Kwang-Wook Park

PRODUCTION OF NUCLEAR TRANSFER-DERIVED SWINE THAT EXPRESS THE ENHANCED GREEN FLUORESCENT PROTEIN

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of “large offspring syndrome” were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts

Abstract Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

Dynamic Events Are Differently Mediated by Microfilaments, Microtubules, and Mitogen-Activated Protein Kinase During Porcine Oocyte Maturation and Fertilization In Vitro

Abstract The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.

PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization

We report a novel alkaline extractable protein of the sperm head that exclusively resides in the post-acrosomal sheath region of the perinuclear theca (PT) and is expressed and assembled in elongating spermatids. It is a protein that shares sequence homology to the N-terminal half of WW domain-binding protein 2, while the C-terminal half is unique and rich in proline. A functional PPXY consensus binding site for group-I WW domain-containing proteins, and numerous unique repeating motifs, YGXPPXG, are identified in the proline-rich region. Considering these molecular characteristics, we designated this protein PAWP for postacrosomal sheath WW domain-binding protein. Microinjection of recombinant PAWP or alkaline PT extract into metaphase II-arrested porcine, bovine, macaque, and Xenopus oocytes induced a high rate of pronuclear formation, which was prevented by co-injection of a competitive PPXY motif containing peptide derived from PAWP but not by co-injection of the point-mutated peptide. Intracytoplasmic sperm injection (ICSI) of porcine oocytes combined with co-injection of the competitive PPXY peptide or an anti-recombinant PAWP antiserum prevented pronuclear formation and arrested fertilization. Conversely, co-injection of the modified PPXY peptide, when the tyrosine residue of PPXY was either phosphorylated or substituted with phenylalanine, did not prevent ICSI-induced fertilization. This study uncovers a group I WW domain module signal transduction event within the fertilized egg that appears compulsory for meiotic resumption and pronuclear development during egg activation and provides compelling evidence that a PPXY motif of sperm-contributed PAWP can trigger these events.

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

Regulation of Mitogen-Activated Protein Kinase Phosphorylation, Microtubule Organization, Chromatin Behavior, and Cell Cycle Progression by Protein Phosphatases During Pig Oocyte Maturation and Fertilization In Vitro

Abstract We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.

Phenotyping of Transgenic Cloned Piglets

Numerous reports list the abnormalities obtained from cloning sheep and cattle. To date, few reports provide detailed information regarding the overall health status and performance data of cloned animals. This report follows three litters totaling 10 transgenic cloned piglets from birth through puberty. Significant findings from physical examinations and response to treatments are included, as well as necropsy data from five of the piglets that died during the study. The birth weights, placental weights, and growth rates for this group of piglets were not different from that of control animals raised in the same environment. Hematology and serum chemistry data were collected at 2 days of age, and at 2, 4, 8, 12, 16, 20, and 24 weeks of age. Results indicated a mild anemia and hypoproteinemia in the cloned piglets from birth through 4 weeks of age, but both conditions were corrected by 8 weeks of age. Echocardiography was performed on seven of the piglets. No anatomical defects were detected, but three of the piglets had decreased cardiac output values. However, both animals are growing and show no evidence of clinical disease. The immune system was evaluated by quantification of serum IgM and IgG levels and by determining the population of B-cells, macrophages, helper T-cells (CD4), cytotoxic T-cell (CD8), and double positive T-cells (CD4/CD8). With the exception of one animal, no abnormalities were detected with the immune system of the examined piglets. During the course of this study, five of the 10 piglets were euthanized or died, indicating there is a high mortality rate among cloned piglets, but the remaining five cloned piglets are free from detectable defects.

High developmental competence of pig oocytes after meiotic inhibition with a specific M-phase promoting factor kinase inhibitor, butyrolactone I.

Abstract Butyrolactone I specifically inhibits M-phase promoting factor activation and prevents the resumption of meiosis. These experiments were conducted to examine effects of butyrolactone I on pig oocytes in a serum-free maturation system. The first experiment was conducted to determine the effect of butyrolactone I (0–100 μM) on nuclear maturation. At concentrations of ≥12.5 μM, germinal vesicle breakdown was prevented in >90% of the oocytes after 24 h of culture. In the second experiment, the kinetics of in vitro maturation of butyrolactone I-treated oocytes was investigated. Oocytes were treated with 0 or 12.5 μM butyrolactone I and FSH for 20 h and then cultured with LH in the absence of butyrolactone I for another 24 h. Fewer butyrolactone I-treated oocytes reached MII stage at 36 h compared with controls (5.8% vs. 62.4%, P < 0.01). However, by 44 h, 83.4% of butyrolactone I-treated oocytes reached MII compared with 88.6% of controls. In the third experiment, butyrolactone I-treated oocytes were fertilized and cultured in vitro. No differences (P > 0.05) were found between controls and treated groups in cleavage rate, blastocyst rate, or mean number of cells per blastocyst. Effects of butyrolactone I on mitogen-activated protein kinase activation and localization of microfilaments and active mitochondria were examined by Western blot analysis and laser scanning confocal microscopy, respectively. The results suggested that although butyrolactone I reversibly inhibited germinal vesicle breakdown and mitogen-activated protein kinase activation, it did not affect mitochondrial and microfilament dynamics. Butyrolactone I is a potent inhibitor of nuclear maturation of porcine oocytes, and the inhibition is fully reversible.

Developmental Potential of Porcine Nuclear Transfer Embryos Derived from Transgenic Fetal Fibroblasts Infected with the Gene for the Green Fluorescent Protein: Comparison of Different Fusion/Activation Conditions

Abstract The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42–44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3–5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 μsec (group A), or one pulse of 1.6 kV/cm for 30 μsec followed in 30 min by one pulse of 1.2 kV/cm for 30 μsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean ± SEM, 68.4% ± 3.9%) was higher (P < 0.05) than in group B (59.4% ± 2.3%). The rates of cleavage (50.1% ± 4.6% to 62.8% ± 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% ± 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% ± 1.7% and 4.9% ± 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% ± 4.2%) was higher (P < 0.05) than in group C (58.9% ± 2.7%). The cleavage rates were not different among control and treatment groups (58.1% ± 8.1% to 73.6% ± 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% ± 1.7%) was lower (P < 0.05) than in controls and group C (11.4% ± 2.0% to 16.4% ± 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% ± 1.4%) was higher (P < 0.05) than in the group without CB (70.9% ± 0.2%). The cleavage rate of the control group (85.5% ± 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% ± 2.7% and 63.9% ± 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% ± 2.5% to 19.1% ± 6.0%) and the mean cell number of blastocysts (29.4 ± 5.2 to 45.7 ± 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.