Yeon Woo Jeong

Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes

In an effort to improve the quality of in vitro produced porcine embryos, we investigated the effect of brain-derived neurotropic factor (BDNF), a neurotropin family member, on in vitro maturation (IVM) of porcine oocytes. The expression of BDNF and truncated isoforms of its receptor, tyrosine kinase B (TrkB), and p75 common neurotropin receptor was detected in both follicular cells and metaphase-I stage oocytes by RT-PCR. However, mRNA of full-length TrkB was not found in oocytes although it was detected in follicular cells. The expression pattern of BDNF and TrkB was confirmed by immunohistochemistry. Supplementation with BDNF (30 ng/ml) during IVM significantly (P < 0.05) increased the first polar body extrusion and glutathione levels in oocytes, whereas the effect of BDNF on nuclear maturation was diminished when gonadotropin and epidermal growth factor (EGF) were added to the culture media. However, treatment with BDNF (30 ng/ml) along with EGF (10 ng/ml) in the presence of gonadotropin significantly (P < 0.05) increased the developmental competence of oocytes to the blastocyst stage after both in vitro fertilization (IVF; 29.1% when compared with control, 15.6%) and somatic cell nuclear transfer (SCNT; 13.6% when compared with control, 3%). This appeared to reflect a stimulatory interaction between BDNF and EGF to enhance the cytoplasmic maturation of oocytes to support successful preimplantation development. In conclusion, BDNFenhanced nuclearand cytoplasmic maturation of oocytes by autocrine and/or paracrine signals. Also, when used together with EGF, BDNF increased the developmental potency of embryos after IVF and SCNT, demonstrating an improved in vitro production protocol for porcine oocytes.

Birth of Beagle dogs by somatic cell nuclear transfer.

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or squeezing method, fused with two DC pulses of 1.75 kV/cm for 15 micros electrical stimulation, chemically activated after 1h of fusion using 10 microM calcium ionophore for 4 min and cultured 4h in 1.9 mM 6-dimethylaminopurine. Finally, 103 or 214 embryos for aspiration or squeezing method were transferred to 6 or 11 naturally synchronized recipients, respectively. A total of 53, 317 and 342 embryos were transferred to 7, 17 and 12 recipients for the group of 4-10, 11-25 and 26-40 embryos, respectively. There was no difference between fusion rate (76.87% vs. 80.15%), full term pregnancy rate (16.66% vs. 27.27%) and percent of live puppies born (0.97% vs. 1.87%) for aspiration and squeezing method (P>0.05). Production efficiency of cloned dogs was significantly affected by the number of embryos transferred to each recipient. No pregnancy was established for the group of 4-10 embryos (n=7) and 26-40 embryos (n=12) while pregnancy was detected in 23.53% recipients received a group of 11-25 embryos (n=17). Among them, five (1.76%) live puppies were born (P<0.05). These data show an increase in the overall efficiency of SCNT in canine species.

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.

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy‐six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase‐like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family. Mol. Reprod. Dev. 76: 483–489, 2009.

The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos

The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; P<0.05), but comparable pseudo-pronuclei formation was observed in both (85.3% vs. 75.8%). None of the inter-species cloned embryos developed beyond the 8-cell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.

Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase

Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.

Effects of Trichostatin A on In vitro Development of Porcine Embryos Derived from Somatic Cell Nuclear Transfer

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9→114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

Senescence is accelerated through donor cell specificity in cloned pigs

Animals cloned by somatic cell nuclear transfer (SCNT) sometimes have abnormalities that result in large offspring syndrome or early death during gestation due to respiratory and metabolic defects. We cloned pigs using two sources of donor cells and observed phenotypic anomalies in three pigs cloned from one type of cell, s-pig fetal fibroblasts. These animals had many wrinkles on their faces and bodies and looked older than age-matched normal pigs. We performed the present study to examine whether the wrinkled phenotype in the cloned pigs was due to senescence, a genetic problem with donor specificity, or epigenetic problems with reprogramming. To address this issue, we investigated biomarkers of senescence, including telomere length and the expression of senescence-associated β-galactosidase (SA-β-gal), glyceraldehyde phosphate dehydrogenase (GAPDH) and β-actin. We also assessed the methylation status of euchromatic PRE-1 repetitive sequences and centromeric satellite DNA, and measured the mRNA levels of six imprinted genes, Copg2, Mest, Igf2R, GNAS, SNRPN and Ube3a. The telomeres of the wrinkled cloned pigs were much shorter than those of the normal cloned pigs and age-matched normal pigs. In the wrinkled cloned pigs, SA-β-gal activity was detected and GAPDH and β-actin were repressed. The mRNA levels of Mest, GNAS and Ube3a were reduced in the wrinkled cloned pigs, although there was no difference between the normal cloned pigs and normal controls. This gene expression analysis indicates that the wrinkled abnormality of our pigs originates from genetic abnormalities in the donor cells used for SCNT.

Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines

Using in vivo-flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9-20 kg), large (>20-40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P < 0.01). Perinatal mortality until weaning was significantly higher in small breeds (33.3%) compared with medium, large, or ultra-large breeds where no mortality was observed. The mean birth weight of cloned pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype.

Protocol for the Recovery of In Vivo Matured Canine Oocytes Based on Once Daily Measurement of Serum Progesterone

The collection of in vivo matured canine oocytes relies on the accurate prediction of ovulation. The present study was designed to develop a protocol for the recovery of in vivo matured canine oocytes based on once daily measurements of serum progesterone (P(4)) concentrations. Blood samples (2 mL) were collected every day at 0900 h, and P(4) concentrations were analyzed using a DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit. The average number of oocytes at the metaphase II (M II) stage was significantly higher at or after 72 h (6.7 to 7.5) compared to 56 h (1.7) following ovulation. The highest numbers of corpora lutea, and therefore the highest numbers of oocytes, were recovered from bitches with initial ovulatory P(4) concentrations ranging from 6.0 to 8.0 ng/ mL (12.2 and 11.4, respectively) compared to from 4.0 to 4.9 ng/ mL (9.6 and 8.8, respectively; p < 0.05). The average number of M II oocytes recovered at 84 h from bitches with initial ovulatory P(4) levels of 5.0 to 5.9 ng/mL (7.7) was higher compared to bitches with P(4) levels of 4.0 to 4.9 ng/ mL (3.5) and 6.0 to 8.0 ng/ mL (4.8; p < 0.05). When oocyte recovery time was adjusted for initial ovulatory P(4) concentration, no significant difference in recovery rates or oocyte quality were observed. In conclusion, once daily measurements of P(4) can be used to predict ovulation in bitches, and oocyte recovery time should be adjusted for initial ovulatory serum P(4) concentrations.