We recently discovered an unexpected phenomenon of somatic cell reprogramming into pluripotent cells by exposure to sublethal stimuli, which we call stimulus-triggered acquisition of pluripotency (STAP). This reprogramming does not require nuclear transfer or genetic manipulation. Here we report that reprogrammed STAP cells, unlike embryonic stem (ES) cells, can contribute to both embryonic and placental tissues, as seen in a blastocyst injection assay. Mouse STAP cells lose the ability to contribute to the placenta as well as trophoblast marker expression on converting into ES-like stem cells by treatment with adrenocorticotropic hormone (ACTH) and leukaemia inhibitory factor (LIF). In contrast, when cultured with Fgf4, STAP cells give rise to proliferative stem cells with enhanced trophoblastic characteristics. Notably, unlike conventional trophoblast stem cells, the Fgf4-induced stem cells from STAP cells contribute to both embryonic and placental tissues in vivo and transform into ES-like cells when cultured with LIF-containing medium. Taken together, the developmental potential of STAP cells, shown by chimaera formation and in vitro cell conversion, indicates that they represent a unique state of pluripotency.

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming.

Abstract. In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos. Key words: Early embryo, MG132, Mouse, Ubiquitin-proteasome system, Zygotic gene activation (J. Reprod. Dev. 56: [655][656][657][658][659][660][661][662][663] 2010) he proteasome is a large protein complex for degradation of most intracellular proteins in eukaryotic cells [1,2]. For proteasomal degradation, the substrate proteins are required for tagging by ubiquitin. Generally, the ubiquitin-proteasome system (UPS) is involved in a wide variety of biological processes, including DNA repair, apoptosis, immune response, signal transduction, transcription, metabolism, protein quality control and developmental programs [3,4].In mammalian oocytes, the UPS is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation [5][6][7]. Ubiquitin enzyme E1 [8], E2 [9] and E3 [10] are active during mammalian spermatogenesis, suggesting that the UPS may play roles during spermatogenesis [11]. In fertilization, ubiquitination of the sperm mitochondrial membranes may assist in destruction of paternal mitochondria at fertilization for promoting the maternal inheritance of mitochondrial DNA in mammals [11,12].In mouse embryos, the onset of zygotic gene activation (ZGA) has been shown to occur after fertilization and is a critical event that governs the maternal-to-zygotic transition (MZT) for embryonic development [13]. The correct regulation of the onset of ZGA is an important process for remodeling of an oocyte to a totipotent zygote. Recently, several studies have implicated the UPS in the regulation of transcription [14][15][16][17]. These observations led us to investigate a potential link between the UPS and ...

SummaryDuring the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin–proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT.

and then transferring them to recipient mice is challenging, and information other than the category will be lost, making detailed analysis difficult. Combining live-cell imaging and single embryo transfer could overcome this problem 13 , and we could directly link the relationship between the type/severity of the result of transplantation. Further, previous studies on the relationship between embryo ploidy and developmental potential used biopsy of blastocysts and subsequent chromosome analysis 14-16 ; in this study, ploidy of blastomeres of 2-cell embryos was investigated by single-cell genome sequencing after live-cell imaging of 1 st mitosis to link the imaging data of chromosome segregation and ploidy of embryo. Through live-cell imaging, single embryo transfer, and genome sequencing at single-cell resolution, we demonstrated that early chromosomal segregation error resulting in aneuploidy in mouse pre-implantation embryos is a developmental risk to the blastocyst, but some blastocysts retain their developmental potential. Methods Animals. This study conformed to the Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Animal Care and Use Committee at the Research Institute for Kindai University (permit number: KABT-31-016). ICR or B6D2F1 (BDF1) strain mice (12-16 weeks old) were obtained from Japan SLC, Inc. (Shizuoka, Japan). Room conditions were standardized, with the temperature maintained at 23 °C, relative humidity at 50%, and a 12-h/12-h light-dark cycle. Animals had free access to water and commercial food pellets. Mice used for experiments were sacrificed by cervical dislocation.