The structural diversity and localization of cell surface glycosphingolipids (GSLs), including gangliosides, in glycolipid-enriched microdomains (GEMs) render them ideally suited to play important roles in mediating cell recognition, adhesion, interactions, receptor function, and signaling. Gangliosides, sialic acid-containing GSLs, are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and these changes are mainly regulated through stage-specific expression of glycosyltransferase genes. However, roles of gangliosides in neuronal differentiation of mesenchymal stem cells (MSCs) is unclear. We previously demonstrated for the first time that the glycosyltransferase genes during mouse embryogenesis. So, we investigated the effects of ganglioside gene in differentiation of adipose-derived MSCs (AD-MSCs). GM2 and GD3 ganglioside synthease were increased during neuronal differentiation of AD-MSCs. This study showed that the differentiation of neuronal marker was decreased on the first step of ganglioside synthase UDP-glucose ceramide glucosyltransferase(UGCG) and knock downed GM2 sythase (B4GALNT1). The result of suggested that GM2 and GD3 might be important roles in the neural differentiation of mini-pig AD-MSCs. This work was carried out with the funding of the cooperative research Program for Agriculture Science & Technology Development[Project No. PJ00999901], the Rural Development Adiministration, the KRIBB Research Initiative Program[KGM4251622].

Flavonoids have a range of biological activities, including anti-allergic, anti-inflammatory, anti-microbial, and anti-cancer activities, as demonstrated by in vitro studies. In this study, we investigated whether luteolin can be applied to suppression of lipopolysaccharide (LPS)-stimulated inflammatory responses in murine macrophages. Luteolin was found to reduce nitric oxide (NO) production in LPS-stimulated Raw 264.7 cells. In addition, expression of inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokine tumor necrotic factor-α (TNF-α) at the mRNA and protein levels were decreased. These inhibitory effects were found to be caused by the blockade of nuclear factor kappa-light- chain-enhancer of activated B cells (NF-κB) activation and phosphorylation of mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAP kinase. In addition, pre-treatment with luteolin resulted in reduced ganglioside expression levels and inhibited expression of GT1b in Raw 264.7 cells. On the basis of these observations, we suggest that luteolin has potential as an anti-inflammatory drug candidate, and ganglioside GT1b may play a role in the inflammatory process.

Germ cell-specific hyaluronidases such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5) are in part responsible for dispersal of the cumulus cell mass, which is a critical step in establishing fertilization in mammals. In this study, we identified two testis-hyaluronidases, SPAM1 and Hyal5, in hamster and rat. These two genes were expressed specifically in the testis. At the protein level, hamster SPAM1 and Hyal5 display 78.7% and 75.4% identity with mouse SPAM1 and Hyal5. Further, the activity of the enzymes with respect to cumulus cell dispersion did not differ, although we observed that the enzymatic activity differed in pH range. These studies suggest that different sperm hyaluronidases are capable of dispersing the cumulus cell mass despite differences in enzyme activity.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes. However, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism (s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress inearly embryos. Collectively, these results suggest that early embryo genesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

Autophagy is known to be involved in a variety of biological processes. However, relatively a little is known regarding oocyte maturation and preimplantation development in mammals. Thus, the current study was conducted to investigate the role of autophagy in oocyte maturation and subsequent preimplantation development in pigs. Porcine oocytes were matured in the presence or absence of 1 μM rapamycin, an autophagy inducing agent, fertilized in vitro, and cultured to blastocyst stage. From Western blotting analysis, we found that active form LC3 was detected during in vitro maturation (IVM) period, suggesting the possible role of autophagy in oocyte maturation. Interestingly, treatment of rapamycin during IVM significantly increased nuclear maturation compared to control group. Importantly, rapamycin-assisted IVM greatly improved monospermic fertilization and blastocyst development rates compared to control embryos. In addition, we also found that cell number and blastomere survival in blastocysts were markedly increased in rapamycin treatment group, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Collectively, these results strongly suggest that induction of autophagy may contribute to the completion of nuclear and cytoplasmic maturation of porcine oocytes.

Interferon-tau (IFNT) is regarded, generally, to be the conceptus protein that signals maternal recognition of pregnancy in ruminants. Although the discovery was made over two decades ago, the molecular mechanisms that regulate IFNT expression are not well understood. Previous studies demonstrated that transcription factors, caudal-related homeobox- 2 (CDX2), JUN, ETS2 and a transcriptional coactivator CREB binding protein (CREBBP) positively influenced IFNT gene expression, while OCT4 may exhibit negative regulation. We and others have observed that both CDX2 and OCT4 coexist during early stages of conceptus elongation but as development proceeds, OCT4 expression diminishes. The objective of this study was to evaluate the stimulatory and inhibitory effects of CDX2 and OCT4, respectively, on IFNT gene transcription when evaluated with other transcription factors. Human choriocarcinoma JEG3 cells were co-transfected with an ovine IFNT (-654 base pair)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. When the reporter construct was co-transfected with either CDX2, ETS2 or CJUN, transcription of -654-oIFNT-Luc increased about two-fold compared to -654-oIFNT-Luc alone. When -654-oIFNT-Luc was co-transfected with both c-jun and Ets-2, activity of -654-oIFNT-Luc was increased about four-fold; cotransfection with JUN, ETS2 and CDX2 increased -654-oIFNT-Luc expression 12X, indicating that the stimulatory activity of the transcription factors was additive. OCT4, when cotransfected with -654-oIFNT-Luc, reduced expression of the later about 40% when compared to -654-oIFNT-Luc alone. When co-transfected with JUN and/or ETS2, OCT4 abolished the stimulatory effect of these transcription factors. OCT4 also inhibited the stimulatory activity of CDX2 alone, but not when CDX2 was combined with JUN and/or ETS2. Therefore, when combined with the other transcription factors, CDX2 over the transcriptional inhibitory activity of OCT4. Conversely, when cells were transfected initially with OCT4 (0h) followed by transfection with CDX2, ETS2 and JUN 24 h later, -654-oIFNT-Luc expression was reduced to control (-654-oIFNT-Luc, alone) levels. Not surprisingly, 12S E1A, an inhibitor of transcriptional coactivator CREBBP, reduced stimulation of -654- oIFNT-Luc expression by CDX2, ETS2 and JUN, in combination, by about 40%, indicating that proper transcription complex formation is required for maximum expression. In conclusion, it is suggested that prior to conceptus elongation, pre-existing OCT4 may inhibit IFNT expression, but as elongation proceeds, IFNT expression increases, resulting from incremental increases in CDX2 expression, diminished OCT4 expression, and possibly proper transcription factor complex formation. Key words) Interferon-tau, CDX2, OCT4, transcription

During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.

Despite of the presence of estradiol-17β (E2) in ovarian follicles, its role(s) in in vitro maturation (IVM) is still largely unknown, especially in pigs. Thus, the current study was conducted to investigate the effect of E2 on in vitro maturation (IVM) of porcine oocytes and subsequent preimplantation development using in vitro fertilization (IVF)- or somatic cell nuclear transfer (SCNT)-derived embryos. To define the effects of E2 on IVM and early embryogenesis, porcine oocytes were matured in the presence or absence of E2, fertilized in vitro and cultivated to blastocyst stage. Compared to control group, the production of MII oocytes was significantly increased by treatment with E2, accompanying with the increase in MPF content and ERK phosphorylation, and monospermic fertilization and blastocyst development rates were also greatly elevated in the E2-treated oocytes. In addition, the advantageous role of E2 was also found in blastomere survival, which was further evidenced by both elevation of anti-apoptotic transcript Bcl-XL and decrease of pro-apoptotic transcript Bax. Furthremore, these positive effects of E2 were highly reproducible in early development of SCNT embryos. Collectively, the current study strongly suggests that E2 can be used as a efficient IVM supplement leading to successful nuclear/cytoplasmic maturatioin in pigs.

Successful early embryogenesis of somatic cell nuclear transfer (SCNT) embryos is very important to produce cloned animals. However, poor preimplantation development of SCNT embryos has been a major obstacle to the generation of cloned animals due to a lack of understanding of developmental events and underlying mechanism(s). In the current study, we show that production of SCNT embryos with high developmental competence is dependent on the fusion method. Electrofusion causes spontaneous egg activation, accompanied by an increase in intracellular Ca2+ and improper nuclear remodeling, whereas Sendai virus (SV)-mediated fusion greatly reduces these events. In addition, SV-SCNT increased the blastocyst development rate and trophectoderm cell number compared to electrofusion-mediated SCNT (E-SCNT). In particular, expression of ER stress-associated genes and blastomere apoptosis were significantly increased in E-SCNT embryos, which could be alleviated by inhibition of ER stress or by using the SV-mediated fusion method. Taken together, these results strongly suggest that SV is a useful fusion material for improvement of preimplantation development of SCNT embryos through reduction of ER stress-associated apoptosis.

Acute ischemic stroke results from sudden decrease or loss of blood supply to an area of the brain, resulting in a coinciding loss of neurological function. The antioxidant action of melatonin is an important mechanism among its known effects to protective activity during ischemic/reperfusion injury. The focus of this research, therapeutic efficacy of melatonin on recovery of neurological function following long term treatment in ischemic brain injured rats. Male Sprague-Dawley rats (n=40; 8 weeks old) were divided into the control group, and MCAo groups (Vehicle, MT7 : MCAo+ melatonin injection at 7:00, MT19 : MCAo+melatonin injection at 19:00, and MT7,19 : MCAo+melatonin injection at 7:00 and 19:00). Rat body weight and neurological function were measured every week for 8 weeks. After 8 weeks, the rats were anesthetized with a mixture of zoletil (40 mg/kg) and xylazine (10 mg/kg) and sacrificed for further analysis. Tissues were then collected for RNA isolation from brain tissue. Also, brain tissues were analyzed by histological procedures. We elucidated that melatonin was not toxic in vital organs. MT7,19 was the most rapidly got back to mild symptom on test of neurological parameter. Also, exogenous melatonin induces both the down-regulation of detrimental genes, such as NOSs and the up-regulation of beneficial gene, including BDNF during long term administration after focal cerebral ischemia. Melatonin treatment reduced the loss of primary motor cortex. Therefore, we suggest that melatonin could be act as prophylactic as well as therapeutic agent for neurorehabilitative intervention.

Clinical arthritis is typically divided into rheumatoid arthritis (RA) and osteoarthritis (OA). Arthritis-induced muscle weakness is a major problem in aged people, leading to a disturbance of balance during the gait cycle and frequent falls. The purposes of the present study were to confirm fiber type-dependent expression of muscle atrophy markers induced by arthritis and to identify the relationship between clinical signs and expression of muscle atrophy markers. Mice were divided into four experimental groups as follows: (1) negative control (normal), (2) positive control (CFA+acetic acid), (3) RA group (CFA+acetic acid+type Ⅱ collagen), and (4) aging-induced OA group. DBA/1J mice (8 weeks of age) were injected with collagen (50 μg/kg), and physiological (body weight) and pathological (arthritis score and paw thickness) parameters were measured once per week. The gastrocnemius muscle from animals in each group was removed, and the expression of muscle atrophy markers (MAFbx and MuRF1) and myosin heavy chain isoforms were analyzed by reverse transcription-polymerase chain reaction. No significant change in body weight occurred between control groups and collagen-induced RA mice at week 10. However, bovine type Ⅱ collagen induced a dramatic increase in clinical score or paw thickness at week 10 (p<0.01). Concomitantly, the expression of the muscle atrophy marker MAFbx was upregulated in the RA and OA groups (p<0.01). A dramatic reduction in myosin heavy chain (MHC)-Iβ was seen in the gastrocnemius muscles from RA and OA mice, while only a slight decrease in MHC-Ⅱb was seen. These results suggest that muscle atrophy gene expression occurred in a fiber type-specific manner in both RA- and OA-induced mice. The present study suggests evidence regarding why different therapeutic interventions are required between RA and OA.

Arthritis is a common disease in aged people, and is clinically divided into rheumatoid arthritis (RA) and osteoarthritis (OA). Although common symptoms such as pain are present, the underlying pathological mechanisms are slightly different. Therefore, the objectives of the present study were to compare joint damage induced by RA and OA by analyzing the major morphological and molecular differences, and to propose a suitable therapeutic intervention based on the pathophysiological conditions of bones and joints. For the RA animal model, 8-week-old DBA1/J mice were immunized with bovine type II collagen emulsified in complete Freund’s adjuvant (CFA). Normal C57BL/6 mice (over 2 years of age) were used for OA. The clinical arthritis score was calculated using a subjective scoring system, and paw thicknesses were measured using calipers. The serum TNFα level was analyzed using an ELISA kit. Micro- CT was used to identify pathological characteristics and morphological changes. In collagen-induced RA mice, there were increased ankle joint volumes and clinical scores (p<0.01). The concentration of TNFα was significantly increased from 3 to 7 weeks after immunization. Micro-CT images showed trabecular bone destruction, pannus formation, and subchondral region destruction in RA mice. OA among aged mice showed narrowed joint spaces and breakdown of articular cartilage. This study suggests that a careful therapeutic intervention between RA and OA is required, and it should be based on morphological alteration of bone and joint.