Protective effects of extracorporeal shockwave on rat chondrocytes and temporomandibular joint osteoarthritis; preclinical evaluation with in vivo99mTc-HDP SPECT and ex vivo micro-CT.

OBJECTIVE: Extracorporeal shockwave therapy (ESWT) has been shown to have chondroprotective effects on arthritic diseases. We investigated the effects of ESWT on temporomandibular joint osteoarthritis (TMJOA) using rat chondrocytes and TMJOA rat models. DESIGN: Cell viability and expression of pro-inflammatory cytokines, cartilage degradation, and apoptosis markers were measured in control, monosodium iodoacetate (MIA)-treated and ESWT plus MIA-treated chondrocytes in vitro, and intra-articular MIA injection (TMJOA) and ESWT on TMJOA rats in vivo. In vivo99mTc-hydroxymethylene diphosphonate (HDP) single-photon emission computerized tomography/computerized tomography (SPECT/CT) and ex-vivo micro-CT and histologic examinations were performed in rat models. RESULTS: ESWT plus MIA-treated chondrocytes showed increased cell viability significantly (P = 0.007), while decreased genetic expression of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6); P < 0.001 for each] and cartilage degradation markers [matrix metalloproteinase-3 (MMP3), matrix metalloproteinase-13 (MMP13), and bone morphogenetic protein 7 (BMP7); P < 0.001 for each], and number of apoptotic cells (P < 0.001) compared to MIA-treated chondrocytes. Changes in cytochrome c and cleaved caspase-3 levels relative to procaspase-3 were decreased over MIA-treated chondrocytes. ESWT on TMJOA rat models was associated with a significant decrease in pro-inflammatory and cartilage degradation markers, as demonstrated by real-time PCR and immunohistochemistry stains (P < 0.001 for each). On 99mTc-HDP SPECT/CT, the ESWT group showed a significantly lower uptake ratio compared to the TMJOA group (P = 0.008). Micro-CT analysis revealed that the ESWT group showed improved structure and bone quality compared to the TMJOA control group. CONCLUSIONS: ESWT was associated with a protective effect on cartilage and subchondral bone structures of TMJOA by reducing inflammation, cartilage degradation, and chondrocyte apoptosis.

Quality improvement of transgenic cloned bovine embryos using an aggregation method: Effects on cell number, cell ratio, embryo perimeter, mitochondrial distribution, and gene expression profile.

The production of cloned embryos using conventional methods has extremely low success rates owing to low embryo quality. To improve the quality of cloned bovine embryos expressing enhanced green fluorescent protein (EGFP), we applied an aggregation culture method. The EGFP gene was transfected into bovine fetal fibroblasts using a retroviral vector system. Somatic cell nuclear transfer was performed using these cells, and the resulting embryos were cultured in aggregates or individually. Gene expression was analyzed by a microarray, and differentially expressed genes were validated by quantitative real-time polymerase chain reaction. The total number of cells per blastocyst and the ratio of inner cell mass cells to trophectoderm cells were higher in aggregated transgenic cloned blastocysts (agBL; 368.7 ± 109.6 and 1:4.8, respectively) than in in vitro-fertilized blastocysts (ivfBL; 189.8 ± 65.8 and 1:2.6, respectively) and nonaggregated transgenic cloned blastocysts (sBL; 113.1 ± 36.3 and 1:1.5, respectively; P < 0.05 and P < 0.01, respectively). Moreover, the blastocyst perimeter was larger in the agBL group than in the ivfBL and sBL groups (1168.8 ± 200.23 vs. 887.33 ± 187.62 and 678 ± 226.1 µm; P < 0.05). In addition, mitochondrial fluorescence intensity was higher in the agBL group than in the ivfBL and sBL groups (P < 0.05). The number of apoptotic cells per blastocyst was lower in the ivfBL and agBL groups than in the sBL group (3.7 ± 2.2 and 3.4 ± 2.1 vs. 6.7 ± 6.8; P < 0.05). The genes identified in the microarray belonged to 18 categories. Expression of the Krüppel-like factor 4 gene, which is associated with cell proliferation, development, and transcription, was 7.2-fold higher in the agBL group than in the ivfBL group (P < 0.05) but did not differ between the sBL and ivfBL groups (P > 0.05). Expression of the heat shock 70-kDa protein 1A gene, which is associated with apoptosis, was 12-fold higher in the sBL group than in the ivfBL and agBL groups (P < 0.05). Expression of a stemness-related gene (octamer-binding transcription factor 4) and trophectoderm-specific genes (homeobox protein CDX2 and keratin 18) was higher in the agBL group than in the sBL group (P < 0.05). However, expression of the stemness gene homeobox protein NANOG did not differ among the groups (P > 0.05). Taken together, these data suggest that the aggregation method improves the quality of cloned embryos expressing EGFP and might be helpful in animal cloning.

Effects of co-culture of cumulus oocyte complexes with denuded oocytes during in vitro maturation on the developmental competence of cloned bovine embryos.

This study evaluated the effects of co-culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co-cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non-co-cultured somatic cell nuclear transfer (SCNT-DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT-DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation-related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT-DO group. Similarly, while the mRNA levels of the deacetylation-related genes HDAC2 and HDAC3 were significantly higher in the SCNT-DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co-culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number.

Effects of flunixin meglumine and prostaglandin F2 α treatments on the development and quality of bovine embryos in vitro.

Assisted reproduction procedures, such as embryo transfer (ET) and artificial insemination (AI), in cattle could induce the secretion of prostaglandin F2 -alpha (PGF2 α) from uterine horns which may in turn interrupt embryo development and implantation. This study investigated the effect of flunixin meglumine (FM), prostaglandin F2 alpha (PGF2α) and FM combined with PGF2α supplementation in culture medium (IVC-II) on the development and quality of in vitro produced bovine embryos. The development rate of embryos was significantly higher in the FM group (33.3%) than in control (24.3%), PGF2 α (23.9%) and FM + PGF2 α groups (24.5%). The percentage of hatched blastocysts was also higher (p < 0.05) in the FM group (41.2%) than in the control (27.8%) and PGF2 α groups (19.8%). While, there was no significant difference in total cell number in all experimental groups, the number of apoptotic cells was significantly higher in the PGF2 α group (8.2 ± 6.6) than in the control (4.7 ± 3.2), FM (4.7 ± 2.5) and FM + PGF2 α (4.9 ± 3.4) groups. Detected by real-time PCR, secreted vesicle seminal protein 1 (SSLP1) and prostaglandin G/H synthase 2 (PTGS2) gene expression decreased (p < 0.05) in the PGF2 α group. However, SSLP1 and PTGS2 gene expression in the FM + PGF2 α group returned to their baseline levels, similar to the control and FM groups. Caspase 3 (CAPS3) gene expression increased in the PGF2 α group compared with other groups (p < 0.05). In conclusion, addition of FM in vitro culture significantly improved embryo development as well as alleviated the negative impact of PGF2 α.

9-cis Retinoic acid inhibits cumulus cell apoptosis during the maturation of bovine cumulus-oocyte-complexes.

Cumulus cell (CC) apoptosis is inversely correlated with embryonic development in vitro. Therefore, inhibition of CC apoptosis is important for proper embryonic development and quality. Retinoic acids (all-transRA and 9-cisRA) are natural components of retinoids, and 9-cisRA is the physiologically active metabolite of retinoic acid in vitro. During in vitro maturation, 9-cisRA enhances oocyte competence through multiple mechanisms affecting the oocyte and preimplantation embryo; however, the effect of 9-cisRA on CC apoptosis has yet to be elucidated. The aim of the present study was to evaluate the effect of 9-cisRA on CC apoptosis and to identify the molecular mechanism underlying that effect. Bovine slaughterhouse cumulus-oocyte complexes (COC) were matured in vitro in the absence or presence of 5 nM 9-cisRA. Cumulus cells were collected from immature and matured COC for the detection of apoptosis and gene expression analysis. Results showed that 9-cisRA reduced the number of apoptotic CC by about 2.7 fold (P < 0.023), compared with control. However, apoptosis is rare in CC of immature COC (0.01% ± 0.001). Transcripts involved in the caspase cascade were down-regulated upon exposure to 9-cisRA, including tumor necrosis factor alpha (TNF-α, 11.1 fold, P < 0.001), tumor necrosis factor alpha receptor 1 (TNFR1, 2.3 fold, P < 0.01), caspase 9 (CASP9, 2.0 fold, P < 0.031), caspase 8 (CASP8, 2.2 fold, P < 0.012), and caspase 3 (CASP3, 2.1 fold, P < 0.006), while antiapoptotic B-cell lymphoma 2 (BCL2) transcript was increased (3.1 fold, P < 0.004), compared with control. In addition, 9-cisRA inhibited mitogen activated protein kinase mRNA expression in CC, including extracellular signal-regulated kinase 1/2 (ERK1, 2.7 fold, P < 0.02; ERK2, 2.7 fold, P < 0.03), and c-Jun N-terminal kinase (JNK, 1.6 fold, P < 0.044), as well as the activator protein-1 (AP1) family members c-jun (1.6 fold, P < 0.041) and c-fos (2.0 fold, P < 0.06). The transcript abundances of TNF-α, TNFR1, CASP9, CASP8, CASP3, ERK1, ERK1, JNK, and BCL2 were increased, while c-fos and c-jun mRNA expression was decreased in the matured CC. On the basis of the data, we suggest that 9-cisRA inhibits CC apoptosis during in vitro maturation of bovine COC.

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development.

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-µl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.

9-cis retinoic acid improves developmental competence and embryo quality during in vitro maturation of bovine oocytes through the inhibition of oocyte tumor necrosis factor-α gene expression.

Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.

Generation of a recloned transgenic cat expressing red fluorescence protein.

Somatic cells from a first-generation red fluorescence protein transgenic cat (first RFP TG cat) were used to produce a recloned RFP transgenic cat (Re-RFP TG cat) (Felis catus) that systemically expressed RFP. A total of 281 RFP cloned embryos were transferred into 13 surrogate mothers (mean=21+/-7.7 embryos/recipient). One surrogate cat was diagnosed pregnant (7.7%) and delivered one live kitten. The presence of the RFP gene in the mRNA and genomic DNA of the Re-RFP TG cat was confirmed by polymerase chain reaction analyses, and red fluorescence was detected in its internal organs and placental tissue samples. Analysis of nine feline-specific microsatellite loci confirmed that the Re-RFP TG cat was genetically identical to the donor cat. To test whether results such as normality of offspring and a low cloning success were due to epigenetic modifications, global methylation of placenta from the two first cloned RFP TG cats (77.08% and 82.29%) and the Re-RFP TG cat (76.38%) were compared by bisulfite mutagenesis sequencing analysis. In conclusion, although cloning efficiency was low, we demonstrated the successful use of a cloned first RFP TG cat as a donor cat to produce a Re-RFP TG cat. These results may facilitate future developments in biomedical models for human therapeutic applications.

Effect of equine chorionic gonadotropin on the efficiency of superovulation induction for in vivo and in vitro embryo production in the cat.

The effects of various dosages of equine chorionic gonadotropin (eCG) on superovulation induction for in vivo and in vitro embryo production were examined in stray cats (Felis catus). Cats (n=286) were allocated into five treatment groups with 0, 50, 100, 200, or 400 IU eCG, followed by 100 IU human chorionic gonadotropin (hCG). In vivo- and in vitro-produced blastocysts were obtained by artificial insemination (AI) and in vitro fertilization (IVF), somatic cell nucleus transfer (SCNT), or parthenogenetic activation (PA). The percentage of cats that developed mature follicles, the percentage of cats with collected embryos, and the mean number of in vivo blastocysts per cat were higher in the 200 IU treatment group (43.9%, 31.8%, and 1.53, respectively) compared with those of the other groups (P<0.05). The percentage of follicular developed cats, the percentage of cumulus-expanded oocytes, and the mean number of collected cumulus-oocyte complexes per cat in the 200 IU (56.7%, 67.8%, and 26.2, respectively) and 400 IU (53.3%, 64.2%, and 26.7, respectively) groups were higher than those in the other groups (P<0.05). Furthermore, the percentage of in vitro-produced blastocyst per cleaved embryos and the average cell number of the blastocysts from IVF (52.7% and 125.8, respectively) was higher than those of the blastocysts from PA (30.1% and 85.2) and higher than those of the blastocysts from SCNT (15.3% and 37.5; P<0.05). In conclusion, the current study demonstrated that in vivo and in vitro embryo production were affected by the dosage of eCG; the best results were obtained with 200 IU.

Prevalence of primary antiretroviral resistance: trends in Korea.

Primary drug resistance is an emerging problem in HIV infections. We have investigated the current prevalence of primary resistance in Korea and compared it with previous data. Drug-naive HIV patients attending the outpatient clinic of Seoul National University Hospital between April and August 2006 were enrolled. A medical interview and a genotypic resistance test were performed for each patient. The International AIDS Society-USA Panel consensus statement issued in 2006 was used to define resistance mutations. Eighty-one drug-naive HIV patients were enrolled. Two (2.5%) were infected with primary drug-resistant virus: M41L and K103N, respectively. In our previous study, conducted between 1998 and 2002, three (6%) of 50 subjects harbored resistant viruses. Thus the frequency of primary resistance was lower in the present sample than in the earlier one, though the difference is not statistically significant (p = 0.37). In view of our findings, routine antiretroviral resistance tests for drug-naive HIV patients are not obligatory in Korea.

ProteoChip-based library screening of integrin alpha5beta1 antagonists from Korean medicinal plant extracts.

Integrins consist of transmembrane glycoproteins noncovalently associated to form alphabeta heterodimers. Various alpha/beta associations determine binding specieficities for cell surface molecules of the immunoglobulin superfamily as well as for extracellular matrix components. Through their cytoplasmic domains, integrins are responsible for the transmission of signals between the intracellular and the extracellular environment. We immobilized an integrin alpha5beta1 microarray on a ProteoChip to screen Korean medicinal plant extracts for binding activity. The microarray preserved the integrin alpha5beta1-fibronectin interaction, and was suppressed by the synthetic RGD peptide. We identified ten extracts with high integrin affinity using a high-throughput, competitive inhibition assay. We also demonstrate the biological function of these extracts in HUVECs.