In MRJ/MpJ mice, there is a genetic mutation of exonuclease 1 (Exol), in which the exon 9 is sometimes deleted. In the present study, to check the gen-eration of the spliced exons, exon 8-intron 8-exon 9 (pCX/Ex/EIE/B and pCX/ Ex/EIE/M) plasmids were temporally transfected in vitro into BALB 3T3 cells, and RT-PCR using appropriate primer pair was carried out 1 day after transfection. In these constructions, pCX/Ex/EIE/B was derived from genomic sequence of C57BL/6 mice, and pCX/Ex/EIE/M was from MRL/MpJ. A spliced band was detected in pCX/Ex/EIE/B , but was present little or very weakly in pCX/Ex/EIE/M . Next, the same spliced band was demonstrated in pCX/Ex/EIE/M(T) plasmid, in which the branchpoint sequence (BPS) of pCX/Ex/EIE/M including the exon 9 was changed into that of pCX/Ex/EIE/B. The splicing did not occur in the del1/B mutant, in which 1960 nucleotides of the intron 8 were deleted, whereas it was detected in the del2/B plasmid deleted 1036 nucleotides in its middle region. These results suggest that the nucleotide T to A mutation of the BPS in the intron 8 is at least a sufficient for generation of splice variants (tr-l and tr-2 Exol).

Although tumor necrosis factor (TNF) alpha is an important key factor in degeneration of equine superficial digital flexor tendon (SDFT), the dynamism of TNF receptors and associated factors on tendinocytes has not been elucidated. To reveal signaling events mediated by TNF-receptors (TNF-Rs) in tendinocytes, we focused on four signaling factors, TNF-R1, TNF-R2, TNF-Rassociated factor 2 (TRAF2) and nuclear factor-kappa B (NE-KappaB) , and investigated the distribution and production of these factors. Cultured tendinocytes were obtained from SDFTS of thoroughbred horses. The tendinocytes were treated with 10ng/ ml equine TNFAlpha medium for 6 hours and then the four factors on tendinocytes were visualized by using an immunohistochemical method, and the amounts of the four factors were determined by Western blot analysis. Although TNF-R1 and TNF-R2 co-localized on the same tendinocyte, in untreated control cells (normal condition), immunoreactivity against TNF-R1 was very weak but TNF-R2 showed a strong reaction. However, TNF-R1 showed the same high level of reaction as TNF-R2 in TNFAlpha-treated cells (inflamed condition). Intense TRAF2 and NF-KappaB were detected at inflamed condition, however both factors were also detected at normal condition. The distinct distributions of the four factors under different conditions (normal and inflamed condition) in uitro not only reflect the dynamism of the cytokines but may also provide important clues for a means to prevent from occurrence of tendonitis and progress of tendon degeneration.

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.