Serum melatonin concentrations were studied in normal pregnant women and in women with several types of pathologic pregnancies, e.g., twins, preeclampsia or intrauterine growth retardation (IUGR). Blood samples were collected from the maternal antecubital vein at 14:00 hr (daytime) and 02:00 hr (nighttime) during pregnancy, and also from the umbilical vein and artery immediately after delivery. Serum melatonin concentrations were measured by radioimmunoassay. Daytime serum melatonin levels in normal (single fetus; singleton) pregnancies were low. While the levels showed an increasing tendency toward the end of pregnancy, no statistically significant changes occurred. On the other hand, the nighttime serum melatonin levels increased after 24 weeks of gestation, with significantly (P < 0.01) high levels after 32 weeks; these values decreased to non-pregnant levels on the 2nd day of puerperium. Nighttime serum melatonin levels were significantly (P < 0.05) higher in twin pregnancies after 28 weeks of gestation than in singleton pregnancies, whereas the patients with severe preeclampsia showed significantly (P < 0.05) lower serum melatonin levels than the mild preeclampsia or the normal pregnant women after 32 weeks of gestation. Melatonin concentrations in umbilical vessels showed a higher tendency in neonates who were born during at night compared with the other neonates; moreover, those in the umbilical artery were generally higher than those in the umbilical vein. The present results indicate that in humans, the maternal serum melatonin levels show a diurnal rhythm, which increases until the end of pregnancy, reflecting some pathologic states of the feto-placental unit. Fetuses may produce melatonin with a circadian rhythm.

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Recently, we have found that the nitrate/nitrite concentrations in preovulatory follicles significantly decrease after hCG injection and that inducible nitric oxide synthase (iNOS) plays a main role in the decrease of the intrafollicular nitric oxide (NO) concentration. The purpose of the present study was to investigate the role of NO on oocyte meiotic maturation and to consider the physiological means of the decrease in intrafollicular NO concentration. Immature rats received 15 IU of eCG, and ovaries were removed under ether anesthesia 48 h later. Each ovary was bluntly divided into five or six pieces containing from four to seven preovulatory follicles under the microscope and then incubated with hCG, aminoguanidine (AG; an iNOS inhibitor), or S-nitroso-L-acetyl penicillamine (SNAP; an NO donor) for 5 h. After incubation, preovulatory follicles were punctured, and germinal vesicle breakdown (GVBD) was observed. Also, cGMP concentrations in these follicles were measured. Next, denuded oocytes were recovered from preovulatory follicles at 48 h after injection of 15 IU of eCG and incubated with SNAP with or without ferrous hemoglobin. Every 30 min up to 12 h, GVBD was observed. Both AG and hCG promoted GVBD, and SNAP prevented this effect. In addition, AG decreased intrafollicular cGMP levels, and the concomitant addition of SNAP prevented this decrease. Finally, SNAP dose-dependently inhibited GVBD in denuded oocyte, and this effect of SNAP was reversed by the addition of hemoglobin. We conclude that the iNOS-NO-(cGMP) axis may play an important role in oocyte meiotic maturation.

We investigated how maternal melatonin is regulated in pregnant rats. To examine the involvement of the conceptus (fetus and placenta) in serum melatonin concentrations, the number of conceptuses was experimentally reduced to one on day 7 of pregnancy (1-conceptus group). Maternal circulating nighttime melatonin levels increased toward day 21 of pregnancy and rapidly decreased to the non-pregnancy levels after parturition, whereas the maternal serum nighttime melatonin levels of the 1-conceptus group on day 21 of pregnancy were significantly lower than normal pregnancy bearing dams more than 10 conceptuses. When the fetuses were removed by fetectomy (all fetuses but not the placentae) on day 12 of pregnancy, serum melatonin concentrations were not decreased. To examine the source of circulating maternal melatonin, mRNA expression of N-acetyltransferase (NAT), which is a late limiting enzyme for melatonin synthesis, was examined in the placenta and fetal pineal. NAT was not expressed in the placenta and was negligible in the pineal gland of the fetus compared with the mother's pineal gland. To examine the effect of placental hormones on maternal melatonin production, a conditioned medium, which was made by incubating placenta of day 20 of pregnancy with medium, was injected into the 1-conceptus dams from day 17 to day 20 of pregnancy. Injection of conditioned medium significantly increased serum melatonin concentrations compared with the control values whereas charcoal treatment abolished the stimulatory effect of conditioned medium. In conclusion, maternal circulating melatonin is from the maternal pineal gland and is increased by placental hormones during pregnancy.

Follicular atresia is characterized by apoptosis of granulosa cells, and telomerase plays an important role in the apoptotic process. To study the relationship between follicular atresia and telomerase activity, we investigated changes in telomerase activity and localization in experimentally induced atretic follicles of immature rats. Immature female Sprague-Dawley rats received 15 IU equine CG (eCG) by subcutaneous injection. Rats were killed under ether anesthesia at 2, 3, 4, or 5 days after eCG injection. Telomerase activity in granulosa cells was measured by telomeric repeat amplification protocol (TRAP) assay, and telomerase localization in the ovary was examined by in situ TRAP assay. Telomerase activity was detected at high levels in granulosa cells on day 2 after eCG injection regardless of follicle size, and levels were significantly decreased in large follicles, with atretic changes, on days 4 and 5. No such decrease was observed in granulosa cells of small follicles. In the next experiment, rats received subcutaneous injections of estradiol (1, 10, or 50 microg/rat) to prevent follicular atresia or sesame oil as a control from day 2 to day 4 after eCG injection and were killed under ether anesthesia on day 5 after eCG injection. The changes observed in the large follicles on days 4 and 5 in oil treated rats were not observed with estradiol treatment. These findings suggest that the telomerase in granulosa cells is likely to play an important role for healthy follicle life and that loss of its activity may be associated with follicular atresia.