TNFα-signal and cAMP-mediated signals oppositely regulate melanoma- associated ganglioside GD3 synthase gene in human melanocytes

Analyses of expression and regulation of ganglioside synthases in melanocytes are important to understand roles of gangliosides in melanomagenesis. In this study, we analyzed the expression and regulatory mechanisms of glycosyltransferase genes responsible for ganglioside synthesis in normal melanocytes. We reported previously that culture supernatants of UVB-irradiated keratinocytes induced upregulation of ganglioside GD3 synthase gene in melanocytes, and mainly TNFα was responsible for it. Then, we found that elimination of dibutyryl cyclic AMP and IBMX from the medium also resulted in upregulation of the GD3 synthase gene. The addition of α-melanocyte-stimulating hormone which increases cAMP, to the medium led to a significant reduction in the GD3 synthase gene expression level, and a PKA inhibitor enhanced the GD3 synthase gene level. These results suggest that signals mediated via TNFα and cAMP oppositely regulate GD3 synthase gene expression in melanocytes. The results of an IKK inhibitor indicate the possibility that TNFα induces GD3 synthase gene expression via NF-κB signaling in melanocytes. When melanoma cells were treated by these factors, no fluctuation in the GD3 synthase gene expression level was observed, although an IKK inhibitor significantly suppressed it, suggesting that ganglioside synthase genes are regulated in distinct manners between melanocytes and melanomas.

43 WARMING TEMPERATURE AFFECTS THE VIABILITY AND MEIOTIC COMPETENCE OF IMMATURE PORCINE OOCYTES VITRIFIED IN A CHEMICALLY DEFINED SOLUTION

The aim of this study was to examine the viability and meiotic competence of porcine oocytes when immature porcine cumulus-oocyte complexes (COC) were pretreated for vitrification at different temperatures (25 and 39°C), vitrified in a chemically defined solution, and warmed at different temperatures (39 and 60°C). Cumulus-oocyte complexes were aspirated from middle-size follicles (3–6 mm in diameter) of abattoir-derived porcine ovaries. After collection, the COC were pretreated with cryoprotectants at different temperatures (25 and 39°C) and vitrified in a serum-free chemically defined solution containing 0.6 mg mL–1 of hydroxypropyl cellulose, basically according to a commercial protocol (Cryotop, Kitazato BioPharma Co. Ltd., Fuji, Japan). The vitrified COC were warmed in 1 M trehalose solution at 39 for 60 s or at 60°C for 30 s. The COC were cultured for in vitro maturation (IVM) in modified porcine oocyte medium (POM) supplemented with 50 μM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dibutyryl cyclic AMP (dbcAMP) for 20 h and then in the fresh medium without hormonal supplements and dbcAMP for another 24 h. Viability of COC was evaluated under fluorescent microscopy after stain with fluorescein diacetate and propidium iodide. Nuclear maturation of the oocytes was evaluated after 44 h of IVM. Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). Although viabilities of vitrified oocytes after 44 h of IVM [6.0% (9/149) to 37.8% (59/155)] were significantly lower than fresh controls [98.8% (158/160)], the viabilities of vitrified oocytes warmed at 60°C [32.0% (49/160) to 37.8% (59/155)] were significantly higher than those warmed at 39°C [6.0% (9/149) to 10.0% (16/160)]. Maturation rates in vitrified oocytes [2.7% (4/149) to 19.8% (31/155)] were also significantly lower than fresh controls [74.8% (120/160)]. Regardless of temperature during pretreatment for vitrification (25 and 39°C), maturation rate of the oocytes warmed at 60°C after vitrification [16.4% (25/154) to 19.8% (31/155)] was significantly higher than that warmed at 39°C [3.1% (5/160) to 2.7% (4/149)]. In conclusion, these results demonstrate that warming at 60°C for 30 s maintains the viability and meiotic competence of immature porcine COC.