• Asian-Australasian Journal of Animal Sciences
• /
• v.22 no.7
• /
• pp.962-968
• /
• 2009

The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.

• Korean Journal of Animal Reproduction
• /
• v.26 no.4
• /
• pp.353-360
• /
• 2002

Although successful pronuclear formation and apposition were seen in porcine oocytes following mouse sperm injection, little is known on the morphology of male and female pronuclei following sperm injection. The objective of this study is to describe the ultrastructure of porcine zygote following murine sperm injection in relation to the chronology of pronuclear S phase. At 40h ~ 44h following in vitro maturation, Cumulus cells were removed in TCM-HEPES with 0.1% hyaluronidase. Then, spermatozoa was injected into the cytoplasm of oocytes. After. injection, all oocytes were transferred to NCSU23 medium and cultured at 39$^{\circ}C$ under 5% $CO_2$ in air. Oocytes were fixed in 2% glutaraldehyde in Dulbeccos phosphate-buffered saline and observed by Transmission Electron Microscopy. Nuclear precursor bodies were observed in each pronucleus. A cluster of large and small granules was attached in the nucleolus precursor body. After the apposition of male and female chromatin, chromatin condensation was observed throughout the nucleoplasm and nucleolus precursor bodies and condensed chromatin in contact with clusters of small and large granules and the nuclear envelope were found in apposed pronuclear regions. These results suggest that non-species specific nuclear cytoplasmic interactions take place during pronuclear formation and apposition following sperm injection.

• Journal of Embryo Transfer
• /
• v.23 no.1
• /
• pp.19-24
• /
• 2008

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

• Journal of Embryo Transfer
• /
• v.32 no.4
• /
• pp.275-285
• /
• 2017

Lysophosphatidic acid (LPA) is an important signaling molecule. Here, the effect and mechanism of LPA on the preimplantation development of porcine embryos during in vitro culture (IVC) was examined. Porcine embryos were cultured in porcine zygote medium (PZM-3) supplemented with $30{\mu}M$ LPA during different days. There was a significantly higher cleavage rate in Day 1-7 and significantly higher total cell number of blastocysts in Day 1-3 and Day 4-7. It was also found that messenger RNA (mRNA) expression level of PCNA, BCL2 and BAX in blastocysts obtained from D1-7 group were significantly higher and BCL2/BAX mRNA ratio in D1-3 group was significantly lower than control group but Day 4-7 and Day 1-7 groups were comparable with control group. Treatment with $20{\mu}M$ PLC inhibitor significantly decreased the embryo cleavage rate and blastocyst formation rate. Moreover, LPA as an activator of PLCs, enhanced the $30{\mu}M$ LPA + $20{\mu}M$ U73122 group embryo cleavage rate which similar with control group. In conclusion, the results suggest that treatment with LPA during IVC improves the porcine early embryo cleavage by activation of PLC signaling pathway and regulate the mRNA expression that contribute to total cell number of blastocysts during blastocyst formation.

• Horticultural Science & Technology
• /
• v.29 no.2
• /
• pp.123-129
• /
• 2011

To identify unintended vertical gene-transfer rates from the developed transgenic plants, rapid and unequivocal techniques are needed to identify event-specific markers based on flanking sequences around the transgene and to distinguish zygosity such as homo- and hetero-zygosity. To facilitate evaluation of zygosity, a polymerase chain reaction technique was used to analyze a transgenic pepper line B20 (homozygote), P915 wild type (null zygote), and their F1 hybrids, which were used as transgene contaminated plants. First, we sequenced the 3'-flanking region of the T-DNA (1,277 bp) in the transgenic pepper event B20. Based on sequence information for the 3'- and 5'-flanking region of T-DNA provided in a previous study, a primer pair was designed to amplify full length T-DNA in B20. We successfully amplified the full length T-DNA containing 986 bp from the flanking regions of B20. In addition, a 1,040 bp PCR product, which was where the T-DNA was inserted, was amplified from P915. Finally, both full length T-DNA and the 1,040 bp fragment were simultaneously amplified in the F1 hybrids; P915 ${\times}$ B20, Pungchon ${\times}$ B20, Gumtap ${\times}$ B20. In the present study, we were able to identify zygosity among homozygous transgenic event B20, its wild type P915, and hemizygous F1 hybrids. Therefore, this novel zygosity identification technique, which is based on PCR, can be effectively used to examine gene flow for transgenic pepper event B20.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.38 no.3
• /
• pp.164-171
• /
• 2005

Codium fragile (Suringar) Hariot, an edible green alga is farmed in Korea by natural blooming zygotes attachment. Experiments were conducted to reveal the conditions for artificial seed production of C. fragile by sexual and asexual reproduction. Growth was compared between zygotes attachment (sexual reproduction) and isolated utricles with medullary filaments (asexual reproduction). Zygotes and isolated utricles with medullary filaments were cultured under different light conditions (10, 20, 40, 60 and $100\;{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$) and temperatures (5, 10, 15, 20 and $25^{\circ}C$) under 16:8LD. Maximum growth of zygote was $261.3{\pm}21.0\;{\mu}m$ under $15^{\circ}C$ and $20\;{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ after 13 days culture. Maximum regeneration of isolated medullary filament was $8.1{\pm}1.7\;mm$ per one isolated utricle under $20^{\circ}C$ and $100\;{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ after 15 days culture. After intermediate culture during two months in the field, morphogenesis occurred in both sexual and asexual reproduction, and growth of young thalli was not significantly different (p>0.05) between the both reproduction methods. Even though seed production of C. fragile is possible in both sexual and asexual reproduction, the mass artificial seed production of asexual reproduction is much more effective than that of sexual reproduction that is too much affected by maturity.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.39 no.5
• /
• pp.419-426
• /
• 2006

This study investigated the ecology and growth of Monostroma nitidum Wittrock in both its natural habitat and the laboratory. The maximum length, width, and weight of M. nitidum in March were 9.0$\pm$4.7 cm, 9.6$\pm$3.6 cm, and 1.52$\pm$1.13 g, respectively. Yellowish-green or yellowish-brown reproductive thalli began to appear in January, and over 80% of the thalli matured by March. The male and female spores were ca. 6 $\mu$m long, and elongate and ovoid in shape. The spores had two flagella and one-eye spot, and tended to swim toward light. Maximum number of spores released from matured thalli was 236 cells/mL after 70 minutes at a light intensity of 100 $\mu$mol/m$^2$/s. The zygote diameter ranged from 3.4-6.0 $\mu$m (mean 4.2 $$m) and increased to 69.8 $\mu$m 14 weeks after culture. The mass release of zoospores was observed from thalli in the dark (3 to 12 days), after 30 min under dry conditions in the shade, at 25$^{\circ}C$, and a light intensity of 100 $\mu$mol/m$^2$/s. The maximum number of zoospores released was 109.8 cells/mL after 60 min of induction. M. nitidum fronds on the net increased to 6.8-7.2 cm in length, and 6.6-8.9 cm in width during the winter.

• Clinical and Experimental Reproductive Medicine
• /
• v.26 no.1
• /
• pp.117-121
• /
• 1999

Although anejaculation is a relatively uncommon occurrence in the general population, over 12,000 new cases are reported annually. Anejaculation may result from spinal cord injury, retroperitoneal lymph node dissection, diabetes mellitus, transverse myelitis, multiple sclerosis, or psychogenic disorders. At least 30% of men with this problem are or will be married and many will seek help to remedy their infertile state. The evolution of technique and instrumentation over the last 30 years has made electroejaculation an accessible and acceptable form of therapy. Recent successes in inducing ejaculation by means of rectal probe electrostimulation or vibratory stimulation combined with assisted reproductive techniques, such as zygote intrafallopian transfer (ZIFT), gamete intrafallopian transfer (GIFT), and in vitro fertilization (IVF), have provided these men means of producing their own biologic offspring. We have experienced a successful pregnancy with electroejaculation and in vitro fertilization in a infertile patient whose husband had an ejaculatory disturbance due to a spinal cord injury. So we report this case with a brief review of literatures.

• Journal of Embryo Transfer
• /
• v.30 no.1
• /
• pp.23-31
• /
• 2015

Catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa Libosch. It possesses a broad range of biological and pharmacological activity including anti-tumor, anti-inflammation and anti-oxidant by acting as a free radical scavenger. Therefore, in this study, the effects of catalpol on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in porcine embryos. After in vitro maturation and fertilization, porcine embryos were cultured for 6 days in porcine zygote medium 3 (PZM-3) supplemented with catalpol (0, 100, 200 and $400{\mu}M$, respectively). Blastocyst development not significantly improved in the catalpol treated group when compared with control group. Otherwise, the intracelluar levels of ROS were decreased and the numbers of apoptotic nuclei were reduced in the catalpol ($100{\mu}M$) treated porcine blastocysts (P<0.05). On the other hand, blastocyst development was significantly improved in the catalpol ($100{\mu}M$) treated group when compared with the untreated catalpol group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress (P<0.05). Otherwise, the intracellular levels of ROS in catalpol ($100{\mu}M$) treated group were significantly decreased in the untreated catalpol group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress (P<0.05). Furthermore, the total cell numbers of blastocysts were significantly increased (P<0.05) in the catalpol ($100{\mu}M$) treated group under $H_2O_2$ ($200{\mu}M$) induced oxidative stress, whereas numbers of apoptoic nuclei were significantly reduced (P<0.05). In conclusion, our results indicate that treatment of catalpol may have important implications for improving developmental competence and preimplantation quality of porcine embryos through its anti-oxidant and anti-apoptotic effect.

• KOREAN JOURNAL OF CROP SCIENCE
• /
• v.31 no.2
• /
• pp.143-149
• /
• 1986

Anatomical changes in the forming and germinating processes of tobacco seeds were investigated to obtain basic information on the ecological characteristics of tobacco seeds. Seed development studied through the longitudinal section of the fertilized ovule clarified that the cell division of the zygote was initiated after 7 days of flowering. After 12 days of flowering, perfect seed constituents such as cotyledon, epicotyle and radicle were formed and those were expressed to recognizable level of germinability. After 15 days of flowering germination rate reached higher than 30% and 17 and 21 days after flowering a perfect seed which have 70% or higher germinability were produced. Seed size was ranged between 0.3-0.6 mm and varietal differences were noted in the given seed size range. Under the light treatment, the morphological changes were observed by elongation of radicle after 2 days of imbibition and apparent germination after 3 days of imbibition. But no responses of the seeds imbibed 6 days under the dark condition were observed.