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Oocyte Maturation (oocyte + maturation)
Selected AbstractsConsequences of Nitric Oxide Synthase Inhibition During Bovine Oocyte Maturation on Meiosis and Embryo DevelopmentREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010KRL Schwarz Contents The importance of nitric oxide synthase (NOS) in bovine oocyte maturation was investigated. Oocytes were in vitro matured with the NOS inhibitor Nw - l -nitro-arginine methyl-ester (10,7, 10,5 and 10,3 m l -NAME) and metaphase II (MII) rates and embryo development and quality were assessed. The effect of l -NAME (10,7 m) during pre-maturation and/or maturation on embryo development and quality was also assessed. l -NAME decreased MII rates (78,82%, p < 0.05) when compared with controls without l -NAME (96%). Cleavage (77,88%, p > 0.05), Day 7 blastocyst rates (34,42%, p > 0.05) and total cell numbers in blastocysts were similar for all groups (146,171 cells, p > 0.05). Day 8 blastocyst TUNEL positive cells (3,4 cells) increased with l -NAME treatment (p < 0.05). For oocytes cultured with l -NAME during pre-maturation and/or maturation, Day 8 blastocyst development (26,34%) and Day 9 hatching rates (15,22%) were similar (p > 0.05) to controls pre-matured and matured without NOS inhibition (33 and 18%, respectively), while total cell numbers (Day 9 hatched blastocysts) increased (264,324 cells, p < 0.05) when compared with the controls (191 cells). TUNEL positive cells increased when NOS was inhibited only during the maturation period (8 cells, p < 0.05) when compared with the other groups (3,4 cells). NO may be involved in meiosis progression to MII and its deficiency during maturation increases apoptosis in embryos produced in vitro. Nitric oxide synthase inhibition during pre-maturation and/or maturation affects embryo quality. [source] Cryotolerance of Bovine Blastocysts is Affected by Oocyte Maturation in Media Containing Palmitic or Stearic AcidREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2009MA Shehab-El-Deen Contents In this study, non-esterified fatty acids (NEFAs) were added during in vitro maturation at concentrations measured previously in follicular fluid (FF) of high-producing dairy cows in a negative energy status to evaluate their subsequent effect on the embryos cryotolerance. Oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of NEFAs dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in synthetic oviduct fluid medium supplemented with 5% FCS. Embryos that had at least reached the blastocyst stage were vitrified by open pulled straw (OPS) vitrification. Addition of palmitic (C16 : 0) or stearic acid (C18 : 0) during oocyte maturation had significant negative effects on embryo cryotolerance, whereas ethanol or oleic acid (C18 : 1) had no effect. These in vitro results suggest that high NEFA concentrations in FF during a period of negative energy balance in high-yielding dairy cows can have carry-over effects on embryo quality. [source] Actions of Tumor Necrosis Factor-, on Oocyte Maturation and Embryonic Development in Cattle,AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 5 2003P. Soto Problem:, Infertility can accompany mastitis in cattle. Involvement of tumor necrosis factor- , (TNF- ,) in this phenomenon is suggested by observations that circulating concentrations of TNF- , are elevated after intramammary infection or infusion of endotoxin. It was hypothesized that (1) TNF- , acts on the oocyte during maturation to decrease the percent of oocytes that cleave and develop following fertilization; (2) exposure of embryos to TNF- , after fertilization reduces development to the blastocyst stage; and (3) TNF- , increases the proportion of blastomeres that undergo apoptosis in a stage-of-development dependent manner. Method of study:, In one experiment, oocytes were matured with various concentrations of TNF- , and then fertilized and cultured without TNF- ,. In another study, embryos were cultured with TNF- , for 8 days beginning after fertilization. Finally, embryos were collected at the two or four-cell stage (at 28,30 hr after insemination) or when ,9-cells (at day 4 after insemination) and cultured ± TNF- , for 24 hr. The proportion of blastomeres undergoing apoptosis was then determined by the TUNEL procedure. Results:, Addition of TNF- , to maturation medium did not affect the proportion of oocytes that cleaved. However, the percent of oocytes that developed to the blastocyst stage at day 8 after insemination was reduced (P = 0.05) at all TNF- , concentrations tested (0.1,100 ng/mL). When added during embryo culture, there was no significant effect of TNF- , on the proportion of oocytes that became blastocysts. In addition, TNF- , did not induce apoptosis in two and four-cell embryos. For embryos ,9-cells, however, 10 and 100 ng/mL TNF- , increased (P < 0.05) the percent of blastomeres labeling as TUNEL-positive. Conclusion:, TNF- , can have deleterious actions on oocyte maturation that compromise development of the resultant embryo. While exposure of fertilized embryos to TNF- , did not inhibit development to the blastocyst stage, TNF- , increased the percentage of blastomeres undergoing apoptosis when exposure occurred for embryos ,9-cells. Increased blastomere apoptosis could conceivably compromise subsequent embryo survival. [source] Parthenogenetic 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] Regulation of oocyte maturation in fishDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2008Yoshitaka Nagahama A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17,, 20,-dihydroxy-4-pregnen-3-one, 17,, 20,-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17,,20,-DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17,,20,-DP in response to LH is correlated with decreases in P450c17 (P450c17-I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17-II) and 20,-hydroxysteroid dehydrogenase (20,-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17,, 20,-DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH2 terminus at lysine 57. [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] Altered gene expression in the brain and ovaries of zebrafish (Danio Rerio) exposed to the aromatase inhibitor fadrozole: Microarray analysis and hypothesis generation,,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2009Daniel L. Villeneuve Abstract As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 ,g/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 ,g/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors. [source] Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1GENES TO CELLS, Issue 7 2005Takashi Sasayama Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis. [source] Reproductive status in females of the Brazilian catfish, Pseudoplatystoma fasciatum reared in cagesJOURNAL OF APPLIED ICHTHYOLOGY, Issue 5 2010E. Romagosa Summary The distinctive morphological features of the ovaries the ,cachara', Pseudoplatystoma fasciatum were characterized macroscopically, and by histology, when reared in cages, from March 2005 to February 2006. Forty eight females (mean total weight = 2.7 kg, mean standard length = 65.1 cm) were allocated to four cages of 2.7 m3 (20 fish/cage) which were installed in four 600 m2 ponds, located at the IP, Pariquera-Açu, São Paulo, Brazil. The monthly, samples were fixed in 4%-buffered formalin before preparation for histological examination, ovaries were removed and weighted. The gonadosomatic index (GSI) was calculated as = 100 × weight ovaries/total fish weight. The ovaries are the cystovarian type and macroscopically, were established three stages of ovarian maturation: Resting, developing Maturation (initial, intermediate, final) and Regression (initial, intermediate, final). Based on morphological criteria of those ovaries, the oocyte development has been divided into distinct stages: (i) oocyte growth (vitellogenesis); (ii) oocyte maturation, along which it goes through different phases of development, before (iii) ovulation and, (iv) spawning. When the P. fasciatum were kept in confinement and not induced to breed occurs fail to attain final oocyte maturation, start the process of degeneration. Consequently, the weight started to decline and 45% of the ovaries showed atresia of vitellogenic follicles. This was considered indicative of a recent cessation of the reproductive activity. Such failure could have been caused by stress of the monthly sampling involving a certain degree of disturbance, and perhaps also by the existence of stressors while in captivity. The synchronous ovary contained oocytes in an unique stage of development and had potential to perform total spawning up to one time a year, with the period reproductive beginning in the end of November to the beginning of February, coinciding with the highest water temperatures in the experimental cages (29.0,31.5°C) and the increase of mean values of GSI. During the regression phase, residual oocytes could be observed together with decrease of the mean values of GSI and, the temperatures. [source] Modulation of O-GlcNAc glycosylation during Xenopus oocyte maturation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2004Tony Lefebvre Abstract O-linked N -acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which is believed antagonises phosphorylation. We have studied the O-GlcNAc level during Xenopus oocyte meiotic resumption, taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation of immature stage VI oocytes using progesterone was followed by a 4.51,±,0.32 fold increase in the GlcNAc content, concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase of O-GlcNAc for the 97 kDa protein, which we identified as ,-catenin was partly related to its accumulation during maturation, as was demonstrated by the use of the protein synthesis inhibitor,cycloheximide. Microinjection of free GlcNAc, which inhibits O-glycosylated proteins,lectins interactions, delayed the progesterone-induced maturation without affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate protein,protein interactions required for the cell cycle kinetic. © 2004 Wiley-Liss, Inc. [source] Diel spawning periodicity of red snapper Lutjanus campechanus in the northern Gulf of MexicoJOURNAL OF FISH BIOLOGY, Issue 3 2006M. W. Jackson Ovaries of red snapper Lutjanus campechanus were examined histologically to determine rates of oocyte maturation, diel spawning periodicity and whether lunar cycle influenced spawning rhythm. Hydration of red snapper oocytes began during the mid-morning hours; c. 5 h was necessary for oocytes to become fully hydrated and ovulation occurred no more than 5 h after oocytes attained full hydration. Appearance of fresh postovulatory follicles after 1330 hours and the absence of hydrated oocytes after 1830 hours signified that red snapper spawning occurred during this 5 h period. In addition, evidence of a peak in spawning was seen near 1600 hours. Postovulatory follicles degenerated within a 24 h time period. A lunar spawning cycle was not evident. [source] Insect gonadotropic peptide hormones: some recent developmentsJOURNAL OF PEPTIDE SCIENCE, Issue 1 2007Mariola Kuczer Abstract Gonadotropic peptides are a new generation of peptide hormone regulators of insect reproduction. They have been isolated from ovaries, oviducts, or brains of insects. The subject of this paper is insect peptides that exert stimulatory or inhibitory effects on ovarian development and oocyte maturation. On the basis of the literature data and the results of our investigations, the structure and biological properties of different groups of peptides are presented. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] Behaviors of ATP-dependent chromatin remodeling factors during maturation of bovine oocytes in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2010Gabbine Wee The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1,hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126,135, 2010. © 2009 Wiley-Liss, Inc. [source] Molecular characterization and polyadenylation-regulated expression of cyclin B1 and Cdc2 in porcine oocytes and early parthenotesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2010Ding-Xiao Zhang Meiotic maturation of mammalian oocytes is controlled by the maturation/M-phase promotion factor (MPF), a complex of Cdc2 kinase and cyclin B protein. To better understand the molecular mechanism of oocyte maturation, we characterized porcine cyclin B1 and Cdc2 genes, both of which are widely expressed in pig tissues. We further analyzed their expression profiles during in vitro maturation of pig oocyte and early embryonic development at both the mRNA and protein level. Two isoforms of cyclin B1, comprising the same open reading frame but differing in 3,-UTR length, were identified. Cyclin B1 transcripts was up-regulated after 30,hr of maturation, while Cdc2 mRNA levels were unchanged during maturation except for a sharp decline at 44,hr. Cyclin B1 protein synthesis increased with oocyte maturation. Cdc2 protein expression was relatively low during 0,18,hr, followed by a higher level of expression up to 44,hr of maturation. Poly(A)-test PCR clearly revealed that both cyclin B1 isoforms underwent cytoplasmic polyadenylation starting around 18,24,hr during maturation, while a substantial de-adenylation and degradation of Cdc2 isoforms were observed in metaphase II oocytes and during embryo development after parthenogenetic activation. Porcine MII oocytes derived from small follicles (,3,mm) and bad quality 2-cell parthenotes showed lower developmental competence and lower levels of cyclin B1 protein, and Cdc2 mRNA or both gene mRNAs, respectively, compared to their control counterparts. These results suggested that cyclin B1 was regulated posttranscriptionally by cytoplasmic polyadenylation during porcine oocyte maturation. Further, the decreased expression of maternal cyclin B1 and Cdc2 at the mRNA or protein level in developmentally incompetent oocytes and embryos was responsible for, at least in part, a profound defect in further embryonic development. Mol. Reprod. Dev. 77: 38,50, 2010. © 2009 Wiley-Liss, Inc. [source] Fatty acid oxidation and meiotic resumption in mouse oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2009Stephen M. Downs We have examined the potential role of fatty acid oxidation (FAO) in AMP-activated protein kinase (AMPK)-induced meiotic maturation. Etomoxir and malonyl CoA, two inhibitors of carnitine palmitoyl transferase-1 (CPT1), and thus FAO, blocked meiotic induction in dbcAMP-arrested cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) by the AMPK activator, AICAR. C75, an activator of CPT1 and FAO, stimulated meiotic resumption in CEO and DO. This effect was insensitive to the AMPK inhibitor, compound C, indicating an action downstream of AMPK. Palmitic acid or carnitine also promoted meiotic resumption in DO in the presence of AICAR. Since C75 also suppresses the activity of fatty acid synthase (FAS), we tested another FAS inhibitor, cerulenin. Cerulenin stimulated maturation in arrested oocytes, but to a lesser extent, exhibited significantly slower kinetics and was effective in CEO but not DO. Moreover, etomoxir completely blocked C75-induced maturation but was ineffective in cerulenin-treated oocytes, suggesting that the meiosis-inducing action of C75 is through activation of FAO within the oocyte, while that of cerulenin is independent of FAO and acts within the cumulus cells. Finally, we determined that long chain, but not short chain, fatty acyl carnitine derivatives were stimulatory to oocyte maturation. Palmitoyl carnitine stimulated maturation in both CEO and DO, with rapid kinetics in DO; this effect was blocked by mercaptoacetate, a downstream inhibitor of FAO. These results indicate that activation of AMPK stimulates meiotic resumption in mouse oocytes by eliminating a block to FAO. Mol. Reprod. Dev. 76: 844,853, 2009. © 2009 Wiley-Liss, Inc. [source] Factors affecting the in vitro action of cumulus cells on the maturing mouse oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2008Li Ge Abstract The removal of cumulus cells (CCs) from oocytes at the germinal vesicle (GV) stage still represents a major limitation in such embryo techniques as GV transfer, somatic cell haploidization, and oocyte cryopreservation. However, no efficient in vitro maturation (IVM) system for CC-denuded oocytes (DOs) has been established in mammalian species. Although follicular cells are considered to play an important role in oocyte maturation, the specific role and mechanisms of action of different cell types are poorly understood. Reports on whether junctional association between CCs and the oocyte is essential for the beneficial effect of CC co-culture on oocyte maturation are in conflict. Our objective was to try to address these issues using the mouse oocyte model. The results indicated that while co-culture with the CC monolayer could only partially restore the developmental potential of DOs without corona cells, it restored the competence of corona-enclosed DOs completely. Culture in medium conditioned with CC monolayer also promoted maturation of DOs. However, co-culture with the monolayer of mural granulosa cells had no effect. The efficiency of CC co-culture was affected by various factors such as density and age of the CCs, the presence of gonadotropin in the maturation medium and the duration for in vivo (IVO) gonadotropin priming. It is concluded that mouse CCs produce a diffusible factor(s) that support DO maturation in a CC-oocyte junctional communication dependent manner. The data will contribute to our understanding the mechanisms by which CCs promote oocyte maturation and to the establishment of an efficient DO IVM system. Mol. Reprod. Dev. 75: 136,142, 2008. © 2007 Wiley-Liss, Inc. [source] Reduced oxygen concentration improves the developmental competence of mouse oocytes following in vitro maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2007Kimberly A. Preis Abstract Reduced atmospheric oxygen concentration is beneficial to embryo development; however, optimal oxygen concentration for oocyte maturation remains undetermined. Likewise, there is no consensus of appropriate medium supplementation during maturation. The objective of this study was to determine whether oxygen tension (20% or 5% O2) and epidermal growth factor (EGF) affect oocyte metabolism and subsequent embryo development. Cumulus-oocyte complexes (COCs) were collected from 28-day-old equine chorionic gonadotropin (eCG) primed or unprimed F1 (C57BL/6xCBA) mice. COCs were matured in defined medium in one of four groups: 20% O2, 20% O2,+,EGF, 5% O2, 5% O2,+,EGF. In vivo matured COCs were also collected for analysis. COCs from unprimed mice, matured in 5% O2,±,EGF or 20% O2,+,EGF had higher metabolic rates than COCs matured in 20% O2 (P,<,0.05). COCs from primed mice had higher metabolic rates when matured in the presence of EGF, regardless of oxygen tension (P,<,0.01). Oxygen uptake and mitochondrial membrane potential were higher for in vivo matured oocytes and oocytes matured under 5% O2 compared to oocytes matured under 20% O2 (P,<,0.05). Blastocyst formation was not different between maturation groups (primed or unprimed); however, embryo cell numbers were 20,45% significantly higher when COCs were matured at 5% O2 (P,<,0.05). Results suggest that oocytes matured in physiological concentrations of oxygen have improved development and metabolic activity, more closely resembling in vivo maturation. These findings have implications for oocyte maturation in both clinical and research laboratories. Mol. Reprod. Dev. 74: 893,903, 2007. © 2006 Wiley-Liss, Inc. [source] Proteomic profiling of murine oocyte maturation,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2007Alejandra M. Vitale Abstract In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin ,2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin ,2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage. Mol. Reprod. Dev. 74: 608,616, 2007. © 2006 Wiley-Liss, Inc. [source] Effects of delayed excision of oviducts/ovaries on mouse oocytes and embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2007De-Qiang Miao Abstract To achieve the best and reproducible results of experiments, effects of delayed excision of oviducts/ovaries on mouse ovarian/ovulated oocytes and embryos have been studied. Oviducts/ovaries were excised at different times after death of mice and effects of the postmortem interval on ovarian/ovulated oocytes and embryos were analyzed. When oviduct excision was delayed 10 min, many ovulated oocytes lysed or underwent in vitro spontaneous activation, and this postmortem effect aggravated with the extension of postmortem interval and oocyte aging. Oocytes from different mouse strains responded differently to delayed oviduct removal. Delayed oviduct excision did not cause lysis of zygotes or embryos but compromised their developmental potential. When ovaries were excised at 30 min after death, percentages of atretic follicles increased while blastocyst cell number declined significantly after oocyte maturation in vitro. Preservation of oviducts in vitro, in intact or opened abdomen at different temperatures and histological analysis of oviducts from different treatments suggested that toxic substance(s) were secreted from the dying oviducts which induced oocyte lysis and spontaneous activation and both this effect itself and the sensitivity of oocytes to this effect was temperature dependent. It is concluded that a short delay of oviduct/ovary removal had marked detrimental effects on oocytes and embryos. This must be taken into account in experiments using oocytes or embryos from slaughtered animals. The data may also be important for estimation of the time of death in forensic medicine and for rescue of oocytes from deceased valuable or endangered mammals. Mol. Reprod. Dev. 74: 468,477, 2007. © 2006 Wiley-Liss, Inc. [source] Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms I. The roles of serine/threonine kinases and phosphatasesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2006Stephen A. Stricker Abstract Unlike in most animals, oocytes of marine nemertean worms initiate maturation (=germinal vesicle breakdown, GVBD) following an increase, rather than a decrease, in intraoocytic cAMP. To analyze how serine/threonine (Ser/Thr) kinase cascades involving mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), cAMP-dependent protein kinase (PKA), and phosphatidylinositol 3-kinase (PI3K) regulate nemertean GVBD, oocytes of Cerebratulus sp. were treated with pharmacological modulators and stimulated with cAMP-elevating drugs or seawater (SW) alone. Both cAMP elevators and SW triggered GVBD while activating MAPK, its target p90Rsk, and MPF. Similarly, neither cAMP- nor SW-induced GVBD was affected by several Ser/Thr phosphatase inhibitors, and both stimuli apparently accelerated GVBD via a MAPK-independent, PI3K-dependent mechanism. However, inhibitors of Raf-1, a kinase that activates MAPK kinase, blocked GVBD and MAPK activation during SW-, but not cAMP-induced maturation. In addition, MPF blockers more effectively reduced GVBD and MAPK activity in SW versus in cAMP-elevating treatments. Moreover, the two maturation-inducing stimuli yielded disparate patterns of PKA-related MAPK activations and phosphorylations of putative PKA substrates. Collectively, such findings suggest that in maturing oocytes of Cerebratulus sp., Ser/Thr kinase cascades differ during cAMP- versus SW-induced GVBD in several ways, including MAPK activation modes, MPF-feedback loops, and PKA-related signaling pathways. Additional differences in cAMP- versus SW-induced oocyte maturation are also described in the accompanying study that deals with the roles of tyrosine kinase signaling during GVBD. Mol. Reprod. Dev. 73: 1578,1590, 2006. © 2006 Wiley-Liss, Inc. [source] Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ,-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, ,-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2006AnilKumar Bettegowda Abstract Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ,-glucuronidase, glyceraldehyde-3-phosphate dehydrogenase, ,-actin, and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2005Laurence Gall Abstract EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus,oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Oocyte-selective expression of MT transposon-like element, clone MTi7 and its role in oocyte maturation and embryo developmentMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Chang-Eun Park Abstract Previously, we found MT transposon-like element, clone MTi7 (MTi7) is highly expressed in the mouse ovary. Here, we show that the MTi7 is expressed in the oocyte from the primordial to the preovulatory follicles. For RNA interference (RNAi), double stranded RNAs (dsRNAs) were prepared for MTi7 and c-mos, a control gene with known functions. Each dsRNA was microinjected into germinal vesicle (GV) stage oocytes or zygotes with pronuclei (PN), after which developmental changes, mRNA expression, and nuclear and microtubular organization were analyzed. We found a 43.4,53% GV arrest in the microinjected oocytes with a concomitant decrease in targeted mRNA expression. In MTi7 dsRNA-injected early and late PN zygotes, a 92.9% 1-cell arrest and 76.9% 2-cell arrest were observed, respectively. This is the first report of an oocyte-selective expression of MTi7 mRNA, and our results strongly suggest that MTi7 involved in the nuclear membrane breakdown during oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 365,374, 2004. © 2004 Wiley-Liss, Inc. [source] Several signaling pathways are involved in the control of cattle oocyte maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Céline Vigneron Abstract The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment. Mol. Reprod. Dev. 69: 466,474, 2004. © 2004 Wiley-Liss, Inc. [source] Expression of peroxiredoxins in bovine oocytes and embryos produced in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2004Gregory Leyens Abstract Peroxiredoxins (PRDXs) form a family of peroxidases involved in antioxidant protection and cell signaling. Due to their peroxide reductase activity, these enzymes might be involved in fine-tuning peroxide levels in embryos during in vitro production. In this study, RT-PCR was used to examine the expression of the six PRDX isoforms (PRDX1 to PRDX6) in bovine oocytes and embryos. PRDXs were detected in oocytes both before and after in vitro maturation. Besides, PRDX6 was up-regulated after maturation. Single embryos were analyzed from the two-cell to the blastocyst stages. PRDX1 and PRDX5 transcripts were detected throughout development. PRDX2, PRDX3, and PRDX6 were not expressed around the 9- to 16-cell stage. PRDX4 transcripts were weakly detected in pools of embryos from the 9- to 16-cell stage onwards. In situ immunodetection of PRDX5, which was previously reported to exhibit the widest subcellular distribution among PRDXs in adult mammalian cells, showed a mitochondrial distribution pattern in the bovine embryo. Finally, the potential modulation by oxidative stress of PRDX expression around the major embryonic genome activation was evaluated by culturing embryos under 20% O2 instead of 5%. No significant difference in the pattern of PRDX expression was observed under 20% O2. In conclusion, our data show for the first time that PRDXs are expressed in mammalian oocytes and early embryos. Moreover, the bovine transcripts exhibit various patterns of expression that might be related to the potential role of PRDXs in oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 243,251, 2004. © 2004 Wiley-Liss, Inc. [source] Maternal chromatin remodeling during maturation and after fertilization in mouse oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Marcella Spinaci Abstract Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation. Mol. Reprod. Dev. 69: 215,221, 2004. © 2004 Wiley-Liss, Inc. [source] Timing of Plk1 and MPF activation during porcine oocyte maturation,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2004Martin Anger Abstract A Polo-like kinase 1 (Plk1) appears involved in an autocatalytic loop between CDC25C phosphatase and M phase promoting factor (MPF) in Xenopus oocytes and leads to activation of MPF that is required for germinal vesicle breakdown (GVBD). Although similar evidence for such a role of Plk1 in MPF activation during maturation of mammalian oocytes is absent, changes in Plk1 enzyme activity correlate with MPF activation, Plk1 co-localizes with MPF, and microinjection of antibodies neutralizing Plk1 delays GVBD. In this study, we exploited the prolonged time required for maturation of porcine oocytes to define precisely the timing of Plk1 and MPF activation during maturation. GVBD typically occurs between 24 and 26 hr of culture in vitro and meiotic maturation is completed after 40,44-hr culture. We find that Plk1 is activated before MPF, which is consistent with its role in activating MPF in mammalian oocytes. Mol. Reprod. Dev. 69: 11,16, 2004. © 2004 Wiley-Liss, Inc. [source] Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001Maria Elena Dell'Aquila Abstract There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P,<,0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P,<0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes. Mol. Reprod. Dev. 60: 79,88, 2001. © 2001 Wiley-Liss, Inc. [source] Germinal vesicle materials are not required for the activation of MAP kinase in porcine oocyte maturationMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2001K. Sugiura Abstract The requirement of the germinal vesicle (GV) for the normal kinetics of mitogen-activated protein (MAP) kinase activity during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated, and the locations of their extracellular signal-regulated kinases 1 and 2 (ERK1/2), major MAP kinases in maturating porcine oocytes, were detected by indirect immunofluorescent microscopy. The MAP kinase activity was assayed as myelin basic protein (MBP) kinase activity, and the phosphorylation states of ERK1/2 were detected by immunoblotting analyses. Translocation of MAP kinase into the GV and association with the spindle were observed in intact oocytes, while MAP kinase in enucleated oocytes was distributed almost uniformly in cytoplasm throughout the culturing period. The phosphorylation and the activation of MAP kinase were induced, and the activity was comparable with that of control denuded oocytes. The high level of activity was maintained through maturation, even in the absence of spindle formation. These results indicate that the presence of nuclear material and translocation into the GV are dispensable for the activation of MAP kinase and that associating with the spindle is not required for maintenance of its activity though porcine oocyte maturation. Mol. Reprod. Dev. 59:215,220, 2001. © 2001 Wiley-Liss, Inc. [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. 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