased, and was significantly lower in the 20 and 100 mM groups compared to the 0, 5, and 10 mM treatments (0...

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51...

The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development...

The quest for porcine pluripotent stem cells (PSCs) was initiated in the early 90s. Initially, it was the intention to benefit from these cells for production of genetically modified pigs using homologous recombination followed by derivation of chimeric offspring; a technology that has been used to produce genetically modified mice since the mid-80s. However, no convincing reports on the generation of bona fide porcine embryonic stem cells or embryonic germ cells resulted from these activities, and with the advent of somatic cell nuclear transfer during the late 90s, alternative methods for creating genetically modified pigs emerged...

The objective of this study was to examine the effects of colcemid treatment during oocyte in vitro maturation (IVM) and embryonic development after parthenogenetic activation (PA) and somatic-cell nucleus transfer (SCNT) in pigs. Immature oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM (designated as COL0-22, COL38-42, and COL0-22/38-42, respectively). The proportion of oocytes reaching the germinal vesicle (GV)/GV breakdown (GVBD) stage after 22 hr of IVM was higher in COL0-22 (98...

Antioxidants are widely used for in vitro production of embryos due to their activity as reactive oxygen species scavengers. Among various antioxidants, resveratrol supplementation in in vitro-maturation (IVM) media and trolox supplementation in in vitro-culture (IVC) media improves oocyte maturation and embryonic development in other species, such as cattle and sheep. Limited information is available, however, on the effect of resveratrol and/or trolox on porcine embryos produced in vitro. In this study, we evaluated the effect of resveratrol supplemented to the media of IVM and trolox treatment during IVC on porcine parthenotes...

The developmental ability of interspecies somatic cell nuclear transfer (iSCNT) embryos decreases as the taxonomic distance between the donor and recipient species increases. Treatment of cat iSCNT embryos using bovine oocytes with 50nM of trichostatin A (TSA) improves in vitro embryonic development (Wittayarat et al. 2013 Cell. Reprogram. 15, 301-308). This study investigated whether the TSA treatment effects differ between the development of cat iSCNT embryos reconstructed with porcine and bovine oocytes...

Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT...

Pluripotent stem cells are possibly the best candidates for regenerative medicine, and somatic cell nuclear transfer (SCNT) is one of the viable options to make patient-specific embryonic stem cells. Till date efficacy of SCNT embryos is very low and requires further improvement like ideal oocyte activation and in vitro culture system. The aim of the present study was to evaluate ideal oocyte activation using different stimulation protocols and to study the effect of cumulus co-culture conditions on embryo development...

Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts...