Mi-Ryung Park

Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes

Genomic reprogramming factors in the cytoplasm of germinal vesicle (GV) stage oocytes have been shown to improve the efficiency of producing cloned mouse offspring through the exposure of nuclei to a GV cytoplasmic extract prior to somatic cell nuclear transfer (SCNT) to enucleated oocytes. Here, we developed an extract of GV stage pig oocytes (GVcyto-extract) to investigate epigenetic reprogramming events in treated somatic cell nuclei. This extract induced differentiation-associated changes in fibroblasts, resulting in cells that exhibit pluripotent stem cell-like characteristics and that redifferentiate into three primary germ cell layers both in vivo and in vitro. The GVcyto-extract treatment induced large numbers of high-quality SCNT-generated blastocysts, with methylation and acetylation of H3-K9 and expression of Oct4 and Nanog at levels similar to in vitro fertilized embryos. Thus, GVcyto-extract could elicit differentiation plasticity in treated fibroblasts, and SCNT-mediated reprogramming reset the epigenetic state in treated cells more efficiently than in untreated cells. In summary, we provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with porcine GVcyto-extract.

Alpha 1,3-galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity.

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

α1,3-Galactosyltransferase Deficiency in Germ-Free Miniature Pigs Increases N-Glycolylneuraminic Acids As the Xenoantigenic Determinant in Pig–Human Xenotransplantation

In this study, we examined whether Hanganutziu-Deicher (H-D) antigens are important as an immunogenic non-α1,3-galactose (Gal) epitope in pigs with a disrupted α1,3-galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The α1,3-galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote α1,3-galactosyltransferase gene knockout (GalT-KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of α1,3-galactosyltransferase activity when compared to those of control. Enzyme-linked lectinosorbent assay showed that the heterozygote GalT-KO pig had more sialylα2,6- and sialylα2,3-linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT-KO pig had a higher N-glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT-KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Intraovarian transplantation of primordial follicles fails to rescue chemotherapy injured ovaries

Busulfan and cyclophosphamide (B/C)-treated mice exhibited a marked increase in apoptosis and a concomitant decrease in the ovarian weight. Histological and RT-PCR analysis indicate that the period of germ cell depletion in the B/C-treated ovaries coincides with decreased expression of genes Figla, Lhx8, Nobox, c-kit, and Sox3. However, depletion of the ovarian germ cells is mediated by autophagy-independent pathways that involve Fas/FasL-, TNF-, and/or p53-signalings. Treatment with B/C resulted in the cease of the reproductive function to produce their offspring during the 15th week post-treatment period in female mice. Furthermore, injection of the 3 × 106 GFP positive primordial follicles into the ovaries of the B/C treated mouse did not show apparent colonization of the transplanted follicles within the recipient ovaries. The present results suggest that B/C treatment is closely associated with an increased risk of premature ovarian failure.

Chitosan nanoparticles cause pre- and postimplantation embryo complications in mice.

ABSTRACT Embryo development is a complex and tightly controlled process. Nanoparticle injury can affect normal development and lead to malformation or miscarriage of the embryo. However, the risk that these nanoparticles may pose to reproduction is not clear. In this study, chitosan nanoparticles (CSNP) of near uniform size, in the range of 100 nm, were synthesized and confirmed by a particle size analyzer and transmission electron microscopy. Morulae-stage embryo exposure to CSNP during in vitro culture caused blastocyst complications that had either no cavity or a small cavity. Furthermore, CSNP-treated embryos showed lower expression of not only trophectoderm-associated genes but also pluripotent marker genes. When blastocysts developed in both media with and without CSNP were transferred to recipients, the percentage of blastocysts resulting in viable pups was significantly reduced. These detrimental effects are linked to the reduction of total cell numbers, enhanced apoptosis, and abnormal blastocoels forming at the blastocyst stage, indicating that CSNP treatment might have long-term adverse biological effects in view of pregnancy outcome.

Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development

Although it is known that the tetraploid embryo contributes only to the placenta, the question of why tetraploid embryos differentiate into placenta remains unclear. To study the effect of electrofusion on the development of mouse tetraploid oocytes, mouse two‐cell embryos were fused and cultured in vitro in Chatot‐Ziomek‐Bavister medium. After electrofusion, two chromosome sets from the tetraploid blastomere were individually duplicated before nuclear fusion. At 8–10 hr after electrofusion, each chromosome set was condensing and the nuclear membrane was breaking down. Around 12–14 hr after electrofusion, the two chromosome sets had combined together and had reached the second mitotic metaphase, at this point with 8n sets of chromosomes. Interestingly, we discovered that expression of OCT4, an inner cell mass cells biomarker, is lost by the tetraploid expanded blastocysts, but that CDX2, a trophectoderm cells biomarker, is strongly expressed at this stage. This observation provides evidence clarifying why tetraploid embryos contribute only to trophectoderm. Developmental Dynamics 240:1660–1669, 2011.

Production of transgenic pig as an Alzheimer’s disease model using a multi-cistronic vector system

Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. We generated an AD Tg pig by somatic cell nuclear transfer (SCNT) using a multi-cistronic vector that harbored three AD-related genes with a total of six well-characterized mutations: hAPP (K670N/M671L, I716V, and V717I), hTau (P301L), and hPS1 (M146V and L286P). Four AD Tg cell lines were established from Jeju black pig ear fibroblasts (JB-PEFs); the resultant JB-PEFAD cells harbored transgene integration, expressed transgene mRNAs, and had normal karyotypes. Tg line #2–1, which expressed high levels of the transgenes, was used for SCNT; cleavage and blastocyst rates of embryos derived from this line were lower than those of Non-Tg. These embryos yielded three piglets (Jeju National University AD-Tg pigs, JNUPIGs) revealed by microsatellite testing to be genetically identical to JB-PEFAD. Transgenes were expressed in multiple tissues, and at especially high levels in brain, and Aβ-40/42, total Tau, and GFAP levels were high in brains of the Tg animals. Five or more copies of transgenes were inserted into chromosome X. This is the first report of an AD Tg pig derived from a multi-cistronic vector.

Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos

The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

Comparative Gene Expression Analysis of Somatic Cell Nuclear Transfer-Derived Cloned Pigs with Normal and Abnormal Umbilical Cords

Gene expression profiling of compromised umbilical cords (CUCs) derived from somatic cell nuclear transfer (scNT) clones was performed to determine why scNT-derived clones often exhibit malformed umbilical arteries. Umbilical cord samples were obtained from 65 scNT piglets, and of these, nine displayed a CUC. Microscopic analyses of the scNT clones with CUCs (scNT-CUCs) revealed complete occlusive thrombi that were not detected in the arteries of scNT clones with normal umbilical cords (scNT-Ns). Moreover, whereas the allantoic ducts of the scNT-Ns contained columnar epithelium, the scNT-CUCs lacked this epithelial layer. Compared to scNT-Ns, the scNT-CUCs exhibited severe histological damage, including tissue swelling and vein and arterial damage with complete occlusive thrombi. To investigate functional abnormality, gene expression profiles were created in duplicate using the Platinum Pig 13K oligonucleotide microarray, which contains 13 610 probes of 70 bp in length and is capable of interrogating 13 297 targets with up to one probe per target. Probe sets were selected according to a 2-fold or greater increase or decrease of gene expression in scNT-CUCs compared to scNT-Ns. Most genes expressed in scNT-Ns were also expressed by scNT-CUCs. However, most genes involved in transcriptional regulation, such as JUN, JUNB, and FOSL2, showed a significant decrease in expression in the scNT-CUCs, which may produce a ripple effect capable of altering the transcriptomes of many other cellular processes, including angiogenesis, antioxidation, and apoptosis. The scNT-CUCs with thrombosis showed extensive apoptosis leading to placental insufficiency and related pathology. Considering that the umbilical cord plays a role in the transportation of metabolites to the fetus, placental insufficiency in scNT-CUCs may be caused by an increase in apoptotic protein expression from scNT-derived umbilical cords with hypoplastic arteries, and our results provide evidence that porcine oligonucleotide microarray analysis is a useful tool for screening scNT-derived abnormalities in pigs.

Identification of lactoferrin and glutamate receptor-interacting protein 1 in bovine cervical mucus: A putative marker for oestrous detection

Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous-specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non-oestrous and controlled internal drug release (CIDR)-induced oestrous-stage mucus proteins were purified and subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor-interacting protein 1 (GRIP1) showed a twofold increase during the CIDR-induced oestrous stage compared to the levels in non-oestrous stage in bovine cervical mucus. The RT-PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non-oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.