Mitochondrial proteome of mouse oocytes and cisplatin-induced shifts in protein profile.

Mitochondria are essential organelles that provide energy for mammalian cells and participate in multiple functions, such as signal transduction, cellular differentiation, and regulation of apoptosis. Compared with the mitochondria in somatic cells, oocyte mitochondria have an additional level of importance since they are required for germ cell maturation, dysfunction in which can lead to severe inherited disorders. Thus, a systematic proteomic profile of oocyte mitochondria is urgently needed to support the basic and clinical research, but the acquisition of such a profile has been hindered by the rarity of oocyte samples and technical challenges associated with capturing mitochondrial proteins from live oocytes. Here, in this work, using proximity labeling proteomics, we established a mitochondria-specific ascorbate peroxidase (APEX2) reaction in live GV-stage mouse oocytes and identified a total of 158 proteins in oocyte mitochondria. This proteome includes intrinsic mitochondrial structural and functional components involved in processes associated with "cellular respiration", "ATP metabolism", "mitochondrial transport", etc. In addition, mitochondrial proteome capture after oocyte exposure to the antitumor chemotherapeutic cisplatin revealed differential changes in the abundance of several oocyte-specific mitochondrial proteins. Our study provides the first description of a mammalian oocyte mitochondrial proteome of which we are aware, and further illustrates the dynamic shifts in protein abundance associated with chemotherapeutic agents.

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor ß Signaling Pathway.

Differentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified. Here, we show that the neuronal repressor RE1-silencing transcription factor (REST) is rich in porcine oocytes and requires for nuclear transfer (NT)-mediated reprogramming through inhibiting TGFß signaling pathway. REST was dramatically degraded after oocyte activation, but the residual REST was incorporated into the transferred donor nuclei during reprogramming in NT embryos. Inhibition of REST function in oocytes compromised the development of NT embryos but not that of IVF and PA embryos. Bioinformation analysis of putative targets of REST indicated that REST might function on reprogramming in NT embryos by inhibiting TGFß pathway. Further results showed that the developmental failure of REST-inhibited NT embryos could be rescued by treatment of SB431542, an inhibitor of TGFß pathway. Thus, REST is a newly discovered transcription factor that is required for NT-mediated nuclear reprogramming.

Alteration of the DNA methylation status of donor cells impairs the developmental competence of porcine cloned embryos.

Nuclear reprogramming induced by somatic cell nuclear transfer is an inefficient process, and donor cell DNA methylation status is thought to be a major factor affecting cloning efficiency. Here, the role of donor cell DNA methylation status regulated by 5-aza-2'-deoxycytidine (5-aza-dC) or 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP) in the early development of porcine cloned embryos was investigated. Our results showed that 5-aza-dC or 5-methyl-dCTP significantly reduced or increased the global methylation levels and altered the methylation and expression levels of key genes in donor cells. However, the development of cloned embryos derived from these cells was reduced. Furthermore, disrupted pseudo-pronucleus formation and transcripts of early embryo development-related genes were observed in cloned embryos derived from these cells. In conclusion, our results demonstrated that alteration of the DNA methylation status of donor cells by 5-aza-dC or 5-methyl-dCTP disrupted nuclear reprogramming and impaired the developmental competence of porcine cloned embryos.

Epigenetic modification agents improve genomic methylation reprogramming in porcine cloned embryos.

Incomplete DNA methylation reprogramming in cloned embryos leads to low cloning efficiency. Our previous studies showed that the epigenetic modification agents 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) could enhance the developmental competence of porcine cloned embryos. Here, we investigated genomic methylation dynamics and specific gene expression levels during early embryonic development in pigs. In this study, our results showed that there was a typical wave of DNA demethylation and remethylation of centromeric satellite repeat (CenRep) in fertilized embryos, whereas in cloned embryos, delayed demethylation and a lack of remethylation were observed. When cloned embryos were treated with 5-aza-dC or TSA, CenRep methylation reprogramming was improved, and this was similar to that detected in fertilized counterparts. Furthermore, we found that the epigenetic modification agents, especially TSA, effectively promoted silencing of tissue specific genes and transcription of early embryo development-related genes in porcine cloned embryos. In conclusion, our results showed that the epigenetic modification agent 5-aza-dC or TSA could improve genomic methylation reprogramming in porcine cloned embryos and regulate the appropriate expression levels of genes related to early embryonic development, thereby resulting in high developmental competence.

Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.

The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

[Telomere lengthening by trichostatin A treatment in cloned pigs].

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging. In this study, we investigated telomere function in cloned or transgenic cloned pigs, including the cloned Northeast Min pigs, eGFP, Mx, and PGC1α transgenic cloned pigs, and found that the telomere lengths of cloned pigs were significantly shorter than the nuclear donor adult fibroblasts and age-matched noncloned pigs (P<0.05), indicating that nuclear reprogramming did not restore cellular age of donor cells after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), an inhibitor of histone deacetylase, has proven to enhance the efficiency of nuclear reprogramming in several species. In order to test whether TSA also can effectively enhance reprogramming of telomeres, TSA (40 nmol/L) was used to treat porcine cloned embryos at 1-cell stage for 24 h. Consistent with previous reports, the developmental rate of SCNT embryos to the blastocyst stage was significantly increased compared with those of the control group (16.35% vs. 27.09%, 21.60% vs. 34.90%, P<0.05). Notably, the telomere length of cloned porcine blastocysts was also significantly elongated (P<0.05). Although TSA did not improve the cloning efficiency (1.3% vs. 1.7%, TSA vs. control), the telomere lengths of cloned pig-lets were significantly longer compared with those of the control group and the donor fibroblasts (P<0.05). In conclusion, telomeres have not been effectively restored by SCNT in pigs but TSA can effectively lengthen the telomere lengths of cloned pigs.