[Show abstract][Hide abstract] ABSTRACT: Cryopreservation of bovine embryos can be performed by a variety of methods with variable degree of success. Here, we report a new, easy to perform, simple, inexpensive, and successful method for vitrification of bovine blastocysts. In vitro produced bovine blastocysts were exposed to vitrification solution (5.5 m ethylene glycol, 10% serum and 1% sucrose) in one single step for 20 s, loaded on a paper container prepared from commonly available non-slippery, absorbent writing paper, and then were directly plunged into liquid nitrogen for storage. Vitrified blastocysts were warmed by serial rinsing in 0.5, 0.25 and 0.125 m sucrose solution for 1 min each. Results showed that one step exposure of bovine blastocysts to cryoprotective agents was sufficient to achieve successful cryopreservation. Under these conditions, more than 95% of blastocysts survived the vitrification-warming on paper containers which was significantly higher than those obtained from other containers, such as electron microscope (EM) grid (78.1%), open pulled straw (OPS; 80.2%), cryoloop (76.2%) or plastic straw (73.9%). Embryo transfer of blastocysts vitrified-warmed on paper container resulted in successful conception (19.3%) and full-term live birth of offspring (12.3%) which were lower (P < 0.05) than those obtained from non-vitrified blastocysts (38.0 and 32.7%) but were comparable (P > 0.05) to those obtained from blastocysts vitrified-warmed on EM grid (23.3 and 14.2%). Our results, therefore, suggest that paper may be an inexpensive and useful container for the cryopreservation of animal embryos.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n=4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10mM) of 3MA for 22h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P<0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2±0.8 v. 54.0±10.1%) or absence (96.5±1.8 v. 41.0±17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9±13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22h by 3MA, were further cultured for 22h in the absence of 3MA, 96.1±1.5% of oocytes reached metaphase II stage at 42h of in vitro maturation and did not differ (P>0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1±1.2 v. 72.7±2.8%), and form blastocyst (15.4±1.5 v. 12.7±0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8±1.7 v. 84.6±5.1%; blastocyst rate: 44.3±12.4 v. 45.1±7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage.
Reproduction Fertility and Development 01/2011; 23(1):230-231. DOI:10.1071/RDv23n1Ab265 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells, can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which, upon testicular transplantation, produce teratomas instead of initiating spermatogenesis. This study evaluated the DNA methylation and expression of imprinted microRNA (miRNA) in mouse GS and maGS cells. The GS and maGS cell lines were established essentially as described earlier (Jung et al. 2010 Mol. Hum. Reprod. PMID: 20610616) and were quantified for maternally (miR-296-3p, miR-296-5p, miR-483) and paternally (miR-127, miR-127-5p) imprinted miRNA by real-time TaqMan(®) MicroRNA assay and for DNA methylation at imprinting control regions of respective miRNA (Gnas-Nespas DMR, Igf2-H19 ICR, and Dlk1-Dio3 IG-DMR) by bisulfite genomic sequencing. Sperm and embryonic stem (ES) cells were used as controls for comparison. Results showed that, similar to sperm, expression of maternally imprinted miRNA was consistently higher (P<0.001), whereas that of paternally imprinted miRNA was consistently lower (P<0.001) in GS cells than in control ES cells. The DNA methylation analyses further confirmed that imprinted miRNA were androgenetic in GS cells. On the other hand, DNA methylation of maGS cells resembled that of ES cells, but the expression pattern of imprinted miRNA was intermediate between that of GS cells and ES cells. The expression of imprinted miRNA in GS and maGS cells was also altered during their in vitro differentiation but varied with both the differentiation stage and the miRNA. In conclusion, our data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNA which changes to an ES cell-like pattern upon their conversion to maGS cells and, therefore, may serve as an epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.
Reproduction Fertility and Development 01/2011; 23(1):248. DOI:10.1071/RDv23n1Ab302 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids and triacylglycerols and the accumulation of hepatic lipids by increasing mRNA expression related to lipid metabolism, and that excess E(2) in the blood leads to activation of E(2) catabolic metabolism (cytochrome P450 3A37)-related mRNA expression.
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor (EGF) has been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes, but inconsistencies exist between earlier studies, probably due to differences in the culture conditions used. Using a serum- and hormone-free in vitro maturation (IVM) medium, this study investigated the specific contribution of EGF on IVM of porcine (Sus scrofa) oocytes and its interactive effects with follicle-stimulating hormone (FSH), porcine follicular fluid (pFF), cumulus cells, and serum. It was noteworthy that EGF functionally mimicked the action of FSH and could completely replace FSH for nuclear maturation (83.2+/-4.4% vs. 55.9+/-5.2%; mean+/-SEM), whereas EGF had a synergistic effect with FSH on cytoplasmic maturation of porcine oocytes (P<0.05). Specific inhibition of EGF receptor (EGFR) by tyrphostin AG 1478 inhibited both EGF- and FSH-induced meiotic resumption (17.9+/-5.2% and 18.2+/-4.4%, respectively), thereby suggesting that EGFR signaling pathway was essential for oocyte reentry into the meiotic cell cycle. Furthermore, it is possible that FSH action occurs via the EGFR signaling pathway to induce meiotic maturation, although alternate pathways could not be excluded. There were also individual or combined effects of cumulus cells, FSH, serum, and pFF with EGF on IVM of porcine oocytes (P<0.05). Although FSH had a synergistic effect with EGF on cytoplasmic maturation, pFF masked the effects of EGF on both nuclear and cytoplasmic maturation of porcine oocytes (P<0.05). Moreover, the presence of cumulus cells was essential for EGF action. In conclusion, a defined system was used to better examine the effects of EGF. We inferred that EGF functionally mimics FSH for nuclear maturation of porcine oocytes, and its exogenous supplementation into IVM medium can optimize the beneficial effects of FSH on cytoplasmic maturation of oocytes to obtain enhanced embryo development in vitro.
[Show abstract][Hide abstract] ABSTRACT: Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We have analysed 1970 high-quality chromatograms (Phred value >or= 30) that were obtained from sequencing the 5' ends of brainstem, liver, neocortex and spleen clones derived from full-length enriched cDNA libraries from Korean native pigs. In addition, 50,000 pig expressed sequence tag (EST) sequence trace files were obtained from Genbank and combined with our sequencing information to facilitate SNP identification in silico. The process generated 8118 contigs, of which 239 included minimum one sequence from Korean native pig and contained 678 putative coding single nucleotide polymorphisms (cSNPs). Of these, 33 putative cSNPs were randomly selected for confirmatory analysis and validated using 20 pigs from four different breeds (Duroc, Landrace, Yorkshire, Korean native pig). Of the 33 putative cSNPs, 20 were confirmed (61%), which was similar to the frequency reported in other studies. We also identified 15 new cSNPs from the validation process, which were not detected by our in silico analysis. Our study shows that analysing genetically diverse pig breeds including the Korean native pig could serve as a useful strategy for generating a large number of cSNPs.
Journal of Animal Breeding and Genetics 05/2009; 126(2):127-33. DOI:10.1111/j.1439-0388.2008.00765.x · 1.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNbeta-Z or LNbeta-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro. Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNbeta-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 +/- 6.4%; 12.0 +/- 5.7%) or EGFP (57.5 +/- 6.3%; 10.1 +/- 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 +/- 8.2%; 12.3 +/- 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.
[Show abstract][Hide abstract] ABSTRACT: Simultaneous quantification of multiple proteins could be a very useful tool in cell signaling studies. Parthenogentic embryos do not develop to term, although their in vitro developmental ability to the blastocyst stage and to form pluripotent stem cells upon culture is comparable to those of sperm- or somatic nucleus-introduced embryos. This study was designed to quantify the expression of eight selected proteins by multiple selected reaction monitoring (mSRM) of trypsinized preparations from porcine zygotes (n = 1500) produced by parthenogenesis. The trypsinized samples were prepared, spiked with isotopically labeled synthetic MAP3K (IPTGTV*HNQAK) peptides as internal standards for quantification (asterisk denotes isotopic amino acid residue), and analyzed by reverse phase LC-MS/MS. Upon the appearance of the target peptide, SRM data were acquired within fragmention mass ±1.5 m/z and each SRM transition, with its respective retention time, was validated as indicative of each specific peptide. Data were processed by integrating the appropriate peaks for the native and internal standard peptide, followed by calculation of the ratio of peak areas to estimate the relative abundance of the peptide. Analysis of data showed that the relative abundance of Janus kinase 2 (JAK2), signal transducer and activator of transcription 1 (STAT1), STAT2, calpastatin, complement cytolysis inhibitor, plakoglobin, serotransferrin, and epsilon-globin were 203.72, 138.52, 60.50, 18.63, 31.23, 0.85, 10.33, and 0.12, respectively. These data were consistent with those observed by reverse phase LC-MS/MS combined with 1-dimensional (1-D) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of cellular lysates from porcine embryos (n = 6000). The data presented here, therefore, provide mSRM of embryos for the first time in any species and suggest that mSRM in combination with reverse-phase LC-MS/MS combined with 1-D SDS-PAGE could be a powerful tool for proteome analysis of porcine embryos to obtain vital new information for understanding the basic biology of embryonic development.
This study was supported by grants from Biogreen 21, RDA, and the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.
Reproduction Fertility and Development 01/2008; 20(1). DOI:10.1071/RDv20n1Ab173 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have demonstrated previously that retroviral-mediated gene transfer is a promising method to produce transgenic avian, porcine, and bovine embryos. This study was designed to evaluate the development potential of transgenic porcine embryos produced by somatic cell nuclear transfer (SCNT) of fetal fibroblast (pFF) cells transfected by a robust replication-defective retroviral vector harboring enhanced green fluorescent protein (EGFP) or β-galactosidase (LacZ) gene. Moloney murine leukemia virus (MoMLV)-based retroviral vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein and harboring EGFP or LacZ under the control of β-actin promoter were produced and used to transfect primary pFF cells that were subsequently used for SCNT of enucleated porcine oocytes matured in vitro. Our results showed that all surviving cells after transfection and antibiotic selection expressed the genes without any evidence of replication-competent retrovirus. The fusion, cleavage, and blastocyst rates were 85.6 ± 6.5, 53.6 ± 6.4, and 12.0 ± 5.7% for EGFP; 83.5 ± 8.2, 57.5 ± 6.3 and 10.1 ± 4.1% for LacZ; and 80.5 ± 4.2, 60.9 ± 8.2 and 12.3 ± 4.0% for controls, respectively. Mosaicism was not observed in any of the group as evidenced by the expression of LacZ or EGFP in individual blastomeres of all embryos upon staining with β-galactosidase (for LacZ) or when visualized under UV illumination of an epifluorescent microscope using the fluorescein isothiocyanate (FITC) filter set (for EGFP). Further recloning of EGFP-expressing blastomeres, obtained from 4-cell-stage cloned embryos produced by SCNT of pFF cells infected with EGFP harboring vector, into enucleated metaphase II (MII) oocytes resulted in consistent expression of EGFP in recloned blastocysts. Interspecies SCNT (iSCNT) of transfected pFF into enucleated bovine oocytes could also result in consistent gene expression without any adverse effect on blastocyst rate (5.5 v. 4.9%) compared with non-transfected pFF. These data indicate that the replication-defective retroviral vector used in the present study is robust and independent of the genes inserted. Furthermore, introduction of transgenes by this method does not influence the in vitro development rate of cloned embryos.
This work was supported by a grant from Biogreen 21 Program, RDA, Republic of Korea.
Reproduction Fertility and Development 01/2008; 20(1). DOI:10.1071/RDv20n1Ab310 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transgenic chickens, ubiquitously expressing a human protein, could be a very useful model system for studying the role of human proteins in embryonic development as well as for efficiently producing pharmaceutical drugs as bioreactors. Human parathormone (hPTH) secreted from parathyroid glands plays a significant role in calcium homeostasis and is an important therapeutic agent for the treatment of osteoporosis in humans. Here, by using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein, we generated transgenic chickens expressing hPTH under the control of a ubiquitous Rous sarcoma virus promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage. After 21 d of incubation, 42 chicks hatched from 473 retrovirus-injected eggs. All 42 living chicks were found to express the vector-encoded hPTH gene in diverse organs, as revealed by PCR and reverse transcription-PCR analysis by using primer pairs specific for hPTH. Four days after hatching, 6 chicks died and 14 chicks showed phenotypic deformities. At 18 wk of age, only 3 G(0) chickens survived. They also released the hPTH hormone in their blood and transmitted the hPTH gene to G(1) embryos. However, although the embryos were alive at d 18 of incubation, none hatched. An electrochemiluminescence immunoassay further showed that the hPTH expression level was markedly elevated in mammalian cells infected by the retrovirus vector. Thus, we demonstrated that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, not only could be an efficient bioreactor for the production of pharmaceutical drugs, but also could be useful for studies on the role of human proteins in embryonic development. To our knowledge, this is the first report on the production of a human protein (hPTH) in transgenic chickens under the control of a ubiquitous promoter by using a replication-defective Moloney murine leukemia virus-based retrovirus vector system.
[Show abstract][Hide abstract] ABSTRACT: Mass spectrometry (MS) is a powerful emerging tool in proteomics for global identification and quantification of proteins and their differential analysis. The aim of this study was to utilize MS for global peptide sequencing and the relative quantification of peptide sequences in porcine embryos produced by parthenogenesis. Oocytes (n = 6000) recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and parthenogenetically activated by a single electric pulse of 1.36 kV cm-1. After 24 h of in vitro culture in NCSU23 medium supplemented with 0.4% BSA, presumptive zygotes were sampled in Laemmli buffer and stored in liquid nitrogen until analysis. SDS-PAGE was used as the first step to separate intact proteins into 16 fractions. Each fraction was then in-gel tryptic-digested and analyzed by nanoLC-nano-ESI-MS/MS to sequence the peptide components in each fraction by LTQ ion-trap mass spectrometer (Finnigan, Fresno, CA, USA) after peptide purification by C18 ZipTips (Millipore, Billerica, MA, USA). Synthetic peptides from Myoglobin (LFTGHPETL*EK) and MAP3 Kinase (IPTGTV*HNQAK) protein having an isotope-labeled carbon (*) were used as internal standards for relative quantification. In total, 54 060 peptides were sequenced. Of these, 259 proteins were identified in the NCBI protein database for Sus scrofa using Spectrum Mill software (Agilent Technologies, Palo Alto, CA, USA). Of these, 199 proteins were identified with high confidence having at least 2 peptide sequences matching each protein. These included heat shock protein families, ribosomal proteins, cytoskeletal proteins, mitochondrial enzymes, disulfide isomerase proteins, glutathione S-transferase, DNA methyltransferase, STAT1, Ras-related protein Rab 1A, voltage-dependent ion channel proteins, zinc finger proteins and elongation factors, etc., which are involved in several important metabolic and cell signaling pathways. Several proteins including DNA methyltransferase 1, peroxidoxin 2, ubiquitin carboxyl-terminal hydrolase L1, serpin, heat shock proteins, cytoskeletal proteins, mitochondrial enzymes, elongation factor 1, bone morphogenetic protein, glucose-related proteins, etc., were among the highly abundant proteins. Our results thus showed that proteomic strategy can be successfully applied to analyze porcine embryo proteome. To our knowledge, this is the first time that several proteins have been sequenced and quantified in porcine parthenotes by proteomic means. In the long run, this study may help the understanding of mechanisms underlying embryonic development and identification of biomarkers of embryo quality.
This work was supported by grants from the Research Project on the Production of Bio-organs (No. 200503030202), Ministry of Agriculture and Forestry, and Korea Health 21 R&D Project (No. A060769), Ministry of Health and Welfare, Republic of Korea.
Reproduction Fertility and Development 01/2007; 19(1). DOI:10.1071/RDv19n1Ab2 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng µL-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5%) was similar to that of non-injected control oocytes (67.8 ± 0.4%). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1%. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1% when the DNA concentration was increased to 40 ng µL−1. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1%). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency.
This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.
Reproduction Fertility and Development 01/2007; 19(1). DOI:10.1071/RDv19n1Ab402 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 ± 0.3 vs. 27.5 ± 0.3%, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 ± 0.9 vs. 33.4 ± 0.3%, respectively). In the SCNT group, however, both cleavage (73.6 ± 0.2 vs. 64.2 ± 0.4%) and blastocyst rate (18.7 ± 0.2 vs. 13.8 ± 0.3%) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 ± 1.8 vs. 14.6 ± 4.9%) than those of control group (P < 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 ± 4.9 vs. 66.5 ± 3.3) as well as SCNT-derived (43.1 ± 2.6 vs. 31.8 ± 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development.
This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.
Reproduction Fertility and Development 01/2007; 19(1). DOI:10.1071/RDv19n1Ab60 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xenotransplantation of porcine organs has the potential to overcome the current critical shortage of allogenic organs for transplantation in humans. However, the existence of porcine endogenous retroviruses (PERVs) presents a problem for the clinical use of xenografts from pigs. In an attempt to understand the molecular characteristics of PERVs, we cloned the PERV env gene from six pig breeds (ie, Berkshire, Duroc, Landrace, Yorkshire, and two types of miniature pigs) in Korea. A total of 141 env clones were isolated and their sequences were analyzed. Phylogenetic analyses of these genes revealed the presence of PERVs, from both classes A and B, in 54% and 46% of the env clones, respectively. Among these clones, 37 isolates had the correct open reading frame (ORF; 27 clones in subclass A and 10 clones in subclass B), while the others had premature termination. These PERV nucleotide sequences can be used in a database for comparisons of PERV distribution among different pig breeds and for monitoring PERV infection using isolates with functional ORFs. Recombinant envelope of subclass A and B with functional ORF was expressed by vaccinia virus systems. Additionally isolated env clones can be used for various experiments, such as PERV control and infectivity tests, and may enhance the understanding of molecular mechanisms through pseudotyped PERV viruses.
[Show abstract][Hide abstract] ABSTRACT: Little is known about the specific functions of the human cortical structures receiving nociceptive input, their relationship to various dimensions of pain, and the modulation of these inputs by attention. We now review studies demonstrating the subdural potentials evoked by a cutaneous laser stimulus which produces a pure pain sensation by selective activation of cutaneous nociceptors (LEPs). These LEPs were localized over human anterior and middle cingulate (A & MCC), somatosensory (S1) and parasylvian (PS) cortices. LEP, lesion and imaging data define pain-related elements within each of these structures: insula and parietal operculum within PS, anterior and middle cingulate cortex, and possibly Brodman's areas 3a, 3b and 1 within SI. LEPs recorded over each of these areas is modulated with laser intensity and evoked pain. Attention to the painful laser produces an increase in the amplitude of LEPs over all three cortical areas and emergence of a late positive potential over ACC alone. These studies provide clear evidence of human cortical structures receiving nociceptive input and the modulation of that input by exogenous (e.g. laser intensity) and endogenous factors (e.g. directed attention).
[Show abstract][Hide abstract] ABSTRACT: Cryopreservation of porcine oocytes has a special importance in reproductive biotechnologies owing to the high lipid content of the oocytes. The objective of this study was to investigate the efficiency of a new and simple method of oocyte vitrification for cryopreservation of porcine oocytes. Abattoir ovary-derived prepubertal porcine oocytes at germinal vesicle (GV) or metaphase II (MII) stage were exposed for 10-15 min to vitrification solution I that contained 4% (v/v) ethylene glycol (EG) in either TCM-199 supplemented with 20% FCS or phosphate-buffered NCSU23 medium supplemented with 0.4% BSA. Oocytes were then washed thrice in EG (35%; v/v)-based vitrification solution II containing sucrose (13.7%; w/v) and polyvinyl pyrrolidone (5%; w/v) and placed in groups of 25-50 oocytes per 1-2 µL droplet onto aluminum foil kept directly over the liquid nitrogen (LN2). After visually observed vitreous formation, they were transferred to cryovials and directly plunged into LN2 for storage. Thawing was performed in sucrose- or EG-based media. After warming, the viability was assessed by fluorescein diacetate (FDA) staining and in vitro maturation (IVM) of GV-stage oocytes was determined based on cumulus expansion and polar body extrusion. In addition, the developmental capacity of oocytes was assessed by in vitro fertilization (IVF). Effects of pretreatment with cytochalasin B (CB) on the post-thaw viability, nuclear maturation, and developmental capacity were also investigated. The results demonstrated that the rate of oocytes having normal morphology was more than 90% in all groups. Viability based on esterase enzyme activity and oolemma integrity, as determined by FDA staining, ranged from 80% (without CB) to 86% (with CB) for MII-stage oocytes and 79% (without CB) to 90% (with CB) for GV-stage oocytes. The IVM rate of pelleted GV-stage oocytes reached 70% based on cumulus expansion and 55% based on polar body extrusion. Sucrose-based thawing media was found to be superior to EG-based media. Although the vitrification method resulted in a high survival rate based on the morphological appearance, FDA staining, and IVM rate, developmental rates of pelleted oocytes were found to be compromised. However, we believe that there is a scope for improving the developmental rates with further modifications in the method.
Reproduction Fertility and Development 01/2006; 18(2). DOI:10.1071/RDv18n2Ab96 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is well known that very early development of the mammalian pre-implantation embryo is regulated by gene transcripts and proteins stored in the oocyte and that the embryonic genome gains control of development following 1 to 3 cleavage divisions. An active transcription and translation is required for chromatin condensation and germinal vesicle breakdown in pig oocyte. The transition from maternal to embryonic control of development is a gradual event, and following this transition, the maternally derived transcripts and proteins are gradually degraded. Successful embryonic development is dependent on the temporal and stage-specific expression of proper genes, but information on specific gene expression during early stages before zygotic gene activation (ZGA) is limited. Before activation of the embryonic genome, mRNA and proteins synthesized during oocyte growth and maturation contribute to early development. In this study, we compared the mRNA transcripts level among porcine immature, in vitro-matured and cleaved 2- to 4-cell stage embryos after in vitro fertilization to identify genes that show differential mRNA transcript levels during maturation and very early embryonic development. For the first strand cDNA synthesis, oligo (dT) primers were added to the total RNA isolated from each sample. Using annealing control primer (ACP)-based GeneFishing PCR, we detected tens of different genes showing differential mRNA transcript level (DRTL) and nine DRTL genes were identified to be KCRF, CAMSAP1, SMP1, FLJ20647, LOC132321, NADH1, NADH6, HERC3, and TEGT. Of 9 DRTL genes, TEGT showed higher mRNA transcript level at the immaturation stage, and mRNA transcript levels of the other 8 genes were increased after in vitro maturation. Therefore, we focused on TEGT (testis enhanced gene transcript), which is highly expressed in testis and also in oocytes before in vitro maturation. Differential mRNA transcripts pattern of CAMSAP1 and TEGT were confirmed using RT-PCR and real-time RT-PCR. Porcine TEGT (pTEGT) was cloned and sequenced to have an ORF of 714 bp nucleotides and to encode an integral membrane protein. When overexpressed in HEK293 cells, pTEGT suppressed apoptosis induced by etoposide. We found that pTEGT, but not TEGT-C (C-terminal deletion mutant), inhibited etoposide- and staurosporine-induced cell death. Next, we found that introduction of TEGT siRNA suppressed the anti-apoptotic effect of TEGT. Interestingly, expression of TEGT suppressed etoposide-induced ERK activation, suggesting that ERK phosphorylation is involved in the anti-apoptotic function of the gene. Several reports showed that apoptosis and MAP kinase signaling pathways play important roles in oocyte maturation and early embryo development. Therefore, the anti-apoptotic effect of TEGT was suggested to play a key role in the normal ooctye maturation and early embryo development.
This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.
Reproduction Fertility and Development 01/2006; 18(2). DOI:10.1071/RDv18n2Ab130 · 2.40 Impact Factor