Estrogen regulation of nitric oxide synthesis in the porcine oocyte.
The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.
Hattori
Masa-aki
MA
Laboratory of Reproductive Physiology and Biotechnology, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka, Japan. mhattori@agr.kyushu-u.ac.jp
Arai
Manabu
M
Saruwatari
Keishi
K
Kato
Yukio
Y
D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006080:Ovarian Follicle / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D013997:Time Factors
D002457:Cell Extracts; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D004967:Estrogens; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D009569:Nitric Oxide; D004958:Estradiol; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III