The effects of progesterone and RU486, a synthetic anti-progesterone, on ovarian 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a key enzyme of progesterone production, were studied during ovulation in immature 22-day-old rats primed with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). Ovarian 3β-HSD activities had increased significantly 4 h after hCG injection. These increases were inhibited at 4 and 6 h after hCG when 20 mg RU486 kg−1 was administered 2 h before hCG. However, RU486 had no influence on the activity of 3β-HSD when administered at the same time as hCG injection. A histochemical study revealed that 3β-HSD activities in the granulosa cell layer, but not in the theca cell layer, were inhibited when RU486 was given 2 h before hCG. Serum progesterone concentrations, but not oestradiol concentrations, were significantly suppressed by RU486 treatment 4 and 6 h after hCG. The effect of progesterone on ovarian 3β-HSD activity was tested by administering graded doses of progesterone exogenously to rats 2 h before hCG injection. Ovarian 3β-HSD activity was increased in a dose-dependent manner, and more than 20 mg progesterone kg−1 significantly stimulated the activity. Although 10 mg progesterone kg−1 did not stimulate ovarian 3β-HSD activities, the RU486-inhibited activities were recovered by the concomitant administration of 10 mg progesterone kg−1 with RU486. These results indicate that ovarian 3β-HSD activity depends on progesterone concentrations, and suggest an autocrine regulation of progesterone production during ovulation in immature rat ovaries stimulated with PMSG and hCG.
N. Tanaka, J. Iwamasa, K. Matsuura and H. Okamura
L. L. Espey, N. Tanaka, V. Winn and H. Okamura
Summary. The ovulatory process was initiated in 25-day-old rats by injecting them with hCG (10 i.u., s.c.) 2 days after the animals had been primed with PMSG (10 i.u., s.c.). At 2-h intervals after hCG, the ovaries were extracted and assayed for glandular kallikrein activity by using a chromogenic substrate H-d-Val-Leu-Arg-p-nitroanilide) which exhibits optical density (at 405 nm) upon hydrolysis. In 0-h control ovaries the activity was 12·5 × 10−3 kallikrein units (KU)/mg protein and it increased to a peak of 56·6 × 10−3 KU/mg at 12 h after hCG, when the follicles first began to rupture. The kallikrein activity was distinguishable from ovarian plasminogen activator activity on the basis of pH optima and response to trypsin inhibitor (SBTI). The activity was inhibited by a s.c. dose of indomethacin of 0·3 mg/rat, or higher, and this dosage inhibited ovulation. The results suggest that kallikrein activity contributes to the degradation of Graafian follicles during ovulation in mammals.
Keywords: ovulation; kallikrein; plasminogen activator; indomethacin; rat
R. Nagai, N. Tanaka, Y. Fukumatsu, H. Katabuchi and H. Okamura
The immunolocalization of prolyl 4-hydroxylase (PHase), a key enzyme of collagen synthesis, and the effects of anti-progesterone RU486 on PHase during the ovulatory process in eCG–hCG-treated immature rat ovaries were studied to investigate the mechanisms of tissue repair in follicle walls after follicular rupture. Immunolocalization of PHase was studied using an anti-rat PHase subunit monoclonal antibody, and the amount of immunoreactive PHase was measured by enzymeimmunoassay. No obvious immunolocalization of PHase was observed in theca cells throughout the ovulatory process except just after follicular rupture. In contrast, in granulosa cells, PHase was first observed at 9 h after the hCG injection, and the staining intensity apparently increased from 9 to 15 h, especially around the apex of preovulatory follicles and the orifice of ruptured follicles. Consistent with these observations, PHase concentration in granulosa cells isolated from the ovaries significantly increased by 9 h (0.45 ± 0.03 pg per cell), and reached a peak at 15 h (0.66 ± 0.06 pg per cell) after the hCG injection. This peak was inhibited when 20 mg RU486 kg−1 was administered at 8 h (0.46 ± 0.05 pg per cell), and the RU486-inhibited PHase concentration was recovered by the concomitant administration of 10 mg progesterone kg−1 (0.65 ± 0.02 pg per cell). The results suggest that PHase expressed in granulosa cells may play an important role in the repair of ruptured follicle walls, via progesterone-dependent PHase production.
N. Tanaka, K. Miyazaki, H. Tashiro, H. Mizutani and H. Okamura
Adenylyl cyclase activity was studied in human endometrium during the menstrual cycle and in human decidua during pregnancy. Higher adenylyl cyclase activity was found in the endometrium than in the myometrium, corpus luteum or Fallopian tubes. In the endometrium, the basal and stimulated activities were highest in the fundus and decreased slightly from the fundus to the isthmus. Prostaglandin-stimulated adenylyl cyclase activity increased gradually from the proliferative phase to the secretory phase, and then quickly reached its highest value in the late secretory phase. Catecholamine-stimulated adenylyl cyclase activity reached a peak in the late proliferative phase and decreased significantly thereafter. Forskolin-stimulated activity was significantly higher throughout the secretory phase than in the proliferative phase. In the decidua, prostaglandin-, catecholamine- and forskolin-stimulated adenylyl cyclase activities in late pregnancy were significantly lower than those in early pregnancy. Our results demonstrate dramatic alterations in adenylyl cyclase activity in human endometrium during the menstrual cycle and in human decidua during pregnancy.
K. Miyazaki, N. Tanaka, S. Kawakami and H. Okamura
Adenylyl cyclase activity was studied in human decidua and myometrium in early pregnancy and at term before and after the onset of labour. Decidual basal, prostaglandin-, catecholamine- and forskolin-stimulated adenylyl cyclase activities at term before the onset of labour were significantly lower than those in early pregnancy. After the onset of labour at term, decidual basal, prostaglandin-, catecholamine-, NaF- and forskolin-stimulated adenylyl cyclase activities significantly increased compared with those at term before the onset of labour. Myometrial prostaglandin- and catecholamine-stimulated activities did not alter during pregnancy, except for basal and forskolin-stimulated activity. Myometrial basal, prostaglandin-, catecholamine-, NaF- and forskolin-stimulated activities at term showed no change after the onset of labour. At term, before the onset of labour, myometrial basal, prostaglandin, catecholamine-, NaF- and forskolin-stimulated activities were the same as those in the decidua. However, after the onset of labour at term, decidual basal and the stimulated activities were significantly higher than those in the myometrium. These results suggest that decidual prostaglandin- and catecholamine-stimulated adenylyl cyclase may play an important role in the initiation or maintenance of human labour or in both processes.
K. Nishimura, N. Tanaka, A. Ohshige, Y. Fukumatsu, K. Matsuura and H. Okamura
The effect of macrophage colony-stimulating factor (M-CSF) on folliculogenesis and ovulation was studied. Folliculogenesis and ovulation were induced in immature female rats with a s.c. injection of equine chorionic gonadotrophin (eCG), followed 48 h later by human chorionic gonadotrophin (hCG). The ovulation rate was measured after the following treatments. (1) Graded doses of human M-CSF (1–300 × 103 iu per rat) were administered i.p. daily for 3 consecutive days. (2) M-CSF (100 × 103 iu) was administered i.p. at designated times between 96 h before and 10 h after hCG injection. (3) Rabbit anti-human M-CSF polyclonal antibody (5 μg) was administered into the left ovarian bursa at designated times between 49 h before and 10 h after hCG injection. In addition, the effect of M-CSF on ovarian macrophages was investigated using immunohistochemistry with mouse anti-rat macrophage monoclonal antibody, TRPM-3. The treatment with M-CSF (> 30 × 103 iu per rat) significantly increased the ovulation compared with controls in a dose-dependent manner. This stimulatory effect of M-CSF was observed when it was administered between 96 h and 49 h before hCG injection. The ovarian intrabursal administration of anti-M-CSF antibody significantly inhibited the number of ovulated ova from the treated ovaries compared with either those from control rats or from the contralateral untreated ovaries between 24 h before and 3 h after hCG injection. The immunohistochemistry revealed that M-CSF increased the number of ovarian macrophages in growing follicles. The results suggest that M-CSF is involved in the process of folliculogenesis and that it promotes ovulation by influencing ovarian macrophages.
K Iwahashi, N Kuji, T Fujiwara, H Tanaka, J Takahashi, N Inagaki, S Komatsu, A Yamamoto, Y Yoshimura and K Akagawa
Syntaxin is an integral membrane protein that is involved in membrane fusion. The exocytosis of the contents of cortical granules, secretory vesicles located in the cortex of an egg, modify the extracellular environment to block additional spermatozoa from penetrating the newly fertilized egg. The aim of this study was to characterize syntaxin expression in mouse oocytes, and to determine the specific isoform that is expressed. Syntaxin was demonstrated in the mouse ovary and in mouse oocytes by both western blot and reverse transcription-polymerase chain reaction analyses. Syntaxin 4 was specifically expressed in metaphase II oocytes. Syntaxin was also immunolocalized within metaphase II oocytes and one-cell embryos with pronuclei using laser scanning confocal microscopy. In metaphase II oocytes, syntaxin was located on the plasma membrane and in the cortex, where cortical granules are present, but was not seen at sites free of cortical granules. In one-cell embryos, no cytoplasmic region was free of syntaxin immunoreactivity. Immunoelectron microscopy detected syntaxin on both the plasma membrane and the vesicle membranes in mouse metaphase II oocytes. In conclusion the results indicate that syntaxin 4 co-localizes with cortical granules and participates in membrane fusion and exocytosis during the cortical reaction.