Zubing Cao

Efficient reprogramming of naive-like induced pluripotent stem cells from porcine adipose-derived stem cells with a feeder-independent and serum-free system.

Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naïve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naïve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naïve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers in vitro and in vivo. Furthermore, nuclear transfer embryos from naïve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naïve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs.

Effects of chemically defined medium on early development of porcine embryos derived from parthenogenetic activation and cloning.

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.

Genome-Wide Dynamic Profiling of Histone Methylation during Nuclear Transfer-Mediated Porcine Somatic Cell Reprogramming

The low full-term developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos is mainly attributed to imperfect epigenetic reprogramming in the early embryos. However, dynamic expression patterns of histone methylation involved in epigenetic reprogramming progression during porcine SCNT embryo early development remain to be unknown. In this study, we characterized and compared the expression patterns of multiple histone methylation markers including transcriptionally repressive (H3K9me2, H3K9me3, H3K27me2, H3K27me3, H4K20me2 and H4K20me3) and active modifications (H3K4me2, H3K4me3, H3K36me2, H3K36me3, H3K79me2 and H3K79me3) in SCNT early embryos from different developmental stages with that from in vitro fertilization (IVF) counterparts. We found that the expression level of H3K9me2, H3K9me3 and H4K20me3 of SCNT embryos from 1-cell to 4-cell stages was significantly higher than that in the IVF embryos. We also detected a symmetric distribution pattern of H3K9me2 between inner cell mass (ICM) and trophectoderm (TE) in SCNT blastocysts. The expression level of H3K9me2 in both lineages from SCNT expanded blastocyst onwards was significantly higher than that in IVF counterparts. The expression level of H4K20me2 was significantly lower in SCNT embryos from morula to blastocyst stage compared with IVF embryos. However, no aberrant dynamic reprogramming of H3K27me2/3 occurred during early developmental stages of SCNT embryos. The expression of H3K4me3 was higher in SCNT embryos at 4-cell stage than that of IVF embryos. H3K4me2 expression in SCNT embryos from 8-cell stage to blastocyst stage was lower than that in the IVF embryos. Dynamic patterns of other active histone methylation markers were similar between SCNT and IVF embryos. Taken together, histone methylation exhibited developmentally stage-specific abnormal expression patterns in porcine SCNT early embryos.

Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer

Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

WDR5 in porcine preimplantation embryos: expression, regulation of epigenetic modifications and requirement for early development†

Abstract WD repeat-containing protein 5 (WDR5), a member of conserved WD40 protein family, is an essential component of the mixed lineage leukemia (MLL) complexes, which are crucial for numerous key biological processes including methylation of histone H3 lysine 4 (H3K4), self-renewal of embryonic stem cells, and formation of induced pluripotent stem cells. The expression pattern and functional role of WDR5 during porcine preimplantation embryonic development, however, remain unknown. Our results showed that the transcripts and protein of WDR5 exhibited stage-specific expression pattern in porcine early embryos. Moreover, blastocyst rate and total cell number per blastocyst were reduced by RNAi-mediated silencing of WDR5 or pharmacological inhibition of WDR5. Knockdown of WDR5 also disturbed the expression of several pluripotency genes. Interestingly, tri-methylation of H3K4 (H3K4me3) level was dramatically increased by WDR5 depletion. Further analysis revealed that loss of MLL3 phenocopied WDR5 knockdown, triggering increased H3K4me3 level. Simultaneously, WDR5 depletion significantly decreased the levels of histone H4 lysine 16 acetylation (H4K16ac) and its writer males absent on the first (MOF). Last but not least, WDR5 knockdown induced DNA damage and DNA repair defects during porcine preimplantation development. Taken together, results of described studies establish that WDR5 plays a significant role in porcine preimplantation embryos probably through regulating key epigenetic modifications and genome integrity. Summary Sentence WDR5 participates in the regulation of porcine embryonic development, likely mediated by regulation of epigenetic modifications (H3K4me3 and H4K16ac) and genome integrity.

DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment for 12 or 24 h significantly enhanced the blastocyst rate of SCNT embryos and dramatically reduced the level of H3K79me2 during SCNT 1-cell embryonic development. Additionally, H3K79me2 level in the EPZ-treated SCNT embryos was similar to that in in vitro fertilized embryos, suggesting that DOT1L-mediated H3K79me2 is a reprogramming barrier to early development of porcine SCNT embryos. qRT-PCR analysis further demonstrated that DOT1L inactivation did not change the expression levels of DOT1L itself but increased the expression levels of POU5F1, LIN28, SOX2, CDX2 and GATA4 associated with pluripotency and early cell differentiation. In conclusion, DOT1L inhibitor improved early developmental efficiency of porcine SCNT embryos probably via inducing the increased expression of genes important for pluripotency and lineage specification.

Maternal histone acetyltransferase KAT8 is required for porcine preimplantation embryo development

K (lysine) acetyltransferase 8 (KAT8), an acetyltransferase that specifically catalyzes histone H4 lysine 16 acetylation, is critical for key biological processes including cell proliferation and maintenance of genome stability. However, the role of KAT8 during preimplantation development in pigs remains unclear. Results herein showed that KAT8 mRNA is maternally derived and it is required for successful development of early embryos. An abundance of KAT8 transcripts are expressed in oocytes and its abundance continuously decreases throughout meiotic maturation and preimplantation development. In addition, KAT8 expression is insensitive to RNA polymerase II inhibitor after embryonic genome activation, suggesting its maternal origin. The levels of KAT8 mRNA and H4K16 acetylation were effectively knocked down by siRNA microinjection. Knockdown of KAT8 significantly reduced the blastocyst formation rate and total cell number per blastocyst. Analysis of trophectoderm lineage and marker of DNA double-strand breaks revealed that the impaired developmental competence and quality of embryos might be attributed to defects in both the first two lineages development and genome integrity. Taken together, these results demonstrate that maternal KAT8 is indispensible for porcine early embryo development potentially through maintaining the proliferation of the first two lineages and genome integrity.

Membrane receptor-independent inhibitory effect of melatonin on androgen production in porcine theca cells

Excessive secretion of androgens including androstenedione and testosterone in theca cells frequently causes female infertility in mammals. Melatonin is a potent inhibitor of androgen production in gonadal cells of several species in a membrane receptor-dependent manner. However, the function of melatonin in steroidogenesis of porcine theca cells remains unclear. Here we report that melatonin inhibits androgen biosynthesis independently of its membrane receptors in pigs. Using flow cytometry, immunofluorescence and RT-PCR we showed that the vast majority of cells isolated from the theca layer of antral follicles are indeed theca cells. Furthermore, we demonstrated that of the two of melatonin membrane receptors encoded in the porcine genome, theca cells exclusively express melatonin receptor 1B. Cell counting analysis indicated that different concentrations of melatonin did not alter the normal viability and proliferation of theca cells. Additionally, hormone radioimmunoassay and qPCR respectively showed that a high concentration of melatonin significantly repressed both androgen production and expression of steroidogenic genes involving StAR, CYP11A1, HSD3β and SET (P < 0.05), but did not impair progesterone production. Interestingly, these effects were not reversed by N-acetyl-2-benzyltryptamin, a melatonin membrane receptor antagonist. Overall, these results demonstrate that melatonin inhibits androgen production in porcine theca cells independently of its membrane receptor.

RNA-seq analysis of lncRNA-controlled developmental gene expression during puberty in goat & rat

BackgroundPuberty is a pivotal stage in female animal development, and marks the onset of reproductive capability. However, little is known about the function of lncRNAs (long noncoding RNAs) in puberty. Therefore, RNA-seq analysis were performed between goats and rats to clarify the roles of lncRNAs and mRNAs in the onset of puberty.ResultsIn the present study, the length of lncRNAs, the length of the open reading frame and the exon count were compared between the two species. Furthermore, functional annotation analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis of lncRNAs target genes and differentially expressed mRNA demonstrated the significantly enriched terms, such as AMPK signaling pathway, oxytocin signaling pathway, insulin secretion as well as pheromone receptor activity, and some other signaling pathways which were involved in the regulation of female puberty. Moreover, our results of siRNA interference in vitro showed the candidate lncRNA XLOC_446331 may play a crucial role in regulating female puberty.ConclusionIn conclusion, the RNA-seq analysis between goat and rat provide novel candidate regulators for genetic and molecular studies on female puberty.