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K Takesue1 , N Nishida1 , M-A Hattori1 *, N Fujihara1
Laboratory of Animal Reproductive Physiology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan 1
There are some evidence that nitric oxide (NO) is synthesized in the ovary, and NO regulates some ovarian functions. However, more studies were required to evaluate its functions in the ovary exactly. A method for real-time detection of NO synthesis may be very valuable to reveal its functions. Present study was designed to detect NO synthesized by the porcine oocyte using 4,5-diaminofluorescein diacetate (DAF-2 DA), a fluorescent indicator for NO. The cumulus-oocyte complexes (COCs) were collected from the small follicles (1-3 mm diameter) of porcine ovaries by aspirating. After removal of cumulus cells, oocytes were incubated for 1 h in Hanks balanced salt solution (BSS), supplemented with 25 mM HEPES and 0.1 % (wt/vol) BSA (pH 7.4), containing 10 M DAF-2 DA, then they were exposed to 1 mM L-arginine (L-Arg), substrate of NO synthase (NOS), or 2 mM NG-Nitro-L-arginine (NNA), NOS inhibitor. The oocytes were observed under a fluorescence microscope with an excitation filter (460-500 nm), a dichroic mirror (505 nm), and a band-pass emission filter (510-560 nm). After exposure to the substrate for 45-60 min, the oocytes began to produce fluorescence strongly in BSS containing L-Arg, but not NNA. On the other hand, when COCs were cultured for 24 h with FSH, the fluorescence was markedly reduced in these oocytes. As revealed by Western blot analysis, inducible NOS (iNOS) decreased during culture, whereas endothelial NOS (eNOS) remained constant. In this study using a fluorescent indicator for NO, we directly detected the porcine oocyte-synthesized NO which was regulated by iNOS and eNOS. In addition, it is strongly suggested that NO is markedly produced by iNOS in the porcine oocyte of immature follicle.
This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.