All-trans retinoic acid (t-RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t-RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus-oocyte complexes (COCs) were matured in vitro in the absence or presence of t-RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real-time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t-RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t-RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t-RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t-RA inhibits the apoptosis of cumulus cells (P < 0.01). t-RA treatment up-regulated the expression of B-cell lymphoma 2 (BCL-2), catalase (CAT) (P < 0.05) and down-regulated the expression of Caspase-8 (P < 0.05). In conclusion, t-RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.
Adipose-derived stem cells (ADSCs), a subset of mesenchymal stem cells, have promising potential for regenerative medicine applications. However, the efficient culture of mouse adipose-derived stem cells (mADSCs) is complicated or impracticable and many properties of mADSCs are still unknown. Here, we report that vitamin C (Vc) is available for the long-term culture of mADSCs in vitro. Compared with that cultured without Vc, mADSCs growing in Vc-added media proliferate faster. The occurrence of replicative senescence and transformation of Vc-treated mADSCs is also postponed. Vc also enhanced the secretory activity of collagen and adipogenic differentiation ability of mADSCs. Moreover, the expression of CD44, Sca-1, and CD105 is higher in Vc-treated mADSCs than nontreated ones. We also found that genes related to proliferation, secretion, and pluripotency are all promoted in Vc-treated mADSCs. However, the adipogenesis ability and expression of CD44, Sca-1, and CD105 decreased when passage increased from very low passages, in parallel to the downregulation of closed-related gene expression. Our results indicate that Vc is essential for the maintenance of original properties of mADSCs in vitro; additional insights into the function of Vc on mADSCs are provided. Furthermore, as the passage increased in six passages, the characteristics of mADSCs with Vc addition were also revealed.
ObjectivecAMP and mature promoting factor (MPF) play critical roles during the maturation of mammalian oocytes. The aim of this study was to produce the offspring from denuded oocytes (DOs) in mice by regulating cAMP and MPF.MethodsIn this study, we used DOs at the germinal vesicle (GV) stage in mice and regulated levels of cAMP and MPF in DOs by adding Forskolin and PD166285 during in vitro maturation without follicle stimulating hormone and luteinizing hormone, respectively.ResultsCombined use of 50 μM Forskolin for 3 h and 2.5 μM PD166285 for additional 21 h enhanced the developmental competence of DOs, maturation rate of DOs was 76.71%± 4.11%, blastocyst rate was 18.33%±4.44% after parthenogenetic activation (PA). The DOs could successfully be fertilized with sperm in vitro, cleavage rate was 17.02%±5.82% and blastocyst rate was 5.65%±3.10%. Besides, 2-cell in vitro fertilization embryos from DOs produced 4 normal live offspring (4/34).ConclusionThe results confirmed that the combination of Forskolin and PD166285 can induce DOs to complete meiosis process and produce normal offspring.
In mammals, the successful development of live offspring by somatic cell nuclear transfer (SCNT) has demonstrated the ability of oocyte or egg cytoplasm to reprogram the differentiated status of somatic DNA. However, the efficiency of development is low, and this has been attributed to incomplete or inappropriate reprogramming of epigenetic status. One such epigenetic marker is methylation of genomic DNA at CpG islands. In SCNT, derived embryo abnormal DNA methylation patterns have been reported by a number of groups; in particular, it has been observed that the methylation pattern of embryonic cells resembles that of the donor cell (Santos et al. 2003 Curr. Biol. 13, 1116-1121). One strategy to improve reprogramming and, hence, development is to erase or reprogram the epigenetic status of the donor cell prior to nuclear transfer. We have previously reported that Xenopus egg and oocyte extracts show a differential effect on transcription. In oocyte extracts Pol I and II transcripts are maintained in the somatic cells; in egg extracts, these are abolished (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). To extend these studies, we have investigated the ability of oocyte and egg extracts to demethylate bovine somatic DNA. Preparation of Xenopus oocyte and egg extracts, culture, permeabilization of donor cells, and incubation conditions were all as previously described (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). Cells were incubated in extracts for 1 and 3 h at 21�C, centrifuged onto glass slides fixed in 4% Para formaldehyde for 15 min, followed by 4 M HCL for 1 h at 39�C, and blocked for 1 h. Cells were stained with mouse monoclonal anti-1MeC (1:50) overnight at 4�C followed by FITC-conjugated goat anti-mouse antibody (1:20) for 1 h at room temperature and mounted in Vectashied containing 10 �g of propidium iodide/mL. Nuclei were scored as positive or negative for 5MeC staining. In control cells, 90% of nuclei stained positively for 5MeC. In both oocyte and egg extracts the number of positive nuclei decreased with time showing demethylation of the somatic DNA 68 and 58% and 38 and 42% positive, respectively, after 1 and 3 h of incubation. Addition of apyrase (2%) to hydrolyze ATP inhibited demethylation in both extracts (90% nuclei positive). High rates of DNA replication were observed in somatic cells in egg extracts in contrast to no replication in oocyte extracts. Aphidicolin (1 �g/20 �L) was added to egg extracts to inhibit DNA replication, and under these conditions, DNA demethylation was abolished, suggesting a passive DNA demethylation mechanism as a result of DNA replication. In conclusion, Xenopus laevis oocyte and egg extracts can demethylate mammalian somatic DNA in an energy-dependent manner. In oocyte extracts, demethylation is independent of DNA replication, suggesting an active mechanism. In egg extracts, DNA replication is required, suggesting a passive mechanism. These studies further demonstrate the differences in reprogramming activities between oocyte and egg cytoplasm and suggest that interspecies extracts may provide a tool for nuclear reprogramming.
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