Mitochondria is energy generating organelle. It synthesizes ATP, which is the essential energy source of many cellular processes. During producing energy, some redox centres leak electrons to oxygen and it is contributory to the reactive oxygen species. Besides, mitochondria have significant functions in metabolism, calcium homeostasis, and fatty acid oxidation. Also mitochondria has importance to the breakdown of the ovarian follicles and could be factor determining oocyte of quality adversely. Increasing evidence shows that the number of mitochondria affect oocyte of developmental competence and maturation detrimentally during aging. Oocyte is the mitochondria-rich cell and enable the organelle to have competence for fertilization and early embryonic development. Occurrence of blastomere depends on distribution change of mitochondria which present in the egg. Lonicera caerulea treatment inhibited ovarian mitochondrial oxidative damage by suppressing mitochondrial reactive oxygen species (mROS) generation, decreasing apoptosis, controlling disintegration of mitochondrial membrane potential and conserving respiratory chain complex activities. The purpose of this study is to identify if mouse accepting treatment with L. caerulea could counter age-induced sterility and ovarian mitochondrial OS in a model organism of ovarian ageing.

Sex hormones including progesterons, androgens, and estrogens are influential in differentiation of ovarian tissues and competence of fertility. These steroid hormones derived from cholesterol are required for cumulus-oocyte complexes(COCs) during oocyte maturation. COCs is a total functional and active entity playing a central role in oocyte. Lipid metabolism in the mammalian COCs is controlled by environmental factors. The intracellular cholesterol contents go through remarkable changes. It plays an important part of oocyte developmental competence. However, heat stress affects steroid hormone by decreasing progesterone, estrogen concentrations, and resumption of meiosis in COCs maturation. Reduction of the hormone and meiotic resumption might lead to the decline of ovarian function, follicle maturation, and subsequent embryogenesis. In the same vein, heat stress also influence on germinal vesicle breakdown, lipolytic variations, and loss of the nuclear envelope in the course of maturation of oocytes. In summary, we examined the effects of thermal stress on oocyte maturation through steroid hormone contents of change identifying the molecular mechanisms of lipids metabolism. It may have the solution to further the therapy methods for disorders regarding sterility.

Mammalian oocytes are sensitive to psychological stress at each period of follicular development. Especially, thermal stress interfere with reproductive condition by inducing formation of reactive oxygen species (ROS) and oxidative stress (OS). ROS lead to oocyte apoptosis, weakening oocyte quality and lowering the fertilization rate. As a result, the pregnancy rate is lowered, leading to infertility. Thermal stress also seems to influence zygotes through physiological changes in the maternal environment surrounding them. Loss of developmental competence suggests hyperthermia-induced oxidative stress in embryos. Interest in organic Lonicera caerulea berries has increased in recent years. They are abundant in various health-improving materials. Berries that found from natural products can be as free as possible from the bioactive toxicity of the active ingredient without side effects, and it can be a big advantage because it can work. Mammalian oocytes are arrested at the first meiotic prophase stage and get their meiotic competence to produce offspring during the development of follicle. A series of nuclear and cytoplasmic maturations are involved in this process and these vary in temperature sensitivity. Our study demonstrated that L. caerulea can relieve the negative effects of maternal hyperthermia by reducing ROS level at the developmental stage.

Lonicera Caerulea(Honey berry) has been used in medical treatment in Russia, Japan, China and Korea. It has high level of vitamin C and polyphenolics. Polyphenolics can improve anti-inflammatory effect and prevent cancer, diabetes mellitus type 2. Also, Vitamin C is a representative anti- oxidant. however, It is still unknown what effect it will have on the oxidation stress of the reproductive system. In previous studies, ROS can be produced when it is exposed to heat stress and has negative effect on sperm's maturation, capacitation, hyperactivation, acrosome reaction and fusion of egg and sperm. Therefore, The purpose of this study is to investigate the antioxidant effects of L.caerulea on the sperm and egg cells of mice. At first, it conducted using ICR mouse(n=20) during 4 weeks. There are four groups of mouse(n=5 per group). Also, L.caerulea was taken by oral gavage. Group Ⅰ(control) kept at 23℃~27℃ and administer D.W(0.5ml/day), Likewise, Group Ⅱ(HB) kept at room temperature but gave HB(0.5ml/day), Group Ⅲ (HB+HS) received heat stress (40℃) using hyperthermia induction chamber and gave HB at same dose. and Group Ⅳ(HS) exposed heat stress only. Mainly, we showed degree of gene expression using Western blot in SOD, HSP 70, 17β-HSD and Real time PCR. It can find correlation between intracellular activity like steroid hormone, apoptosis under ROS and antioxidant activity of L.caerulea.

Previous studies have shown that Lonicera caerulea has a chemical protective effect. Phenolic and vitamin C contained in Lonicera caerulea prevent cancer, diabetes and cardiovascular disease, lower blood pressure and delay the aging process. However, the antioxidant mechanism of male reproductive system to heat stress is still unknown. Male reproductive system is very sensitive to heat. When scrotum temperature increase, oxidative stress can occur. Oxidative stress affects sperm motility and spermatogenesis, resulting in infertility. Therefore, we investigated the antioxidant effect of L. Caerulea in male genitalia by inducing oxidative stress by artificially exposing the testicles to heat at 42 ° C. The experiment was performed by dividing the ICR mouse into four groups. Each group is n = 5. Control group (C) and heat stress group (HS) were oral gavage administered D.W. Honey berry group (HB) and Honeyberry / heat stress group (HB + HS) were oral gavage administered honey berry (250mg / kg / day). HS groups (n=5, per n=5) received heat stress by exposing their lower bodies in the water bath at 42℃ for 30 minutes. We confirmed that there was a significant difference in the motility, morphology and the number of sperms using CASA(computer-assisted semen analysis). Lipid peroxidation assay results showed heat causes oxidative stress in serum. This study is conducting to investigate the antioxidant effect of L. Caerulea. Histologically analyzed the testicular form of each group, the activity level of heat shock protein and the level of reactive oxygen species were measured by Western blot and the level of catalase and HSP-90 was examined by RT-PCR analysis. Thus, studies of testicular morphology, sperm kinetics, hormone levels, heat shock protein expression and antioxidant enzymes under heat stress have shown that L. Caerulea ingestion has Anti-oxidant and thermal protective activity on the testis by heat damage.

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Recently, polymorphisms reported to be significant association with sperm MOT. This study was conducted to evaluate the SNP in the coding region of ESR1 (g.672C>T inexon 1) as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results,The g.672C>T was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP Also, the SNP was low significantly associated with ALH.Therefore, we suggest that theSNP in the coding region of ESR1 (g.672C>T in exon 1) may be used as a molecular marker for Duroc boar Post-thawed semen quality.

Estrogen receptor 2 (ESR2) is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 126 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.35547A>G) was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine ESR2 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the ESR2 in spermatogenesis within the reproductive tracts, and will shed light on ESR2 as a candidate gene in the selection of good sperm quality boars.

To test the muscle cell specific gene expression, we examined the ability of human α-skeletal muscle actin (ACTA) promoter or human myoglobin (hMb) promoter to direct the expression of the GFP gene in both muscle and non-muscle cells, respectively. C2C12 cells, a mouse myoblast cell line, provide a powerful model to study skeletal muscle differentiation in vitro. We intended to use this cell line as a model for skeletal muscle-specific gene expression during myogenic differentiation from myoblast to myotubes. We compared marker gene expression profiles of proliferating and differentiated C2C12 cells using RT-PCR and fluorescent microscopy analysis. Also, we found that the expression of PCK1 gene under the control of ACTA promoter was proportionally increased as C2C12 differentiated into myotube form. PCK1 is involved in the regulation of gluconeogenesis. In previous research, transgenic mice with overexpressing PCK1 in skeletal muscle showed a greatly enhanced level of physical activity, which extends well into old age. This is due, in part, to an increased number of mitochondria and a high concentration of triglyceride in their skeletal muscles. These mice also had very little body fat, despite eating 60% more than controls. We also constructed a mesenchymal stem cell line and fetal fibroblast cell line for the experiments aiming to make transgenic animals in which the PCK1 gene is specifically expressed in muscle tissue. Accumulated knowledge of this approach could be applicable to a variety of related biological areas including transgenic animal research, gene function study, anti-aging study, etc. This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Export Promotion Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (316002-5).

Variance of conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major conceptus protein that signals the process of maternal recognition of pregnancy in ruminants, essential for the maintenance of early pregnancy. Similar to other IFN genes such as IFNA and IFNB, multiple IFNT genes are present. However, some kinds of IFNT genes actively transcribed and regulated in bovine conceptuses have not been well characterized. In this study, during the course of bovine IFNT gene transcription through the use of next generation sequencer SOLiD3, revealed that among 38 IFN genes registered, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses during early pregnancy. Also, to identify a transcription factor(s) involved in the regulation of IFNT genes, mRNAs for various known transcription factors were investigated by real-time PCR in conceptus tissues, respectively. Furthermore, compared to the IFNT genes, IFNT1 and IFNTc1 had same active levels, which were previously shown to correlate with the appearance of effective antiviral activity. However, the expression levels of these Luc activities differed. Bovine ear fibroblast (EF) cells were cotransfected with luciferase reporter constructs carrying upstream (–631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1(JUN), ETS2 and/or cAMP-response element binding protein (CREB)-binding protein (CREBBP). CDX2, either alone with the other 2 transcription factors, was found to increase luciferase activity approximately 14- and 11-folds, respectively. The degree of transcriptional activation of the IFNTc1 gene was not similar to that IFNT1 gene by AP1, ETS2 or/and CREBBP, expression plasmid. These results suggest that two isoforms of bovine conceptus IFNT genes are regulated differently in conceptuses during early pregnancy.

Transglutaminase (TGM2) belongs to a family of cross-linking enzymes responsible for catalyzing Ca2+-dependent acyl-transfer reactions between the substrate proteins. TGM2 is a cytosolic protein that has also been observed in the nucleus and can be expressed to the cell surface or extracellular matrix. Despite ubiquitous expression, its functions are poorly understood and still need to be elucidated. Moreover, there is no clear data regarding the role of transglutaminase in mammalian oocytes. So, in this study, we have patterned the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity in heat stressed mouse oocytes. We have collected mouse oocytes from the (6–9 weeks old) mouse and in vitro matured for 20 h. Immunocytochemistry was performed to checked the transglutaminase 2 (TGM2) and anti-N epsilon gamma glutamyl lysine (AB424) activity after 6 h of heat stress (HS) at 39.1 ℃. Both TGM2 and AB424 expression were significantly (P < 0.05) higher compared to control when oocytes were subjected to HS at 6 h of IVM at 39.1 ℃. Our hypothesis is that TGM2 and AB424 activity may be correlated with the cellular regression and also involvement in apoptosis. We hope that, our study will help to elucidate the normal function of mouse oocyte and also identification of the principal proteins as well as the pathogenic mechanism of altered physiology. These results suggest that the nuclear accumulation of the transglutaminase may play an important role in nuclear remodeling during folliculogenesis and early embryonic development

Acute kidney injury (AKI) is a disorder that is manifested by a sudden decrease of renal function within several hours, and AKI remains a significant cause that can lead to increasing morbidity and mortality. Although AKI has been extensively studies in animal models, translating the results from animal studies into clinical use has not been successful due to various factors including basal etiology of kidney deficiency and comorbidities and the complexity of this pathology. As a golden parameter, measuring serum creatinine (SCr) and blood urea nitrogen (BUN) has been conventionally used for determining the renal function, however, these biomarkers has been regarded suboptimal to identify renal injuries in early stages. In this study, we attempted to screen other serum biomarkers in early AKI event using cynomolgus monkeys. Two male monkeys, aged 60 months, were subjected to ischemic injury by unilateral clamping of renal pedicles for forty five minutes and then subsequently reperfused; the unclamped kidney was regarded as non-injured controls. Compared with control kidneys, we have found that the concentration of several inflammatory proteins including MCP1, TGFα, GSTα, were higher in the renal vein of injured kidney compared with control side after 24 and 48 hours of AKI. However, changes of serum level of KIM-1, which is one of the most-widely studied marker in rodent studies, were not different after AKI. Our results provide an useful information while developing a novel marker in AKI.

The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each 12.58 ± 8.31 and 13.25 ± 7.86. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen (3.75 ± 1.98 vs. 8.23 ± 6.07, P<0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P<0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p<0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

Until recently, there have been many researches about the freezing methods and several methods of cryopreservation. Hypothermic preservation has been used to complement the embryo freezing technology. There is a study to show the successful results for long-term hypothermic preservation. For that reason, FBS and BSA are commonly added to the culture medium to support embryo development. We investigated the effectiveness of hypothermic preservation method at 4℃ according to embryonic developmental stages for Hanwoo embryos and evaluated the effect of FBS and BSA on Hanwoo embryos as a supplemental reagent in hypothermic preservation medium after recovering preserved embryos from hypothermic preservation. The present study reported that survival and hatching rates of embryos at morula stage following storage at 4℃ is Day 7 group was significantly higher (p < 0.05) compared than those of other groups (p < 0.05). As a result, the survival and hatching rates of embryos at the blastocyst stage following storage at 4℃ result is showed that significantly higher (p < 0.05) survival rates than those of other groups an Day 6. The result showed that hatching rate at Day 6 and 7 were significantly lower (p < 0.05) compared with other groups. The result regarding the survival and hatching rates of bovine embryos following storage at 4℃ for 72 h in various concentrations of BSA are shown The results showed that survival rate of 1% BSA group was not significantly different (p < 0.05) compare with control (FBS) group. Also, the results showed that hatching rate of control (FBS) and 1% BSA were significantly different (p < 0.05) compared with other groups. In conclusion, our result demonstrated that the hypothermic preservation did not effect on the survival and hatching rates of embryos after recovering. In addition, the supplementation of BSA in preservation medium showed no difference in the embryo developmental competence after hypothermic preservation compared to FBS treatment. With that, BSA can be an alternative reagent for the hypothermic preservation medium as an energy source and pH buffer.

Cryopreservation of bovine embryos is used to efficiently implant surrogate mothers. It has been widely accepted that high lipid content in the oocyte interrupts its survival during freeze-thaw cycles. Serum component in the culture medium is thought to increase the embryo`s lipid contents. Conversely, L-carnitine stimulates lipid metabolism by transporting long chain fatty acids into the mitochondria. Objective of this study was to analyze the effect of L-carnitine supplementation in IVM medium and defined IVC medium on the development, lipid contents and the cryosurvival of bovine IVF embryos. 0.0, 1.5, 3.0 and 6.0 mM L-carnitine was supplemented in IVM medium, respectively (IVM-LC 0.0, LC 1.5, LC 3.0 and LC 6.0). Development rate from the 2cell to the morula stages was higher in IVM-LC 3.0 groups than those of IVM-LC 6.0 (p<0.05). But there were no significant differences among the other groups in the blastocyst rates and lipid content results. When 0.0, 1.5, 3.0 and 6.0 mM L-carnitine were supplemented in IVC medium (IVC-LC 0.0, LC 1.5, LC 3.0 and LC 6.0), development competence was not significantly different between those embryos. Lipid contents of embryos treated L-carnitine (IVC-LC 1.5, 3.0 and 6.0) were significantly lower than embryos of non-treated group. L-carnitine was supplemented 0.0, 1.5, 3.0, 6.0 mM during IVM and 3.0 mM during IVC (LC 0.0 - 3.0, LC 1.5 – 3.0, LC 3.0 – 3.0, LC 6.0 – 3.0) and cryosurvival of blastocysts confirmed after freezing-thawing. There were no significant differences on development, but LC 3.0 – 3.0 was significantly lower lipid contents than other groups. And LC 3.0 – 3.0 had better survival rates and hatched rates of blastocysts than LC 0.0 – 0.0. In conclusion, supplementation of L-carnitine in defined IVC medium decreases lipid contents. And L-carnitine supplementation improves cryosurvival and developmental ability of bovine IVF embryos.