Effects of nitrous oxide on preimplantation mouse embryo cleavage and development

Preimplantation mouse embryos were exposed to nitrous oxide for 30 min to determine its effects on subsequent development after short durations of exposure. Two-cell mouse embryos were exposed to 60% nitrous oxide/40% oxygen at 6-7 h, 3-4 h, or 0-1 h prior to the expected onset of their first cleavage in vitro, or at the 4-cell or morula stages. Effects of nitrous oxide were not observed except in 2-cell embryos treated within 4 h of the expected in vitro cleavage. At 3-4 h and 0-1 h prior to the onset of cleavage, exposure to 60% nitrous oxide/40% oxygen resulted in blastocyst development rates of 27.7% and 4.7%, respectively, while control rates ranged from 75% to 77%. The majority of affected embryos were halted at the 2-cell stage before completing cell division. Similar effects were obtained with 80% nitrous oxide/20% oxygen. Thus, we conclude that brief exposure of mouse preimplantation embryos to nitrous oxide may be deleterious to subsequent embryo cleavage, but this effect is highly dependent on the developmental stage at which exposure occurs.     

Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo

This study was an investigation of the interaction of lactate on pyruvate and glucose metabolism in the early mouse embryo. Pyruvate uptake and metabolism by mouse embryos were significantly affected by increasing the lactate concentration in the culture medium. In contrast, glucose uptake was not affected by lactate in the culture medium. At the zygote stage, the percentage of pyruvate taken up and oxidized was significantly reduced in the presence of increasing lactate, while at the blastocyst stage, increasing the lactate concentration increased the percentage of pyruvate oxidized. Lactate oxidation was determined to be 3-fold higher (when lactate was present at 20 mM) at the blastocyst stage compared to the zygote. Analysis of the kinetics of lactate dehydrogenase (LDH) determined that while the V(max) of LDH was higher at the zygote stage, the K(m) of LDH was identical for both stages of development, confirming that the LDH isozyme was the same. Furthermore, the activity of LDH isolated from both stages was reduced by 40% in the presence of 20 mM lactate. The observed differences in lactate metabolism between the zygote and blastocyst must therefore be attributed to in situ regulation of LDH. Activity of isolated LDH was found to be affected by nicotinamide adenine dinucleotide(+) (NAD(+)) concentration. In the presence of increasing concentrations of lactate, zygotes exhibited an increase in autofluorescence consistent with a depletion of NAD(+) in the cytosol. No increase was observed for later-stage embryos. Therefore it is proposed that the differences in pyruvate and lactate metabolism at the different stages of development are due to differences in the in situ regulation of LDH by cytosolic redox potential.     

Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses

The effects of two metabolic inhibitors, methyl palmoxirate (MP) and amino-oxyacetate (AOA), on mouse preimplantation embryo development and cell number, and inner cell mass (ICM) cell metabolism have been examined. Two-cell embryos were cultured in media supplemented with either MP, which inhibits fatty acid oxidation, or AOA, which inhibits the transamination of glutamate into α-ketoglutarate. Embryos were scored for development daily. On day 5, expanded blastocysts were differentially labeled with fluorochromes to visualize TE and ICM cell nuclei, or the ICMs isolated by immunosurgery and their energy metabolism determined using microfluorometric methods. Embryos exposed to the two inhibitors developed into fully expanded blastocysts, although cell numbers of both the TE and ICM cells were significantly reduced compared to controls. The uptake of glucose in the presence of 1 mM MP or AOA did not differ from the controls, but less glucose was accountable for by lactate production. MP significantly reduced lactate production. In the presence of 4 mM AOA, the amount of glucose oxidized and the amount of lactate formed by ICMs were significantly reduced. The results indicate that the fuels used by isolated mouse ICMs vary in response to substrate availability and that fatty acids may be a potential energy source. © 1996 Wiley-Liss, Inc.     

Effects of cordycepin on macromolecular synthesis and development in the preimplantation mouse embryo

Investigations were conducted to test the effects of cordycepin, a naturally-occurring analog of adenosine, on gene activity in preimplantation mouse embryos. Embryos were explanted into culture at the 2-cell, morula and blastocyst stages, and incubated in the absence or presence of cordycepin (5–100 μg/ml) to determine the effects of the drug on continued development and macromolecular synthesis. Cordycepin at concentrations exceeding 10 μg/ml caused a dose-responsive inhibition of cleavage and blastulation of embryos in culture. Exposure of morulae and blastocysts to cordycepin concentrations of 10–100 μg/ml produced a dose- and time-dependent suppression of RNA synthesis as measured by incorporation of [³H]uridine. Suppression in blastocyst-stage embryos was enhanced by preincubation, and reached 70% after 4 h at 100 μg/ml. Cordycepin (50–100 μg/ml) reduced synthesis of major RNA components detected by electrophoresis, blocked incorporation of radioactivity into fractions bound by olido(dT)-cellulose, and produced a time- and dose-dependent reduction of protein synthesis in blastocysts, causing a maximum inhibition of 25% after 4 h of preincubation at 50 μg/ml.     

Role of glutathione in reproductive tract secretions on mouse preimplantation embryo development

We investigated the hypothesis that glutathione (GSH) in reproductive tract secretions (RTS) protects the preimplantation embryo from endogenous reactive oxygen species and is important for normal development during the embryo's sensitive period when it is incapable of synthesizing GSH de novo. Mice were administered buthionine sulfoximine (BSO) to inhibit GSH synthesis and decrease GSH concentration in RTS. Embryos were then allowed to develop either in vivo or in vitro in the presence of RTS and the GSH concentration of the embryos was quantified by HPLC and embryonic development was recorded. GSH concentration in RTS did not differ over the phases of the estrous cycle, but there were significant decreases in GSH concentration on Day 2 of gestation and due to BSO treatment. Embryos allowed to develop in vivo and in vitro in RTS with decreased GSH concentration did not exhibit decreased development or GSH concentration. Oocytes exposed to BSO during maturation in vivo experienced a significant decrease in GSH concentration and an increase in percent of degenerate embryos when compared with control. These data suggest that most of the GSH in RTS does not play a critical role in normal preimplantation embryo development but that GSH stored in the oocyte during maturation has an important role in subsequent embryo development. Our studies do not exclude the possibility that GSH in RTS plays an important role in protection of the preimplantation embryo during exposure to some toxicants.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

The effect of progesterone and exogenous gonadotropin on preimplantation mouse embryo development and implantation

The aim of this study was to evaluate the effects of progesterone and ovarian stimulation on the development and implantation rate of mouse embryos. Two-cell embryos were collected from superovulated mice and cultured in the presence of different concentrations of progesterone (0, 5, 10 and 20 ng/ml). Also other mice were rendered pregnant in unstimulated, unstimulated progesterone-injected, superovulated and superovulated progesterone-injected groups to collect the blastocysts. The number of blastocysts and implantation sites were recorded on the 4th and 7th day of pregnancy, respectively. The diameter and cell number of blastocysts were analyzed in the in vitro and in vivo groups. After 120 h culture, the percentage of hatched blastocyst embryos in control and 5, 10 and 20 ng/ml progesterone-injected groups were 63.9%, 64.2%, 64.2% and 75.6% respectively. There were significant differences between the developmental rates of embryos in the presence of 20 ng/ml progesterone and the control and other concentrations of progesterone-injected groups (P< or =0.001). The in vivo blastocyst survival rate (97.68%) and implantation rate (92.06%) in the unstimulated and progesterone-injected groups were higher than in the other groups. Blastocyst cell numbers in the superovulated (128.62 +/- 1.30) and superovulated progesterone-injected groups (126.88 +/- 1.60) were significantly different from the control (P<0.001). The progesterone injection without ovarian induction improved the embryo survival and implantation rates, but after superovulation it did not ameliorate the negative effects of superovulation on the implantation rate.     

Arp3 is required during preimplantation development of the mouse embryo

The role of Arp3 in mouse development was investigated utilizing a gene trap mutation in the Arp3 gene. Heterozygous Arp3(WT/GT) mice are normal, however, homozygous Arp3(GT/GT) embryos die at blastocyst stage. Earlier embryonic stages appear unaffected by the mutation, probably due to maternal Arp3 protein. Mutant blastocysts isolated at E3.5 fail to continue development in vitro, lack outgrowth of trophoblast-like cells in culture and express reduced levels of the trophoblast marker Cdx2, while markers for inner cell mass continue to be present. The recessive embryonic lethal phenotype indicates that Arp3 plays a vital role for early mouse development, possibly when trophoblast cells become critical for implantation.     

Effect of gossypol on bovine embryo development during the preimplantation period

The purpose of this study was to evaluate the effect of varying doses of gossypol acetic acid on early bovine embryo development in vitro. One hundred and forty-eight excellent and good quality bovine morulae were randomly cultured in 0, 1.0, 5.0, 10.0, 30.0 mug gossypol acetic acid (GAA) in normal steer serum and Ham's F-10 media. Bovine embryo development was assessed at 12-h intervals for 96 h. Sixty-seven percent of embryos developed in 0 mug GAA to the hatched blastocyst stage, while 43, 19, 4 and 0% had comparable development in 1.0, 5.0, 10.0 and 30.0 mug GAA, respectively. Embryos in 5.0 mug GAA had a delayed development to the blastocyst stage compared to embryos in 1.0 mug GAA. Development time to expanded blastocyst stage was longer for 10.0 mug GAA embryos than 0, and 1.0 GAA-treated embryos. No embryo cultured in 30.0 mug GAA advanced past the morula stage. Final developmental scores were highest for embryos in 0 mug GAA (4.06) and lowest for embryos cultured in 10.0 and 30.0 mug GAA (0.44 and -0.02, respectively). Embryos cultured in higher doses of GAA degenerated sooner than embryos cultured in 0 mug GAA. These data show a dose-dependent detrimental action of GAA on early bovine embryo development and suggest a direct action on the embryo itself.     

Effect of heterogeneous ventilation and nitric oxide production on exhaled nitric oxide profiles.

Elevated exhaled nitric oxide (NO) in the breath of asthmatic subjects is thought to be a noninvasive marker of lung inflammation. Asthma is also characterized by heterogeneous bronchoconstriction and inflammation, which impact the spatial distribution of ventilation in the lungs. Since exhaled NO arises from both airway and alveolar regions, and its level in exhaled breath depends strongly on flow, spatial heterogeneity in flow patterns and NO production may significantly affect the exhaled NO signal. To investigate the effect of these factors on exhaled NO profiles, we developed a multicompartment mathematical model of NO exchange using a trumpet-shaped central airway segment that bifurcates into two similarly shaped peripheral airway segments, each of which empties into an alveolar compartment. Heterogeneity in flow alone has only a minimal impact on the exhaled NO profile. In contrast, placing 70% of the total airway NO production in the central compartment or the distal poorly ventilated compartment can significantly increase (35%) or decrease (-10%) the plateau concentration, respectively. Reduced ventilation of the peripheral and acinar regions of the lungs with concomitant elevated NO production delays the rise of NO during exhalation, resulting in a positive phase III slope and reduced plateau concentration (-11%). These features compare favorably with experimentally observed profiles in exercise-induced asthma and cannot be simulated with single-path models. We conclude that variability in ventilation and NO production in asthmatic subjects impacts the shape of the exhaled NO profile and thus impacts the physiological interpretation.