• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.63-68
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• 2005

Cloned calves derived from somatic cell nuclear transfer (SCNT) have been frequently lost by sudden death at 1 to 3 month following healthy birth. To address whether placental anomalies are responsible for the sudden death of cloned calves, we compared protein patterns of 2 placentae derived from SCNT of Korean Native calves died suddenly at two months after birth and those of 2 normal placentae obtained from AI fetuses. Placental proteins were separated using 2-Dimensional gel electrophoresis. Approximately 800 spots were detected in placental 2-D gel stained with coomassie-blue. Then, image analysis of Malanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between normal and SCNT placentae. In the comparison of normal and SCNT samples, 8 spots were identified to be up-regulated proteins and 24 spots to be down-regulated proteins in SCNT placentae, among which proteins were high mobility group protein HMG1, apolipoprotein A-1 precursor, bactenecin 1, tropomyosin beta chain, $H^+-transporting$ ATPase, carbonic anhydrase II, peroxiredoxin 2, tyrosine-rich acidic matrix protein, serum albumin precursor and cathepsin D. These results suggested that the sudden death of cloned calves might be related to abnormal protein expression in placenta.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.249-254
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• 2012

This study aimed at investigating whether a porcine follicular fluid (pFF) supplementation positively affects the characteristics of donor cells and the developmental competence of porcine cloned embryos. Ear fibroblast cells (donor cell) from an Massachusetts General Hospital miniature pig were cultured in different culture methods: (1) Dulbecco's modified Eagle's medium (DMEM)+10% FBS (Control); (2) DMEM+0.5% FBS (SS); and (3) DMEM+10% FBS+10% pFF (pFF) for 72 h. In each conditioned medium, the concentrations of 4 amino acids (Thr, Glu, Pro, and Val) in the pFF group were significantly different from those in the control group (p<0.05 or p<0.01). The proliferation of the cells cultured in the SS group was significantly lower than that of the other treatment groups (p<0.01). The population of apoptotic and necrotic cells in the SS group was significantly higher than that of either the control or the pFF group (p<0.01). The number of embryos that cleaved (p<0.05) and developed into blastocysts (p<0.01) in the SS group was significantly lower than that of either the control or the pFF group. Compared to other groups, the blastocysts produced from the donor cells in the pFF group had higher total cells and lower apoptotic cells (p<0.05). It can be concluded that pFF supplementation in the donor cell culture medium positively affects cell death, cell cycle and quality of the cloned blastocyst.

• Toxicological Research
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• v.12 no.2
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• pp.203-211
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• 1996

Methanol has been widely used as an industrial solvent and environmental exposure to methanol would be expected to be increasing. In humans, methanol causes metabolic acidosis and damage to ocular system, and can lead to death in severe and untreated case. Clinical symptoms are attributed to accumulation of forrnic acid which is a metabolic product of methanol. In humans and primates, formic acid is accumulated after methanol intake but not in rodents due to the rapid metabolism of methanol. Neverthless, the developmental and reproductive toxicity were reported in rodents. Previous reports showed that perinatal exposure to ethanol produces a variety of damage in human central nervous system by direct neurotoxicity. This suggests that the mechanism of toxic symptoms by methanol in rodents might mimic that of ethanol in human. In the present study I hypothesized that methanol can also induce toxicity in neuronal cells. For the study, primary culture of rat hippocampal neurons and glias were empolyed. Hippocampal cells were prepared from the embryonic day-17 fetuses and maintained up to 7 days. Effect of methanol (10, 100, 500 and 1000 mM) on neurite outgrowth and cell viability was investigated at 0, 18 and 24 hours following methanol treatment. To study the changes in proliferation of glial cells, protein content was measured at 7 days. Neuronal cell viability in culture was not altered during 0-24 hours after methanol treatment. 10 and 100 mM methanol treatment significantly enhanced neurite outgrowth between 18-24 hours. 7-day exposure to 10 or 100 mM methanol significantly increased protein contents but that to 1000 mM methanol decreased in culture. In conclusion, methanol may have a variety of effects on growing and differentiation of neurons and glial cells in hippocampus. Treatment with low concentration of methanol caused that neurite outgrowth was enhanced during 18-24 hours and the numbers of glial cell were increased for 7 days. High concentration of methanol brought about decreased protein contents. At present, the mechanism responsible for the methanol- induced enhancement of neurite outgrowth is not clear. Further studies are required to delineate the mechanism possibly by employing molecular biological techniques.

• Development and Reproduction
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• v.14 no.1
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• pp.7-11
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• 2010

This study attempted to verify the possibility of using germ cell aspiration (GCA) method as a non-fatal technique in studying the life-history of equilateral venus, Gomphina veneriformis (Veneridae) and granular ark, Tegillarca granosa (Arcidae). Using twenty-six gauge 1/2" (12.7mm) needle, GCA was carried out in equilateral venus through external ligament. In granular ark, GCA was performed by preventing closure of the shells by inserting a tongue depressor between the shells while still open. The success rate of sex identification using the GCA method was 95.6% for the equilateral venus (n=650/680) and 94.3% for the granular ark (n=707/750). Mortality of equilateral venus, which spent 33 days under wild conditions, was 13.8% (n=90/650) while the mortality of granular ark, which spent 390 days under wild conditions, was 2.4% (n=17/707). Although we believe that GCA does not appear to cause death in equilateral venus or granular ark, the success rate in employing of this methodology may differ depending on the level of proficiency of the researcher and reproductive stage of the bivalve. This study concludes that GCA is a convenient non-fatal methodology, which can be employed to identify sex and investigate gonadal maturity in Gomphina veneriformis and Tegillarca granosa.

• 대한생식의학회:학술대회논문집
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• 2002.05a
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• pp.11-17
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• 2002

Animal clones derived from somatic cells have been successfully produced in a variety of mammalian species such as sheep, cattle, mice, goats, pigs, cat and rabbits. However, there are still many unsolved problems in the present cloning technology. Somatic cell nuclear transfer has shown several developmental aberrancies including high rate of abortion in early gestation and increased perinatal death. These developmental failures of cloned embryos may arise from abnormal reprogramming of donor genome and/or incomplete cloning procedure. We have found that overall genomic methylation status of cloned bovine embryos is quite different from that of normal embryos in various genomic regions, suggesting that the developmental failures of cloned embryos may be due to incomplete reprogramming of donor genomic DNA. Many of the advances in understanding the molecular events for reprogramming of donor genome will more clarify the developmental defects of cloned embryos.

• Development and Reproduction
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• v.5 no.2
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• pp.93-100
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• 2001

Precise mechanism by which the fetus can escape from mother's immune rejection is not well understood yet over the last 50 years. The clarification of immune mechanism at the feto-maternal interface is very important, because this can be a common pathogenesis of various pathologic conditions including spontaneous abortion, habitual abortion fetal growth restriction preeclampsia, implantation failure after assisted reproductive techniques, and fetal death. In this review, current hypothetical contents were described with the priority of importance: 1) The center of this mechanism is cross-talk between the expression of HLA-C, E, G on the extravillous cytotrophoblasts and their receptors on decidual NK cell, 2) immunomodulation, 3) innate immunity is the main immunologic mechanism, 4) various mechanisms besides HLA system(eq. complement) may be associated. The overall balance of immunomodulation among these mechanisms should result in the outcome of each pregnancy. Further researches regarding the regulation of HLA system, roles of cytokines, complements should be followed in the future.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.4
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• pp.311-317
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• 2019

Reproductive potential decreases with age. A decrease in male fertility is due to a combination of morphological and molecular alterations in the testes. Transient receptor potential vanilloid receptor-1 (TRPV1) is associated with aging and lifespan, and its activation causes apoptotic cell death in various cell types. However, the effect of TRPV1 on testicular apoptosis in aged mice has not yet been reported. TRPV1 knockout (KO) mice had a longer lifespan than that of wild-type (WT) mice. Lifespan was increased by 11.8% in male TRPV1 KO mice compared to that in WT mice. TRPV1 KO males lived approximately 100 days longer than WT males on average, and the maximum lifespan was markedly extended in TRPV1 KO mice compared with that in WT mice. The TRPV1 expression levels were highly increased in the testes of older mice. TRPV1 was expressed in the entire testes region of the old mice. In addition, old TRPV1 KO mice had lower testicular apoptosis than that of WT mice. Our results show that TRPV1 induces testicular apoptosis and suggest that TRPV1 may be associated with testicular aging.

• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.27-33
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• 1999

In the mammalian ovary, follicular development and atresia continuously occur during the reproductive cycles. Follicular atresia occurs through granulosa cell apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2 and bcl-xLong are known as inhibitors of apoptosis, whereas bax and bcl-xShort are known as inducer of apoptosis. We thought that bcl-2 protein is associated with follicular development and atresia. But it is not known that the distribution of cells containing bcl-2 protein during follicular development and atresia. Therefore, to examine the distribution of cells with bcl-2 protein during ovarian follicular development and atresia, the immunohistochemistry was used in the rat ovary. Bcl-2 immunoreactivity was localized in the interstitial cells, theca externa cells and granulosa cells around of antrum. All positive signals were observed in the cytoplasm of these cells. Positive signals were strongly observed in the interstitial and theca externa cells of growing antral follicles. While, positive signals were weakly observed in these cells from atretic antral follicles. Positive signals were very weakly observed in the granulosa cells of growing and atretic antral follicles. According to these data, we suggested that bcl-2 proteins which were strongly expressed in the interstitial cells and theca externa cells of growing antral follicles inhibit follicular atresia. And we purposed that bcl-2 proteins regulated follicular development and atresia through the action of bcl-2 gene family.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.267-271
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• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.257-262
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• 2009

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen-thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2-cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time-dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.