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PubMed1988

Cytogenetic study of parthenogenetically activated bovine oocytes matured in vivo and in vitro.

Three experiments were performed to evaluate the potential for parthenogenetic activation of bovine oocytes in in vitro fertilization systems and to determine the chromosome complement of the resulting parthenogenotes. In the first experiment, immature oocytes from slaughtered cattles were matured in vitro in Defined Medium (DM) for 24 h to simulate in vitro fertilization conditions. Subsequently, a portion was fixed, and the remainder were transferred to rabbit oviducts. Oocytes were then cultured for 6-8 h or for 24 h with Colcemid present during the last 6 to 8 h and fixed on slides and examined. In the second experiment, mature oocytes were collected from the preovulatory follicles, and the oocytes were subjected to the same culture as in experiment I. In the third experiment, oocytes were treated as in experiment II, except that instead of transfer to rabbit oviducts, they were cultured an additional 48 h in vitro. In experiment I, 131 oocytes were fixed after culture in DM. Of the 79 oocytes analyzed in the pre-rabbit group, 71 (90%) were at the second meiotic metaphase (MII), and 8 (10%) were at pre-MII stage; none were activated. After transfer to rabbits, 291 were fixed. Of these, 80 were analyzed; 37 (46.3%) were MII, 7 (8.6%) were pre-MII, and 36 (45%) were activated. Of the 36 activated oocytes, 26 (72.2%) were haploid, 4 (11.1%) were diploid, 1 (12.8%) was tetraploid, and 5 (13.8%) were in the process of endoreduplication. In experiment II, 51 oocytes were fixed after culture in DM of which 36 (70.6%) could be analyzed; 30 (83.3%) were MII, and 6 (16.7%) were pre-MII. After culture in the rabbit, 68 were fixed of which 27 (39.7%) could be analyzed. Of these 27, 20 (74.1%) were MII, and 7 (25.9%) were activated; 6 were haploid, and 1 was endoreduplicating. In experiment III, 30 oocytes were fixed at the end of the culture period; only 10 could be analyzed of which 8 (80%) were MII and 2 (20%) were pre-MII. In all, 46% of in vitro and 26% of in vivo matured oocytes were activated, based on chromosomal analysis. Of those activated, the majority (74.4%) were haploid, suggesting that activation occurs at or after completion of MII. Endoreduplication appears to be one of the mechanisms leading to the formation of diploid and polyploid parthenogenotes.

Animals↗

PubMed1988

Development of enucleated parthenogenones that received pronuclei or nuclei from fertilized mouse eggs.

This study compares the developmental potential of enucleated mouse parthenogenones that received pronuclei from fertilized eggs with those that received nuclei from late two-cell embryos. The proportion of reconstituted parthenogenones, which received pronuclei at the one-cell stage, that developed to blastocysts in vitro and to live fetuses after transfer to recipients was significantly lower than that of reconstituted control eggs. However, the in vitro and in vivo developmental potential of reconstituted parthenogenones that received nuclei at the two-cell stage from fertilized late two-cell embryos was not different from that of reconstituted control eggs. These results were contrary to those reported previously by Mann and Lovell-Badge (1984), who showed that parthenogenetic eggs receiving pronuclei from fertilized eggs developed well both in vitro and in vivo.

Animals↗

PubMed1989

Parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro.

The present study was undertaken to determine conditions for parthenogenetic activation of mouse and rabbit eggs by electric stimulation in vitro. The cumulus-free eggs were submitted to square direct current pulses at output voltage of 1.0 to 2.5 kV/cm for 25 to 200 microseconds. The best conditions for the activation of mouse eggs were 1.5 kV/cm for 100 microseconds, in which 78% of eggs were activated, 32% of which developed to blastocysts in vitro. When the nonelectric solution (0.3M mannitol) was used for electric stimulation, the activation rate was quite low (16%). Optimal conditions for activation of rabbit eggs were 1.5 kV/cm for 200 microseconds, in which 77% of eggs were activated, 25% of which developed to blastocysts. Unlike mouse eggs, rabbit eggs frequently had three pronuclei after electric stimulation. It is clearly shown that electric stimulus can induce parthenogenetic activation of the mouse and rabbit eggs in vitro.

Animals↗

PubMed1989

Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation.

Protein phosphorylation activity, chromosome segregation, and cortical granule exocytosis (CGE) have been studied in mouse eggs activated parthenogenetically by specific PKC stimulators such as 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (OAG), or by agents inducing an immediate increase in cytosolic calcium concentration ([Ca2+]i) such as ethanol and Ca-ionophore A23187. When protein phosphorylation activity of mouse eggs was analyzed 10 min after different activation treatments, the phosphorylation of a 32 kDa polypeptide was a feature common to all different parthenogenetic agents used. The appearance of such labeling was independent of an increasing [Ca2+]i, as indicated by direct measurements of 1) cytosolic Ca2+ concentration with fura-2 and 2) exogenous Ca2+ entrance into activated eggs. Emission of the second polar body was blocked in PMA-elicited parthenogenones, whereas it was apparently normal in OAG-treated eggs, unless the eggs were continuously exposed to OAG. CGE was almost immediate in ethanol-activated eggs, but in PMA-treated cells, it occurred significantly later, with a timing corresponding to that found for the appearance of sustained Ca2+ oscillations in this system. Here, we propose that in mammalian eggs 1) PKC stimulation represents an early regulatory step in egg activation; 2) this kinase activity is turned off before the second meiotic cleavage; and 3) cytosolic free Ca2+ rise is essential for CGE occurrence.

Animals↗

PubMed1989

Parthenogenetic activation of unfertilized mouse oocytes by exposure to 1,2-propanediol is influenced by temperature, oocyte age, and cumulus removal.

Cumulus-intact and -denuded unfertilized oocytes from two mouse strains were exposed to 1.5 M ethanol (EtOH) or two cryoprotectant solutions, 1.5 M propanediol (PROH) or 1.5 M dimethylsulfoxide (DMSO), for 4.5 min at 27 degrees C, and the proportion of activating or degenerating oocytes studied. Exposure to DMSO did not significantly increase activation above that of oocytes not exposed to DMSO. Treatment of oocytes in PROH resulted in the activation of up to 87% of viable oocytes. This was significantly higher (P less than .01) than in control oocytes and comparable to the rate of activation after treatment with EtOH (59-96% activation). In solutions at 1 degree C, 47% of control oocytes were activated, which was not significantly different from the rate of activation in EtOH (36%) or PROH (50%) at 1 degree C. Following treatment with PROH, up to 87% of oocytes degenerated within a period of 6 h in vitro. The age of the oocytes (h post hCG) and the time of cumulus removal with the enzyme hyaluronidase, relative to the time of exposure to the chemicals, influenced the level of degeneration in most groups. Significantly fewer oocytes degenerated when cumulus cells were removed before treatment (0-31%) than when the cumulus was left intact throughout the treatment and 6 h culture period (10-87%). Exposure to PROH at 1 degree C reduced oocyte degeneration to 5%. We conclude that PROH causes significantly greater losses of oocytes as a result of parthenogenetic activation and degeneration than of exposure to DMSO.

Animals↗

PubMed1989

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs.

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3.5, and 8 h after activation, and in series B, 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively; chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anucleate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclear growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.

Animals↗

PubMed2002

Mouse Xist expression begins at zygotic genome activation and is timed by a zygotic clock.

The imprinted mouse Xist (X-inactive specific transcript) gene is involved in the initiation of X-chromosome inactivation. Only the paternal Xist is expressed in preimplantation development beginning from the 4-cell stage, preceding and in correlation with paternal X-inactivation in the extraembryonic lineage of the blastocyst. To better understand the mechanisms regulating Xist expression in early development, we investigated the precise timing of its onset. We set up a single-cell RT-PCR for the simultaneous analysis on single embryos of Xist and Hprt (internal control) cDNAs and a Y-chromosome specific DNA sequence, Zfy (for embryo sexing). Applying this procedure, we demonstrate that Xist expression begins at the G2-phase of 2-cell female embryos, earlier than previously reported and at the same time of the major wave of zygotic genome activation (ZGA). We then examined, if Xist expression at the 2-cell stage is dependent on the lapse of time spent since fertilization, as previously reported for zygotic genes. One-cell embryos at the G2-phase of the first cell-cycle were cultured with cytochalasin D (inhibitor of cytokinesis but not of DNA synthesis or nuclear progression) for a time equivalent to the 4-cell stage in control, untreated embryos. We show that Xist activation occurs at a scheduled time following fertilization despite the embryos being blocked at the 1-cell stage, suggesting the existence of a zygotic clock involved in the regulation of the transcription of this imprinted gene.

Animals↗

PubMed2002

Parthenogenetic activation and subsequent development of rat oocytes in vitro.

Studies were undertaken to determine whether electrical stimulation, or ethanol treatment alone or in combination with 6-dimethylaminopurine (6-DMAP) influenced the rate of parthenogenetic activation of rat oocytes. The percentages of activated oocytes with pronuclei (89-91%) and those developed to the two-cell stage (68-72%) were significantly higher after electrical stimulation with direct current (DC) at 100 V/mm, 99 microsec once or twice, than when other DC voltages (75, 150, and 200) were applied or when ethanol or 6-DMAP treatment was given alone. However, none of the activated oocytes developed beyond the four-cell stage. The percentages of activated oocytes with pronuclei (100%) that developed to the two-cell (100%), eight-cell (89%) and blastocyst stages (50%) were significantly higher when electrical stimulation was followed by treatment with 2 mM 6-DMAP for 4 hr than when other combined procedures were applied. In conclusion, the results of the present study clearly showed that combined treatment of electrical stimulation or ethanol with 6-DMAP induces parthenogenetic activation and subsequent development of rat oocytes in vitro.

Adenine↗

PubMed2004

Optimization of parthenogenetic activation protocol in porcine.

The effects of the electrical field strengths, number of pulses, and post-activation media on chromatin conformation and parthenogenetic development were studied to optimize the activation protocol for porcine nuclear transfer. In experiment 1, electrical field strengths were examined. Oocytes were subjected to square direct current pulses at output voltages of 1.2, 1.7, 2.2, and 2.7 kV/cm for 1 x 30 microsec. The voltage resulting from experiment 1 was 2.2 kV/cm, in which 50.0% of activated oocytes developed to blastocysts in vitro. In experiment 2, the influence of 1, 2, and 3 pulses on blastocyst development was tested using field strengths and post-activation medium described in experiment 1. Oocytes activated by a single 30 microsec pulse of 2.2 kV/cm DC yielded a higher blastocyst rate (56.3%) than oocytes activated by 2 or 3 pulses (<42.5%). In experiment 3 and 4, we investigated the effects of cytochalasin B (CB), cycloheximide (CH), and CB + CH on nuclear development stages and parthenogenetic development following a single 30 microsec pulse of 2.2 kV/cm DC. The percentage of activated oocytes was not different among CB (93.3%), CB + CH (98.3%), control (80.0%), and CH (80.0%) groups 12 hr after activation. Treatment with CB (57.5%) or CB + CH (53.8%) enhanced the blastocyst rate compared with other groups, CH (23.8%) treated- and control group (18.8%). The results demonstrated that a single 30 microsec pulse of 2.2 kV/cm DC followed by culturing in post-activation medium with CB for 5 hr were effective parameters for parthenogenetic activation and blastocyst formation of in vitro matured porcine oocytes which suggests that a single calcium rise is sufficient to activate pig oocytes and to achieve high rate of blastocyst development.

Animals↗

PubMed2004

Differential effect of recipient cytoplasm for microtubule organization and preimplantation development in rat reconstituted embryos with two-cell embryonic cell nuclear transfer.

In the present study, we examined the developmental ability of enucleated zygotes, MII oocytes, and parthenogenetically activated oocytes at pronuclear stages (parthenogenetic PNs) as recipient cytoplasm for rat embryonic cell nuclear transfer. Enucleated zygotes as recipient cytoplasm receiving two-cell nuclei allowed development to blastocysts, whereas the development of embryos reconstituted with MII oocytes and parthenogenetic PNs was arrested at the two-cell stage. Previous observations in rat two-cell embryos suggested that the distribution of microtubules is involved in two-cell arrest. Therefore, we also examined the distribution of microtubules using immunofluorescence. At the two-cell stage after nuclear transfer into enucleated zygotes, microtubules were distributed homogeneously in the cytoplasm during interphase, and normal mitotic spindles were observed in cleaving embryos from the two- to four-cell stage. In contrast, embryos reconstituted with MII oocytes and parthenogenetic PNs showed aberrant microtubule organization. In enucleated zygotes, fibrous microtubules were distributed homogeneously in the cytoplasm. In contrast, dense microtubules were localized at the subcortical area in the cytoplasm and strong immunofluorescence intensity was observed at the plasma membrane, while very weak intensity was detected in the central part of enucleated MII oocytes. In enucleated parthenogenetic PNs, high-density and fibrous microtubules were distributed in the subcortical and central areas, respectively. Pre-enucleated parthenogenetic PNs also showed lower intensity of microtubule immunofluorescence in the central cytoplasm than zygotes. In conclusion, the results of the present study showed that zygote cytoplasm is better as recipient than MII oocyte and parthenogenetic PNs for rat two-cell embryonic cell nuclear transfer to develop beyond four-cell stage. Furthermore, microtubule organization is involved in the development of reconstituted embryos to overcome the two-cell arrest.

Animals↗

PubMed2004

Isolation and characterization of a bovine trophectoderm cell line derived from a parthenogenetic blastocyst.

A bovine trophectoderm cell line was established from a parthenogenetic in vitro-produced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first 10 days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 weeks. Thereafter, the cell culture was passaged at 1:10-1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n = 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measured by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.

Animals↗

PubMed2004

Association of female aging with decreased parthenogenetic activation, raised MPF, and MAPKs activities and reduced levels of glutathione S-transferases activity and thiols in mouse oocytes.

This study aims to determine in the mouse whether oocytes from reproductively old females exhibit a different susceptibility to be parthenogenetically activated when compared to oocytes from young females. At the age of 10-12 (young-female group) or 60-62 (old-female group) weeks, hybrid female mice were superovulated using pregnant mare's serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later. After removing the cumulus cells, oocytes were exposed to any of two different activating protocols: (a) 6-min exposure to 8% ethanol; and (b) treatment with 200 microM thimerosal for 15 min followed by 8 mM dithiothreitol (DTT) for 30 min. Oocytes from old female mice displayed (1) lower total percentage of parthenogenetic activation and extrusion of the second polar body after treatment with either thimerosal + DTT or ethanol; (2) higher M-phase-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) activities; and (3) lower intracytoplasmic levels of glutathione S-transferases (GSTs) activity and thiols than oocytes from young females. These data show that female aging is associated with higher resistance of oocytes to be parthenogenetically activated, higher MPF and MAPKs activities and lower intracytoplasmic levels of GSTs activity and thiols.

Aging↗

PubMed2005

Identification of maternal mRNAs in porcine parthenotes at the 2-cell stage: a comparison with the blastocyst stage.

Successful embryonic development is dependent on the temporal and stage-specific expression of appropriate genes. Currently, information on specific gene expression during early cleavage-stage embryos before zygotic gene activation (ZGA) is limited. In the present study, we compare gene expression between porcine 2-cell and blastocyst stage parthenotes to identify genes that are specifically or predominantly expressed by employing annealing control primer (ACP)-based GeneFishing PCR. Using 60 ACPs, we identified and sequenced nine differentially expressed genes (DEGs). A BLAST search revealed that cloned genes or ESTs (GDI-2, MTMR3, MKLN1, NUP88, ePAD, CIRHIM, UPF3B, ITGA2, and CGI-140) had significant sequence similarities with known genes (78-95%) of other species in the GenBank/EMBL database. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) data disclosed that these genes were regulated upstream in metaphase II (MII) oocyte, 1-cell, and 2-cell stage embryos during early pre-implantation. Similarly, upregulation was observed in MII mouse oocytes and 1-cell stage embryos before ZGA, suggesting that these nine differentially expressed orthologous genes play important roles during early cleavage before ZGA. Further analysis of the differentially expressed genes identified in this report should provide the basis for research on early cleavage and activation of the embryonic genome.

Animals↗

PubMed2005

Polyamines inhibit apoptosis in porcine parthenotes developing in vitro.

Polyamines, namely putrescine, spermidine, and spermine, are biogenic low-molecular-weight aliphatic amines which play essential roles in cell growth and proliferation. The aim of this study was to determine the effects of polyamines on the viability and development of porcine diploid parthenotes developing in vitro. The addition of 0.1 or 1.0 microM of putrescine, spermidine, or spermine, individually, to the culture medium did not enhance the development of 2-cell parthenotes to the blastocyst stage and did not change the total number of nuclei in the blastocysts. However, combined addition of these three compounds increased developmental rate to blastocyst and total cell numbers. Apoptosis in blastocyst stage parthenotes was decreased in the presence of exogenous polyamines. Real time PCR revealed that addition of polyamines to the culture media decreased the ratio of mRNA expression of Bak/Bcl-xL, Fas/Bcl-xL, and caspase 3, and enhanced mRNA expression of ornithine decarboxylase (ODC) and spermidine synthase, enzymes of polyamine biosynthesis. In the presence of L-alpha-difluoromethyl ornithine (an inhibitor of ODC) or cyclohexylamine (an inhibitor of spermidine synthase) development of porcine parthenotes decreased, apoptosis increased, and mRNA expression of the ratio of Bak/Bcl-xL and Fas/Bcl-xL, and caspase 3 increased. These results suggest that exogenous polyamines in the culture medium prevent apoptosis of porcine parthenotes and results in the net enhancement of porcine embryo viability.

Animals↗

PubMed2005

Effects of MAP kinase pathway and other factors on meiosis of Urechis unicinctus eggs.

The eggs of Urechis unicinctus Von Drasche, an echiuroid, are arrested at P-I stage in meiosis. The meiosis is reinitiated by fertilization. Immunoblotting analysis using anti-ERK2 and anti-phospho-MAPK antibodies revealed a 44 kDa MAP kinase species that was constantly expressed in U. unicinctus eggs, quickly phosphorylated after fertilization, and dephosphorylated slowly before the completion of meiosis I. Phosphorylation of the protein was not depressed by protein synthesis inhibitor Cycloheximide (CHX), but was depressed by the MEK1 inhibitor PD98059. Under PD98059 treatment, polar body extrusion was suppressed and the function of centrosome and spindle was abnormal though GVBD was not affected, indicating that MAP kinase cascade was important for meiotic division of U. unicinctus eggs. Other discovery includes: A23187 and OA could parthenogenetically activate U. unicinctus eggs and phosphorylated 44 kDa MAP kinase species, indicating that the effect of fertilization on reinitiating meiosis and phosphorylation of 44 kDa MAP kinase specie is mediated by raising intracellular free calcium and by phosphorylation of some proteins, and that phosphotase(s) sensitive to OA is responsible for arresting U. unicinctus eggs in prophase I. diC8, an activator of PKC, accelerated the process of U. unicinctus egg meiotic division after fertilization and accelerated the dephosphorylation of 44 kDa MAP kinase specie, which implied that the acceleration effect of PKC on meiotic division was mediated by inactivation of MAP kinase cascade. Elevating cAMP/PKA level in U. unicinctus eggs had no effect on meiotic division of the eggs.

1-Methyl-3-isobutylxanthine↗

PubMed2005

Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer.

Fragmentation occurs during early developmental stages of electrically activated oocytes and nuclear transfer (NT) embryos. It might contribute to the low developmental rate of porcine NT embryos. The present study was conducted to investigate whether the addition of sugars such as sorbitol or sucrose suppresses fragmentation and supports the development of electrically activated oocytes and NT embryos. The activated oocytes were cultured in Porcine Zygote Medium-3 (PZM-3) supplemented with sorbitol or sucrose for 2 days after electric activation, and then cultured in the PZM-3 for the remaining 4 days. The osmolarities of PZM-3, PZM-3 supplemented with 0.05 or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose were 269 +/- 6.31, 316 +/- 3.13, 362 +/- 4.37, and 315 +/- 5.03 mOsm, respectively. When parthenogentically activated oocytes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate were observed. Interestingly, addition of sugar to PZM-3 for 2 days reduced the fragmentation rate compared to PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 increased the fusion rate (84.2% +/- 6.07 vs. 95.1% +/- 2.52), cleavage rate (67.6% +/- 5.80 vs. 77.3% +/- 3.03), and developmental rate to the blastocyst stage (10.2% +/- 0.79 vs. 19.4% +/- 1.77). There was no significant difference between treatments for the number of the blastocysts. In addition the fragmentation rate was reduced compared to PZM-3 without sorbitol (26.1 +/- 4.30 vs. 14.5 +/- 1.74). In conclusion, increasing the osmolarity of PZM-3 through addition of either sorbitol or sucrose for 48 hr increased the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate through increasing osmolarity.

Animals↗

PubMed2005

Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments.

Previous studies mainly evaluated the effect of culture conditions on preimplantation embryo apoptosis. In order to inhibit apoptosis of nuclear transfer (NT) embryos, putative apoptosis inhibitors were used to treat donor cells. However, little is known about the effect of activation treatments on embryo apoptosis. We firstly investigated the effect of various parthenogenetic activation (PA) treatments on embryo development, blastocyst cell number, and apoptosis, and then one of these activation treatments proved to be most efficient was selected for activation rabbit NT embryos. The activation by electrical pulses and 30 min later, electroporation with 25 muM D-myoinositol 1,4,5-trisphosphate (IP3) in Ca(2+)- and Mg(2+)-free PBS, then exposure to 2.0 mM 6-dimethylaminopurine (6-DMAP) for 3 hr effectively activated rabbit oocytes, and resulted in significantly a higher blastocyst development rate (72.7%) and total cell number (175 +/- 14.1), and markedly lower apoptosis level of blastocyst (4.3 +/- 0.5) than all the other groups. When the same activation protocol was applied in NT embryo activation, we found that exposure of the embryos to 6-DMAP for 3 hr could decrease the apoptosis level of blastocyst and increase blastocyst rate and cell number. The results demonstrate that oocyte activation affects not only embryo development and quality but also embryo apoptosis.

Adenine↗

PubMed2005

Utility of ultrasound stimulation for activation of pig oocytes matured in vitro.

The present study was carried out to examine the development of pig oocytes after exposing to ultrasound under various conditions. When oocytes were exposed to ultrasound in the sorbitol medium, the blastocyst formation rate was significantly (P < 0.01) higher than that of oocytes exposed in HEPES-TLP-PVA. Optison, an echo-contrast microbubble, prevented the development into blastocysts of oocytes exposed to ultrasound in the sorbitol medium (P < 0.01). The mean number of cells in the blastocysts developed from oocytes exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that obtained by using ultrasound with 50% duty cycle. The blastocyst formation rate of oocytes exposed to ultrasound for 30 sec was significantly (P < 0.05) higher than that exposed for 10 sec. There were no significant differences in the rates of oocytes developed to the blastocyst stage and the mean numbers of cells in the blastocysts among different intensities of ultrasound. The pronuclear formation and second polar body extrusion rates of oocytes exposed to ultrasound did not differ from eclectically activated oocytes. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from oocytes activated by exposing to ultrasound was significantly (P < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the nuclear activation and parthenogenetic development of pig oocytes matured in vitro.

Albumins↗