• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.10
• /
• pp.1412-1421
• /
• 2002

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. In situ hybridization demonstrated that the transfection rate of sperm cells with and without BSA was 41 and 68% respectively, when the cells were treated with liposome/DNA complexes and 13% for DNA alone. LSC analysis showed that the binding of exogenous DNA was greatly reduced by DNase I treatment which digests DNA bound onto spermatozoa, suggesting that some of the DNA was internalized into the sperm membrane. To find out whether transfected DNA was internalized into sperm intracytomembrane, sperm DNA was amplified by inverse PCR. No PCR products were detected from sperm cells, indicating that the foreign DNA was simply bound onto the sperm membrane. To investigate transfer rates of exogenous DNA into oocytes via sperm cells, we used immunofluorescene method to follow the distribution of foreign DNA via spermatozoa: a few exogenous DNA was located in the cytoplasm of early embryos (13/60, 21.7% for DNA+/liposome+/BSA) and was not located in the pronucleus and/or nucleus. These results suggest that most of the transfected sperm cells could carry the foreign DNA into the egg by in vitro fertilization, but that the transferred DNA is degraded in the developing embryos without stable integration into the zygote genome. Therefore, we have directly injected with transfected sperm cell into oocyte cytoplasm and observed that some of the exogenous DNA was detected in preimplantation embryonic cytoplasm and expressed at preimplantation stages, suggesting that exogenous DNA in early zygote has their integrity. In this study, we have not identified a noble mechanism that interfering transportation of foreign DNA into zygote genome via spermatozoa. Our data, however, demonstrated that inverse PCR and immunofluorescene methods would be used as a new tool for follow-up of gene distribution in oocyte via sperm cells.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.40 no.6
• /
• pp.387-393
• /
• 2007

We examined the conditions for separating and preserving the male and female gametophytes of Kjellmaniella crassifolia. The highest percentage of zygote germination (85%) was on semi-solid medium composed of 1.0% transfer gel agar at $15\;^{\circ}C$ and $20\;{\mu}mol/m^2/s$ after a 4-week culture. Zygote germination in PESI liquid medium was 93.5% at $20\;^{\circ}C$ and $20\;{\mu}mol/m^2/s$. The maximum zygote growth was $252{\pm}19.7\;{\mu}m$ on 1.0% transfer gel agar at $15\;^{\circ}C$ and $40\;{\mu}mol/m^2/s$ after 5-week culture, and was $76.7{\pm}2.8\;{\mu}m$ in PESI liquid medium at $20\;^{\circ}C$ and $40\;{\mu}mol/m^2/s$. The respective numbers of separated male and female gametophytes from germinated zygotes were 157 and 93 on 1.0% transfer gel agar and 14 and 28 in PESI liquid medium. The maximum growth of separated male and female gametophytes was $575{\pm}28.3\;{\mu}m$ at $5\;^{\circ}C$ and $60\;{\mu}mol/m^2/s$ and $686{\pm}35.4\;{\mu}m$ at $20\;^{\circ}C$ and $20\;{\mu}mol/m^2/s$ in PESI liquid medium after 3 weeks, respectively. The highest percentage fertilized was $93.3{\pm}5.8%$ at $15\;^{\circ}C$ and $20\;{\mu}mol/m^2/s$ in PESI liquid medium. These results show that the best conditions for the separation and preservation of gametophytes (male and female) consisted of culturing on 1.0% transfer gel agar at $15\;^{\circ}C$ and $20\;{\mu}mol/m^2/s$.

• 대한생식의학회:학술대회논문집
• /
• 1996.11a
• /
• pp.63.1-63.1
• /
• 1996

• 대한생식의학회:학술대회논문집
• /
• 2003.12a
• /
• pp.122.2-123
• /
• 2003

• Journal of Embryo Transfer
• /
• v.27 no.2
• /
• pp.121-126
• /
• 2012

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and $10{\mu}M$ ${\beta}$-mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

• Applied Biological Chemistry
• /
• v.10
• /
• pp.101-105
• /
• 1968

The effect of concentration of carbon and nitrogen sources on the sporulation and the life cycles of three strains of Zygosaccharomyces was investigated. The results are as follows: 1) The good sporulation of Zygosaccharomyces bisporus, delbruekii, and Z. steineri was obtained on solid medium containing 0 to 0.001% of nitrogen and 10 to 20% of glucose. The high content of nitrogen was detrimental to sporulation and asci were formed under 0.01% of nitrogen. 2) It is widely accepted that the life cycle of Zygosaccharomyces proceeds in the following way: Ascospore...Vegetative cells...Conjugation of vegetative cells...Sporulation...Ascopores But zygotes of Z. bisporus proceeded to vegetative cells when transfered to the suitable medium for vegetative reproduction, and then formed asci.

• Journal of Embryo Transfer
• /
• v.30 no.4
• /
• pp.299-303
• /
• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

• Journal of Korean Medical classics
• /
• v.3
• /
• pp.1-150
• /
• 1989

Oriental medicine thinks life and death as the following. 1. The universe seems to be a kind of organism which is divided into 3 branches, as Heaven, Earth and Man. Man is not created from nihil by the Creator. Heaven and Earth by their interaction operate to produce man. This is similiar that zygote is not created from nihil, and that sperm and ovum are transformed into zygote by their interaction. The symbolic meaning of sperm is Heaven, and that of ovum is Earth. Mind and body, as well as spirit and body, are not the real, but artificial words for the purpose of observing and expressing one man. So there is not spiritual substance as distinct from body. The expected life span of man is subjected to change, and is always becoming through life. Fate, the Creator and the world to come cannot be said to be. 2. After one's death, man is transformend into Heaven and Earth. Dying is this process of transformation. Although man comes into existence and closes one's life, the total life of the universe does not change. The criteria of determination of death is not in cell death, but in somatic death. Somatic death divided into 2 branches, one is heart-lung death, the other is brain death. For the standard of health changes ceaselessly as time goes by, aging and dying is not the process of losing health. Because of mind cannot be seperated from body, we'll feel at ease bodily and mentally in healthy dying. The completion of lifetimes is the value of healthy dying. 3. From the viewpoint of these, we must think to let a person die healthily is the right medical ethics. The way to let a person die healthily is divided into 3 branches, one is treatment, another is prevention and the other is promotion of health. We should treat and prevent death of sickness, but take care of healthy dying.

• Clinical and Experimental Reproductive Medicine
• /
• v.25 no.3
• /
• pp.261-268
• /
• 1998

The present study was to assess the effect of ultrarapid freezing on the development of 1-cell mouse zygote using cryoprotectants, DMSO (dimethyl sulfoxide) or PROH (1,2-propanediol). We investigated the effect of the type and concentration of cryoprotectant, and of the temperature and time of prefreezing equilibration on their capacity to develop to the blastocyst stage in vitro. The concenration, the equilibration temperature, and the exposure time seemed to serve as an important factor in ultrarapid freezing of 1-cell mouse zygotes. In addition to the exposure time and the concentration of cryoprotectant appeared to playa key role in the development of the embryo. In general, the development of the embryo was more effective at $3^{\circ}C$ than $23^{\circ}C$ and 4.5 M than 3 M for 3 to 5 minutes. At $23^{\circ}C$ the development of the embryo was stimulated by DMSO while at $3^{\circ}C$ it was stimulated by PROH. Thus it has been suggested that there exists a correlation between the concentration of cryoprotectants and exposure time in the development of the embryo. In conclusion, we found that for ultrarapid freezing of mouse 1-cell embryos in DMSO, or PROH-based solution, viability shown optimum depending on the cryoprotectant, the concentration of the cryoprotectant and on the temperature and the duration of equilibration.

• Journal of Species Research
• /
• v.6 no.1
• /
• pp.68-75
• /
• 2017

We investigated the effects of sodium hypochlorite (NaOCl) and culture medium on embryo swelling and germination of Calanthe discolor Lindl., and established a method for determining the swelling and protocorm formation of C. discolor seeds via in vitro examination of immature seeds. Treatment of immature seeds with NaOCl greatly enhanced the extent of embryo swelling and protocorm formation of immature zygote embryos compared to seeds without NaOCl treatment. The effects of the culture media were also evaluated with regard to embryo swelling and protocorm formation of in vitro cultured seeds with and without NaOCl treatment. Additionally, the effects of white fluorescent light and red and blue LEDs lights on seedling growth in in vitro culture were examined. The most suitable condition for seedling growth after 12 weeks of culture was the red LEDs light with POM medium. These results show effective asymbiotic germination and growth of C. discolor.