Search

Free fatty acids (FFA) are produced in the intestine by microbial fermentation. Recently, a family of G protein-coupled receptors (GPR) acting as FFA transporters has been reported including GPR120, which is expressed by intestinal epithelial cells. The GPR120 has been reported to affect the expression of glucagon-like peptide (GLP)-1 as well as function as a control point for anti-inflammatory effects. The aim of the present study was to evaluate whether 12 selected intestinal bacteria, representing the 4 major phyla present in the intestine, affect intestinal epithelial cell GPR120 and GLP-1 mRNA abundance. Supernatants of the 12 bacteria were added to differentiated Caco-2 intestinal epithelial cells cultured on filter inserts in concentrations corresponding to a cell:bacteria ratio of 1:200. After 4 h of incubation, changes in cellular mRNA of GLP-1 and GPR120 by bacterial supernatant were examined using real-time reverse transcriptase polymerase chain reaction. The abundance of GLP-1 mRNA decreased when cells were exposed to 4 of the 12 supernatants (P ≤ 0.05) compared with cells without bacteria added. Supernatants from 8 of the 12 bacteria analyzed increased the mRNA level of GPR120 (P ≤ 0.05) compared with cells without bacteria added. The alteration in cellular GPR120 mRNA was observed with bacteria categorized as either probiotics or bacteria capable of inducing an anti-inflammatory effect. The beneficial effect of these bacteria may very well be mediated by regulation of GPR120. The regulation of GPR120 by intestinal microbiota represents a direct signaling pathway for gut bacteria to affect host health and metabolism.

Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338-344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143-150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus-oocyte complexes (COCs) were matured for 41-42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle's salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL-1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL-1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT Modified environmental stress was reported to improve the developmental competence and cryotolerance of porcine oocytes, such as high hydrostatic pressure (HHP; Du et al. 2008 Cloning Stem Cells, Epub ahead of print) and osmotic stress (Lin et al. 2008 Reprod. Biomed. Online, in press). HHP also improved the cryotolerance of bovine and murine blastocysts (Pribenszky et al. 2005a Reprod. Dom. Anim. 40, 338-344; Pribenszky et al. 2005b Anim. Reprod. Sci. 87, 143-150). In the present study we compared the effects of NaCl with that of concentrated solutions of two non-permeable osmotic agents, sucrose and trehalose on in vitro maturated oocytes. A total of 2050 slaughterhouse-derived porcine cumulus-oocyte complexes (COCs) were matured for 41-42 h, and then put into 800 μL T2 (HEPES-buffered TCM-199 [Earle's salts] with 2% cattle serum) supplemented with additional NaCl, sucrose or trehalose with the same osmotic level (588 mOsmol) in 4-well dishes and incubated for 1 h at 38.5°C in air. COCs incubated in T2 under the same conditions without supplementation were used as controls. Subsequently COCs were incubated in IVM medium for 1 h at 38.5°C in 5% CO2 with maximum humidity. After this recovery period cumulus cells were removed with 1 mg mL-1 hyaluronidase and pipetting, and oocytes were used as recipients for somatic nuclear transfer with handmade cloning (HMC) method. Porcine fetal fibroblasts were used as nuclear donor cells. Embryo culture was performed in PZM-3 medium (Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) in 5% CO2, 5% O2 and 90% N2 and maximum humidity. Cleavage and blastocyst rates were checked on Day 1 and Day 6, respectively. Cell numbers were counted after fixation in glycerol containing 20 μg mL-1 Hoechst 33342 fluorochrome on Day 6. t-test was performed for statistical calculations with SPSS 11.0 program (SPSS, Chicago, IL, USA). Results are shown in Table 1. Osmotic stress with both permeable and non-permeable agents increased developmental competence of porcine IVM oocytes. NaCl seems to be more appropriate for the purpose, as the other two components resulted in decreased cell number in blastocysts after somatic cell nuclear transfer (SCNT). In conclusion, a simple NaCl pre-treatment of oocytes has improved the in vitro efficiency of porcine SCNT

High hydrostatic pressure has been reported to improve the fertilizing or developmental ability of mammalian spermatozoa, oocytes and embryos. This study investigated the effect of another stress, temporarily increased NaCl concentration, on cryotolerance and developmental competence of porcine oocytes. In Experiment 1, survival rates were compared after 1 h exposure to seven elevated NaCl concentrations and 1 h recovery time. In Experiment 2, oocytes were exposed to 593 and 1306 mOsmol NaCl, subsequently recovered, vitrified, then subjected to parthenogenetic activation. Both cleavage and blastocyst rates increased after NaCl treatment compared with untreated controls. In Experiment 3, oocytes were treated with 593 mOsmol NaCl followed by 1 and 2 h recovery, respectively, then used as recipients for somatic cell nuclear transfer (SCNT). Cleavage rates were not different from those in untreated controls, but blastocyst rates increased in both NaCl-treated groups. In conclusion, treatment of porcine oocytes with elevated NaCl concentrations improved their developmental competence after vitrification and parthenogenetic activation or SCNT. Further experiments are required to investigate in-vivo consequences, and the effect on gametes and embryos of different mammalian species. Udgivelsesdato: March High hydrostatic pressure has been reported to improve the fertilizing or developmental ability of mammalian spermatozoa, oocytes and embryos. This study investigated the effect of another stress, temporarily increased NaCl concentration, on cryotolerance and developmental competence of porcine oocytes. In Experiment 1, survival rates were compared after 1 h exposure to seven elevated NaCl concentrations and 1 h recovery time. In Experiment 2, oocytes were exposed to 593 and 1306 mOsmol NaCl, subsequently recovered, vitrified, then subjected to parthenogenetic activation. Both cleavage and blastocyst rates increased after NaCl treatment compared with untreated controls. In Experiment 3, oocytes were treated with 593 mOsmol NaCl followed by 1 and 2 h recovery, respectively, then used as recipients for somatic cell nuclear transfer (SCNT). Cleavage rates were not different from those in untreated controls, but blastocyst rates increased in both NaCl-treated groups. In conclusion, treatment of porcine oocytes with elevated NaCl concentrations improved their developmental competence after vitrification and parthenogenetic activation or SCNT. Further experiments are required to investigate in-vivo consequences, and the effect on gametes and embryos of different mammalian species.