No abstract

The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.

Curcumin, a natural polyphenol antioxidant extracted from the root of turmeric (Curcuma longa), can induce apoptosis and DNA demethylation in several types of cancer cells. However, the mechanism of its anticancer potentials and DNA demethylation effects and the potential relationships between these outcomes have not been clearly elucidated. In the present study, the effects of curcumin on the proliferation, colony formation, and migration of human gastric cancer cells (hGCCs) were explored. Reactive oxygen species (ROS) levels, mitochondrial damage, DNA damage, and apoptosis of curcumin-treated hGCCs were analyzed. Changes in the expression of several genes related to DNA damage repair, the p53 pathway, cell cycle, and DNA methylation following curcumin treatment were also evaluated. We observed that curcumin inhibited the proliferation, colony formation, and migration of hGCCs in a dose- and time-dependent fashion. A high concentration of curcumin elevated ROS levels and triggered mitochondrial damage, DNA damage, and apoptosis of hGCCs. Further, curcumin-induced DNA demethylation of hGCCs was mediated by the damaged DNA repair-p53-p21/GADD45A-cyclin/CDK-Rb/E2F-DNMT1 axis. We propose that the anticancer effect of curcumin could largely be attributed to its prooxidative effect at high concentrations and ROS elevation in cancer cells. Moreover, we present a novel mechanism by which curcumin induces DNA demethylation of hGCCs, suggesting the need to further investigate the demethylation mechanisms of other DNA hypomethylating drugs.

ABSTRACT. Bovine mammary epithelial stem cells (MESCs) are very important in agricultural production and bioengineering. In the present study, we compared different isolation and culture methods for MESCs and observed their growth and differentiation characteristics. MESCs have an extremely weak proliferation capacity, and it is very difficult to obtain and prolong subculture of a bovine mammary epithelial stem cell line. We obtained some multipotent MESC aggregates that looked like spherical colonies. These colonies were only derived from suspension culture and were induced to differentiate into epithelial-like cells, myoepithelial-like cells and secretory cells and to establish a ductal-like structure. In contrast, MESCs cultured in adherent culture displayed low morphogenetic competence and only differentiated into epithelial-like cells. MESCs are often identified by testing their differentiation in vivo; however, herein, we have demonstrated the in vitro differentiation potential of bovine MESCs. In our study, beta 1-integrin and alpha 6-integrin which are expressed by human epidermal stem cells, were found in bovine, which shows that bovine MESCs share the same molecular signature as human MESCs.

Mitochondria are the key generators of cellular ATP, and contain extranuclear genome-mitochondrial DNA (mtDNA). In the process of nuclear transfer (NT), heteroplasmic sources of mtDNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. Previous studies showed inconsistent patterns of mtDNA inheritance in offspring and early fetuses generated through interspecies NT. The quantitative analysis of mitochondrial RNA (mtRNA) in interspecies cloned embryos is useful for better understanding the fate of two types of mitochondria. The components of nicotinamide adenine dinucleotide (NADH) dehydrogenase were coded by both nuclear DNA (nDNA) and mtDNA. The Subunit 1 (ND-1) is one of seven NADH dehydrogenase subunits coded by mtDNA. In present study, using real-time and reverse-transcription PCR, the copy number of species-specific ND-1 mRNA was examined in goat-sheep cloned embryos of various developmental stages, and was applied to evaluate the expression pattern of species-specific mtDNA. The results of showed that (1) the expression of mtDNA derived from goat fetal fibroblast (GFF) decreased from 1-cell stage (immediately after fused) to 2-cell stage, and could not be detected from 4-cell stage onward to blastocyst stage; (2) the expression of mtDNA derived from sheep oocyte was roughly constant from 1-cell stage to the 8-cell stage, increased gradually from 16-cell stage, and sharply at morula and blastocyst stage. Moreover, we strongly argued a mechanism, that is GFF-derived mitochondria were degraded for the depression of bioenergetic functions, and then selectively eliminated during the embryogenesis of goat-sheep cloned embryos.

Enhancer of zeste homolog 2 (EZH2), a component of the PRC2 complex, trimethylates H3K27, a transcriptionally repressive histone mark. EZH2 is encoded by a dormant maternal mRNA and inhibiting the maturation-associated increase in EZH2 activity using either a combined siRNA/ morpholino approach or a small molecule inhibitor (GSK343) inhibits development of diploidized parthenotes to the blastocyst stage but not inseminated eggs, with longer GSK343 treatments leading to progressively greater inhibition of development. GSK343 treatment also results in a decrease in H3K27me3 and a decrease in global transcription in 2-cell parthenotes but not 2-cell embryos derived from inseminated eggs. RNA-sequencing revealed the relative abundance of~100 zygotically-expressed transcripts is decreased by GSK treatment in parthenotes, but not in embryos, with many of the affected transcripts encoding proteins involved in transcription. A previous study found that parthenotes deficient in maternal Ezh2 readily develop to the blastocyst stage. To reconcile these differences we propose that the H3K27me3 state present in the zygote needs to be faithfully propagated following DNA replication in at least one pronucleus, otherwise development is compromised.

It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT). Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1) cultured to 70–80% confluency (control group), (2) cultured to full confluency, (3) starved in low serum medium for 4 d, or (4) cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in G0/G1 phase, and the short-term treatment of nuclear donor cells with these two methods could improve the efficiency of SCNT.

Somatic cell nuclear transfer can be used to produce embryonic stem (ES) cells, cloned animals, and can even increase the population size of endangered animals. However, the application of this technique is limited by the low developmental rate of cloned embryos, a situation that may result from abnormal expression of some zygotic genes. In this study, sheep-sheep intra-species cloned embryos, goat-sheep inter-species cloned embryos, or sheep in vitro fertilized embryos were constructed and cultured in vitro and the developmental ability and expression of three pluripotency genes, SSEA-1, Nanog and Oct4, were examined. The results showed firstly that the developmental ability of in vitro fertilized embryos was significantly higher than that of cloned embryos. In addition, the percentage of intra-species cloned embryos that developed to morula or blastocyst stages was also significantly higher than that of the inter-species cloned embryos. Secondly, all three types of embryos expressed SSEA-1 at the 8-cell and morula stages. At the 8-cell stage, a higher percentage of in vitro fertilized embryos expressed SSEA-1 than occurred for cloned embryos. However, at the morula stage, all detected embryos could express SSEA-1. Thirdly, the three types of embryos expressed Oct4 mRNA at the morula and blastocyst stages, and embryos at the blastocyst stage expressed Nanog mRNA. The rate of expression of Oct4 and Nanog mRNA at these developmental stages was higher in in vitro fertilized embryos than in cloned embryos. These results indicated that, during early development, the failure to reactivate some pluripotency genes maybe is a reason for the low cloning efficiency found with cloned embryos.