Coenzyme Q(10) in submicron-sized dispersion improves development, hatching, cell proliferation, and adenosine triphosphate content of in vitro-produced bovine embryos.
(73/4036)
Coenzyme Q(10) (CoQ(10)) is an essential component of the plasma membrane ion transporter (PMIT) system and of the electron transport chain in the inner mitochondrial membrane. Because of its intrinsic functions in cell growth and energy metabolism (ATP synthesis), and its protective effects against oxidative stress, CoQ(10) is a good candidate for supporting growth of cells in culture. However, because of its quinone structure, CoQ(10) is extremely lipophilic and practically insoluble in water. We used a specific technology to prepare a submicron-sized dispersion of CoQ(10), inhibiting re-crystallization by a stabilizer. This dispersion, which exhibits a very large specific surface area for drug dissolution, was tested as a supplement for the in vitro culture of bovine embryos in a chemically defined system. The rate of early cleavage of embryos (5- to 8-cell stages) was evaluated 66 h postinsemination (hpi) and was highest in medium supplemented with 30 or 100 microM CoQ(10) (66.5 +/- 0.8% and 68.7 +/- 1.1%, respectively) and lowest in 10 microM CoQ(10) (55.3 +/- 0.8%). The proportions of oocytes developing to blastocysts by 186 hpi were 19.0 +/- 0.6% and 25.2 +/- 0.3% in medium supplemented with 10 microM and 30 microM CoQ(10), respectively, and were significantly (p < 0.001) higher than those obtained with the equivalent amounts of stabilizer (9.9 +/- 0.4% and 11.3 +/- 0.4%). In the presence of 30 microM CoQ(10), significantly (p < 0.001) more blastocysts hatched by 210 hpi than in the equivalent amount of stabilizer (31.8 +/- 1.3 vs. 8.4 +/- 2.2). Expanded blastocysts produced in the presence of 30 microM CoQ(10) had significantly (p < 0.01) more inner cell mass cells and trophectoderm cells, and a significantly (p < 0.001) increased ATP content as compared to expanded blastocysts produced in the presence of the corresponding amount of stabilizer. Our results show that noncrystalline CoQ(10) in submicron-sized dispersion supports the development and viability of bovine embryos produced in a chemically defined culture system. (+info)
Laser-assisted hatching in assisted reproduction.
(74/4036)
AIM: The use of a 1.48 um diode laser for assisted hatching was investigated in animal experimentation. Laser assisted hatching was offered to patients with advanced maternal age to evaluate a possible benefit. METHODS: Using the Fertilase(r) system we investigated the impact of openings with different size in the zona of mouse embryos on the hatching process, as well as that of two openings. Laser-drilling was performed at the blastocyst stage to look for differences in timing and efficacy of hatching. The possible benefit of assisted hatching was studied in 24 couples with advanced maternal age (38.8+2.1 years) and compared to a control group (37.8+2.5 years) treated in the same time period but without assisted hatching. RESULTS: A certain diameter of a laser drilled opening in the zona pellucida is necessary for efficient hatching. When two openings are present in the zona, the embryo will use both openings for hatching and subsequently become trapped. Laser-drilling at th e expanded blastocyst stage causes an immediate collapse of treated blastocysts and the onset of hatching is retarded. Assisted hatching in 24 patients with advanced maternal age resulted in a significant increase (p<0.01) in the implantation rate when compared to 24 untreated patients. CONCLUSION: The use of a 1.48 microm diode laser to drill an opening into the zona pellucida provides a good alternate to conventionally applied techniques. The procedure is efficient and safe as long as it is applied properly. In a human in vitro fertilization program, selected patients will have a benefit form assisted hatching. (+info)
Expression of Fgfr2 in the early mouse embryo indicates its involvement in preimplantation development.
(75/4036)
We report that the IIIc transcriptional alternative of Fgfr2 is transcribed in the unfertilized egg and that during early zygotic transcription, messages encoded by both Fgfr2 alternatives (IIIc and IIIb) are present. The Fgfr2 protein was first detected in peripheral blastomeres of compacted morulae. Trophectoderm specificity of Fgfr2 became obvious in the early blastocyst and with maturation its localization underwent further specification, Fgfr2 concentration increased at the abembryonic pole and decreased at the embryonic pole. Moreover Fgfr2 expression became markedly asymmetrical along the animal-vegetal axis of the mature blastocyst. Our observations indicate a role for Fgfr2 in trophectoderm growth and specification and in the orientation and polarity of the preimplantation conceptus. (+info)
Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation.
(76/4036)
The mRNA expression of five enzymes: catalase, Cu-Zn-superoxide dismutase (Cu-Zn-SOD), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GPX), and gamma-glutamylcysteine synthetase (GCS) each involved in protection against free radicals was studied in human and mouse oocytes. In the mouse, oocytes were collected at different stages of maturation in order to determine the storage of these transcripts. For the human, germinal vesicle (GV) oocytes harvested during intracytoplasmic sperm injection (ICSI) procedures and failed fertilized metaphase II (MII) oocytes were analysed. Human and mouse were compared in order to determine whether the differential developmental capacity of mouse and human preimplantation embryos in culture could be explained by the variations in the patterns of expression for these enzymes. mRNA expression for these enzymes was examined using reverse transcription-polymerase chain reaction (RT-PCR). In the mouse, all transcripts (except for catalase) were detected, whatever the maturation stage. No qualitative differences were detected between GV and MII oocytes. In human, all the enzymes (except for catalase) were expressed in MII oocytes and Cu-Zn-SOD was particularly highly expressed. Transcripts corresponding to GPX and Mn-SOD were not detected at GV stage but only at MII stage, suggesting that storage could occur between GV and MII stages. However, using 3' end-specific primers for GPX and Mn-SOD, instead of the oligo(dT)(12-18) primer, for the reverse transcription reaction, the transcripts for these antioxidants enzymes have been detected in human oocytes at the GV stage. This suggests the presence of maturation-specific polyadenylation of these transcripts. These enzymes can be considered as markers of cytoplasmic maturation. (+info)
Stage-specific expression of estrogen receptor subtypes and estrogen responsive finger protein in preimplantational mouse embryos.
(77/4036)
In hope of understanding possible roles of estrogen during early embryogenesis, we examined the expression of both estrogen receptor alpha (ER alpha) and ER beta, a recently cloned novel subtype, in mouse oocytes and preimplantation embryos by means of reverse transcription polymerase chain reaction (RT-PCR). To investigate whether estrogen actually exerts its action, we further determined the expression of efp (estrogen-responsive finger protein), a newly characterized estrogen responsive gene belonging to the RING finger family. ER alpha mRNA was detected in whole ovaries, cumulus-oocyte complexes, denuded oocytes, 2-cell and 4-cell embryos, whereas it was undetected in 8-cell embryos. Interestingly it reappeared in morulae and blastocysts. ER beta mRNA was detected similarly to ER alpha except for the absence of ER beta mRNA in morulae. The efp mRNA was detected in whole ovaries, cumulus-oocyte complexes, 4-cell embryos, morulae and blastocysts. The stage specific expression of ER alpha and ER beta along with detection of the product of the estrogen responsive gene in early preimplantation embryos may indicate the possible physiological roles of estrogen in early embryogenesis. (+info)
Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.
(78/4036)
Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus. (+info)
A maternal form of the phosphatase Cdc25A regulates early embryonic cell cycles in Xenopus laevis.
(79/4036)
In mammalian cells the Cdc25 family of dual-specificity phosphatases has three distinct isoforms, termed A, B, and C, which are thought to play discrete roles in cell-cycle control. In this paper we report the cloning of Xenopus Cdc25A and demonstrate its developmental regulation and key role in embryonic cell-cycle control. Northern and Western blot analyses show that Cdc25A is absent in oocytes, and synthesis begins within 30 min after fertilization. The protein product is localized in the nucleus in interphase and accumulates continuously until the midblastula transition (MBT), after which it is degraded. Upon injection into newly fertilized eggs, wild-type Cdc25A shortened the cell cycle and accelerated the timing of cleavage, whereas embryos injected with phosphatase-dead Cdc25A displayed a dose-dependent increase in the length of the cell cycle and a slower rate of cleavage. In contrast, injection of the phosphatase-dead Cdc25C isoform had no effect. Western blotting with an antibody specific for phosphorylated tyr15 in Cdc2/Cdk2 revealed a cycle of phosphorylation/dephosphorylation in each cell cycle in control embryos, and in embryos injected with phosphatase-dead Cdc25A there was a twofold increase in the level of p-tyr in Cdc2/Cdk2. Consistent with this, the levels of cyclin B/Cdc2 and cyclin E/Cdk2 histone H1 kinase activity were both reduced by approximately 50% after phosphatase-dead Cdc25A injection. The phosphatase-dead Cdc25A could be recovered in a complex with both Cdks, suggesting that it acts in a dominant-negative fashion. These results indicate that periodic phosphorylation of Cdc2/Cdk2 on tyr15 occurs in each pre-MBT cell cycle, and dephosphorylation of Cdc2/Cdk2 by Cdc25A controls at least in part the length of the cell cycle and the timing of cleavage in pre-MBT embryos. The disappearance of Cdc25A after the MBT may underlie in part the lengthening of the cell cycle at that time. (+info)
Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts.
(80/4036)
The developmental potential of bovine fetal fibroblasts was evaluated using nuclear transfer. Fibroblasts from a 37-day-old fetus were fused to enucleated oocytes before activation. Nuclei of starved (cultured for 8 days in medium containing 0.5% serum) fibroblasts supported the development of reconstructed embryos to the blastocyst stage significantly better than those of non-starved fibroblasts (39% versus 20%; P < 0.05). When nuclear transfer morulae derived from starved or non-starved fibroblasts were used for re-cloning, the proportion of blastocysts (52 and 55%, respectively) obtained with these embryonic nuclei was significantly higher than it was with fibroblast nuclei used in the first round of nuclear transfer (P < 0.05 and P < 0.001, respectively). After transfer of blastocysts derived from non-starved and starved fibroblasts, respectively, 33% (1/3) and 78% (7/9) of recipients were pregnant on day 30 as assessed by ultrasonography. On day 90, the corresponding pregnancy rates were 33% (1/3) and 63% (5/8). Two live male twin calves, derived from non-starved fibroblasts, were delivered by Caesarean section at day 281 of gestation. This study demonstrates a positive effect of serum starvation on the efficiency of nuclear transfer using bovine fetal fibroblasts. The efficiency of nuclear transfer could be further increased by recloning. (+info)