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PubMed2006

Developmental competence of porcine parthenogenetic embryos relative to embryonic chromosomal abnormalities.

Parthenogenetically activated (PA) embryos exhibit delayed development, a lower blastocyst rate, and less successful development in vitro compared to in vitro fertilized (IVF) embryos. To investigate the possible mechanisms for unsuccessful parthenogenetic development, this study analyzed the chromosome abnormalities and developmental potential of porcine PA embryos. Mature oocytes were electrically activated and cultured in Porcine Zygote Medium-3 (PZM3) supplemented with 3 mg/ml BSA for 6, 7, or 8 days. The percentage of PA blastocysts was lower than that of IVF embryos on days 6 and 7 (16.4 +/- 7.4 vs. 28.7 +/- 3.7; 10.9 +/- 2.8 vs. 21.5 +/- 4.7, P < 0.05; respectively), and the PA blastocysts had significantly fewer nuclei than IVF blastocysts (23.2 +/- 1.8 vs. 29.7 +/- 0.8; 29.7 +/- 3.3 vs. 32.0 +/- 2.4, P < 0.05). The percentage of abnormal PA embryos (including embryos with condensed nuclei, arrested embryos and fragmented embryos) was higher than that of IVF embryos (PA: 52.9 +/- 12.8 vs. 16.4 +/- 7.4 on day 6), and increased with culture time (71.9 +/- 12.1 vs. 10.9 +/- 2.8. on day 7,and 75.0 +/- 22.6 vs. 12.1 +/- 2.3 on day 8, P < 0.05). The Day-6 PA blastocysts (n = 147) were divided into three classes according to the total number of nuclei (<20, 20-39, >40) and into three groups according to the morphological diameter (<150, 150-180, >180 microm). Of the haploid blastocysts, 56.1% had less than 20 nuclei, and 71.5% were less than 150 microm in diameter. Of all (114) blastocysts suitable for analysis, 55.5% displayed chromosomal abnormalities. Among chromosomal abnormalities in PA blastocysts, haploid blastocysts were most prevalent (43.6%), while polyploidy (4.4%) and mixoploidy (7.7%) embryos were less prevalent. Chromosomal abnormalities of porcine PA embryos might contribute to a higher rate of abnormal embryonic development. We suggest that a careful consideration should be given when using the blastocysts with smaller size, and establishing the optimum culture condition for PA embryos development in vitro.

Animals↗
PubMed2006

The effect of 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and nuclear transfer embryos.

The effects of activation by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) on the development and chromosomal complement of sheep parthenogenetic and SCNT embryos were investigated. The results revealed that the blastocyst development of parthenogenetic embryos was significantly higher (P < 0.05) in 6-DMAP activated oocytes, compared to those activated with CHX (21.0 +/- 0.9 vs. 14.9 +/- 0.5, respectively). In contrast, the blastocyst frequencies did not significantly differ (P > 0.05) between the two activation treatment groups for SCNT embryos. The 6-DMAP or CHX treatment did not result in any significant difference in the blastocyst total cell number in either parthenote or SCNT embryos. The chromosomal analysis revealed that all the parthenogenetic embryos (100.0%) derived from 6-DMAP treatment, were chromosomally abnormal whereas in CHX-treated embryos, it was significantly lowered (93.6%, P < 0.05). Conversely, the proportions of chromosomally abnormal SCNT embryos did not significantly differ (P > 0.05) among the 6-DMAP and CHX- treated embryo groups (60.0% vs. 56.2%, respectively). This study demonstrated that oocyte activation agents such as DMAP and CHX have differing effects on meiotic or mitotic nuclei. The study also highlighted the feasibility of using bovine X and Y chromosome specific painting probes in sheep embryos.

Adenine↗
PubMed2005

Inactivation of MPF and MAP kinase by single electrical stimulus for parthenogenetic development of porcine oocytes.

This study was conducted to examine the activities of maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase in the porcine oocytes after artificial activation. To determine optimal electrical activation condition, oocytes were exposed to single DC pulse in a variety of electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 microsec). After the artificial activation, 40-50 oocytes were cultured in a 50 microl drop of NCSU23 medium supplemented with 0.4% BSA at 39 degrees C, 5% CO2 in air for 6 days. No difference was detected in the preimplantation development of pocine oocytes and the mean nuclei number of blastocysts between electric field strengths. Under the 180 V/mm electric field strength, short pulse durations (15 and 30 microsec) showed a higher preimplantation developmental rate of the oocytes and mean nuclei number of blastocysts than an extended electric pulse (60 microsec) (P < 0.05). Single electrical stimulus (180 V/mm, 15 microsec) resulted in higher preimplantation development of porcine oocytes as compared to other chemical stimulators (P < 0.01). Western blot analyses showed the decrease of MPF and MAP kinase in the electrically-activated oocytes. After single electrical stimulus, the amounts of both cdc2 and ERK in porcine oocytes were remarkably reduced by 4 hr and then further decreased by 8 hr. However, the chemically-stimulated oocytes did not show any significant change at the levels of MPF and MAP kinase. Our results indicate that the optimal single electrical pulse is effective on the inactivation of MPF and MAP kinase, eventually leading to the parthenogenetic development of porcine oocytes.

Animals↗
PubMed2006

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.

Animals↗
PubMed2006

Effects of amino acid substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on fertilization and parthenogenetic development in mature bovine oocytes.

Integrins have been shown to be involved in the process of fertilization and many integrin-ligand interactions are mediated through the recognition of an arginine-glycine-aspartic acid (RGD) sequence. Despite the fact the RGD domain is a principal player in determining the functional characteristics of an adhesive protein, increasing evidence has accumulated implicating the amino acids flanking the RGD sequence in determining the functional properties of the RGD-containing protein. A set of linear peptides in which the amino acid sequence in and around the RGD tri-peptide was modified was synthesized to better understand the specificity of the RGD-receptor interaction. Mature oocytes were fertilized in vitro in the presence of RGD-containing and RGD-modified peptides. Both the RGD-containing and RGD-modified peptides impaired the ability of sperm to fertilize bovine oocytes, illustrated by a reduction in cleavage. The linear modified RGD containing peptides were also examined for their ability to induce parthenogenetic development with the objective of providing a linear RGD peptide with greater biological activity than the one (GRGDSPK) used previously (Campbell et al., 2000). The data demonstrate the specificity of the receptor for the RGD sequence, further implicate the involvement of integrins in the process of bovine fertilization, and illustrate the importance of the amino acids surrounding the RGD sequence in determining the binding and functional properties of RGD-containing peptides. The data support the findings that a linear RGD peptide can block fertilization and that amino acids around the RGD sequence have an impact on the biological activity of the receptor.

Amino Acid Substitution↗
PubMed2007

Effects of arginine-glycine-aspartic acid (RGD) containing snake venom peptides on parthenogenetic development and in vitro fertilization of bovine oocytes.

The ability of synthetic arginine-glycine-aspartic acid (RGD)-containing peptides to induce intracellular calcium transients similar to those observed at fertilization by spermatozoa in the bovine has been reported (Campbell et al., 2000: Biol Reprod 62:1702-1709; Sessions et al., 2006. Mol Reprod Dev). These results also indicated the ability of synthetic RGD-containing peptides to induce activation and subsequent parthenogenetic development to the blastocyst stage, although, at numbers lower than observed with control in vitro fertilization (IVF). Evidence has been provided indicating the important effect of surrounding regions on the biological activity of the RGD sequence (Zhu and Evans, 2002; Sessions et al., 2006). The current experiments were designed to use natural RGD-containing sequences (disintegrins) to understand their effects. A total of three RGD-containing snake venom peptides (Kistrin (K), Elegantin (Ele), and Echistatin (Ech)) and one nonRGD-containing venom (Erabutoxin B (EB; control) were used at three concentrations (0.1, 1, and 10 micro g /ml) to induce parthenogenetic development to the blastocyst stage and in conjunction (1.0, 5.0, and 10 micro g/ml) with spermatozoa to evaluate competitive inhibition of fertilization and subsequent development. A (P < 0.01) higher number of bovine oocytes developed to the blastocyst stage after incubation with K, Ele and Ech at 1.0 micro g/ml, and was not different (P > 0.01) from IVF control. Fertilization was significantly reduced (P < 0.01) at all concentrations of K, Ele and Ech as compared to IVF control. No reduction (P > 0.05) was observed in EB (nonRGD) treated oocytes. These results support the involvement of a disintegrin-integrin interaction at fertilization in the bovine resulting in activation and subsequent development.

Amino Acid Sequence↗
PubMed2006

Developmental potential of aged oocyte rescued by nuclear transfer following parthenogenetic activation and in vitro fertilization.

Mouse oocyte aged in vitro cannot develop normally following activation. To investigate the roles of nucleus or cytoplasm elements in oocyte aged in vitro process and their subsequent development capability following activation, we reconstructed oocytes with MII chromosome spindle and cytoplasm from aged and fresh oocytes by nuclear transfer. The subsequent developmental potential after parthenogenetic activation (PA) or in vitro fertilization (IVF) was evaluated. After nuclear transfer, more than 75.6% of karyoplast and cytoplast pairs can be fused and reconstructed oocytes have a normal haploid karyotype. Following PA, aged oocytes cannot develop beyond four-cell stage, reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with a low percentage (9.1%). Instead, blastocyst formation rate of reconstructed oocyte from aged nucleus and fresh cytoplasm was higher (60.0%). Following IVF, zygote with diploid karyotype can be formed from zona pellucida (ZP)-free oocyte. After cultured in vitro, aged oocytes cannot develop beyond two-cell; reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with low percentage (15.0%). However, high blastocyst formation rate (86.2%) can be obtained from reconstructed oocytes from aged nucleus and fresh cytoplasm. Furthermore, after embryo transfer, three viable pups have been obtained, although the efficiency is very low. These observation demonstrated that cytoplasm is more crucial than nucleus to aging process. Fresh cytoplasm could partly rescue nucleus susceptibility to apoptosis from aging in vitro.

Animals↗
PubMed2007

Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin.

In vitro production of porcine embryos has become routine in most laboratories but the yield and quality of the resultant blastocysts remain suboptimal. Phytohemagglutinin (PHA) is an N-acetylgalactosamine/galactose sugar-specific lectin with a wide variety of biological activities including mitogenesis, mediation of cell recognition, and agglutination of cells. This study was therefore, designed to investigate the effect of PHA on the preimplantation embryo development and quality of in vitro produced porcine parthenotes. Parthenogenetic presumptive diploid zygotes were produced in vitro by electrical activation and cultured in the absence or presence of PHA at different concentrations (0, 5, 10, 15, 20 microg/ml). There were no significant differences in the cleavage rate of porcine parthenotes in control and treatment groups at all tested concentrations of PHA (P < 0.05). However, supplementation of PHA at the concentration of 15 microg/ml significantly improved the blastocyst rate (68.9 +/- 1.5% vs. 43.1 +/- 4.1%), hatching rate (25.8 +/- 3.1% vs. 8.9 +/- 2.0%), and total nuclei number (95.5 +/- 9.3 vs. 63.4 +/- 4.3) when compared to control group (P > 0.05). TUNEL labeling revealed that blastocysts in PHA group were less predisposed to biochemical apoptosis than in control group while total apoptosis and nuclear fragmentation remained unaltered. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis further revealed that PHA decreased the expression ratio of BAX/BCL-XL and enhanced the relative abundance of IGF2 transcripts. Therefore, our study suggests that PHA improves the blastocyst yield and quality by enhancing blastocyst expansion, hatching, and total cell number and decreasing the apoptosis by positively modulating the expression of embryo survival related genes.

Animals↗
PubMed2007

Pronuclear stage porcine embryos do not possess a strict asymmetric distribution of lysine 9 dimethylation of histone H3 based solely on parental origin.

Epigenetic modification of core histone proteins plays an important role in gene transcription and chromatin structure. It is known that the methylation status of the lysine 9 residue of histone H3 (H3K9) is closely linked with repression of transcription and heterochromatin formation. It has been reported that a strict asymmetric distribution of the dimethylated form of H3K9 exists between the two parental genomes in murine embryos. The aim of this study was to characterize the H3K9 dimethylation pattern in cleavage stage porcine embryos. Indirect immunocytochemical analysis showed that this modification is present throughout the nuclei of pronuclear, 2-cell, 4-cell and blastocyst stage porcine embryos. However, at the pronuclear stage the pronuclei within in vitro-fertilized porcine embryos possessed an asymmetric distribution of the dimethylated form of H3K9; this distribution was not based solely on the parent-of-origin. These results show that dimethylation of H3K9 is differentially regulated in pronuclear stage porcine embryos.

Androgens↗
PubMed2007

Serial nuclear transfer improves the developmental potential of mouse embryos cloned from oocytes matured in a protein-free medium.

Germinal vesicle (GV) oocytes matured in vitro are an alternative source for cytoplasmic recipients of nuclear transfer (NT). However, the developmental potential of oocytes matured in vitro is limited. In this study, we developed a protein-free maturation medium for mouse GV oocytes. Following parthenogenetic activation, the oocytes matured in the protein-free medium develop to blastocyst stage with a high efficiency, even up to the rate obtained from in vivo MII-oocytes (90.6% vs. 92.8%). Using the oocytes matured in the protein-free medium as the recipient, NT embryos develop to the blastocyst stage (17.6%). To further improve the developmental potential of NT embryos, we performed serial NT and compared the effect of three different activated cytoplasm samples derived from in vitro matured oocytes as the second recipient, that is, the effect of in vitro fertilized (IVF) zygote, the preactivated cytoplast and the IVF cytoplast, on the development of NT embryos. We found that when the pronucleus of NT zygote was transferred into the cytoplasm of the IVF zygote, the blastocyst formation increased to 39.4%. This is the first report to demonstrate the IVF zygote from oocytes matured in protein-free medium can be used successfully as the recipient for serial NT to enhance the developmental potential of mouse NT embryos from oocytes matured in the protein-free medium.

Animals↗
PubMed2007

Heat stress-induced apoptosis in porcine in vitro fertilized and parthenogenetic preimplantation-stage embryos.

Decades worth of research have consistently shown the adverse effects of elevated temperatures on reproductive parameters of livestock species. The objective of this study was to evaluate the developmental and apoptotic responses of porcine in vitro fertilized (IVF) and parthenogenetically activated (PA) embryos heat stressed at the late 1-cell stage. Embryos were heat stressed (HS) at 42 degrees C for 9 hr starting 22 hr after insemination or artificial activation stimulus. Non heat-stressed (NHS) control embryos were maintained at 39 degrees C for the duration of the experiments. TUNEL staining on Day 5 of development demonstrated that heat stress elicited a significant apoptotic response in IVF embryos (45.6% of HS embryos and 26.7% of NHS embryos were apoptotic; P<0.05), but not in PA embryos (51.1% and 39.9% for HS and NHS embryos, respectively; P>0.1). And, while IVF embryos were highly susceptible to heat-induced developmental perturbations (20.6% and 8.8% development to blastocyst for NHS and HS embryos, respectively; P<0.05), elevated temperatures did not affect blastocyst rates in PA embryos (22.2% for NHS PA embryos and 21.2% for HS PA embryos; P>0.1). These findings indicate that, as in other systems studied, IVF pig embryos are directly affected adversely by heat stress conditions. Parthenogenetic embryos, though, appear to be surprisingly tolerant of the elevated temperatures. The differences between IVF and PA embryos in their response to heat stress warrants further investigation.

Animals↗
PubMed2004

Species differences in the regulation of tyrosine hydroxylase in Cnemidophorus whiptail lizards.

Evolution of behavioral phenotype involves changes in the underlying neural substrates. Cnemidophorus whiptail lizards enable the study of behavioral and neural evolution because ancestral species involved in producing unisexual, hybrid species still exist. Catecholaminergic systems modulate the expression of social behaviors in a number of vertebrates, including whiptails, and therefore we investigated how changes in catecholamine production correlated with evolutionary changes in behavioral phenotype by measuring the size and number of catecholamine producing (tyrosine hydroxylase-immunoreactive, or TH-ir) cells across the reproductive cycle in females from two related whiptail species. Cnemidophorusuniparens is a triploid, parthenogenetic species that arose from hybridization events involving the diploid, sexual species C. inornatus. Prior to ovulation, females from both species display femalelike receptive behaviors. However, after ovulation, only parthenogenetic individuals display malelike mounting behavior. In all nuclei measured, we found larger TH-ir cells in the parthenogen, a difference consistent with species differences in ploidy. In contrast, species differences in the number of TH-ir cells were nucleus specific. In the preoptic area and anterior hypothalamus, parthenogens had fewer TH-ir cells than females of the sexual species. Reproductive state only affected TH-ir cell number in the substantia nigra pars compacta (SNpc), and C. uniparens individuals had more TH-ir cells after ovulation than when previtellogenic. Thus, species differences over the reproductive cycle in the SNpc are correlated with species differences in behavior, and it appears that the process of speciation may have produced a novel neural and behavioral phenotype in the parthenogen.

Animals↗
PubMed2007

Parthenium hysterophorus induced changes in neurotransmitter levels in mouse brain.

Parthenium hysterophorus (10 mg/0.1 ml/kg body weight) was administered daily to 20 adult male mice and levels of 5-hydroxytryptamine, noradrenaline and dopamine in total brain were quantified on days 31 and 61 after administration. All neurotransmitter levels were decreased significantly, with the effects more prominent in the later part of the experiment. The results suggest that Parthenium hysterophorus interferes in the functioning of the hypothalamo-hypophyseal axis by impairing the biogenic amine levels which may in turn affect the physiology of the peripheral endocrine glands.

Animals↗
PubMed1999

A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation.

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Early skeletal muscle development proceeds normally in parthenogenetic mouse embryos.

In mouse chimeras with parthenogenetic cell contribution, the skeletal musculature appears to be largely devoid of parthenogenetically derived cells. To analyze the appearance and early distribution of myotomal cells in parthenotes, we determined the expression of the muscle-specific transcription factors myogenin, MYF-5, and MYF-6 by in situ hybridization in somites of Day 10 and 11 embryos. Here, we report that these myogenic regulatory proteins are expressed in parthenogenetic animals together with desmin, one of the early muscle-specific structural proteins. We also show that parthenogenetic cells contribute equally to dermatome, sclerotome, and myotome in Day 10 and 11 chimeras. These results suggest that early myotomal cells expressing the myogenic control proteins develop and allocate normally in parthenogenetic embryos and in parthenogenetic<==>normal chimeras. The underrepresentation in older chimeras may therefore be due to selective elimination. These data also argue against imprinting of the myogenic factor genes myogenin, Myf-5, and Myf-6.

Animals↗
PubMed1994

Inhibition of protein kinases after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion.

We have examined the response of bovine oocytes matured in vitro for 24 hr to parthenogenic activation using compounds that increase intracellular calcium (ionomycin) or inhibit protein phosphorylation (6-dimethylaminopurine, DMAP). Treatment with ionomycin alone caused resumption of meiosis (57.8 +/- 7.8%) but not pronuclear formation (8.9 +/- 7.3%). DMAP alone did not cause resumption of meiosis or pronuclear formation. Sequential treatment with ionomycin (5 microM for 4 min) immediately followed by DMAP (1.9 mM for 5 hr) resulted in activation that led to pronuclear formation (80.5 +/- 13.1%). Completion of meiosis, however, was bypassed as evidenced by only one polar body and one pronucleus present in activated parthenogenones. It was necessary to incubate the oocytes for at least 3 hr in DMAP to obtain high rates of activation (76.6 +/- 9.8%) and development to blastocysts (21.1 +/- 1.5%). Temporal separation of the two treatments resulted in a decrease in oocytes with one pronucleus and one polar body (uniformly diploid parthenogenones) and an increase in a mixture of diploid and haploid parthenogenones since DMAP was capable of causing transition to interphase of all chromatin configurations after anaphase commenced and prior to metaphase arrest. Parthenotes produced with ionomycin and DMAP that developed to the blastocyst stage had high cell numbers (70 to 88 cells) and were able to cause extended cycles in 33.3% of recipient cattle after nonsurgical transfer to the uterus. Response of the bovine oocyte arrested in metaphase II to different activation stimuli was also found to show age-dependent changes in pattern of activation response and developmental competence.

Adenine↗