Eri Kotani

Biological roles of angiotensin II via its type 2 receptor during rat follicle atresia

Type 1 angiotensin II (ANG II) receptors play crucial roles in the regulation of blood pressure and fluid osmolarity, whereas the physiological roles of type 2 ANG II receptors (AT2) remain unclear. Because AT2 is expressed in atretic follicles where granulosa cells undergo apoptosis, we examined the space and time relationship between AT2 expression and follicle atresia in vivo and the effect of AT2 on follicle-stimulating hormone (FSH) actions in vitro. Binding studies, autoradiography, and RT-PCR of AT2 revealed that the AT2 content in granulosa cells was time dependently increased at both protein and mRNA levels in equine chorionic gonadotropin-treated immature female rats. This increase paralleled the progression of atresia. ANG II suppressed FSH-caused prevention of DNA fragmentation, increases in luteinizing hormone receptor content, and estrogen production through AT2 in cultured granulosa cells. Moreover, FSH-induced stimulation of extracellular signal-regulated kinase activity, critical for cell survival, was inhibited by AT2 stimulation. These results suggest that AT2 mediates the progression of follicle atresia through granulosa cell apoptosis by inhibiting FSH actions.

Tokishakuyakusan Effect on DNA Polymerase α Activity in Relationship to DNA Synthesis before and/or after the LH/FSH Surge in Rats

DNA polymerase alpha activity in ovaries of mature cycling rats during the normal estrous cycle changed in a cyclic manner with a peak at 1800 h in proestrus. Tokishakuyakusan (TS) in vivo did not affect the changes in DNA polymerase alpha and beta activities during the estrous cycle. LH and FSH at 1000 or 1700 h in proestrus increased DNA polymerase alpha activity, but the DNA polymerase alpha activity induced by LH or FSH was not significantly affected by the addition of TS. DNA polymerase beta activity did not change with LH, FSH or TS. In PMS-treated or -untreated immature rats, TS enhanced ovarian DNA polymerase alpha activity but had no significant effect on LH or FSH action. In ovaries, incubated in vitro, in untreated mature or immature rats, TS enhanced ovarian DNA polymerase alpha activity but had no significant effect on LH or FSH action. These results suggest that TS stimulates ovarian DNA polymerase alpha activity in relationship to DNA synthesis and does not affect the effect of LH or FSH on the activity by preovulatory follicle before and/or after the LH/FSH surge.

Effects of follicle-stimulating hormone on endothelin receptors in cultured rat ovarian granulosa cells.

Two subtypes of the endothelin (ET) receptor (ETA and ETB) were studied in cultured ovarian granulosa cells. Immature 21-day-old female Wistar-Imamichi rats were implanted with diethylstilbestrol (DES) pellets for 5 days and granulosa cells were collected by repeated puncturing. Viable cells (2.5 or 5 x 10(5)) were cultured with 50--400 ng/ml of ovine NIH follicle-stimulating hormone (FSH) in the presence or absence of [125I-Tyr13]ET-1 (50 pM) in 1 ml McCoys 5a medium for 72 h. FSH gradually increased the [125I-Tyr13]ET-1 binding to granulosa cells, whereas FSH-untreated granulosa cells had no significant changes. The dose of 200 ng/ml of FSH was most effective for [125I-Tyr13]ET-1 binding for 48-h culture, thereafter revealing a plateau. After 48 h of culture with 200 ng/ml of FSH, granulosa cells were further incubated with [125I-Tyr13]ET-1 (10 pM-1 nM) and/or [125I]IRL1620, the selective ETB receptor agonist (10 pM-1 nM) for 2 h for equilibrium study, and then the dissociation constant and the maximal binding capacity between receptors and ligands were determined by saturation curve and Scatchard plot analysis. ETA + ETB, ETB, and ETA (sites/cells) showed a 4.4-, 2.6-, and 7.5-fold increase, respectively. As for steroidogenesis, ET-1 (100 nM) or ET-3 (100 nM) suppressed FSH-induced progesterone and 17 beta-estradiol production. These results indicate that FSH upregulates both ETA and ETB receptors in DES-treated immature rat granulosa cells, with no significant differences between ET-1 and ET-3, and that ET-1 or ET-3 suppresses FSH-induced steroidogenesis. ETs may affect the granulosa cell function through the ETA and ETB receptors, and the increase in amount of ET binding does not reflect ET effects on granulosa cell function. The ET receptor plays an important role in the development of the ovary.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary.

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10 ,100 and 1000 ng/ml). Following 48 hours of incubation ,the levels of progesterone ,20α-hydroxy-4-pregnene-3-one (20α-OH-P) and adenosine 3′ ,5′-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20α-OH-P in a dose-dependent manner (p < 0.01 and 0.05 ,respectively ,ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each ,ANOVA). Conversely ,LH-stimulated 20α-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20α-OH-P production in LH-stimulated cells ,PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.

Biological roles of angiotensin ii via its type 2 receptor during rat follicle atresia

P4.17.01 BIOLOGICAL ROLES OF ANGIOTENSIN II VIA ITS TYPE 2 RECEPTOR DURING RAT FOLLICLE ATRESIA E. Kotani (l), K. Kondo (l), M Saitoh (l), S. Usuki (l), T. Kubo (l), K. Song (2), M. Miyazaki (2), H. Miyazaki (3) (1) Dept OBIGYN, institute of Clinical Medicine, University of Tsukuba, Tsukuba-shi, Ibaraki, Japan (2) Dept of Pharmacology, Osaka Medical College, Takatuki-shi, Osaka, Japan (3) Gene Experiment Center, University of Tsukuba, Tsukuba-shi, Ibaraki, Japan