Ok-Jae Koo

Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells.

Abstract:u2002 Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription‐polymerase chain reaction (RT‐PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus‐oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10u2003ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT‐PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin‐treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.

Developmental competence of porcine oocytes after in vitro maturation and in vitro culture under different oxygen concentrations

In this study, we investigated the effect of two oxygen concentrations (5 and 20%) during in vitro maturation (IVM) and during in vitro culture (IVC) on porcine embryo development and analysed differences in gene expression between cumulus-oocyte complexes matured under 5 or 20% oxygen and the resulting blastocysts cultured under 5% or 20% oxygen following parthenogenetic activation. There was no significant difference in oocyte maturation rate. However, the numbers of resulting blastocysts were significantly increased in the 5% IVC group compared with the 20% IVC group. Moreover, the M20C5 treatment group (23.01%) supported greater blastocyst development compared with the M5C5 (14.32%), M5C20 (10.30%), and M20C20 (17.88%) groups. However, total cell numbers were not significantly different among groups. According to mRNA abundance data of multiple genes, each treatment altered the expression of genes in different patterns. GLUT1, G6PD and LDHA were up-regulated in cumulus cells that had been matured in low oxygen, suggesting a higher glucose uptake and an increase in anaerobic glycolysis, whereas cyclin B1 (CCNB) and MnSOD (Mn-superoxide dismutase) were upregulated in cumulus cells that had been matured in high oxygen, which suggests a higher activity of mitosis-promoting factor and antioxidant response. In spite of these differential effects on cumulus cells, oocytes could mature normally regardless of different oxygen concentrations. Therefore, it can be concluded that high oxygen concentration during in vitro maturation and low oxygen during in vitro culture may alter the expression of multiple genes related to oocyte competence and significantly improves embryo development (p < 0.05) but not blastocyst quality.

Oxamflatin improves developmental competence of porcine somatic cell nuclear transfer embryos.

Abstract Aberrant epigenetic nuclear reprogramming of somatic nuclei is a major cause of low success in cloning. It has been demonstrated that treatment of histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos by alteration of epigenetic status. The aim of the present study was to investigate the effect of oxamflatin, a novel HDACi, on the developmental competence of porcine SCNT embryos. Treatment with 1u2009μM oxamflatin for 9u2009h after activation of SCNT embryos increased both in vitro and in vivo developmental competence. Treatment of SCNT embryos with 1u2009μM oxamflatin significantly increased blastocyst rate and total cell number in blastocysts (33.3±6.0 and 73.1±1.6, respectively) than that of controls (10.3±3.7 and 54.1±3.5, respectively) or scriptaid (16.4±4.6 and 64.4±2.1, respectively). Moreover, oxamflatin showed significant higher overall cloning efficiency from 0.9% to 3.2%, whereas scriptaid demonstrated 0% to 1.8%. In conclusion, these results indicate that oxamflatin treatment improves the developmental competence of porcine SCNT embryos.

Electrical activation induces reactive oxygen species in porcine embryos.

The objectives were to determine factors affecting generation of reactive oxygen species (ROS) in porcine embryos after electrical activation of oocytes, and the effects of an antioxidant and chemical agent on ROS generation. Greater ROS were induced by electrical activation compared to IVF (mean+/-S.E.M., 14.6+/-0.8 vs. 9.2+/-0.4, P<0.05). Furthermore, ROS generation in embryos after electrical activation was significantly increased by higher intensity and longer duration electrical pulses and by higher exogenous Ca(2+) concentrations. Cleavage rate and blastocyst formation rate were not directly related to the level of ROS. Supplementation of the IVC medium with 0.5mM glutathione (GSH) reduced ROS (9.2+/-0.4 vs. 14.7+/-0.9, P<0.05). Treatment with the chemical activation agent, 6-dimethylaminopurine (6-DMAP) for 3h did not induce further ROS generation in combination with electrical activation, but it improved blastocyst formation rate (53.8+/-1.1 vs. 23.7+/-3.5, P<0.05). We concluded that generation of ROS should be considered for optimizing electrical activation and that supplementing an antioxidant or combining electrical and chemical activation induced lower ROS generation in electrically activated porcine embryos.

Influence of oocyte donor and embryo recipient conditions on cloning efficiency in dogs

To determine factors that affect the efficiency of dog cloning by somatic cell nuclear transfer, the present study was performed to investigate 1) the effects of surgical history (non-operated/operated) and parity (nullipara/multipara) on the recovery of in vivo canine oocytes; 2) the effects of surgical history and parity of recipients on the pregnancy and delivery; and 3) the effects of synchronization state (AA, advanced asynchrony; SY, synchrony; RA, retarded asynchrony) between oocytes donor and recipient on the pregnancy and delivery. Oocyte recovery rate was significantly higher in non-operated dogs compared to operated dogs (93.8 vs. 89.6%, P < 0.05) and not different between nulliparous dogs and multiparous dogs. Delivery rate was also significantly higher in non-operated dogs compared to operated dogs (2.8 vs. 1.0%, P < 0.05) and in nulliparous dogs than multiparous dogs (3.0 vs. 1.7%, P < 0.05). Even though SY showed increased pregnancy and delivery rate (20.0% and 3.0%) compared to AA (15.0% and 2.0%) and RA (0.0% and 0.0%), there was no significant difference. In conclusion, we recommend non-operated dogs as experimental dogs and nulliparous dogs as recipient dogs to increase delivery rate after transfer of somatic cell nuclear transferred embryos, but further study is needed to find out appropriate synchrony status at the transfer.

Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels

Quercetin is a plant-derived flavonoid found in fruits or vegetables that has antioxidant properties and acts as a free radical scavenger. We investigated the effects of quercetin on porcine oocyte nuclear maturation and embryonic development after parthenogenetic activation. We then evaluated the antioxidant activities of quercetin by measuring reactive oxygen species (ROS) levels in matured oocytes. Immature oocytes were untreated or treated with 1, 10, and 50 µg/mL quercetin during in vitro maturation (IVM). Quercetin treatment did not improve oocyte nuclear maturation, but significantly higher blastocyst rates (p < 0.05) of parthenogenetically activated oocytes were achieved when the IVM medium was supplemented with an adequate concentration of quercetin (1 µg/mL). However, cleavage rates and blastocyst cell numbers were not affected. Oocytes treated with 1 or 10 µg/mL quercetin had significantly lower (p < 0.05) levels of ROS than the control and group treated with the highest concentration of quercetin (50 µg/mL). Moreover, this highest concentration was detrimental to oocyte nuclear maturation and blastocyst formation. Based on our findings, we concluded that exogenous quercetin reduces ROS levels during oocyte maturation and is beneficial for subsequent embryo development.

Effect of roscovitine-treated donor cells on development of porcine cloned embryos.

Synchronization of the donor cell cycle is an important factor for successful animal cloning by nuclear transfer. To improve the efficiency of porcine cloning, in the present report, we evaluated effects of contact inhibition, serum starvation and roscovitine treatment of donor cells on in vitro and in vivo developmental potency of cloned porcine embryos. Fibroblasts derived from a porcine foetus at day 30 of gestation were isolated and cultured to 70% confluency. Then, cells were either cultured to 100% confluency for contact inhibition, or cultured in 0.5% serum for 72u2003h for serum starvation or with 15u2003μM roscovitine for 24u2003h. Cells were most effectively synchronized at G0/G1 in the serum starvation group (87.5%) compared with the contact inhibition and roscovitine treatment groups (76.3% and 79.9% respectively pu2003<u20030.05). However, after somatic cell nuclear transfer followed by in vitro culture, the serum starvation group showed a significantly lower blastocyst formation rate (5.6%) compared with the contact inhibition and roscovitine treatment groups (11.6% and 20.0% respectively). Differential expression of apoptosis-related genes and the level of apoptosis in each treatment group explain the variation in developmental competence among the groups. Significantly higher level of apoptosis was observed in the serum starvation group. On the other hand, the roscovitine treatment group shows the lowest level of apoptosis and the best in vitro development among the groups. Cloned embryos derived from roscovitine-treated donor cells were transferred to surrogate pigs. Three healthy live piglets were produced. In conclusion, we suggest that roscovitine treatment of donor cells improves development of cloned porcine embryos and can raise the efficiency of cloned piglet production.

Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

As shown by the birth of the first cloned dog Snuppy, a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.

Influence of Ovulation Status, Seasonality and Embryo Transfer Method on Development of Cloned Porcine Embryos

To improve pig cloning efficiency, the present study evaluated the effect of ovulation status, seasonality and embryo transfer (ET) method on in vivo development of cloned porcine embryos. Cloned embryos were transferred to surrogate mothers on the same day of somatic cell nuclear transfer. In pre-ovulation stage (PO), pregnancy rate (PR) and delivery rate (DR) were 36.3% and 9.4%, respectively. In post-ovulation stage, 22.7% PR and 2.1% DR were recorded (both PR and DR are significantly higher in PO). When ET was performed during winter (December-February), spring (March-May), summer (June-August) and autumn (September-November), the PRs were 13.4%, 37.3%, 24.6% and 51.0%, while DRs were 0%, 12.7%, 4.3% and 7.8%, respectively. The highest PRs were recorded in autumn groups. However, DRs were significantly lower in autumn (7.8%) group compared with spring (12.7%) group. The PR was the lowest and no piglets were born in winter group, which might be because of the effect of low temperature during ET. To overcome the low PR in winter group, 0.25 ml straws were used for ET to minimize exposure time of embryos to ambient temperature. The straw ET group showed significantly higher PR in the winter group (23. 9%) compared with the conventional catheter-loading group (7.7%). We suggest that using PO recipient and ET in spring is the best condition for pig cloning. In addition, alternative method to reduce cold shock during ET in winter is necessary.

Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre- loxP

It is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with a plasmid that contained a red fluorescent protein marker (pCALNL-DsRed) and a floxed neomycin-resistance gene. Cells were selected with 750 μg/ml neomycin for 2 weeks following transfection but did not express DsRed after visualization under a fluorescence microscope. Expression was achieved only after transient transfection with plasmid DNA that expressed the Cre recombinase enzyme. The cells that expressed DsRed were used for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) were seen to have cleaved. Six blastocysts developed after SCNT and expressed DsRed. Deletion of the floxed neomycin-resistance gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in cloned blastocysts. This study demonstrated that Cre-loxP recombination can be conducted successfully in miniature pig fibroblasts and that the sequentially transformed cells can develop to the pre-implantation embryo stage via SCNT.