|Budget Amount *help
¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
In order to investigate the role of oxygen super oxide and nitric oxide (NO) in regulation of luteal function, we studied in vivo mRNA expression of Mn-SOD, eNOS and iNOS in rat corpus luteum and interaction between Mn-SOD and NO in regulation of luteal function in cultured rat luteinized granulosa cells.
In the in vivo study, Wistar Imamichi rats were stimulated with hCG, and mRNA and protein expression of Mn-SOD and progesterone (P4) in the corpus luteum were detected with Northern blot hybridization, ELISA and RIA, respectively, at 0 to 48 hours after the hCG injection or during 2 to 20 days of pregnancy. Expression of eNOS and iNOS was detected with competitive RT-PCR.
In the in vitro study, granulosa cells collected 48 hours after PMSG stimulation, were cultured in CIT medium. After addition of NO donors, SNAP and SIN-i, and Mn-SOD, P4 concentration in the supernatant was measured with RIA.DNA fragmentation was detected as well. Further more, effect of SNAP and DIN-I on the mRNA exp
ression of Mn-SOD was also evaluated with Northern blot hybridization.
Results showed that Mn-SOD mRNA was detectable during the whole process of superovulation, reaching peak value at 7 hours after the hCG injection. Mn-SOD protein started to increase at 12 hours after hCC injection, reaching peak at 24 hours. P4 showed the same profile as Mn-SOD protein did. The level of eNOS mRNA gradually increase after hCG injection for up to 15- hours and decreased afterwards iNOS mRNA maintained a high level after hCG injection, reaching peak at 15 hours. In pregnancy, mRNA of Mn-SOD was detectable during the whole process of pregnancy, reaching peak at the l4th day of pregnancy, while Mn-SOD proteins reached peak at the 16th day. P4 showed a similar pattern with Mn-SOD protein up to the 12th day of pregnancy when it reached peak, but gradually decreased afterwards.. While mRNA of eNOS had the tendency to increase gradually during pregnancy, mRNA of iNOS rapidly increased from the 12th day and reached peak at the end of pregnancy.
In cultured rat granulosa cells, production of P4 was suppressed by SNAP and SIN-I in a dosage-dependent manner. Addition of Mn-SOD partially reversed the suppression by 22% and 29%, respectively (p<0.0 1). Similarly, fragmentation of DNA was induced by addition of SIN-I and SNAP, but was inhibited by addition of Mn-SOD.Further more, mRNA expression of Mn-SOD in cultured granulosa cells treated With SNAP and SIN- I were increased 1.6 fold and 1.3 fold compared with the untreated control.
We conclude that Mn-SOD is involved in stimulation, whereas NO plays a role in suppression of luteal function. In addition, Mn-SOD and NO interact with each other in regulating the function of corpus luteum. Less