No abstract

In vivo post-ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca(2+) oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca(2+) homeostasis mainly through direct effects on the machinery involved in intracellular Ca(2+) storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca(2+)-oscillation-dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 microM hydrogen peroxide (H(2)O(2)) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H(2)O(2)-treated fresh oocytes. The frequency of Ca(2+) oscillations was significantly increased whereas the amplitude of individual Ca(2+) transients was lowered in the aged and H(2)O(2)-treated fresh oocytes. The rates of rise and decline in individual Ca(2+) transients were decreased in these oocytes, indicating impaired Ca(2+) handling. When lipid peroxidation was assessed using 4,4-difluoro-5-(4-phenyl-1,3-buttadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) in unfertilized oocytes placed in a 5% CO(2) in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca(2+) oscillations play a key role in the development of fertilized aged oocytes.

Extracellular Ca2+ is normally required for myometrial cells to contract. Ca2+ enters muscle cells mainly through voltage-dependent Ca2+ channels (VDCCs) that open in response to action potentials. The synthesis of myometrial VDCCs may change during pregnancy to alter excitation-contraction coupling. We investigated the mRNA levels for the alpha 1- and beta-subunits of the L-type VDCC in rat myometrium to determine whether alterations are associated with term or preterm labor. RNA isolated from myometrial tissues was analyzed by reverse transcription-polymerase chain reaction (PCR) using specific primers designed according to the published sequences of the VDCC subunits. From pregnant rat myometrium, two distinct PCR products were obtained for the alpha 1-subunit: one of the expected size at 372 bp and a smaller at 339 bp. Sequence analysis of the larger product revealed a 99.5 or 88% sequence homology between rat myometrium and rat aorta or rabbit heart, respectively, and the smaller product had an identical sequence to a 33-bp deletion. The two alpha 1-products followed the same trend throughout pregnancy. VDCC alpha 1-mRNA levels increased gradually to 6.9-fold just before labor on day 22 but decreased during labor. However, the beta-subunit mRNA level increased sharply on day 22 and then also declined during labor. Progesterone treatment from day 19 to day 22 inhibited term delivery and prevented the significant increase in alpha 1-mRNA levels. In contrast, antiprogesterone (onapristone, ZK-98.299) treatment on day 17 caused a statistically significant increase in the alpha 1- and beta-VDCC subunit mRNA after 8 and 15 h, respectively, then a decrease during preterm labor at 24 h. We conclude that mRNA levels for the VDCC subunits increase before term and preterm labor but decline during periods when VDCCs are likely at their peaks. The increase in levels of mRNA for VDCC likely reflects changes in expression of VDCCs during periods of term and preterm labor that may facilitate uterine contractility required for this process. Progesterone withdrawal or blockade appears to be responsible for regulating levels of mRNA for VDCC in the myometrium in preparation for labor.

Postovulatory aging of oocytes significantly affects embryonic development. Also, altered Ca2+ oscillation patterns can be observed in fertilized, aged mouse oocytes. Because Ca2+ oscillations depend on Ca2+ release and reuptake in the endoplasmic reticulum, and the latter relies on ATP availability, we simultaneously measured changes in intracellular ATP concentration ([ATP]i) and Ca2+ oscillations in fresh and aged mouse oocytes. We continuously assessed changes in [ATP]i from intracellular free Mg2+ concentration measured by fluorescent dye Magnesium Green (MgG) while intracellular Ca2+ concentration ([Ca2+]i) was monitored by Fura-PE3. At fertilization, MgG fluorescence was transiently increased concomitant with the first transient elevation in [Ca2+]i, indicating a relative decrease in [ATP]i. In fresh oocytes, it was quickly followed by a significant decrease below baseline, indicating a relative increase in [ATP]i. In contrast, in aged oocytes, such a decrease in MgG fluorescence was not observed. In a separate experiment, ATP content in fresh and aged oocytes was determined in vitro by the luciferin-luciferase assay. Intracellular ATP contents measured in vitro were comparable in unfertilized fresh and aged oocytes. Intracellular ATP content at 5 h after fertilization was increased in both oocytes, where fresh oocytes showed a significantly higher intracellular value than aged oocytes. These findings suggest that aged mouse oocytes fail to readjust the level of intracellular ATP at fertilization. Relative deficiencies of ATP at fertilization might lead to an altered Ca2+ oscillation pattern and poor developmental potency, which is commonly noted in aged oocytes.

One hundred and forty-three women in the early first trimester of gestation were examined 364 times using transvaginal sonography, and the development of embryonic heart rate was studied. In each case gestational age was revised retrospectively by either recorded basal body temperature or ultrasound crown-rump length dating between 9 and 10 weeks. Embryonic cardiac activity could be detected as early as 37 days of gestation. In 133 continuing pregnancies, embryonic heart rate rose from an average of 97.7 beats per min at 36–38 days to 174.7 beats per min at 60–62 days. A significant correlation was seen between gestational age and embryonic heart rate (p < 0.001). The regression equation for heart rate was as follows: heart rate = 3.850 × gestational age (days) ––54.64 (r = 0.908, n = 347), in short, embryonic heart rate continued to rise about 4 beats per min every day until 8 weeks of gestation. In this series, 10 pregnancies resulted in spontaneous abortion in the first trimester, and all of them showed relative bradycardia. Embryonic heart rate measurements in 8 of them were below the 95% prediction intervals for normal heart rate plotted against gestational age. This study suggests that embryonic heart rate measurement by ultrasound may be a new method for dating early first trimester, and that first trimester bradycardia may be associated with a poor prognosis for the pregnancy.