Fusion Medium Research Articles

DEGENERACY EFFECT ON THE COMPTON SCATTERING POWER

The Compton scattering is one of the loss processes in fusion media. In this paper the Compton cross-section is calculated in three limits of temperature, non-relativistic, relativistic and ultra-relativistic temperatures. By considering the electron distribution function for all the temperature limits, we found the power of Compton scattering in degenerate media. These results show that the Compton scattering power increases with decreasing electron temperature. So, in degenerate conditions, the Compton loss processes decrease in fusion media.

Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin (MSTN) is a negative regulator of skeletal muscle development and responsible for limiting regeneration. It binds with high affinity to the activin type IIB receptor (ActRIIB). Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the MSTN signal with expression of a dominant negative mutant of ActRIIB (dnActRIIB). In vitro, blocking MSTN activity with a lentivirus carrying dnActRIIB increased proliferation and fusion of human myoblasts because MSTN regulates the expression of several myogenic regulatory factors. In vivo, myoblasts infected with the dnActRIIB lentivirus were transplanted in immunodeficient dystrophic mice. Dystrophin immunostaining of tibialis anterior (TA) cross-sections of these mice 1 month post-transplantation revealed more human dystrophin-positive myofibers following the transplantation of dnActRIIB myoblasts than of control myoblasts. Thus, blocking the MSTN signal with dnActRIIB improved the success of myoblast transplantation by increasing the myoblast proliferation and fusion and changed the expression of myogenic regulatory factors.

MitoQ10 induces adipogenesis and oxidative metabolism in myotube cultures

Coenzyme Q(10) (CoQ(10)) plays an essential role in determination of mitochondrial membrane potential and substrate utilization in all metabolically important tissues. The objective of the present study was to investigate the effect of Coenzyme Q analog (MitoQ(10)) on oxidative phenotype and adipogenesis in myotubes derived from fast-glycolytic Pectoralis major (PM) and slow-oxidative Anterior latissimus dorsi (ALD) muscles of the turkey (Meleagris gallopavo). The myotubes were subjected to the following treatments: fusion media alone, fusion media+125 nM MitoQ(10), and 500 nM MitoQ(10). Lipid accumulation was visualized by Oil Red O staining and quantified by measuring optical density of extracted lipid at 500 nm. Quantitative Real-Time PCR was utilized to quantify the expression levels of peroxisome proliferator-activated receptor (PPARγ) and PPARγ co-activator-1α (PGC-1α). MitoQ(10) treatment resulted in the highest (P<0.05) lipid accumulation in PM myotubes. MitoQ(10) up-regulated genes controlling oxidative mitochondrial biogenesis and adipogenesis in PM myotube cultures. In contrast, MitoQ(10) had a limited effect on adipogenesis and down-regulated oxidative metabolism in ALD myotube cultures. Differential response to MitoQ(10) treatment may be dependent on the cellular redox state. MitoQ(10) likely controls a range of metabolic pathways through its differential regulation of gene expression levels in myotubes derived from fast-glycolytic and slow-oxidative muscles.

High Hydrostatic Pressure Treatment of Porcine Oocytes Induces Parthenogenetic Activation

An innovative technique called high hydrostatic pressure (HHP) treatment has recently been reported to improve the cryosurvival of gametes and embryos in certain mammalian species, including the mouse, pig, and cattle. In the present study the parthenogenetic activation (PA) of pig oocytes caused by HHP treatment was investigated in different holding media with or without Ca(2+). The efficiency of activation was tested at different pressure levels and media including T2 (HEPES-buffered TCM-199 containing 2% cattle serum), and mannitol-PVA fusion medium with (MPVA + Ca(2+)) or without Ca(2+) and Mg(2+)(MPVA). The results showed that HHP cannot induce PA in T2, but only in MPVA + Ca(2+) with low Ca(2+) concentration and MPVA without Ca(2+). The highest activation efficiency was achieved with 10 min HHP treatment using 100 or 200 bars for oocytes in MPVA + Ca(2+) or MPVA, respectively. In the light of these results, the possible source of Ca(2+) during activation was investigated. It was found that even after a total of 30-min wash with TL-HEPES-PVA buffer without Ca(2+) before HHP treatment in MPVA, the oocytes could still be activated, indicating the possibility of an intracellular Ca(2+) source caused cytoplasmic free Ca(2+) elevation. In conclusion, parthenogenetic activation could be induced by HHP in certain holding media with low or zero Ca(2+) content. Further experiments are needed to identify the exact mechanisms of activation.

Definition of fusion medium and electric parameters for efficient zygote electrofusion in the Pacific oyster ( Crassostrea gigas)

Cell electrofusion has been widely used in the induction of tetraploidy in mammals, but little attention has been paid in molluscs. This work pursued the establishment of fusion medium (ionic vs. non-ionic) and electric parameters in the electrofusion of Pacific oyster zygotes (prior to the completion of the first mitotic division), minimizing all deleterious effects possible to D-larval stage. The tested combinations of electric field intensity (Vcm −1) and number of square DC pulses applied (for 50 μs each) were (Voltage × N° pulses): 400 × 1, 400 × 2, 400 × 3 and 600 × 1, 600 × 2, 600 × 3. When pulses were applied for first time, it was determined that an ionic fusion medium (microfiltered seawater) offered better conditions than the non-ionic fusion media previously used (0.6 M sucrose or 0.6 M mannitol) in terms of embryo survival and lysis rates. In this fusion medium, two different combinations of electric parameters (3 square DC pulses of 400 Vcm −1 for 50 μs each at 26 °C and 1 square DC pulse of 600 Vcm −1 for 50 μs at 26 °C) offered the best technical results of fusion (57 and 79% respectively) and survival until D-larva (44 and 41% respectively). In conclusion, these electric parameters could be established, using seawater as electrofusion medium, for further approaches to evaluate individual ploidy and survival beyond spat.

Isolation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc.)

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5)/g mycelia and 2.8x10(5)/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Isolation and fusion of protoplasts from the phytopathogenic fungus sclerotium rolfsii(sacc.)

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x105/g mycelia and 2.8x105/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

72 PREGNANCY OUTCOME AFTER OVIDUCTAL TRANSFER OF ZONA-FREE 1-CELL-STAGE PORCINE EMBRYOS PRODUCED BY HANDMADE CLONING

The pig is an important animal model for the study of human diseases. An important step for better use of this species in biomedical research is to obtain genetically identical individuals by procedures such as somatic cell nuclear transfer (SCNT). As the in vitro culture environment is usually sub-optimal for embryo development, the oviductal transfer of cloned embryos at the 1-cell stage may be more efficient for the establishment of pregnancies. However, the transfer at such an early stage usually requires the presence of zona pellucida or agar embedding to protect embryos from the recipient’s immune system (Loi et al. 1999 Livest. Prod. Sci. 60, 281-294). This study aimed to evaluate the developmental viability of 1-cell-stage porcine handmade cloned embryos directly transferred to the oviduct of female recipients without the zona pellucida or agar embedding. After 40 h of IVM in TCM-199 +10% follicular fluid, COCs obtained from sows were denuded, selected for the presence of a polar body (459/689), and submitted to a 0.2% pronase solution in 25% fetal bovine serum (FBS) for partial zona removal, followed by rinses in manipulation medium and pure FBS. Subsequent to oocyte splitting by manual bisection in a 5 μg mL-1 cytochalasin B solution (CCB), hemi-oocytes (87.1%) were screened under fluorescent microscopy using Hoechst 33 342 stain, resulting in 57.6% enucleated halves (461/800). A somatic cell culture established from a fetal clone pig biopsy (Adam et al. 2007 Oncogene 26, 1038-1045) at passage 4 was used for embryo reconstruction, which was done in a 0.05% phytohemagglutinin (PHA) solution, by sticking 2 cytoplasts and a somatic cell in a linear orientation. Reconstructed couplets, rinsed in calcium- and magnesium-free fusion medium, were electrofused in a fusion chamber after exposure to a 30-V AC pulse for 20 s for alignment, followed by two 24-μs-long DC fusion pulses of 1.3 kV cm-1. Fused couplets (154/214) were exposed for 10 min to a solution containing 5 μg mL-1 CCB and 10 μg mL-1 cycloheximide, followed by electrical activation (two 24-μs-long DC pulses of 0.9 kV cm-1) in fusion medium containing calcium and magnesium. Activated embryos were cultured in vitro for 12 h in 500 μL of PZM-3 medium in the well of the well (WOW) system, in a plastic bag filled with gas mixture (90% N2, 5% O2, 5% CO2), at 38.5°C. Then, a total of 70 and 80 non-agar-embedded, zona-free 1-cell-stage cloned porcine embryos were transferred directly to the oviducts of a sow and a gilt, respectively, both synchronous at approximately 12 h before ovulation. The recipient sow was diagnosed pregnant by ultrasonography on Day 66 of gestation. Although the sow was diagnosed open on Day 72, this study demonstrates that the transfer of 1-cell-stage zona-free embryos directly to the oviduct of a synchronous sow can result in pregnancy.

59 PRODUCTION OF HANDMADE CLONED GOAT EMBRYOS WITH TWO TYPES OF DONOR CELLS AND CULTURED IN THREE MEDIA

The study was carried out to see the developmental efficiency of handmade cloned goat embryos with 3 different media: RVCL (Research Vitro Cleave, Cook, Brisbane, Australia), EDM (Embryo Development Media) and modified SOF (mSOF) and 2 types of donor cells: fetal fibroblast and adult fibroblast. Oocytes were isolated from abattoir goat ovaries, matured in maturation medium, and incubated in 5% CO2 in air at 38.5°C for 24 h. Then, the oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona free by pronase (2 mg mL-1). Protrusion cone formation in oocytes was found 95 to 100% in T20 (TCM-199 + 20% FBS). The zona-free oocytes were bisected with an ultra-sharp micro blade on the basis of visible protrusion cones on the surface of oocytes using T20 medium containing 2.5 μg mL-1 cytochalasin-B. Fetal and adult fibroblast cells were used from confluent monolayer at passage 5 after trypsinizing in 0.25% trypsin-EDTA. One somatic cell was attached with one enucleated demioocyte by phytohemagglutinin and further fused with another enucleated demioocyte through electric pulse with a combination of alternating current (4 V) and direct current (2.10 kV cm-1 for 5 μs with a single pulse) in fusion medium (0.3 M mannitol, 0.1 mM MgCl2, 0.05 mM CaCl2, and 3 mg mL-1 BSA). Then, triplets were chemically activated with 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h and cultured in the 3 media. Cleavage and morulae formation were observed at Day 7 from 183 triplets with fetal fibroblasts as donor cells in media RVCL (78.60 ± 2.23, 38.97 ± 2.1), mSOF (72.62 ± 1.89, 33.81 ± 1.9), and EDM (73.96 ± 1.66, 26.20 ± 2.04), respectively. Simultaneously, cleavage and morulae formation were observed at Day 7 from 203 triplets with adult fibroblasts as donor cells in media RVCL (73.97 ± 3.57, 33.14 ± 2.68), mSOF (76.22 ± 4.36, 26.15 ± 0.99), and EDM (65.97 ± 3.11, 20.78 ± 2.77), respectively. Among the 3 media, morulae formation was significantly higher in RVCL. Hence, in the subsequent experiment, RVCL medium was used exclusively in culture for 172 triplets. Cleavage and morulae formation at Day 7 was not significantly different (P &lt; 0.05) in 2 types of donor cells; fetal fibroblasts (77.46 ± 3.65, 38.70 ± 2.66) and adult fibroblasts (75.74 ± 3.04, 33.77 ± 1.43), respectively. The data were analyzed using SYSTAT 7.0 (Systat, SPSS Inc., Chicago, IL, USA) after arcsine transformation, one-way ANOVA followed by Fisher’s LSD test. PCR analysis was performed with highly polymorphic 286-bp fragment of MHC-II DRB gene of cloned embryo and its donor cell. Similar bands were observed in both the cloned embryos and fibroblast cells in agar gel electrophoresis. In conclusion, development of handmade cloned embryos was higher in RVCL medium compared with the other two media tested, and efficiency of morulae formation was similar in both types of donor cells. Further study is required to optimize blastocyst production.

A novel trial of nuclear transfer for repairing an aged oocyte

OBJECTIVE: Nuclear transfer into the metaphase-II(M-II) oocytes shows promise as a means of repairing female infertility due to ooplasmic deficiency and abnormalities. We therefore conducted nuclear transfer of in vitro matured metaphase-II oocytes (recipient oocytes) into enucleated freshly ovulated metaphase-II oocytes(donor oocyte). DESIGN: The fertilization rate, cleavage rate and blastocyst formation rate were investigated following nuclear transfer. MATERIALS AND METHODS: Recipient and donor oocytes were placed in a microdrop containing 5 microgram/ml of cytochalasinB (CCB). The aspirated M-II karyoplast of recipient oocytes was transferred into the perivitelline space of an enucleated donor oocyte. The grafted oocyte was transferred in Zimmerman cell fusion medium. Membrane fusion was facilitated by electrical stimulation (10V for 1 second AC + 10V for 45 microsecond DC) with an electro cell fusion generater (LF 201). After fusion, the constructed oocytes were cultured in HTF medium for 2 hours and ICSI was performed. RESULTS: The percentage of identification of M-II chromosome was 91.1 % (41 out of 45) in freshly ovulated oocytes and 96.0% (48 out of 50) in vitro-matured oocytes. The M-II karyoplast was removed successfully in 35 of 41 (85.4%) of the donor oocytes and 40 of 48 (83.3%) of the recipient oocytes. All of 35 karyoplasts of recipient oocytes were replaced in the periviteleine space of enucleated donor oocytes and 28 of these 80.0% were fused to form a reconstituted oocyte.The fertilization rate, cleavage rate and blastocyst formation rate following ICSI for constructed oocytes and recipients oocytes were(77.1%(27/35), 65.7%(23/35), 25.7%(9/35)),(59.0%(58/98), 26.1%(25/98), 3.4%(3/98))respectively. Chromosomal analysis of 4 embryos following nuclear transfer indicated they were all diploid sets of 46 chromosome. CONCLUSIONS: These results demonstrate that this technique can be applied to the treatment of female infertility due to ooplasmic deficiency and abnormalities in aged oocytes

Latrunculin A Dramatically Improves the Developmental Capacity of Nuclear Transfer Embryos Derived from Gene-Modified Clawn Miniature Pig Cells.

Cells, tissues, and organs of gene-modified miniature pigs produced by somatic cell nuclear transfer (SCNT) are useful for xenotransplantation and biomedical research. However, the efficiency of SCNT still remains low, when the gene-modified cells are used as donor nuclei. The objective of this study is to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo developmental ability of cloned embryos derived from gene-modified miniature pig cells. Fetal fibroblasts were obtained from a female cloned fetus (Day 21 of pregnancy) of Clawn miniature pigs derived from porcine alpha 1-3 galactosyltransferase gene knockout cells. Pig oocytes with compact cumulus mass collected from ovaries of a local slaughterhouse were matured for 40-42 h and in vitro-matured oocytes were enucleated by aspiration with a thin glass pipette. Then a single fibroblast cell was introduced into the perivitelline space of an enucleated oocyte and electrically fused using needle-type electrodes in fusion medium. Following fusion treatment, reconstructed embryos were electrically activated. After activation, reconstructed embryos were cultured for 2 h in modified PZM3 supplemented with or without a cytoskeletal inhibitor [0.5 µM LatA, 1 µM LatA, or 10.4 µM cytochalasin B (CB)]. Following postactivation treatment, cloned embryos were cultured in modified PZM3 for further development and assessed for cleavage and blastocyst formation at 2 and 7 days of culture, respectively. To determine the in vivo developmental ability of cloned embryos, some embryos exposed to 0.5 µM LatA were transferred to the oviducts of a recipient miniature pig (24 h of culture) and lapalotomy was carried out on Day 21 of pregnancy. The cleavage rate was significantly higher (P<0.05) in embryos exposed to 0.5 µM LatA (86.7%) than those in embryos exposed to CB (74.1%) and without a cytoskeletal inhibitor (70.9%). Moreover the blastocyst formation rate was significantly higher (P<0.05) in embryos exposed to 0.5 µM (33.7%) or 1 µM LatA (32.9%) than those in embryos exposed to CB (18.5%) and without a cytoskeletal inhibitor (11.4%). In addition, five fetuses were obtained from recipient uteri after embryo transfer. The results of this study show for the first time that postactivation treatment with LatA is effective to improve the in vitro developmental capacity of gene-modified cloned miniature pig embryos and the embryos treated with LatA have the ability to develop into fetuses. (poster)

Cloning Missy: Obtaining Multiple Offspring of a Specific Canine Genotype by Somatic Cell Nuclear Transfer

The present study was undertaken to evaluate two activation methods for somatic cell nuclear transfer (SCNT), namely, fusion and simultaneous activation (FSA, fusion medium contains calcium), versus fusion followed by chemical activation (F+CA, fusion medium does not contain calcium), and to evaluate the effects of parity of recipient dogs on the success of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells collected from an 11-year-old female dog named Missy. In the FSA method, oocytes were fused and activated at the same time using two DC pulses of 1.75 kV/cm for 15 microsec. In the F+CA method, oocytes were fused with two DC pulses of 1.75 kV/cm for 15 microsec, and then activated 1 h after fusion by 10 microM calcium ionophore for 4 m and cultured for 4 h in 1.9 mM 6-dimethylaminopurine for postactivation. Activation method had a significant impact on the production efficiency of cloned dogs. There was a significant difference in full-term pregnancy rate and percentage of live puppies between the two methods (6.3% and 38.5% for FSA and F+CA, respectively). In our study, four out of five live offspring produced by F+CA survived versus FSA, which did not result in any surviving puppies. Overall, as few as 14 dogs and 54 reconstructed embryos were needed to produce a cloned puppy. In addition, the parity of recipient bitches had no effect on the success of SCNT in canine species. Both the nullipara and multipara bitches produced live puppies following SCNT-ET.

Optimal Concentration of Calcium and Electric Field Levels Improve Tetraploid Embryo Production by Electrofusion in Mice

Polyploid embryo production is an important technique in generating mice directly from embryonic stem (ES) cells. The present study was designed to assess the effect of different calcium concentrations and electric field intensities on the production of tetraploid embryos with higher developmental potential by electrofusion. Two-cell mouse embryos were electrofused in fusion solution containing different concentrations of calcium ion (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mM). The rates of blastomere fusion, and subsequent cleavage and development of tetraploids to the blastocyst stage were highest when two-cell embryos were electrically stimulated in a fusion medium containing 1.0 mM calcium. Therefore, we tested electric field intensities (0.6, 0.8, 1.0, 1.2 and 1.4 kV/cm) for electrofusion of two-cell embryos and subsequent development to the blastocyst stage in 1.0 mM calcium. The highest rates of fusion and blastocyst formation were observed when the electric field strength was 0.8 kV/cm. The present results showed that mouse two-cell embryos stimulated with 0.8 kV/cm in a fusion medium containing 1.0 mM calcium had the highest rates of fusion and development to the blastocyst stage.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Rescue of in-vitro matured oocytes following metaphase-II nuclear transfer in humans

OBJECTIVE: Nuclear transfer into the metaphase-II (M-II) oocytes shows promise as a means of repairing either mitochondrial diseases or female infertility due to ooplasmic deficiency and abnormalities. We therefore conducted nuclear transfer between in vitro matured metaphase-II oocytes.DESIGN: The fertilzation rate, cleavage rare and blastocyst formation rate were investigated following nuclear transfer.MATERIALS AND METHODS: Recipient oocytes were derived from immature oocytes in ICSI treatment and cultured in vitro until the first polar body extrusion was observed. Donor oocytes were prepared from IVF or ICSI patients who had consented to participate in these experiments. Two oocytes were placed in a microdrop containing 5 microgram/ml of cytochalasinB (CCB). A 10-12 micrometer innerdiameter glass pipette was inserted into the perivitelline space and the cytoplasm contatining M-II chromosome was aspirated. The karyoplast of donor oocytes was removed using the same procedure that was used for recipient oocytes and transferred into the perivitelline space of an enucleated donor oocyte. Insertion of the karyoplast, the grafted oocyte was washed for 60 minutes in HTF medium to remove CCB, and then transferred in Zimmerman cell fusion medium. Membrane fusion between oocyte and karyoplast was facilitated by electrical stimulation (10V/sec AC + 15V/45 μsec DC) with an electro cell fusion generater (LF 101). After fusion, the constructed oocytes were cultured in HTF medium for 2 hours and ICSI was performed.RESULTS: The percentage of identification of M-II chromosome was in 42 of the 44 freshly ovulated oocytes (95.5%) and in 38 of the 40 in vitro-matured oocytes [92.7% (55/55)]. The M-II karyoplast was removed successfully in 38 of 42 (90.5%) of the donor oocytes and 45 of 51 (88.2%) of the recipient oocytes. All of 38 karyoplasts of recipient oocytes were replaced in periviteleine space of enucleated donor oocytes and 30 of these 78.9% were fused to form a reconstituted oocyte. The fertilization rate, cleavage rate and blastocyst formation rate following ICSI for constructed oocytes and recipients oocytes were (76.7% (23/30), 66.7% (20/30), 33.3% (10/30))respectively. Chromosomal analysis of 4 embryos following nuclear transfer were diploid sets of 46 chromosome.CONCLUSIONS: These results suggest that normal embryonic development can occur after the transfer of karyoplast between in vitro-matured metaphase-II oocytes. OBJECTIVE: Nuclear transfer into the metaphase-II (M-II) oocytes shows promise as a means of repairing either mitochondrial diseases or female infertility due to ooplasmic deficiency and abnormalities. We therefore conducted nuclear transfer between in vitro matured metaphase-II oocytes. DESIGN: The fertilzation rate, cleavage rare and blastocyst formation rate were investigated following nuclear transfer. MATERIALS AND METHODS: Recipient oocytes were derived from immature oocytes in ICSI treatment and cultured in vitro until the first polar body extrusion was observed. Donor oocytes were prepared from IVF or ICSI patients who had consented to participate in these experiments. Two oocytes were placed in a microdrop containing 5 microgram/ml of cytochalasinB (CCB). A 10-12 micrometer innerdiameter glass pipette was inserted into the perivitelline space and the cytoplasm contatining M-II chromosome was aspirated. The karyoplast of donor oocytes was removed using the same procedure that was used for recipient oocytes and transferred into the perivitelline space of an enucleated donor oocyte. Insertion of the karyoplast, the grafted oocyte was washed for 60 minutes in HTF medium to remove CCB, and then transferred in Zimmerman cell fusion medium. Membrane fusion between oocyte and karyoplast was facilitated by electrical stimulation (10V/sec AC + 15V/45 μsec DC) with an electro cell fusion generater (LF 101). After fusion, the constructed oocytes were cultured in HTF medium for 2 hours and ICSI was performed. RESULTS: The percentage of identification of M-II chromosome was in 42 of the 44 freshly ovulated oocytes (95.5%) and in 38 of the 40 in vitro-matured oocytes [92.7% (55/55)]. The M-II karyoplast was removed successfully in 38 of 42 (90.5%) of the donor oocytes and 45 of 51 (88.2%) of the recipient oocytes. All of 38 karyoplasts of recipient oocytes were replaced in periviteleine space of enucleated donor oocytes and 30 of these 78.9% were fused to form a reconstituted oocyte. The fertilization rate, cleavage rate and blastocyst formation rate following ICSI for constructed oocytes and recipients oocytes were (76.7% (23/30), 66.7% (20/30), 33.3% (10/30))respectively. Chromosomal analysis of 4 embryos following nuclear transfer were diploid sets of 46 chromosome. CONCLUSIONS: These results suggest that normal embryonic development can occur after the transfer of karyoplast between in vitro-matured metaphase-II oocytes.

An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies

We sought to develop and optimize a hybridoma-based technology for generating human hybridomas that secrete virus-specific monoclonal antibodies for clinical diagnosis and therapy. We developed a novel electrofusion protocol for efficiently fusing Epstein-Barr virus (EBV)-transformed human B cells with myeloma partners. We tested seven myeloma cell lines and achieved highest efficiency when the HMMA 2.5 line was used. We optimized the electrofusion process by improving cell treatments before and after electrofusion as well as varying cell ratios, fusion medium and other experimental parameters. Our fusion efficiency increased remarkably to 0.43%, a significant improvement over the efficiency of previous PEG-based or other electrofusion methods. Using the optimized protocol, we obtained human hybridomas that secrete fully human monoclonal antibodies against two major human respiratory pathogens: respiratory syncytial virus (RSV) and an influenza H3N2 vaccine virus strain. In conclusion, we have developed an efficient and routine approach for the generation of human hybridomas secreting functional human virus-specific monoclonal antibodies.

Use of a novel collagen matrix with oriented pore structure for muscle cell differentiation in cell culture and in grafts

Tissue engineering of skeletal muscle from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds. Our study describes the application of artificial scaffolds (collagen sponges, CS) consisting of collagen-I with parallel pores (width 20–50 μm) using the permanent myogenic cell line C2C12. CS were infiltrated with a high-density cell suspension, incubated in medium for proliferation of myoblasts prior to further culture in fusion medium to induce differentiation and formation of multinucleated myotubes. This resulted in a parallel arrangement of myotubes within the pore structures. CS with either proliferating cells or with myotubes were grafted into the beds of excised anterior tibial muscles of immunodeficient host mice. The recipient mice were transgenic for enhanced green fluorescent protein (eGFP) to determine a host contribution to the regenerated muscle tissue. Histological analysis 14–50 days after surgery showed that donor muscle fibres had formed in situ with host contributions in the outer portions of the regenerates. The function of the regenerates was assessed by direct electrical stimulation which resulted in the generation of mechanical force. Our study demonstrated that biodegradable CS with parallel pores support the formation of oriented muscle fibres and are compatible with force generation in regenerated muscle.

Myostatin preferentially down-regulates the expression of fast 2x myosin heavy chain in cattle

Myostatin is involved in an inhibitor of muscular growth and differentiation. Myoblasts derived from double-muscled Japanese shorthorn cattle (DM myoblasts) with absence of functional myostatin had higher abilities to proliferate and differentiate than myoblasts derived from normal-muscled cattle (NM myoblasts). In DM myoblasts, mRNA expressions of fetal myosin heavy chain (MyHC) in growth medium and of fast 2a and 2x MyHC in fusion medium were significantly greater than that in NM myoblasts. No significant difference existed in expressions of embryonic and slow MyHC mRNA between DM and NM myoblasts. The expression of MyoD mRNA was suppressed in myoblasts by administration of myostatin. Two cloned DM myoblast strains (DMc) were established. Addition of myostatin for DMc resulted in less myotube formation and suppression of mRNA expression of fast 2x MyHC. These findings suggest that the endogenous myostatin preferentially down-regulates the expression of the fast 2x MyHC and participates in differentiation of myofiber types during early bovine myogenesis.

Effects of culture conditions and nuclear transfer protocols on blastocyst formation and mRNA expression in pre-implantation porcine embryos

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis ( BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P < 0.05), number of inner cell mass cells (13.3 ± 4.3 vs. 7.6 ± 2.2, P < 0.05), and total cells (57.9 ± 19.5 vs. 36.3 ± 8.2, P < 0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0 mM calcium in fusion/activation medium (1.0C), and 7.5 μg/mL cytochalasin B treatment (0.1C&CB) yielded higher rates ( P < 0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1 mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased ( P < 0.05) in 1.0C (77.4 ± 28.9) compared to the control (58.5 ± 22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1C&CB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes ( BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

Development and calcium level changes in pre‐implantation porcine nuclear transfer embryos activated with 6‐DMAP after fusion

This study investigated the effect of treatment with 6-dimethylaminopurine (6-DMAP) following fusion on in vitro development of porcine nuclear transfer (NT) embryos. Frozen thawed ear skin cells were transferred into the perivitelline space of enucleated oocytes. Reconstructed oocytes were fused and activated with electric pulse in 0.3 M mannitol supplemented with either 0.1 or 1.0 mM CaCl(2). In each calcium concentration, activated oocytes were divided into three groups. Two groups of them were exposed to either ionomycin (I + 6-DMAP or 6-DMAP alone. In experiment 2, fused NT embryos in 0.3 M mannitol containing 1.0 mM CaCl(2) were exposed to 6-DMAP either immediately or 20 min after fusion/activation. For 0.1 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed a higher (P < 0.05) developmental rate to the blastocyst stage than those activated with an electric pulse alone (26.7 and 22.5 vs. 12.5%). For 1.0 mM CaCl(2), oocytes activated with either I + 6-DMAP or 6-DMAP alone showed significantly higher (P < 0.05) developmental rate to the blastocyst stage (35.6 and 28.3 vs. 19.8%). Developmental rate to the blastocyst stage was (P < 0.05) increased in NT embryos activated with 6-DMAP 20 min after fusion. 6-DMAP made a higher and wider Ca(2+) transient compared to that induced by electric pulses (Fig. 3). The fluctuation lasted during the time that oocytes were cultured in 6-DMAP. Regardless of Ca(2+) concentration in fusion medium, activation with 6-DMAP following electric pulses supported more development of porcine NT embryos. Activation of NT embryos with 6-DMAP after fusion in the presence of 1.0 mM CaCl(2) could support better developmental rate to the blastocyst stage.