The mammalian oocyte possesses powerful reprogramming factors, which can reprogram terminally differentiated germ cells (sperm) or somatic cells within a few cell cycles. Although it has been suggested that use of oocyte-derived transcripts may enhance the generation of induced pluripotent stem cells, the reprogramming factors in oocytes are undetermined, and even the identified proteins composition of oocytes is very limited. In the present study, 7,000 mouse oocytes at different developmental stages, including the germinal vesicle stage, the metaphase II (MII) stage, and the fertilized oocytes (zygotes), were collected. We successfully identified 2,781 proteins present in germinal vesicle oocytes, 2,973 proteins in MII oocytes, and 2,082 proteins in zygotes through semiquantitative MS analysis. Furthermore, the results of the bioinformatics analysis indicated that different protein compositions are correlated with oocyte characteristics at different developmental stages. For example, specific transcription factors and chromatin remodeling factors are more abundant in MII oocytes, which may be crucial for the epigenetic reprogramming of sperm or somatic nuclei. These results provided important knowledge to better understand the molecular mechanisms in early development and may improve the generation of induced pluripotent stem cells.germinal vesicle | metaphase II | zygote | protein | reprogramming R eprogramming of patient-specific somatic cells into pluripotent stem cells has attracted wide scientific and public interest because of the great potential value in both research and therapy. Recent advances in induced pluripotent stem cell (iPSC) research have clearly indicated that a small number of transcription factors can reverse the cell fate of differentiated somatic cells; however, the reprogramming process remains slow, and the efficiency is low. Typically, 1% of cells are reprogrammed, but this process requires at least 7 d to 2 wk (1-8). In contrast, reprogramming during somatic cell nuclear transfer (SCNT) occurs within one or two cell cycles and often in a majority of embryos (9-14). The oocytederived transcripts that promote this more efficient reprogramming remain unidentified; however, it has been suggested that their inclusion with the four transcription factors (Oct4, Sox2, Klf4, and c-Myc) may increase the speed and efficiency of the reprogramming process (15). As a step to identification of these factors, this project sought to define the proteome of mouse oocytes at three stages of development, which will also provide us important information on the factors regulating developmental competence of oocytes.During mammalian oogenesis, the oocyte undergoes two cell cycle arrests at the dictyate or germinal vesicle (GV) stage and the metaphase II (MII) stage (16,17). MII oocytes have been widely used to reprogram somatic cell nuclei, because during normal reproduction, sperm and oocyte nuclei are reprogrammed by the MII oocyte to produce totipotent zygotes. By contrast, results from our previous nucl...

Mouse oocytes undergo two successive meiotic divisions to generate one large egg with two small polar bodies. The divisions are essential for preserving the maternal resources to support embryonic development. Although previous studies have shown that some small guanosine triphosphatases, such as RAC, RAN, and CDC42, play important roles in cortical polarization and spindle pole anchoring, no oocytes undergo cytokinesis when the mutant forms of these genes are expressed in mouse oocytes. Here, we show that the ADP-ribosylation factor 1 (ARF1) plays an important role in regulating asymmetric cell division in mouse oocyte meiosis. Microinjection of mRNA of a dominant negative mutant form of Arf1 (Arf1(T31N)) into fully grown germinal vesicle oocytes led to symmetric cell division in meiosis I, generating two metaphase II (MII) oocytes of equal size. Subsequently, the two MII oocytes of equal size underwent the second round of symmetric cell division to generate a four-cell embryo (zygote) when activated parthenogenetically or via sperm injection. Furthermore, inactivation of mitogen-activated protein kinase (MAPK) but not MDK (also known as MEK) has been discovered in the ARF1 mutant oocytes, and this further demonstrated that ARF1, MAPK pathway plays an important role in regulating asymmetric cell division in meiosis I. Similarly, ARF1(T31N)-expressing, superovulated MII oocytes underwent symmetric cell division in meiosis II when activation was performed. Rotation of the MII spindle for 90 degrees was prohibited in ARF1(T31N)-expressing MII oocytes. Taken together, our results suggest that ARF1 plays an essential role in regulating asymmetric cell division in female meiosis.

Meckel-Gruber syndrome type 3 is an autosomal recessive genetic defect caused by mutations in TMEM67 gene. In our previous study, we have identified a homozygous TMEM67 mutation in a Chinese family exhibiting clinical characteristics of MKS3, which provided a ground for further PGD procedure. Here we report the development and the first clinical application of the PGD for this MKS3 family. Molecular analysis protocol for clinical PGD procedure was established using 50 single cells in pre-clinical set-up. After whole genomic amplification by multiple displacement amplification with the DNA from single cells, three techniques were applied simultaneously to increase the accuracy and reliability of genetic diagnosis in single blastomere, including real-time PCR with Taq Man-MGB probe, haplotype analysis with polymorphic STR markers and Sanger sequencing. In the clinical PGD cycle, nine embryos at cleavage-stage were biopsied and subjected to genetic diagnosis. Two embryos diagnosed as free of TMEM67 mutation were transferred and one achieving normal pregnancy. Non-invasive prenatal assessment of trisomy 13, 18 and 21 by multiplex DNA sequencing at 18 weeks’ gestation excluded the aneuploidy of the analyzed chromosomes. A healthy boy was delivered by cesarean section at 39 weeks’ gestation. DNA sequencing from his cord blood confirmed the result of genetic analysis in the PGD cycle. The protocol developed in this study was proved to be rapid and safe for the detection of monogenic mutations in clinical PGD cycle.

Adult stem cells (ASCs) are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues, which are the powerful sources for cell therapy and tissue engineering. Bone marrow-derived mesenchymal stem cells (BMSCs), adipose tissue-derived mesenchymal stem cells (ADSCs), and peripheral blood monocytes (PBMCs) are the common ASCs, and many studies indicated that ASCs isolated from various adult tissues could be induced to hepatocyte-like cells in vitro. However, the isolation, culture protocols, characterization of ASCs and hepatocyte-like cells are different. This review aims to describe the isolation and culture procedures for ASCs, to summarize the molecular characterization of ASCs, to characterize function of hepatocyte-like cells, and to discuss the future role of ASCs in cell therapy and tissue engineering.