Haijing Zhao

Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer

Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis

Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality.

Genetic evidence of ‘genuine’ empty follicle syndrome: a novel effective mutation in the LHCGR gene and review of the literature

Empty follicle syndrome (EFS) is a reproductive disorder in which no oocytes are retrieved during IVF. The existence of genuine EFS (GEFS) is still controversial, and to date, only one missense mutation of Luteinizing Hormone/Choriogonadotropin Receptor (LHCGR) has been reported to be associated with this disease. Here, we describe a GEFS patient in a non-consanguineous family from China. A 27-year-old woman presented with a 5-year history of primary infertility and LH resistance-like ovaries of unequal sizes, but with normal levels of circulating LH. In spite of a satisfactory ovarian reserve and response, no oocytes were retrieved after two cycles of IVF. Her condition did not appear to be failure of the hCG injection. It is more likely to be a genetic cause. A novel homozygous mutation in LHCGR gene, c.1345G>A (p.Ala449Thr), was detected in this patient. Each of her parents is heterozygous for this change, and the change was absent from 407 control subjects. Alanine at this amino acid position was highly conserved and replacement of threonine was predicted to disrupt the third transmembrane helix of the rhodopsin-like G protein-coupled receptor domain. Protein localization studies revealed that a portion of the mutant LHCGR protein molecules was retained intracellularly. Signalling studies demonstrated that this mutation had differing effects on the response of LHCGR to hCG or LH at different concentrations. Specifically, at a concentration <1 IU/ml, the mutant was activated by hCG stimulation but partially resistant to LH stimulation; at a higher concentration (>1 IU/ml), the mutant was activated by both hCG and LH. These data suggest that screening for mutations in the LHCGR gene may assist in the diagnosis of patients with GEFS. The literature describing the relationship between phenotype and genotypes in females is reviewed, and possible aetiologies and treatment options for this disease are proposed based on our and other studies.

Exogenous expression of OCT4 facilitates oocyte‐mediated reprogramming in cloned porcine embryos

OCT4 is a well‐established regulator of pluripotency and nuclear reprogramming. To determine if improving OCT4 abundance can facilitate oocyte‐mediated reprogramming in cloned porcine embryos, we artificially increased OCT4 levels by co‐incubating donor cells with 50 ng/µl OCT4 plasmid. We observed higher rates of blastocyst formation (P < 0.05) and lower levels of blastocyst apoptosis in nuclear‐transfer‐derived embryos carrying OCT4‐incubated donor nuclei (OCT4‐SCNT). The beneficial effect caused by exogenous expression of OCT4 involves epigenetic changes, wherein increased histone acetylation (AcH3K9) appeared in OCT4‐SCNT embryos at the one‐cell and blastocyst stages and reduced histone methylation (H3K9me2) was observed at the one‐cell stage (P < 0.05). There was a transient increase in exogenous OCT4 and an up‐regulation of endogenous OCT4 level in OCT4‐SCNT embryos (P < 0.05), while the expression pattern of epigenetic enzymes was changed. These modifications were accompanied by an up‐regulation of CDX2, whose interaction with OCT4 is instrumental for implantation, and a down‐regulation of XIST, a negative indicator of reprogramming (P < 0.05). Taken together, our results support a role for exogenous expression of OCT4 in improving the efficiency of nuclear reprogramming while establishing a convenient and timesaving method to improve nuclear‐transfer outcomes. Mol. Reprod. Dev. 81: 820–832, 2014.

Damage of zona pellucida reduces the developmental potential and quality of porcine circovirus type 2–infected oocytes after parthenogenetic activation

The present aimed to study if porcine circovirus type 2 (PCV2), which adhered to zona pellucida (ZP), was able to enter mature porcine oocytes with intact and damaged ZP. Four groups, including uninfected ZP-intact oocytes (UOZI), uninfected ZP-damaged oocytes (UOZD), PCV2-infected ZP-intact oocytes (POZI), and PCV2-infected ZP-damaged oocytes (POZD) were studied. The oocytes were incubated with 1 mL minimum essential medium, containing 3.1 × 10(8) copies of PCV2 DNA for 1 hour. Mechanical procedure of the insertion by microneedle induced injuries to the ZP of porcine oocytes. At the blastocyst stage, the percentage of PCV2-infected embryos and the ratio of viral antigen-positive cells per embryo were determined by indirect immunofluorescence. To assess the effect of ZP injury on the developmental competence and quality of porcine PCV2-infected oocytes after parthenogenetic activation, blastocyst formation rates and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were analyzed. Moreover, real-time polymerase chain reaction was used to evaluate the expression of genes related to apoptosis and pluripotency at different developmental stages. The results of indirect immunofluorescence showed that only POZD group presented PCV2-infected embryos and viral-positive cells. The blastocyst rate of POZD group dropped down to approximately half of POZI groups (7.1 ± 1.5 vs. 14.5 ± 3.3). At the blastocyst stage, ZP injury increased apoptotic index of PCV2-infected embryos. The relative expression levels of Caspase 3 were higher in POZD group than the ones in POZI group at the two- and four-cell stages (not statistically significant). Compared with the one in POZI group, the ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of the ability to resist apoptosis, was lower in POZD group at the one-cell stage, but higher at the two- and four-cell stages. Expression levels of Oct4 and Nanog associated with pluripotency were lower in POZD group than the ones in POZI group at the morula stage (not statistically significant). Noteworthily, the expression of Nanog was significantly lower in POZD group versus POZI group (P < 0.05), whereas relative expression of Oct4 was significantly higher in the former at the blastocyst stage (P < 0.01). In conclusion, PCV2, which attached to ZP, was able to enter mature porcine oocytes with damaged ZP and subsequently reduced the developmental competence and quality of the oocytes after parthenogenetic activation.

Generation of CD44 gene-deficient mouse derived induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) show good promise for the treatment of defects caused by numerous genetic diseases. Herein, we successfully generated CD44 gene-deficient iPSCs using Oct4, Sox2, Klf4, and vitamin C. The generated iPSCs displayed a characteristic morphology similar to the well-characterized embryonic stem cells. Alkaline phosphatase, cell surface (SSEA1, NANOG, and OCT4), and pluripotency markers were expressed at high levels in these cells. The iPSCs formed teratomas in vivo and supported full-term development of constructed porcine embryos by inter-species nuclear transplantation. Importantly, incubation with trichostatin A increased the efficiency of iPSCs generation by increasing the histone acetylation levels. Moreover, more iPSCs colonies appeared following cell passaging during colony picking, thus increasing the effectiveness of iPSCs selection. Thus, our work provides essential stem cell materials for the treatment of genetic diseases and proposes a novel strategy to enhance the efficiency of induced reprogramming.

Susceptibility of porcine preimplantation embryos to viruses associated with reproductive failure.

In the modern biological area, the applications of pig as a laboratory model have extensive prospects, such as gene transfer, IVF, SCNT, and xenotransplantation. However, the risk of pathogen transmission by porcine embryos is always a topic to be investigated, especially the viruses related to reproductive failure, for instance, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, and porcine circovirus type 2. It should be mentioned that the zona pellucida (ZP) of porcine embryos can be a barrier against the viruses, but certain pathogens may stick to or even pass through the ZP. With intact, free, and damaged ZP, porcine preimplantation embryos are susceptible to these viruses in varying degrees, which may be associated with the virus-specific receptor on embryonic cell membrane. These topics are discussed in the present review.

Ectopic expression of reprogramming factors enhances the development of cloned porcine embryos

Inefficient cloning by somatic cell nuclear transfer (SCNT) is largely attributed to defects in epigenetic reprogramming. Reprogramming factors (RFs) (Oct4, Sox2, Klf4, c-Myc, Lin28 and Nanog; OSKMLN) can achieve epigenetic reprogramming, suggesting that these might facilitate reprogramming of oocytes. Here, porcine mesenchymal stem cells (pMSCs) treated with exogenous OSKMLN or OSKM were selected as nuclei donors for SCNT. The resulting embryos displayed significantly better development than controls in terms of cleavage rates and blastomere numbers. OSKM treatment improved pluripotency status and regulation of epigenetic factors in modified pMSCs. These changed gene patterns promoted H3K9Ac both in modified pMSCs and their SCNT-derived embryos. Thus, higher histone acetylation levels in donor cells might favor subsequent clone development. Application of exogenous RFs in SCNT offers a novel way for improving cloning efficiency.