• Korean Journal of Animal Reproduction
• /
• v.27 no.1
• /
• pp.69-75
• /
• 2003

The objective of this study is to improve the efficiency of embryo transfer in generation of transgenic mice using inbred mouse (C57BL/6J) embryos. The embryos of C57BL/6J and BCF1 mice were superovulated by the standard protocol. One-cell stage of embryos were microinjected and the resulted one-or two-cell were transferred into one-or two-side oviducts of foster mother, respectively. When one-cell stage of embryos were transferred into one-side oviduct of 0.75 d.p.c. foster mother, the results were not ideal because of showing pregnancy ratios of 68.8$\pm$7.83% for C57BL/6J and 48.3$\pm$14.22% for BCF1, and development ratios of pups vs transferred embryos of 11.9$\pm$5.51% for C57BL/6J and 10.5$\pm$8.03% fur BCF1 . However, when two-cell stage of embryos were transferred into two-side oviducts of 0.5 d.p.c. foster mother, we got better results of 94.4$\pm$9.64% and 100$\pm$0% pregnancy ratio, and 22.1$\pm$0.4% and 21.8$\pm$0.38% development ratio for C57BL/6J and BCF1, respectively. Therefore, transferring two-cell stage of C57BL/6J embryos into two-side oviducts of 0.5 d.p.c. foster mother may be competitive to the result in BCF1 embryos. Even if there are a lot of other factors affecting these results, we conclude from these experiments that transfer of two-cell embryos into two-side oviducts of 0.5 d.p.c. foster mother is a more efficient and safe method than others in generating transgenic mice using inbred mice embryos.

• Applied Microscopy
• /
• v.30 no.1
• /
• pp.45-59
• /
• 2000

Cis-platin is a widely used anticancer drug against certain solid tumors such as malignant ovarian tumor, malignant carcinoma of head and neck, bladder cancer and cervical cancer of uterus, and its major mechanism of action is inhibition of DNA synthesis of the tumor cell. To investigate the inhibitory effects of cis-platin on the ciliogensis of the ciliated cells in the mucosa of oviduct, the author pursued the alterations of $\alpha-tubulin$, which is the main constituent of the microtubles in cilia, after cis-platin treatment. To eliminate the possible variations due to ovarian cycle, female Spargue-Dawley rats ($150\sim200gm$ in B.W.) were pretreated with estradiol benzoate (20 mg/kg, once a day, for 4 consecutive days). Animals were administrated with cis-platin (6 mg/kg, i.p.) and sacrificed at 1day, 3days, 5days and 7days after treatment, respectively. Immunohistochemistry for $\alpha-tubulin$ using mouse anti-rat $\alpha-tubulin$ monoclonal antibody as primary antibody was done. Immunogold electronmicroscopy for intracellular distributions of $\alpha-tubulin$ was also performed with same primary antibody and Goat anti- mouse IgM which is preconjugated with gold particles of 15 nm as secondary antibody. The results obtained were as follows; 1. Strong immunoreactivity of $\alpha-tubulin$ was observed in ciliated cells of oviducts at 1, 3 and 5 days after estradiol pretreatment. 2. Weak immunoreactivity of $\alpha-tubulin$ was observed in ciliated cells of oviducts at 1 and 3 days after cis-platin treatment but it was recovered to strong immunoreactivity in 5 days 3. In immunogold electronmicroscopy, density of gold particles for $\alpha-tubulin$ reactions was decreased in apical cytoplasm, but few changes were observed in basal body or cilia at 1 and 3 days after cis-platin treatment. From these above results, it is indicated that synthesis of $\alpha-tubulin$ in ciliated cells of rat oviduct is inhibited by cis-platin treatment.

• Journal of Embryo Transfer
• /
• v.25 no.1
• /
• pp.85-87
• /
• 2010

A necropsy of a primiparous, 4-year-old, Himalayan tahr (Hemitragus jemlahicus) was performed in September 8, 2009. Typical appearances of ovaries, oviducts, uterine horns, cervix and caruncles were well shown. Five cervical folds were present in the cervix. Cervical opening can be reached in the length of less than 15 cm from the entrance of vagina. Development of two follicles was found in the left ovary even though breeding season of this species naturally starts in late November. The reproductive organs of a primiparous, 3-year-old, Corriedale sheep showed that differences in the length and morphological appearance of cervix exist between two species. Comparative understanding of reproductive systems in Himalayan tahr and Corriedale sheep could help advancing assisted reproductive technologies in feral goats.

• Korean Journal of Animal Reproduction
• /
• v.17 no.2
• /
• pp.121-125
• /
• 1993

Early development of bovine oocytes fertilized in vitro in the medium with caffeine and heparin was examined in different culture systems. When the oocytes were transferred into culture medium 8 h after insemination, 12%(7/60) of penetrated oocytes cleaved to 4-cell stage 24 h after insemination. The proportions of oocytes cleaved to 80to 16-cell stage 48 h after insemination had also a to be higher in oocytes transferred into culture medium 8 h (29%) than 16 h(10%) or 24 h(4%) after insemination. 52% of the 4-cell embryos developed to morula and blastocyst stages when they were co-cultured with oviductal epithelia, whereas only 5% of embryos cultured without the epithelial cells(P<0.001). In another experiment, embryos were co-cultured with ampulla, isthmus or utero-tubal junction of oviducts. There are no significant differences in the proportions of embryos developed to morula and blastocyst stage.

• Korean Journal of Animal Reproduction
• /
• v.17 no.4
• /
• pp.375-383
• /
• 1994

The human growth hormone (hGH) gene uder the control of the rat $\beta$-casein promoter gene was designed to produce transgenic mouse expressed hGH gene in only mammary gland. One hundred seventy two eggs microinjected were transferred to the oviducts of pseudopregnants and 43 offspring were delivered. By Southern blotting hybridization, 3 were transgenic with rat $\beta$-casein/hGH gene. The copy numbers of three transgenic founder were 1, 5, and 15, respectively. A radioimmunoassay was developed to quantitate the amount of expression of the hGH gene in mammary gland of transgenic mice. The amount of hGH was 13.3ng/ml in the lactating milk of one transgenic line, showing predominantly higher than 3.0ng/ml in milk of control mice. Therefore, our findings suggested that $\beta$-casein promoter may induce the tissue specific expression of structural gene.

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.65-72
• /
• 1995

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4$^{\circ}C$ for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.

• Journal of Embryo Transfer
• /
• v.15 no.3
• /
• pp.295-302
• /
• 2000

The objective of this study was to compare different superovulation treatments using PMSG or PG600$^{ }$ and to determine the optimal time of oocyte recovery after hCG administration. A total of 90 prepubertal Yorkshire x Landrace gilts crossed with Duroc, 6~7 months old and 100~120 kg of body weight, were used. PMSG (1,500 IU/head) or 5~7.5 ml of PG600$^{ }$(400 IU of PMSG and 200 IU of hCG) were administrated subcutaneously, and then 1,000 IU of hCG were administered intramuscularly at 72 hours after PMSG or PG600$^{ }$ injection. At carious time of 44, 46, 48 and 50 hours after hCG injection, superovulated gilts were slaughtered in a local abattoir. Ovaries together with oviducts were excised from the body immediately after slaughtered and transported to laboratory in 39$^{\circ}C$ saline. Ovaries were examined fur the number of corpus hemorrhagicum and unovulated follicles present in the surface of ovary. The unovulated follicles were categorized into small (1~3 mm in diameter) and large (4~8 mm) groups according to their diameter. Oocytes were recovered by flushing both oviducts with micropipette tip (1~100 $\mu$l) attached to a 10-ml disposable syringe. The number of CH on ovary and recovered oocytes at 46, 48 and 50 hr after hCG injection in PG600$^{ }$ treated groups were significantly higher than the other group. Group of phCG 50 hr among PMSG treated groups had a greater number of CH and recovered oocytes(P<0.05). The number of CH on ovary and recovered oocytes at 50 hr after hCG injection in 1$\frac{1}{2}$ vial(7.5 ml) of PG600$^{ }$ treated groups was significantly higher than 1 vial(5 ml) of PG600$^{ }$ treated group(P<0.05). In conclusions, considering a number of corpus hemorrhagicum and recovered oocytes after superovulation in gilts, effective time of oocyte recovery by treatment with PMSG and hCG was post-hCG 50 hr and with PG600$^{ }$ plus hCG was post-hCG 46, 48 and 50 hr. Also, admini-stration of 1$\frac{1}{2}$ vial(7.5 ml) of PG600$^{ }$ treated group had a great number of CH and recovered oocytes.covered oocytes.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.10
• /
• pp.1403-1411
• /
• 2002

The aims of this study were, firstly, to analyze the biochemical compositions of serum and follicular fluid (FF) from prepubertal gilts after PMSG (1,000 IU) treatment. The concentrations of total proteins, lipids, cholesterol, glucose and sex hormones (progesterone, $P_4$; estradiol-$17{\beta}$, $E_2$; testosterone, T) were measured. Secondary, the effects of porcine FF (pFF) addition (40% and 100%) in IVM media and different culture conditions [Exp. 1: mBMOC-2+20% porcine serum (PS), fresh IVM medium, filtered IVMconditioned medium, or rabbit oviducts; Exp. 2: mBMOC-2+20%PS or stepwise medium replacement procedures (SMRP) cocultured with or without cumulus cells] on the in vitro development (IVD) of porcine oocytes were also examined. Results showed that no significant differences were found in total protein levels between serum and pFF from different sizes (large, >7 mm; medium, ~5-7 mm; small, <3-5 mm) of follicles (75-85 and 49-90 mg/dl; p>0.05). Total lipid concentrations remained constant in serum (395-472 mg/dl), and reduced significantly in the pFF from large follicles (287 mg/dl) at 132 h after PMSG treatment when compared to those at other time points (441-480 mg/dl). Basal cholesterol levels in serum and pFF at 12 h were similar (153-161 mg/dl), but increased at 36 h (186-197 mg/dl). Basal P4 and E2 levels in serum (0.1 ng/ml and 5.5 pg/ml) were low, but increased from 0.34 ng/ml and 12.13 pg/ml at 24 h to 0.81 ng/ml and 61.70 pg/ml at 98 h, respectively, after PMSG treatment (p<0.05). P4 levels increased linearly in pFF from large follicles during 12 through 132 h (138-1,288 ng/ml). A similar increase was also observed in $E_2$ levels (22-730 pg/ml) before 60 h post PMSG treatment, and then dropped afterwards (730-121 pg/ml). The development of the oocytes fertilized in 40% pFF-medium was greater than that in 100% pFF-medium group without gonaodtropin addition (31% vs 10%, p<0.05). However, both were lower than those in mBMOC-2+20%PS and in rabbit oviducts (p<0.05). When cocultured with cumulus cell monolayers, a greater cleavage rate was observed in the group cultured in filtered IVM-conditioned medium than the SMRP group (36% vs 18%, p<0.05). A similar phenomenon was also observed in the culture without cumulus cell monolayers (33% vs 19%, p<0.05). It is concluded that neither the fresh IVM nor filtered IVM-conditioned medium has positive effect on the IVD of oocytes. Coculture with cumulus cell monolayers and the SMRP were not beneficial to the development of IVF pig oocytes.

• Korean Journal of Animal Reproduction
• /
• v.13 no.2
• /
• pp.70-78
• /
• 1989

Mouse eggs recovered from oviducts at one hourly intervals between 10 and 20 hours after administration of hCG were fixed, stained and then investigated the rate of in vitro fertilization and nuclear maturation. In case of out- bred ICR mice, ovulations were occured between 11 and 13 hours after hCG injection. The stages of in vitro maturation of eggs recovered from female mice at various times after hCG injection were metaphase I, anaphase I, telophase I and metaphase II. However the majority was metaphase I(17.6 to 44.4%) and metaphase II(42.9 to 80.0%) stage. When the eggs were inseminated with epididymal spermatozoa, the fertilization rate was declined as the egg recovery time after hCG administration was delayed. That is, the proportion of eggs undergoing fertilization became higher(68.1 to 77.4%) in the eggs at 12 to 15hr after injection of hCG than those(17.5 to 56.4) at 16 to 20 hr after injection of hCG. Also, when nuclear maturation of the unfertilized eggs were observed at 8 hours after insemination, the majority was in metaphase I and metaphase II and no anaphase I and telophase I were observed.

• Korean Journal of Animal Reproduction
• /
• v.18 no.2
• /
• pp.133-139
• /
• 1994

Two human lactoferrin expression vectors(pCChcLf and pCChcLf-1) were constructed using rat $\beta$-casein gene and human lactoferrin cDNA. The recombinant DNAs containing human lactoferrin cDNA were microinjected into the fertilized eggs of hybrid mice (BDF1 : C57BL$\times$DBA) and the DNA-injected eggs were treansferred into the oviducts of foster mothers. Genomic DNAs were isolated from the tails of mice born from the microinjected eggs and analyzed by Southern blot analysis. As a result, 5 and 9 transgenic mice with CChcLf and CChcLf-1 gene were produced, respectively. To determine tissue-specificity of transgene expression, Northern blot analysis was performed. Female transgenic mice were killed at day 10 of lactation and total RNAs from various tissues were isolated. Based on Northern blot analysis, it was shown that transgene was mainly expressed in the mammary glands of transgenic mice. In addition, the human lactoferrin in milk was detected by enzyme-linked immunosorbent assay. For this study, milk was obtained from the mammary glands of the transgenic mice at day 10 of lactation. In line #2 of CChcLf and line #7 of CChcLf-1 transgenic mice, human lactoferrin was secreted into the milk at concentration levels of 340ng/ml and 60ng/ml, respectively.