Partheno Embryo's research is known to play a very important role in identifying the development of embryonic cells or analyzing the genetic mechanisms of embryonic development, but the information on apoptosis formed during the early stage of development on Partheno Embryo is very little. Therefore, this study analyzed whether the embryonic cell death of unit embryos can be inhibited by adding Scriptaid, one of HDACi, which plays a role in demethylation of histone proteins as a method of regulating the cell cycle in the early embryo development of Partheno Embryo. As a result, the differentiation rate was higher in the group that added Scriptaid and FBS, but the cellular development was higher in the group that added pregnant serum to Scriptaid. As a result of analyzing the expression of the gene through IF and PCR, the group with the addition of gestational serum increased the expression of BCL2 and PCNA, which affects the anti-Casp3 action in cell survival. In addition, it is interpreted that treatment of Scriptaid for 16 hours, rather than 24 h treatment lowers the expression of Casp-3, a representative factor of apoptosis, and also increases embryonic development, thus affecting early embryo development. Therefore, it is concluded that the 16-hour treatment of Scriptaid and the use of gestational serum will inhibit cell death in the early embryonic development and increase the development rate of the embryo.

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

In this study, we investigated whether infusion of colorectal cancer cell line and PMSG could increase endometrial cancer. As a result, our study confirmed that the injection of colorectal cancer can cause inflammation and cancer in the uterus and increase the VEGF gene in the uterus. The study also found that endometrial cancer was associated with PMSG.

Our study has analyzed whether inappropriate gonadotropin secretion affects the morphological changes due to the activation of intrauterine MMP. Methods A total of each 6 mice were injected with PMSG, Progesterone, and Androgen in 5 IU of intraperitoneal injection every 2 days after estrus synchronization, and morphological and MMPs expression patterns were compared after inducing hormone secretion. Also, cell survival and death related genes were compared and analyzed. The endometrium was highly developed in the PMSG, and the androgen was not developed at all. In particular, the diameter of the uterus of the Androgen group was also very narrow. MMPs activity assay in the case of PMSG was confirmed that showed low activity, whereas, progesterone and androgen In showed high activity and, in particular, very high activity of MMPs in the case of androgen in glandular cell. The expression of VEGF in the tissues of each group was different from that of MMPs. In the PMSG group, the activity of VEGF was increased in both the Myo-metrium and the endo-metrium, whereas the progesterone group showed low overall expression in the endo-metrium. Therefore, the present study showed that the activities of the endo-metrial cells and the restructuring of the endometrial cells differed according to the type of the abnormal secretory hormone. In particular, the secretion of androgen increased the activity of MMPs throughout the uterus, The endo-metrial epithelial cells are affected by the progesterone group. In conclusion, this study suggests that inappropriate gonadotropin secretion increases the functional changes of the uterus and this reconstruction may be caused by increased activity of MMPs

The expression of MMPs in the development of the fertilized egg has a very important role in cell configuration. Objective To evaluate the clinical, the effect of differentially expressed MMPs on serum and serum - free medium on the maturation of blastocysts. The expression patterns of MMPs in serum and serum-free medium were compared at 6 h, 18 h and at the blastocyst stage using real-time PCR, ELISA and immunofluorescence. The results showed that the expression of MMPs was increased in the embryos of the serum medium, as a result of analysis of MMPs and TIMPs, MMP-2 was expressed in the cytoplasm of embryos in the serum-free medium, And it was found to be higher in expression than MMP-9. The serum medium was different from the bloodless badge: overall, TIMPs showed a higher expression in the ovarian cells than cyanosis, and TIMP-3 was more pronounced. Development rate of blastocyst according to in vitro culture method was higher than that of serum - free medium (61.22% 60/98) and serum - free medium (48.28% 28/58). Analysis of the protein release locations of MMPs and TIMPs showed that MMPs and TIMPs are highly expressed in serum mediums, focusing on the inner cell mass. However, very low expression appeared in the tropoblast. On the other hand, serum - free medium showed different expression from serum medium and TIMPs expression was generally low. Therefore, in the case of serum media, the expression of MMPs is highly expressed in the cytoplasm of the fertilized egg, increasing the reconstruction of cells.

In the present study of this experiment was to understand the expression of apoptotic gene expression in the ovary of miniature pigs and pigs on the 15th day of estrus. Also the compare and analyze of programmed cell death type(Apoptosis and autophagy) expression pattern during mature oocyte on the miniature and normal pig cells. Analysis of mRNA gene expression of ovary in miniature and normal pigs on the 15th day of estrus showed that the expression of genes related to Autophagy (ATG13, MAP1LC3, Beclin1) was high in normal pigs but the expression of ATG1 and ATG5 genes was low. In addition, the expression of genes related to apoptosis (Casp-3, BAX) was high in the mini pigs, and the gene related to the LH hormone was high in the miniature pigs, whereas the expression of the gene related to the FSH hormone was high in the normal pigs. On the other hand, the result of muture oocyte on the miniature and normal pig cells is the expression of Casp-3 protein was moust high from treatment of FL+rapa (FSH+LH and Rapamycin) of the oocyte on the miniature pig cell. However, MAP1LC3 expression was higher in the oocytes of treatment of rapanycine treatment on the nomal pig cells. There was no gene expression in cumulus cells of matured oocytes in mini pig cells, whereas MAP1LC3 expression was higher in oocyte cumulus cells matured in normal pig cells. It was confirmed that the miniature and normal pigs showed different programmed cell death patterns, In the case of oocytes matured in miniature pig cells, MAP1LC3 gene expression was found to be low in spite of treatment with Autophagy regulator.

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for 30 μsec. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in 5% CO₂ and 95% air at 39℃ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; 64.9±4.8% in 1.1 kV/cm and 62.7±4.0% in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was 12.6±2.5, 20.0±5.0, and 34.9±4.3% in control, 2 mM 6-DMAP, and 7.5 μg/ml cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for 30 μsec) and 7.5 μg/ml cytochalasin B treatment was effective for production of the porcine SCNT embryos.

The purpose of this study is to expression pattern of melanogenesis associate genes on cultured melanocyte layer cells in Korean Brindle Cattle(Dark, Brindle and Yellow) were analyzed to evaluate the effects of sex hormones on the control of melanogenesis pathways. Korean Brindle Cattle(Dark, Brindle and Yellow) melanocyte in the skin cells was collected. after the addition of estrogen and testosterone, the culture was analyzed for expression of cell activity and melanin genes for 72 hours. For the analysis of estrogen in different coat color other than the melanogenesis-related genes it is increasingly yellow showed low expression. in particular, the cells of the brindle coat color is low active and expression of genes. However, the testosterone was low, the expression of cell activity inhibiting MMP-2. the expression of melanin genes actually showed a tendency to increase gradually, which is testosterone compared with the estrogen to be considered that affect the skin cell layer brindle coat color. In this study, stimulation with estrogen triggered the inhibition of MC1R of the melanocyte in brindle coat color, but testosterone is induced MC1R in melanocyte. Therefore, considered the eumelanin or phaeomelanin activation are controlled caused by differential expression of sex hormones on melanocyte in Korean Brindle Cattle.

ompared the expression of MMPs in these oocytes and cumulus cell throughout oocytes maturated. In an attempt to investigate the effect of MMP activation and inhibitors in total protein of cumulus cell and, oocytes during oocytes maturation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), TIMPs (TIMP-2 and TIMP-3), as well as their expression profiles (Real-time PCR, Gelatin Zymography and ELISA). Our results that the bovine oocytes MMP-2 and MMP-9 level was significantly associated with the rate of maturity of oocytes (P<0.05). In cumulus cell, MMP-2 was highly expressed in all stages of the oocyte’s maturation. The final oocytes maturation exhibited strong gelatinase activity. There was no significant correlation between cumulus cell MMP-9 and the maturation rate of oocytes. However, for the oocyte cytoplasm MMP-9 expression was significant correlation to the maturation oocytes. There was no significant correlation between cumulonimbus cells MMP-9 and oocyte maturation rates; however, for oocyte cytoplasm, MMP-9 expression was significantly correlated with mature oocyte. However, the TIMP-1 and TIMP-2 protein expression patterns are not correlated with the maturation rate of the oocyte. Our results suggest that MMP different expression pattern may regulate the morphological remodeling of oocyte's in the cumulus cell. Further, the MMP-2 expression has a strong relation with a higher maturation rate of the oocyte.

The major focus of this study is to analyze the expression of bovine MMPs and to monitor their activity during the estrus cycle and pregnancy. During pregnancy, MMP-2 expression was detectable around 30 days but became insignificant by 60 days, then started to increase again around 90 days and reached the maximum at 250 days. The activity of MMP-2 protein changed in accordance with its expression level. As expected, the level of TIMP-2 exhibited a reverse pattern. About MMP-9, high level expression was observed as early as 30 days and gradually increase until 90 days. Then started to decrease after 250 days. Again, the sites of MMP-9 expression were similar to those of MMP-2. On the other hand, expression of TIMP-3 remained low until 90 days but showed a small and temporal increase around 250 days. In summary, expression of different MMPs were differentially regulated during estrus cycle and pregnancy. While the expression of MMP-2 was high in estrus cycle, MMP-9 slowly takes over with the progression of pregnancy. These results indicated that the luteal tissue perform distinct functions during pregnancy and estrus. Perhaps the activity of MMP-2 is required for the structural remodeling of luteum, resulting the suppression of P4 inflow from blood. On the other hand, steady maintenance of MMP-9 throughout luteal development is important for the activation of cell proliferation, maturation and angiogenesis.

The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

The coat color of mammals is determined by the melanogenesis pathway, which is responsible for maintaining the balance between black-brown eumelanin and yellow-reddish pheomelanin. The melanogenesis-associated genes controlling pigmentation act as a complex and interact with each other to cause phenotypic and genotypic variations in cattle. That the MC1R genotype of Korean native cattle with dark muzzle was e/e or E+/e, while the genotype of Korean native cattle with light muzzle was E+/E+, which is a variant of the MC1R genotype in the Korean native cattle. Especially, the MC1R expression type is shows how much pigmentation, important factor in deciding its status in the coat and nose colours. However, information regarding the coat or nose colours-associated gene regulation of korean cattle is not yet unknown. Therefore, in this study was to investigate the expression patterns of melanogenesis-associated genes in black dot nose(korea brindle cattle) and normal nose(korea native cattle). Using microarray clustering and real-time polymerase chain reaction techniques, we analysed that the expression of genes involved in the mitogen-activated protein kinase (MAPK) and Wnt signaling pathways is distinctively regulated in the dark and light muzzle tissues. Differential expression of tyrosinase was also noticed, although the difference was not as distinct as those of MAPK and Wnt. We hypothesize that emphasis on the MAPK pathway in the Korea brindle cattle induces eumelanin synthesis through the activation of cAMP response elementbinding protein and tyrosinase, while activation of Wnt signaling counteracts this process and raises the amount of pheomelanin in the native cattle. Regarding the increasing interest in the genetic diversity of cattle stocks, genes we identified for differential expression in the brindle cattle vs. native cattle may serve as novel markers for genetic diversity among cows based on the coat and muzzle color phenotype.

In all mammalian species, progesterone is essential to both the preparation for, and maintenance of, pregnancy. The 20α-hydroxysteroid dehydrogenase (20α-HSD) enzyme predominantly converts progesterone into its biologically inactive form 20α-hydroxyprogesterone, thereby regulating its activity. Thus, to directly assess sexual maturation in the MediKinetics micropig®, we analyzed the concentration of the steroid hormones progesterone and estradiol during the estrous cycle.Our results show that the progesterone level exhibited by the analyzed micorpig® was low at the beginning of the estrous cycle, and then abruptly increased to 30.32±10.0 ng/mL and 46.37±11.0 ng/mL by days 9 and 11 of the cycle, respectively. It reached the highest level 55.87±3.5 ng/mL on day 13 of the estrous cycle, before decreasing to 46.58±13.1 ng/mL and 10.0±7.6 ng/mL by days 15 and 17 of the cycle, respectively. In contrast, the estradiol level was shown to be highest (27.13±11.2 ng/mL) at the initiation of the estrous cycle, after which point it decreased to 13.29±6.5 ng/mL and 10.94±5.9 ng/mL by days 4 and 5 of the estrous cycle, respectively. By day 17 of the estrous cycle, the estradiol level decreased to 4.13±7.6 ng/mL.We anticipate that these results will provide useful information to enable the study of human ovulation and reproductive physiology using the MediKinetics micoripig® as a model system. We recommend further investigation to elucidate the functional mechanisms underlying the regulation of sexual maturation in the MediKinetics micropig®.

Recently, the consumption of chickens meat has been gradually increased in Korea. However, most of the chickens breeds in Korea were imported from overseas. This study was conducted to evaluate the genetic distances and single nucleotide polymorphism by the mtDNA D-loop control analysis method for the Korean native chickens(red plumage, red-line plumage, Ogol) and white leghorn. For the initial investigation of the relationships between Korean native chickens(red plumage(KCR), red-line plumage(KCRD), Ogol(KCO)) with white leghorn breeds, the sequences from D-loop control region in mitochondrial DNA (mtDNA) was used. The results from phylogenetic analysis indicated that both KNC and white leghorn breeds were classified well with wild duck breeds. However, KCR was not discriminated well with KCRD. The haplotype analysis indicated that KCR and KCO have ten different haplotypes with nineteen SNPs. Three haplotypes (haplotype 1, 2, 3, 5, 17) were shared both in KNC(KCR, KCRD) and KCO. On the other hand, haplotype 4, 6, 7 was appeared only KCR and haplotype 13, 16, 18 were identified only in KCRD population. In addition, haplotype 8, 9, 11, 12, 14, 15, 19 was appeared only KCO. With further verifications, the results presented here can be used for the on servation and commercialization of the Korean native chickens. This work was supported by a grant from the “Livestock Preservation of Genetic Resources", Rural Development Administration, Republic of Korea.

The nature of molecular mechanisms governing embryonic cell block is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to determine effects of programmed cell death on porcine oocytes development in vitro after parthenogenesis. Among the blastocysts matured in 3MA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of Cyst < 3MA < RP. However, Casp-3 and TNF-r RNA gene expression level decreased in the order of RP < 3MA < Cyst. Expression of mTOR within the RP-cultured blastocyst was the most highly to the inner cell mass, while 3MA-cultured blastocyst showed very lowest expression in inner cell mass. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. When the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each RP treatment group, with the level of another treatment group being relatively higher. Analyses of TIMP-2 and TIMP-3 revealed that their expression was higher in groups that did not receive RP treatment. More specifically, the level of TIMP-2 was not affected by Cyst treatment, while the level of TIMP-3 was higher in 3MA and RP treatment group. There was highly cell division activation efficiency of parthenogenesis on cultured system of RP supplement IVC medium. Therefore, these results suggest that embryo development was significantly increased in conditional culture medium with active autophagy as compared to common cultured condition. Further investigation of this distinction may enable the development of innovative improvements for the production of porcine somatic cell nuclear transfer.

The main purpose of this study is to estimate the effect of adding Tea-N-Tris (TES) to the freezing buffer for miniature pig sperm. In particular, we attempted to identify the association between the MMPs expression and the fertility and viability of frozen sperm from each extender (LEY (Lactose Egg-Yolk), TLE (TES + LEY), TFGE (TES + Fructose + Glucose Egg-Yolk)). In accordance with this, Hypoosmotic Swelling Test (HOST) respond test was the lowest among sperms frozen in LEY while the highest HOST respond was observed among sperms frozen in TLE. Furthermore, we observed MMPs expression in all sperm groups, with pro-MMP showing lower expression than active MMPs. The expression of MMP-9 and MMP-2 was the highest in sperms frozen in LEY, Meanwhile, sperms from the TFGE and TLE group showed lower level of MMP-9 and MMP-2 expression in the order of TLE being the lowest. LEY group showed lower rate of blastocyst development than the TES supplement group, although the difference was not statistically significant. Meanwhile the rate of blastocyst development appeared similar when sperms from TLE and TFGE group were used for IVF. Together, these results indicate that adding Tea-N-Tris to the sperm freezing buffer only suppresses MMPs protein activation but also maximize in-vitro fertility, providing a means to improve the success rate in the in vitro manipulation of miniature pig sperm.

To describe the macroscopic anatomy and ovarian-physiological difference of the genital organs of the female Korean water deer, organs from captured animals in a wild area of Korea were dissected. The ovary of estrus group was about 1.10 ± 0.02 mm long and weighed about 0.50 ± 0.02 g. And pregnant group was about 1.3 ± 0.10 mm long and weighed about 0.40 ± 0.05 g. And the crowns of corpora lutea were found in the estrus group, but we couldn't find crowns at the pregnant group. Especially, the estrus ovaries tended (p=0.04) to be heavier than the ovaries during pregnancy. The MMP-9 activity was higher at the Graafian follicles of pregnant group than that in estrus group. However, with regard to follicles of estrus group, MMP-2 level was higher than that in pregnant group. Furthermore, apoptosis detection marker (Casp-3) was highly expressed in Graafian follicle of the pregnant group and the corpora lutea of estrus group. Thus, the differential expression of MMPs observed in this study suggests that the reflected the mechanisms underlying of monovulatory in estrus and/or pregnancy. Our results may be very useful as it provides with information that may be considered for the development of reproductive biotechnologies in endangered animals.

Bovine coat color is decided by the melanocortin receptor 1 (MC1R) genotype mutation and melanogenesis. Specially, in the various cattle breeds, dominant black coat color is expressed by dominant genotype of ED, red or brown is expressed in the frame shift mutation of recessive homozygous e by base pair deletion and wild type of E+ is expressed in various coat colors. However, not very well known about the effected of MC1R genotype mutation on the coat color through family lines in KBC. Therefore, this study were to investigate effect of MC1R genotype mutation on the coat color, and to suggest mating breed system in accordance with of MC1R genotype for increased on brindle coat color appearance. Parents (sire 2 heads and dam 3 heads) and offspring (total : 54 heads) from crossbreeding in KBC family line with the MC1R genotype and phenotype records were selected as experimental animals. The relationship between melanocortin 1 receptor (MC1R) genotypes expression verified by PCR-RFLP, and brindle coat color appearance to the family line of the cross mating breed from MC1R genotype pattern was determined. As a result, 4MC1R genetic variations, E+/E+ (sire 1), E+/e (sire 2 and dam 3), E+/e with 4 bands of 174, 207 and 328 bp (dam 1) and E+/e with 3 bands of 174, 207, 328 and 535 bp (dam 2) from parents (sire and dam) of KBC. However, 3 genetic variations, e/e (24%), E+/E+ (22%) and E+/e (56%) were identified in offspring. Also, brindle coat color expressrated was the e/e with the 0%, E+/E+ with 67% and E+/e with 77% from MC1R genotype in offspring on the cross mating of KBC. Furthermore, when the sire had E+/e genotype and the dam had E+/E+ with the 3 bands or E+/e genotype, and both had whole body-brindle coat color, 62% of the offspring had whole body-brindle coat color. Therefore, the seresults, the mating system from MC1R genotype patterns of the sires (E+/e) and dams (E+/E+ with the 3 bands or E+/e) with brindle coat color may have the highest whole body-brindle coat color expression in their offspring.

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.