Earticle

Zinc is an extremely important trace element that plays important roles in several biological processes. In this study, we investigated the role of zinc during meiotic resumption and metaphase arrest in in vitro matured porcine oocytes. Oocytes which arrest at GV or MII stage were treated with TPEN, a Zn2+ chelator, respectively. Meiotic resumption and activation were assayed. Effect of PMA, a PKC activator, on GVBD blocking and oocytes activation results from TPEN treatment were checked. Depletion of zinc with TPEN blocked meiotic resumption and results in failure of metaphase II arrest. The p34cdc2 activity in both MII oocytes and GVBD oocytes which treated with TPEN was decreased. Phosphorylated MAPK also be decreased in GVBD stage after TPEN treatment which might be explained by the low expressions of C-mos Cyclin B1 and Cdc2 in GVBD stage. But treated the oocytes with PKC agonist PMA rescued the meiotic resumption and increased MAPK and increases p34cdc2 activity. Treatment oocytes with PMA in GV stage also increased the zinc content in cytoplasm, showed that zinc regulate meiotic resumption is a PKC dependent event. However, although TPEN treatment reduced phosphorylation of PKC substrates in both meiotic resumption and MII stage, rescue the PKC substrates phosphorylation with PMA didn’t prevent the activation of oocytes caused by zinc depletion. These data demonstrate zinc regulates meiotic resumption via a PKC dependent pathway, but independent of that in maintain metaphase arrest in porcine oocytes.

Actin polymerization is essential for various stages of mammalian oocyte maturation, including asymmetric spindle migration, cortical actin cap formation, polar body extrusion, and cytokinesis. The heterodimeric actin capping protein (CP) is an essential element of the actin cytoskeleton. It binds to the fast-growing (barbed) ends of actin filaments, thereby blocking their elongation, and plays essential roles in various actin-mediated cellular processes. However, the roles of CP in mammalian oocyte maturation are poorly understood. We investigated the roles of CP in mouse oocytes, and found that CP is essential for correct asymmetric spindle migration and polar body extrusion. CP colocalized with cortical actin in immature oocytes, but its localization changed during maturation. Experiments in which CP was knocked down or ectopically overexpressed revealed that this protein is critical for Perturbations in the level of CP impaired spindle migration and polar body extrusion during oocyte maturation. Taken together, this study shows that CP is an essential component of the actin cytoskeleton machinery that plays crucial roles in oocyte maturation.

In meiosis, Emi2 plays important role as CSF (Cytostatic Factor) to make the oocyte arrested in mII stage by the inhibition of APC/C (anaphase promoting complex/cyclosome). Once the oocyte fertilized, Emi2 was destabilized and degraded. For the degradation of Emi2, calcium signaling activate calmodulin-dependent protein kinase (CaMK) and phosphorylate emi2. Phosphorylated emi2 is recognized by polo-box domain of polo-like kinase 1 (Plk1) and further degradated by ubiquitin-dependent proteolysis. But recognition of Plk1 and emi2 is unknown. In this works, we determined the high-resolution crystal structure of polo-box domain of Plk1 and phosphorylated emi2 peptide at 1.90Å. Determined structure revealed that several unique features, including binding of Phe169 in the tyrosin-rich hydrophobic pocket. This is the first report of crystallization that Plk1-emi2 complex. Based on the complex structure, we designed the peptide analogs which pontentially inhibits recognition of Emi2 by Plk1 and assessed its biological activity in oocyte maturation and pathernogenetic activation. Injection of AB103-8, the inhibitor of Plk1 Polo-box domain, in mouse oocytes, induced the maturation arrest in GV stage and the delay in mII parthenogenetic activation. Further investigations of the mechanism that Plk1 involved into the Emi2 mII arrest are underway.

CREB/ATF bZip transcription factor (CREBZF, SMILE) is a member of the mammalian ATF/CREB family of transcription factors and an orphan nuclear receptor. Recent studies have suggested that CREBZF is a novel co-repressor that is involved in nuclear receptor signaling; however, the role of CREBZF in osteoblast differentiation has not discovered yet. This study demonstrates that CREBZF suppressed osteoblast differentiation by regulating activity of Runt-related transcription factor-2 (RUNX2). Tunicamycin, a stimulator of ER-stress, induced expression of CREBZF. Bone morphogenetic protein-2 (BMP2) inhibits the expression of CREBZF, and BMP2-induced expression of alkaline phosphatase and osteocalcin, osteogenic marker genes, was suppressed by CREBZF. The molecular mechanism of CREBZF in differentiation of osteoblast was also determined. An immunoprecipitation assay revealed a physical interaction between CREBZF and RUNX2 that significantly impaired RUNX2-dependent activation of the osteocalcin gene. A ChIP assay revealed that CREBZF repressed the ability of RUNX2 to bind to the osteocalcin gene promoter. Taken together, these findings demonstrate that CREBZF negatively regulates osteocalcin via a direct interaction with RUNX2.

The CRISPR/Cas9 system has been proven to be an efficient gene-editing tool for genome modification of cells and organisms. Specific gene knock out and knock in system in pig holds a bright future for the study of complex disease. Using CRISPR/Cas9 system we targeted OCT4gene specifically knock-out the OCT-4 gene during a preimplantation stage. We injected both Cas9 RNA and sgRNA (OCT-4 targeted in exon2) for deletion of specific site, and also injected Cas9 RNA and two sgRNAs (OCT-4 targeted in exon2 and exon5) for deletion of large scale site in OCT-4 sequence. Injection of cas9 and sgRNA against Oct4 decrease percentages of Oct4-positive embryo to 37～50% of total embryo, while ~100% of control embryo show clear Oct4 immunostaining We checked the presence of mutation near guide sequence using PCR and DNA sequencing, significant portion of clones (20% in Exon 2 and 50% in Exon 5) has insertion/deletion near protospacer adjacent motif(PAM). Different target sites have several frequency of deletion but, different concentration of sgRNA was no difference. Oct4 mRNA levels is dramatically decreased at 8 cell stages and barely detectable in BL stages, while other genes including Sox2, Nanog and CDX2 mRNA levels did not affected. Also, the combination of two sgRNAs can be large scale deletion(about 1.8kb) in the same chromosome. In conclusion, CRISPR/CAS9 system provides a good tool for gene function study by deleting target gene in pig.

Glial fibrillary acidic protein (GFAP) is a class-Ⅲ intermediate filament protein specifically expressed in astrocyte of central nerve system (CNS). Studies have demonstrated that approximately 2.2 kilo-base pairs of the human and mouse GFAP promoter region are sufficient to directly regulate GFAP protein or transgene expression in astrocytes and it has been used in numerous studies dealing with the nerve system development, CNS injury-induced process, and disease models. Based on the notion that a pig displays greater taxonomic resemblance to human than a rodent, here we sought to validate a GFAP promoter sequence of a pig as a better representative of that of human. We compared the promoter, mRNA and protein sequences of human, mouse, and swine GFAP and observed that pig and human GFAP show the greatest amount of sequence resemblance. This suggests that the regulatory mechanism underlying swine GFAP expression is more similar to that of human GFAP. A 5.2kb DNA fragment of miniature swine GFAP promoter was isolated from a genomic DNA of miniature pig, Sus scrofa domesticus. In order to validate the key regulatory element of swine GFAP expression, the isolated DNA fragment was further divided into three segments. These partial and full GFAP sequences were then inserted into luciferase reporter systems and their activities were measured by GFAP inducer treatment. The increased expression of swine GFAP-luciferase transgenes was observed upon the treatment of inducer and this suggests that the swine GFAP promoter can be used to induce the expression of a target gene specifically in swine astrocytes. Furthermore, the promoter may be applied to carry out various scientific procedures such the investigation of physiological mechanism underlying gliosis or the generation of CNS disease model.

After somatic cell nuclear transfer (SCNT), epigenetic state of a differentiated donor cell nucleus must be reversed to the embryonic state. Incomplete epigenetic reprogramming is thought to be causing the low cloning efficiency. The present study was carried out to investigate the effects and mechanism of scriptaid, a novel histone deacetylase inhibitor (HDACi), on the in vitro development of porcine SCNT embryos. We found that treating SCNT embryos with 300 or 500 nM scriptaid for 20 h increased the development of embryos to the blastocyst stage and total cell numbers in per blastocyst( p<0.05). Apoptosis in blastocyst was decreased (p<0.05) in the presence of scriptaid. Scriptaid treatment significantly (p<0.05) increased the levels of H3-acK9 and 5-hmc, and the levels of H3-m3K9 and 5-mc were decreased at the pronuclear stage. Expression level of mir-152 was significantly (p<0.05) increased in scriptaid treated embryos. Moreover, treating embryos with 300 nM scriptaid increased the expression level of the genes that play important roles during embryonic development (OCT4 and CDX2). Additionally, mRNA expression of Dnmt1, Cas3 and Bak were significantly decreased and Bcl-xL was increased in scriptaid treated group compared to control. In conclusion, scriptaid improves the developmental capacity, prevent apoptosis through improving the epigenetic reprogramming in porcine SCNT embryos.