Development of osteoporosis animal model using micropigs.

Osteoporosis is a known major health problem and a serious disease of the bone, there has been a great need to develop more and newer animal models for this disease. Among animal models used for testing drug efficacy, the minipig model has become useful and effective due to its close similarity with humans (validity), particularly with the pharmacokinetics of compounds via subcutaneous administration, the structure and function of the organs, the morphology of bone and the overall metabolic nature. Based on these advantages, we sought to develop a new animal model of osteoporosis using micropig, which differs from other miniature pigs in the genetic background. Female micropigs were used for the induction of a moderate osteoporosis model by bilateral ovariectomy (OVX) and compared with shamoperated animals. For osteoporosis evaluation, clinical biomarkers such as blood osteocalcin (OSC) and parathyroid hormone (PTH) levels were measured, as well as bone mineral density (BMD) using micro-computed tomography (micro-CT). Compared to sham, OVX animals have decreased blood OSC level, while the blood PTH level increased in blood sera. In addition, we observed the significantly decreased BMDs of tibia region in OVX animals. Based on these results, we report that the micropig model developed in this study can be used to develop a new and effective medical method for diagnosis and treatment of osteoporosis.

Metformin enhances the action of insulin on porcine granulosa-lutein cells in vitro.

Metformin is an oral antidiabetic drug extensively used to treat the polycystic ovary syndrome in women. Metformin increases insulin-stimulated glucose uptake and has direct effects on ovarian steroidogenesis in humans. However, the molecular mechanisms of metformin' action on the ovary are not clear. To investigate the effects of this drug on the insulin-signaling pathway in porcine granulosa cells as an alternative model for human research, we examined the mRNA expressions of porcine insulin receptor (INSR), insulin-like growth factor-1 receptor (IGF-1R), insulin receptor substrate-1 (IRS-1), and the protein activity (activation and phosphorylation) of downstream targets including Raf, mitogen-activated protein kinase (MEK)1/2, extracellular signal regulated kinase (ERK), phosphoinositide-dependent 1 kinase (PDK1), mammalian target of rapamycin (TOR), p70, and nuclear factor-κB (NF-κB) in a primary culture system consisting of porcine granulosa-lutein cells (pGLs) incubated with 10(-5)M metformin and/or 100ng/ml insulin for 24h in a serum-free medium. We also investigated the luciferase activity of transcription factors activator protein-1 (AP-1) and NF-κB. Metformin with insulin significantly increased mRNA expressions of INSR, IGF-1R, and IRS-1, while metformin alone had no significant effect. And metformin with insulin had the significant effect on the protein activity (activation and phosphorylation) of downstream targets of INSR signaling pathway. Metformin with insulin significantly elicited an induction of luciferase activity in the transfection of AP-1 and NF-κBreporter, while metformin alone did not. In conclusion, we examined the activity of metformin and insulin on pGLS in vitro and metformin enhanced the action of insulin on the intracellular signaling pathways. These results suggest that metformin could change the function of ovarian granulosa cells.

Development of a type II diabetic mellitus animal model using Micropig®.

Diabetes, which has shown an explosive increase in terms of its incidence, is regarded as a serious disease that must be overcome for the sake of human life. Among animal models used for testing of drug efficacy, the mini-pig model has shown a rapid upload due to its many similarities with human, particularly concerning the pharmacokinetics of compounds after subcutaneous administration, the structure and function of the gastrointestinal tract, the morphology of the pancreas, and overall metabolic status. Based on these various advantages, we sought to develop an animal model of type II diabetic mellitus using the Micro-pig, which differs from other miniature pigs. We used six male Micro-pigs for induction of a moderate insulin deficient model with nicotinamide (NIA)/streptozotocin (STZ) treatment and three animals for control. For evaluation of incidence of type II diabetes, we measured blood glucose level, and performed oral glucose tolerance test and immunohistochemistry on pancreatic tissue using insulin antibody. Compared to control animals, all animals treated with NIA/STZ showed high levels of glucose and low levels of insulin. In addition, we observed the partially destroyed beta cell population from tissue of the pancreas in treated animals. Based on these results, we report that the Micro-pig model developed in this study can be used for testing of the efficacy of therapeutic agents for treatment of Type 2 diabetic mellitus.

Oxidative stress in the testis induced by tamoxifen and its effects on early embryo development in isogenic mice.

Oxidative stress induced by tamoxifen (TAM) in male testis and its effects on fertility and early embryo development were investigated. TAM was orally administered for 4 weeks repeatedly to two isogenic male mice strains, inbred strain of C57BL/6J (B6) mice and hybrid strain of C57BL/6J x CBA F1 (B6CBAF1) mice. Oxidative stress in mice testis was measured based on the level of lipid peroxidation (LPO). The LPO level was significantly increased in inbred strain of B6 mice (p < 0.05), but not hybrid strain of B6CBAF1 mice. Paternal exposure to TAM led to a significant decrease in the fertilization rate in B6 mice (p < 0.05), but not their B6CBAF1 counterparts. Interestingly, TAM had no impact on the cell number and apoptosis status in blastocysts. These results indicate that susceptibility to TAM-induced oxidative stress in the testis differs between isogenic mice strains, and genetic variations play an important role in promoting differential degrees of toxic response.

Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development.

BACKGROUNDS: Previous studies suggested that endocrine disruptors (ED) are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. METHODS: Bisphenol A (BPA) and Aroclor 1254 (polychlorinated biphenyls) were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations. RESULTS: At 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro. CONCLUSIONS: In conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors.

Temporal effects of alpha-tocopherol and L-ascorbic acid on in vitro fertilized porcine embryo development.

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.

The beneficial effects of insulin and metformin on in vitro developmental potential of porcine oocytes and embryos.

To investigate whether insulin and/or metformin improve the developmental competence of porcine oocytes and embryos, insulin (100 ng/ml) and/or metformin (10(-5) M) were supplemented during in vitro maturation (IVM) and/or in vitro culture (IVC) periods. Insulin (33 to 34% vs. 21%) or insulin plus metformin (27 to 39% vs. 21%) significantly (P < 0.01) increased the rates of blastocyst formation, whereas metformin alone had no effect when added during the first half (0-22 h), the latter half (22-44 h), or the entire (0-44 h) maturation period. No additional effect of insulin plus metformin on increasing blastocyst formation was observed compared to insulin alone. When supplemented during IVC, insulin (34% vs. 23%) or insulin plus metformin (35% vs. 23%) significantly (P < 0.05) increased the rates of blastocyst formation, whereas metformin alone had no effect. Compared to insulin alone, no additional effect of insulin plus metformin on increasing blastocyst formation was observed. When added during the entire IVM and IVC, insulin (40% vs. 24%) or insulin plus metformin (52% vs. 21%) significantly increased the rates of blastocyst formation, whereas metformin alone had no effect. In addition, the effect of insulin was enhanced when supplemented with metformin compared to insulin alone (52% vs. 40%). After IVM, oocyte glutathione (GSH) content and tyrosine kinase activity were measured. Insulin significantly (P < 0.01) increased oocyte GSH content (6.2 pmol vs. 4.3 pmol) and metformin significantly (P < 0.01) enhanced the action of insulin on GSH content (7.3 pmol vs. 6.2 pmol) and tyrosine kinase activity (1.9 arbitrary units [AU] vs. 1.5 AU) when compared to insulin alone. In conclusion, insulin increased the developmental potential of porcine oocytes and embryos, and metformin enhanced the action of insulin when supplemented during the entire IVM and IVC. The effects of insulin and metformin were associated with oocyte GSH content and tyrosine kinase activity.