Shichao Liu

Sox2 is the faithful marker for pluripotency in pig: Evidence from embryonic studies

Background: Mammalian first lineage segregation generates trophectoderm (TE) and pluripotent inner cell mass (ICM), which provides an ideal model for studying the mechanisms of maintenance and loss of pluripotency. In mouse, the transcription factor OCT4 restricts to ICM and plays a key role in TE/ICM specification and pluripotent regulatory networks. However, in pig, OCT4 does not restrict to ICM cells, suggesting a different molecular basis in TE/ICM specification and pluripotent regulatory networks. Results: To explore molecular basis of porcine TE/ICM specification and pluripotent regulatory networks, we examined expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2, and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress, whereas OCT4 expressed in almost all the cells at the same time. Consistently, analysis of spatiotemporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts, whereas no correlation was observed between OCT4 and CDX2 at the same stage. Conclusions: Our results provide a molecular basis in porcine embryonic patterning and a clue for further studying porcine pluripotent regulatory networks. Developmental Dynamics 244:619–627, 2015.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two‐cell embryos

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN‐, MERVL‐, and ERVK‐associated and essential for mouse embryonic development beyond the two‐cell stage. LincGET is expressed in late two‐ to four‐cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two‐cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA–protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis‐regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two‐cell stage. Thus, as the first functional ERV‐associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

Efficient generation of mouse ESCs-like pig induced pluripotent stem cells

Dear Editor, Porcine induced pluripotency stem cells (piPSCs) are promised in basic research, animal husbandry and regenerative medicine. However, the efficiency of the piPSCs induction has been low and the generated piPSCs varied in cell morphology and cell characteristics. Here we report a novel approach to improve efficiency of piPSCs generation. The induced piPSCs are dome-shaped mouse embryonic stem cells (ESCs)-like and display molecular properties of mouse ESCs. Electroporation study reveals that mouse ESCslike status facilitates genetic manipulating of piPSCs. Importantly, we demonstrate that the domed piPSC colonies are more suitable as donor cells for nuclear transfer (NT) to generate reconstructed embryos than those flattened piPSCs. The potential applications of the newly generated piPSCs in ungulate pluripotent research are discussed.

CDX2 is essential for cell proliferation and polarity in porcine blastocysts.

The role of CDX2 in trophectoderm (TE) cells has been extensively studied, yet the results are contradictory and species specific. Here, CDX2 expression and function were explored in early porcine embryos. Notably, siRNA-mediated gene knockdown and lentivirus-mediated TE-specific gene regulation demonstrated that CDX2 is essential for the maintenance of blastocyst integrity by regulating the BMP4-mediated blastocyst niche and classic protein kinase C (PKC)-mediated TE polarity in mammalian embryos. Mechanistically, CDX2-depleted porcine embryos stalled at the blastocyst stage and exhibited apoptosis and inactive cell proliferation, possibly resulting from BMP4 downregulation. Moreover, TE cells in CDX2-depleted blastocysts displayed defective F-actin apical organization associated with downregulation of PKCα (PRKCA). Collectively, these results provide further insight into the functional diversity of CDX2 in early mammalian embryos. Summary: The transcription factor CDX2 functions in the trophectoderm of pig embryos to maintain polarity and integrity of the blastocyst, revealing further diversity in CDX2 activities across mammals.

Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

Background: Oocyte factors can reprogram the somatic nucleus efficiently, but these factors still need to be defined. Results: Maternal vimentin acts as a genomic protector and results in p53 down-regulation during nuclear reprogramming. Conclusion: Maternal vimentin is crucial for nuclear reprogramming. Significance: We report the first evidence of vimentin as a reprogramming factor. Nuclear reprogramming of somatic cells can be induced by oocyte factors. Despite numerous attempts, the factors responsible for successful nuclear reprogramming remain elusive. In the present study, we found that porcine oocytes with the first polar body collected at 42 h of in vitro maturation had a stronger ability to support early development of cloned embryos than porcine oocytes with the first polar body collected at 33 h of in vitro maturation. To explore the key reprogramming factors responsible for the difference, we compared proteome signatures of the two groups of oocytes. 18 differentially expressed proteins between these two groups of oocytes were discovered by mass spectrometry (MS). Among these proteins, we especially focused on vimentin (VIM). A certain amount of VIM protein was stored in oocytes and accumulated during oocyte maturation, and maternal VIM was specifically incorporated into transferred somatic nuclei during nuclear reprogramming. When maternal VIM function was inhibited by anti-VIM antibody, the rate of cloned embryos developing to blastocysts was significantly lower than that of IgG antibody-injected embryos and non-injected embryos (12.24 versus 22.57 and 21.10%; p < 0.05), but the development of in vitro fertilization and parthenogenetic activation embryos was not affected. Furthermore, we found that DNA double strand breaks dramatically increased and that the p53 pathway was activated in cloned embryos when VIM function was inhibited. This study demonstrates that maternal VIM, as a genomic protector, is crucial for nuclear reprogramming in porcine cloned embryos.

Telomere Elongation Facilitated by Trichostatin A in Cloned Embryos and Pigs by Somatic Cell Nuclear Transfer

Telomere attrition and genomic instability are associated with organism aging. Concerns still exist regarding telomere length resetting in cloned embryos and ntES cells, and possibilities of premature aging of cloned animals achieved by somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), a histone deacetylase inhibitor, effectively improves the developmental competence of cloned embryos and animals, and recently contributes to successful generation of human ntES cells by SCNT. To test the function of TSA on resetting telomere length, we analyzed telomeres in cloned blastocysts and pigs following treatment of SCNT embryos with TSA. Here, we show that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however TSA significantly increases telomere lengths in cloned pigs. Telomeres elongate in cloned porcine embryos during early cleavage from one-cell to four-cell stages. Notably, TSA facilitates telomere lengthening of cloned embryos mainly at morula-blastocyst stages. Knockdown of pTert by shRNA in donor cells reduces telomerase activity in cloned blastocysts but does not abrogate telomere elongation in the TSA-treated embryos (p > 0.05). However, genes associated with recombination or telomerase-independent mechanism of alternative lengthening of telomeres (ALT) Rad50 and BLM show increased expression in TSA-treated embryos. These data suggest that TSA may promote telomere elongation of cloned porcine embryos by ALT. Together, TSA can elongate telomeres in cloned embryos and piglets, and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with TSA.

Cdx2 represses Oct4 function via inducing its proteasome-dependent degradation in early porcine embryos.

Reciprocal repression of inner cell mass specific factor OCT4 and trophectoderm specific factor CDX2 promotes mouse first lineage segregation. Studies in mouse embryonic stem (ES) cells revealed that they bind to each others regulatory regions to reciprocally suppress transcription, additionally they form protein complex for mutual antagonism. However, so far the molecular interaction of Oct4 and Cdx2 in other mammals early embryo is not yet investigated. Here, over-expression of Cdx2 in early porcine embryo showed CDX2 represses Oct4 through neither the transcriptional repression nor forming repressive complex, but promoting OCT4 nuclear export and proteasomal degradation. The results showed novel molecular regulation of CDX2 on Oct4, and provided important clues for clarifying the mechanism of interaction between CDX2 and Oct4 in embryo of mammals other than mouse.

Morphological changes and germ layer formation in the porcine embryos from days 7-13 of development

Morphogenesis and identification of embryonic differentiation in porcine embryos are crucial issues for developmental biology and laboratory animal science. The current paper presents a study on the asynchronous development of hatched porcine embryos from days 7 to 13 post-insemination. Examination of semi-thin sections of the hypoblast showed that it had characteristics similar to those of the mouse anterior visceral endoderm during embryonic disc formation. Also, a cavity appeared in the epiblast, which was similar to a mouse proamniotic cavity. With the gradual disappearance of Raubers layer, the cavity opened and contacted the external environment directly, all of which formed the embryonic disc. To confirm the differentiation characteristics, we performed immunohistochemical analyses and showed that GATA6 was detected clearly in parietal endoderm cells during embryonic disc establishment. OCT4 was expressed in the inner cell mass (ICM) and trophoblast of hatched blastocysts and in the epiblast during formation of the embryonic disc. However, OCT4 showed comparatively decreased expression in the posterior embryonic disc, primitive streak and migrating cells. SOX2 was present in the ICM and epiblast. Therefore, both SOX2 and OCT4 can be used as markers of pluripotent cells in the porcine embryonic disc. At the start of gastrulation, staining revealed VIMENTIN in the posterior of the embryonic disc, primitive streak and in migrating cells that underlay the embryonic disc and was also expressed in epiblast cells located in the anterior primitive streak. Together with serial sections of embryos stained by whole mount immunohistochemistry, the mesoderm differentiation pattern was shown as an ingression movement that took place at the posterior of the embryonic disc and with bilateral migration along the embryonic disc borders.

A Novel Role for DNA Methyltransferase 1 in Regulating Oocyte Cytoplasmic Maturation in Pigs

Maternal factors are required for oocyte maturation and embryo development. To better understand the role of DNA methyltransferase 1 (Dnmt1) in oocyte maturation and embryo development, small interfering RNA (siRNA) was conducted in porcine oocytes. In this study, our results showed that Dnmt1 localized in oocyte cytoplasm and its expression displayed no obvious change during oocyte maturation. When siRNAs targeting Dnmt1 were injected into germinal vesicle (GV) stage oocytes, Dnmt1 transcripts significantly decreased in matured oocytes (P<0.05). After Dnmt1 knockdown in GV stage oocytes, the significant reduction of glutathione content, mitochondrial DNA copy number, glucose-6-phosphate dehydrogenase activity and expression profiles of maternal factors and the severely disrupted distribution of cortical granules were observed in MII stage oocytes (P<0.05), leading to the impaired oocyte cytoplasm. Further study displayed that Dnmt1 knockdown in GV stage oocytes significantly reduced the development of early embryos generated through parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (P<0.05). In conclusion, Dnmt1 was indispensable for oocyte cytoplasmic maturation, providing a novel role for Dnmt1 in the regulation of oocyte maturation.

Identification and characterization of L1-specific endo-siRNAs essential for early embryonic development in pig

Small noncoding RNAs (sncRNAs) play important roles in RNA interference (RNAi). In addition to microRNA (miRNA) and Piwi-interacting RNA (piRNA), one key member of sncRNAs group is endogenous small interfering RNA (endo-siRNA). Some studies do show the role of endo-siRNAs in Dicer and/or Ago mutants, however, the biological functions of specific endo-siRNAs remains mostly unanswered. In the study, we have performed a comparative analysis of endo-siRNAs present in porcine sperms, oocytes and zygotes, identified by deep sequencing and bioinformatics analysis. Further, we observe a large amount of endo-siRNAs specific binding on ORF2 and 3′ UTR of porcine L1 (L1-siRNAs). And, 9 L1-siRNAs generated from a dsRNA formed between L1 transcript and a newly identified an antisense noncoding RNA was characterized. We show the L1-siRNAs regulate early embryonic development by inhibiting the activity of L1 retrotransposition. This work can contribute to understanding the functional role of abundant endo-siRNAs in embryonic development.