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Immature Oocytes (immature + oocyte)
Selected AbstractsParthenogenetic Induction of Canine Oocytes by Electrical Stimulation and Ca-EDTAREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2009SR Lee Contents In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca-EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kV/cm) for 50 ,s with single DC pulse following 1 mM Ca-EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kV/cm after 1 mM Ca-EDTA treatment, the rate of pronucleus and two-cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca-EDTA treatment could cause canine oocytes to be degenerated. However, two-cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested that electrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed. [source] Increase in multidrug transport activity is associated with oocyte maturation in sea stars,DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2006Troy A. Roepke In this study, we report on the presence of efflux transporter activity before oocyte maturation in sea stars and its upregulation after maturation. This activity is similar to the multidrug resistance (MDR) activity mediated by ATP binding cassette (ABC) efflux transporters. In sea star oocytes the efflux activity, as measured by exclusion of calcein-am, increased two-fold 3 h post-maturation. Experiments using specific and non-specific dyes and inhibitors demonstrated that the increase in transporter activity involves an ABCB protein, P-glycoprotein (P-gp), and an ABCC protein similar to the MDR-associated protein (MRP)-like transporters. Western blots using an antibody directed against mammalian P-gp recognized a 45 kDa protein in sea star oocytes that increased in abundance during maturation. An antibody directed against sea urchin ABCC proteins (MRP) recognized three proteins in immature oocytes and two in mature oocytes. Experiments using inhibitors suggest that translation and microtubule function are both required for post-maturation increases in transporter activity. Immunolabeling revealed translocation of stored ABCB proteins to the plasma cell membrane during maturation, and this translocation coincided with increased transport activity. These MDR transporters serve protective roles in oocytes and eggs, as demonstrated by sensitization of the oocytes to the maturation inhibitor, vinblastine, by MRP and PGP-specific transporter inhibitors. [source] Molecular Reproduction & Development: Volume 76, Issue 11MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 11 2009Article first published online: 11 SEP 200 Accessory cells participate to the maintenance of the meiotic arrest by transferring small molecules to the oocyte through gap junctions connecting the two compartments. In fact, a loss of gap junctional communication triggers germinal vesicle breakdown in mammalian immature oocytes. Silvestre et al. (this issue) demonstrate a lack of junctional permeability between the oocyte and the follicle cells in the ascidian Ciona intestinalis oocytes using the small fluorescent dye Lucifer Yellow (pseudocolor). [source] Ion current activity and molecules modulating maturation and growth stages of ascidian (Ciona intestinalis) oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 11 2009Francesco Silvestre Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre-vitellogenic) oocytes exhibited the highest L-type Ca2+current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na+ currents that remained high during the maturation, up to the post-vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca2+ and Na+ currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin-like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians. Mol. Reprod. Dev. 76: 1084,1093, 2009. © 2009 Wiley-Liss, Inc. [source] Changes in global histone acetylation pattern in somatic cell nuclei after their transfer into oocytes at different stages of maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2008Helena Fulka Abstract In our study, we have examined the pattern of global histone modification changes in somatic cell nuclei after their transfer into mouse oocytes at different stages of maturation or after their parthenogenetic activation. While germinal vesicle (GV) staged immature oocytes are strongly labeled with anti-acetylated histone H3 and H4 antibodies, the signal is absent in both metaphase I and metaphase II oocytes (MI, MII). In contrast, the oocytes of all maturation stages show a presence of trimethylated H3/K4 in their chromatin. When somatic cells were fused to intact or enucleated GV oocytes, both the GV and the somatic cell nucleus showed a very strong signal for all the antibodies used. On the other hand, when somatic cells nuclei that are AcH3 and AcH4 positive before fusion are introduced into either intact or enucleated MI or MII oocytes, their acetylation signal decreased rapidly and was totally absent after a prolonged culture. This was not the case when anti-trimethyl H3/K4 antibody was used. The somatic cell chromatin showed only a slight decrease in the intensity of labeling after its transfer into MI or MII oocytes. This decrease was, however, evident only after a prolonged culture. These results suggest not only a relatively higher stability of the methylation modification but also some difference between the oocyte and somatic chromatin. The ability to deacetylate the chromatin of transferred somatic nuclei disappears rapidly after the oocyte activation. Our results indicate that at least some reprogramming activity appears in the oocyte cytoplasm almost immediately after GV breakdown (GVBD), and that this activity rapidly disappears after the oocyte activation. Mol. Reprod. Dev. 75: 556,564, 2008. © 2007 Wiley-Liss, Inc. [source] Expression of Apoptosis Regulatory Genes and Incidence of Apoptosis in Different Morphological Quality Groups of In Vitro-produced Bovine Pre-implantation EmbryosREPRODUCTION IN DOMESTIC ANIMALS, Issue 5 2010MG Melka Contents Apoptosis occurs during early development in both in vivo - and in vitro -produced embryos, and is considered as one of the causes of embryonic loss. The objectives of this study were, therefore, investigating stage-specific expression profiles of apoptosis regulatory genes in three quality groups of in vitro -produced bovine pre-implantation embryos; and analysing the relationship between cell number and DNA fragmentation with expressions of those genes. The relative abundance of mRNA of 9 pro- (Bax, caspase-9, Bcl-xs, P53, Caspase-3 and Fas) and anti- (Bcl-w and Mcl-1) apoptotic genes was analysed. Differential cell staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling were performed to analyse the variation in cell numbers and detect apoptotic nuclei respectively. Expression of Bax and Caspase-3 genes was significantly (p < 0.05) higher in poor quality pre-implantation embryos as compared with that of morphologically good quality embryos of the same developmental stages. Moreover, Mcl-1 expression was significantly higher in good quality immature oocytes than that in the poor quality group. Moreover, higher DNA fragmentation was evidenced in morphologically poor quality blastocysts. In conclusion, our study demonstrates that Bax, caspase-3 and Mcl-1 can be used as potential markers of embryo quality to evaluate in vitro -produced bovine embryos. Further studies are required to investigate specific molecular signatures that can be used in evaluating in vivo -derived embryos. [source] In Vitro Production of Equine Embryos: State of the ArtREPRODUCTION IN DOMESTIC ANIMALS, Issue 2010K Hinrichs Contents In vitro embryo production is possible in the horse both clinically and for research applications. Oocytes may be collected from excised ovaries post-mortem, or from either immature follicles or stimulated pre-ovulatory follicles in the live mare. In vitro maturation of immature oocytes typically yields approximately 60% mature oocytes. As standard in vitro fertilization is not yet repeatable in the horse, fertilization is performed by intracytoplasmic sperm injection. Embryo culture requires medium with high glucose, at least during blastocyst development, and rates of blastocyst development similar to those for cattle (25% to 35%) may be obtained. Pregnancy rates after transfer of in vitro -produced blastocysts are similar to those for embryos recovered ex vivo. [source] Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA MicroarrayREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010OM Kandil Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source] In Vitro Compaction of Germinal Vesicle Chromatin is Beneficial to Survival of Vitrified Cat OocytesREPRODUCTION IN DOMESTIC ANIMALS, Issue 2009P Comizzoli Contents The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8,16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes. [source] Mitochondrial Activity, Distribution and Segregation in Bovine Oocytes and in Embryos Produced in VitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2006AM Tarazona Contents Bovine oocytes and embryos produced in vitro were studied to determine the mitochondrial pattern of distribution, segregation and activity using DIOC 6 and Jc-1 fluorescence. The highest fluorescence level observed in mature oocytes was taken as 100% activity and six activity levels were estimated as follows: (1) 0%, (2) 1,15%, (3) 16,30%, (4) 31,50%, (5) 51,75% and (6) 76,100%. Three patterns of mitochondrial distribution were found: (1) diffused throughout the cytoplasm in oocytes and embryos, (2) pericytoplasmic in oocytes and embryos, and (3) perinuclear only in embryos. The segregation of mitochondria in blastomeres showed two distinct patterns: (1) symmetrical with an even mitochondrial population, and (2) asymmetrical with different numbers of mitochondria in each blastomere. In immature oocytes, mitochondrial activity was very low and the distribution was diffuse or negligible, while in mature oocytes the activity was high and the distribution was diffuse or pericytoplasmic. Competent embryos up to the 16-cell stage showed intermediate levels of activity (16,50%) but activity decreased thereafter up to the blastocyst stage. Non-competent embryos showed low levels of activity (1,15%) at all stages. These results suggest that mitochondria might play an important role during early development and that a minimum threshold of activity regulates the potential competence for reaching the blastocyst stage. [source] Improving in vitro Maturation of Oocytes in the Human Taking Lessons from Experiences in Animal SpeciesREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2001J Smitz One to three per cent of infertile women develop severe ovarian hyperstimulation syndrome after superovulation for assisted reproduction treatment (ART). This severe complication can be avoided when oocytes are obtained at an immature stage (germinal vesicle stage) out of small or medium-sized follicles. This hypothesis has been tested in several infertile women, but clinical pregnancies are disappointlingly low. This new approach in ART is still at an experimental phase and this treatment has still to be improved before routine clinical application. Experimental work in animals and humans suggest a beneficial effect in providing a short preliminary pretreatment with follicle-stimulating hormone to select for a developing cohort of follicles. The aspiration of oocyte cumulus complexes is carried out with a short needle applying reduced aspiration pressure. A crucial point is to provide the appropriate culture environment for the immature oocytes. An optimal cumulus-enclosed human oocyte culture system needs to be defined. The composition of the culture medium could be suggested by in vitro work carried out in animal models. As developmental competence is established during the latest phases of oocyte growth and is dependent on the storage of RNA, a prolonged in vitro maturation period (before inducing nuclear maturation) could provide the necessary transcriptional and translational changes. The conditions to achieve this improved cytoplasmic maturation by prolonging the in vitro culture remain to be defined. More objective noninvasive parameters for oocyte maturity are also needed to pursue research in this field. [source] Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytesANIMAL SCIENCE JOURNAL, Issue 4 2010Yukio NISHIMURA ABSTRACT Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic-polyadenylation-element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT-PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant-negative mutant pCPEB1 (AA-CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA-CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A. [source] Block to DNA replication in meiotic maturation: a unified view for a robust arrest of cell cycle in oocytes and somatic cellsBIOESSAYS, Issue 4 2003Yumiko Kubota Under certain conditions, the cell cycle can be arrested for a long period of time. Vertebrate oocytes are arrested at G2 phase, while somatic cells arrest at G0 phase. In both cells, nuclei have lost the ability to initiate DNA synthesis. In a pair of recently published papers,1,2 Méchali and colleagues and Coué and colleagues have clarified how frog oocytes prevent untimely DNA synthesis during the long G2 arrest. Intriguingly, they found only Cdc6 is responsible for the inability of immature oocytes to replicate DNA. Cdc6 is a key component for replication licensing, and for G0 cells to re-enter the proliferative stage. Strikingly similar strategies for preventing the untimely replication in both cells suggest that the suppression of replication licensing is a universal mechanism for securing the prolonged arrest of the cell cycle. BioEssays 25:313,316, 2003. © 2003 Wiley Periodicals, Inc. [source] | ||||||||||