Earticle

In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.

Galactose-α1,3-galactose (α1,3-Gal) epitope is synthesized at a high concentration on the surface of pig cells by α1,3-galactosyltransferase gene (GGTA1). The α1,3-Gal is responsible for hyperacute rejection in pig-to-human xenotransplantation. The generation of transgenic pigs as organ donors for humans is necessary to eliminate the GGTA1 gene that synthesize Galα(1,3)Gal. To prevent hyperacute graft rejection in pig-to-human xenotransplantation, previously, we developed α1,3-galactosyltransferase gene-knock-out somatic cell by homologous recombination. In this study, we established cell lines of α1,3-GT knock-out expressing hDAF and hHT gene from minipig fibroblasts to apply somatic cell nuclear transfer. The hDAF and hHT mRNA were expressed in the knock-in somatic cells and α1,3-GT mRNA was suppressed. However, the knock-in somatic cells were increased resistance to human serummediated cytolysis.

In a conventional sense, dried-spermatozoa are all dead and motionless due to the lost of their natural ability to penetrate oocytes both in vivo and in vitro. However, their nuclei are completely able to contribute to normal embryonic development even after long-term preservation in a dried state when the dried-spermatozoa are microinjected into the oocytes. In this sense, dried spermatozoa must still be alive. Thus, defining spermatozoa as alive or dead seems rather arbitrary. Several drying method of sperm including freeze-drying, evaporative/convective-drying and heat-drying were represented in this review. Although the drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genetic resources.

This study was carried out to evaluate the effects of embryonic stage and toxicities of cryoprotectant on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology. Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or EG. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO (68.1±24.1) at the 2-cell stage was significantly lower than that were treated with EG (81.2±27.0) or the control (99.0±18.3) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the EG treated group (p<0.05). Both DMSO (15.4±1.5) and EG (10.2±1.4) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.1±0.9, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the EG treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or EG at room temperature. When comparing two cryoprotective agents, EG appeared to be less toxic than DMSO at least in a murine embryo model.

The present study was devised to determine the effects of vitamin E and selenium (Selevit) on body weight, organ weight, hematological values and biochemical parameters in the orchidectomized (Orch) rats. Intact group (n=15) received no treatment and operation. Orch＋Selevit received operation and Selevit. The body weights of each group increased, but that of the Orch＋Selevit group were significantly lower than those of all the other groups. There were significantly different decreased (p＜0.001) of body weights between Orch＋Selevit group and all the other groups. Also, organ weights such as heart, liver, spleen, kidney, lung and skeletal muscle were measured. The heart and liver weights in the Orch＋Selevit group were significantly different decreased (p＜0.001) in comparison with those in the Intact and Sham groups. The kidney weights in the Orch＋Selevit group were significantly different decreased (p ＜0.01, p＜0.001) in comparison with those in all the other groups. The number of white blood cell (WBC) was significantly higher (p＜0.05) in the Orch＋Selevit group than in all the other groups. The hematological values of 12 parameters were not significantly different in any of the groups. The concentrations of serum total protein, albumin and alkaline phosphatase only increased significantly (p＜0.05, p＜0.01) in the Orch＋Selevit group as compared to that in the Orch group. We conclude that Selevit was significantly decreased the body weight in the Orch rats. Our findings suggest that Selevit may influence the process of lipid packaging and absorption in the Orch rats.

The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.