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.
R. Nagai, N. Tanaka, Y. Fukumatsu, H. Katabuchi and H. Okamura
T. Kawano, H. Okamura, C. Tajima, K. Fukuma and H. Katabuchi
Summary. A dose of 30 mg RU 486/kg, an antiprogesterone, was administered to pregnant rats on Day 2 (Group 1) or Day 4 (Group 2) of pregnancy. RU 486 significantly changed serum progesterone and oestradiol concentrations and luteal 3β-HSD and 20α-HSD activities in Group 1, and implantation was significantly inhibited. The luteal 3β-HSD activity in Group 2 rats on Day 6 was significantly (P < 0·01) lower than the control value (7·5 ± 0·6 and 10·1 ± 0·6 mU/mg protein respectively). This decline in the 3β-HSD activity was followed by a marked decrease in the serum progesterone concentration, resulting in a significant decrease of the progesterone/oestradiol ratio and implantation was completely inhibited. The 20α-HSD activity, which could not be detected on Day 6 in the control rats, was twice as great in Group 2 than in Group 1 rats (17·5 ± 1·2 and 7·4 ± 3·1 mU/mg protein respectively). Ultrastructural examination of corpora lutea of Group 2 rats confirmed luteolysis. These results suggest that RU 486 has a luteolytic effect and its anti-implantation effect is concomitant with luteolysis of the corpora lutea of pregnancy.
Keywords: RU 486; corpora lutea; luteolysis; 3β-HSD; 20α-HSD; rat
Y. Fukumatsu, H. Katabuchi, M. Naito, M. Takeya, K. Takahashi and H. Okamura
Summary. The effect of macrophages on proliferation of granulosa cells was examined in gonadotrophin-primed immature female rats. The mouse anti-rat macrophage monoclonal antibodies TRPM-2 and TRPM-3 were used and macrophages were observed in the granulosa layer and antrum of follicles and in corpora lutea and stroma around follicles. There was no difference in distribution between TRPM-2-positive cells and TRPM-3-positive cells. Macrophages with some cytoplasmic vacuoles of various sizes were also demonstrated in growing follicles. The average ratios of macrophages to granulosa cells in preantral, antral and mature follicles were 0·008, 0·007 and 0·002, respectively. Labelling with [3H]thymidine of granulosa cells cultured with peritoneal macrophages was significantly greater and the labelling index peaked to 25·0% when the ratio of macrophages to granulosa cells was 0·01, compared with the value of 14·2% when the granulosa cells were cultured alone. This ratio of macrophages to granulosa cells was similar to that in the preantral and antral follicles in vivo. These results suggest that macrophages participate in promoting proliferation of granulosa cells as local mediators in growing follicles.
Keywords: macrophage; granulosa cell proliferation; immunohistochemistry; culture; rat