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Sample records for cumulus-oocyte complex maturation

  1. Regulation of fatty acid oxidation in mouse cumulus-oocyte complexes during maturation and modulation by PPAR agonists.

    PubMed

    Dunning, Kylie R; Anastasi, Marie R; Zhang, Voueleng J; Russell, Darryl L; Robker, Rebecca L

    2014-01-01

    Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of ³H₂O from [³H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid

  2. In vitro maturation impacts cumulus oocyte complex metabolism and stress in cattle.

    PubMed

    Del Collado, Maite; da Silveira, Juliano Coelho; Oliveira, Marcelo Luna Freire; Alves, Barbara Monteiro da Silva Marques; Simas, Rosineide Costa; Godoy, Adriana Teixeira; Coelho, Mirela Batista; Marques, Lygia Azevedo; Carriero, Mateus Maldonado; Nogueira, Marcelo Fábio Gouveia; Eberlin, Marcos Nogueira; Silva, Luciano A; Meirelles, Flávio Vieira; Perecin, Felipe

    2017-10-02

    The influence of in vitro maturation (IVM) in oocytes is still not totally understood. The aim of this study was to determine the influence of IVM on the metabolism and homeostasis of bovine cumulus-oocyte complexes. In the present study, we demonstrated that IVM leads to accumulation of neutral lipids associated with differential levels of the mono-, di-, and tri-acylglycerols in both cumulus cells and oocytes. We observed that in vitro-matured oocytes exhibited decreased glutathione and reactive oxygen species levels and a lower ATP/ADP ratio when compared to in vivo-matured oocytes, with no significant differences in metabolism and stress related mRNA or miRNA levels. Moreover, in addition to an increase in lipids in in vitro-matured cumulus cells, fatty acid synthesis and accumulation as well as glycolysis pathway genes were upregulated, whereas those affiliated with the -oxidation pathway were decreased. Our gene expression data in cumulus cells suggest the disruption of endoplasmic reticulum stress, apoptosis, and cellular stress response pathways during IVM. Furthermore, a total of 19 miRNAs were significantly altered by the maturation process in cumulus cells. These results indicate some new negative influences of the in vitro system in cumulus-oocyte complexes, demonstrating the occurrence of functional disruption in lipid metabolism and stress pathways and showing evidences suggesting the occurrence of altered mitochondrial activity and energy metabolism during IVM, with a massive dysregulation of the corresponding transcripts in the surrounding cumulus cells.

  3. Fibroblast growth factor 10 markedly improves in vitro maturation of porcine cumulus-oocyte complexes.

    PubMed

    Son, Yeo-Jin; Lee, Seung-Eun; Hyun, Hyuk; Shin, Min-Young; Park, Yun-Gwi; Jeong, Sang-Gi; Kim, Eun-Young; Park, Se-Pill

    2017-01-01

    Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67-75, 2017. © 2016 Wiley Periodicals, Inc.

  4. Steroidogenesis during in vitro maturation of bovine cumulus oocyte complexes and possible effects of tri-butyltin on granulosa cells.

    PubMed

    Schoenfelder, M; Schams, D; Einspanier, R

    2003-02-01

    Steroids are known as important factors on the route of oocytes development and cumulus oocyte complexes (COC) as well as follicular granulosa cells (GC) are suggested to be themselves involved in steroidogenesis. The aim of this study was to characterize such a local sex steroidogenic system during in vitro maturation (IVM) of bovine COCs according to the production of estradiol (E), testosterone (T) and progesterone (P). The expression of two steroid-converting key-enzymes was measured in parallel by quantitative RT-PCR. Furthermore, possible effects of the environmental pollutant tri-butyltin (TBT) were elucidated for the first time on bovine COC and GC in vitro concerning that steroidogenic system. During IVM of bovine COCs concentrations of P increased continuously, corresponding with steady-state levels of 3-beta-hydroxy-steroid-dehydrogenase (HSD) transcripts. In contrast, E together with P450 aromatase mRNA (ARO) increased in the first hours of IVM but declining thereafter, whereas T reached almost balanced levels. However, TBT showed only slight effects during IVM of COC. In cultured GC, LH caused highest P- and E-production within 24h and treatment with 50pM TBT induced a significant decrease of E in contrast to 100pM TBT and the control. These results indicate, that (1) COCs were able to modulate their steroidogenic environment in vitro and that (2) TBT may possibly influence or disturb steroidogenesis in the cows reproductive tract shown here for GC.

  5. Oxidative stress as a damage mechanism in porcine cumulus-oocyte complexes exposed to malathion during in vitro maturation.

    PubMed

    Flores, Diana; Souza, Verónica; Betancourt, Miguel; Teteltitla, Mario; González-Márquez, Humberto; Casas, Eduardo; Bonilla, Edmundo; Ramírez-Noguera, Patricia; Gutiérrez-Ruíz, María Concepción; Ducolomb, Yvonne

    2017-06-01

    Malathion is one of the most commonly used insecticides. Recent findings have demonstrated that it induces oxidative stress in somatic cells, but there are not enough studies that have demonstrated this effect in germ cells. Malathion impairs porcine oocyte viability and maturation, but studies have not shown how oxidative stress damages maturation and which biochemical mechanisms are affected in this process in cumulus-oocyte complexes (COCs). The aims of the present study were to determine the amount of oxidative stress produced by malathion in porcine COCs matured in vitro, to define how biochemical mechanisms affect this process, and determine whether trolox can attenuate oxidative damage. Sublethal concentrations 0, 750, and 1000 µM were used to evaluate antioxidant enzyme expressions, reactive oxygen species (ROS production), protein oxidation, and lipid peroxidation, among other oxidation products. COCs viability and oocyte maturation decreased in a concentration-dependent manner. Malathion increased Cu, Zn superoxide dismutase (SOD1), glutathione-S-transferase (GST), and glucose 6 phosphate dehydrogenase (G6PD) protein level and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) protein level. Species reactives of oxygen (ROS), protein oxidation and Thiobarbituric acid reactive substances (TBARS) levels increased in COCs exposed to the insecticide, but when COCs were pre-treated with the trolox (50 µM) 30 min before and during malathion exposure, these parameters decreased down to control levels. This study showed that malathion has a detrimental effect on COCs during in vitro maturation, inducing oxidative stress, while trolox attenuated malathion toxicity by decreasing oxidative damage. © 2017 Wiley Periodicals, Inc.

  6. Activation of Mouse Cumulus-Oocyte Complex Maturation In Vitro Through EGF-Like Activity of Versican.

    PubMed

    Dunning, Kylie R; Watson, Laura N; Zhang, Voueleng J; Brown, Hannah M; Kaczmarek, Adrian K; Robker, Rebecca L; Russell, Darryl L

    2015-05-01

    In vitro maturation of oocytes is suboptimal to in vivo maturation with altered gene expression and compromised oocyte quality. The large proteoglycan versican is abundant in mouse cumulus-oocyte complexes (COCs) matured in vivo but is absent in cultured COCs. Versican is also positively correlated with human oocyte quality. Versican contains an epidermal growth factor (EGF) motif, and based on EGF-like activities in other systems we hypothesized that versican acts as an EGF-like signaling factor during COC maturation. Here, we purified recombinant versican and compared its function with that of EGF during in vitro maturation (IVM). Versican significantly increased cumulus expansion and induced cumulus-specific genes Ptgs2, Tnfaip6, and Has2, which was blocked by EGF receptor antagonist. Microarray analysis revealed that versican has overlapping function with EGF; however, a subset of genes was uniquely altered following 6 h of IVM with either treatment. Following 6 h of IVM, both Areg and Ereg were significantly increased by both treatments, whereas Egln3, Nr4a1, Nr4a2, Nr4a3, and Adamts5 were significantly higher following versican treatment compared with EGF. In contrast, Sprr1a and Aqp3 were increased after 6 h of EGF but not versican treatment. To determine whether there were temporal differences, COCs were cultured with EGF or versican for 0-12 h. Versican-induced expression occurred later but remained elevated for longer compared with EGF for Ptgs2, Ereg, and Nr4a3. The unique expression profiles of Aqp3 and Nr4a3 during IVM were similarly regulated in vivo. These data indicate that versican has EGF-like effects on COC gene expression, but with distinct temporal characteristics.

  7. Motility contrast imaging of live porcine cumulus-oocyte complexes

    NASA Astrophysics Data System (ADS)

    An, Ran; Turek, John; Machaty, Zoltan; Nolte, David

    2013-02-01

    Freshly-harvested porcine oocytes are invested with cumulus granulosa cells in cumulus-oocyte complexes (COCs). The cumulus cell layer is usually too thick to image the living oocyte under a conventional microscope. Therefore, it is difficult to assess the oocyte viability. The low success rate of implantation is the main problem for in vitro fertilization. In this paper, we demonstrate our dynamic imaging technique called motility contrast imaging (MCI) that provides a non-invasive way to monitor the COCs before and after maturation. MCI shows a change of intracellular activity during oocyte maturation, and a measures dynamic contrast between the cumulus granulosa shell and the oocytes. MCI also shows difference in the spectral response between oocytes that were graded into quality classes. MCI is based on shortcoherence digital holography. It uses intracellular motility as the endogenous imaging contrast of living tissue. MCI presents a new approach for cumulus-oocyte complex assessment.

  8. In Vitro Maturation of Cumulus-Oocyte Complexes for Efficient Isolation of Oocytes from Outbred Deer Mice

    PubMed Central

    Choi, Jung Kyu; He, Xiaoming

    2013-01-01

    Background The outbred (as with humans) deer mice have been a useful animal model of research on human behavior and biology including that of the reproductive system. One of the major challenges in using this species is that the yield of oocyte isolation via superovulation is dismal according to the literature to date less than ∼5 oocytes per animal can be obtained so far. Objective The goal of this study is to improve the yield of oocyte isolation from outbred deer mice close to that of most laboratory mice by in vitro maturation (IVM) of cumulus-oocyte complexes (COCs). Methods Oocytes were isolated by both superovulation and IVM. For the latter, COCs were obtained by follicular puncture of antral follicles in both the surface and inner cortical layers of ovaries. Immature oocytes in the COCs were then cultured in vitro under optimized conditions to obtain metaphase II (MII) oocytes. Quality of the oocytes from IVM and superovulation was tested by in vitro fertilization (IVF) and embryo development. Results Less than ∼5 oocytes per animal could be isolated by superovulation only. However, we successfully obtained 20.3±2.9 oocytes per animal by IVM (16.0±2.5) and superovulation (4.3±1.3) in this study. Moreover, IVF and embryo development studies suggest that IVM oocytes have even better quality than that from superovulation The latter never developed to beyond 2-cell stage as usual while 9% of the former developed to 4-cells. Significance We have successfully established the protocol for isolating oocytes from deer mice with high yield by IVM. Moreover, this is the first ever success to develop in vitro fertilized deer mice oocytes beyond the 2-cell stage in vitro. Therefore, this study is of significance to the use of deer mice for reproductive biology research. PMID:23457518

  9. The importance of having zinc during in vitro maturation of cattle cumulus-oocyte complex: role of cumulus cells.

    PubMed

    Anchordoquy, J M; Anchordoquy, J P; Sirini, M A; Picco, S J; Peral-García, P; Furnus, C C

    2014-10-01

    The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus-oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 μg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.

  10. A pre-in vitro maturation medium containing cumulus oocyte complex ligand-receptor signaling molecules maintains meiotic arrest, supports the cumulus oocyte complex and improves oocyte developmental competence.

    PubMed

    Santiquet, Nicolas W; Greene, Alison F; Becker, John; Barfield, Jennifer P; Schoolcraft, William B; Krisher, Rebecca L

    2017-09-01

    Can a pre-in vitro maturation (pre-IVM) medium containing signaling molecules rather than chemical/pharmaceutical agents, sustain meiotic arrest and improve developmental competence of in vitro matured oocytes in CF1 outbred mice? A short 2 h period of pre-IVM prevents spontaneous meiotic resumption, improves mitochondria activity in subsequently matured oocytes, and increases developmental competence, pregnancy rate and implantation of resulting embryos. Spontaneous resumption of meiosis in vitro is detrimental for oocyte developmental competence. Pre-IVM systems that prevent spontaneous meiotic resumption with chemical/pharmaceutical agents are a promising approach to improving IVM oocyte competence; however, the success of these methods has proven to be inconsistent. This study consisted of a series of experiments using cumulus oocyte complexes (COC) derived from outbred mice following ovarian stimulation. The study was designed to examine if a novel, ligand/receptor-based pre-IVM treatment could sustain meiotic arrest in vitro and improve oocyte developmental competence, compared to control IVM. Two pre-IVM durations (2 h and 24 h) were evaluated, and the effect of the mitochondrial stimulator PQQ during 24 h pre-IVM was studied. Murine (outbred CF1) immature COC were cultured in vitro in the presence of C-type natriuretic peptide (CNP) (30 nM), estradiol (100 nM), FSH (1 × 10-4 IU/ml) and bone morphogenic protein 15 (BMP15) (100 ng/ml) for 2 h or 24 h prior to IVM. Meiotic status during pre-IVM and IVM was analyzed using orcein staining, and functionality of gap junction communication was confirmed using the functional gap junction inhibitor carbenoxolone (CBX). Oocytes exposed to pre-IVM treatment were compared to control oocytes collected on the same day from the same females and undergoing standard IVM. Developmental competence and embryo viability was assessed by oocyte mitochondrial activity and ATP concentration, in vitro embryo development following

  11. 182 MATURATION OF BOVINE CUMULUS-OOCYTE COMPLEXES WITH FOLLICLE FLUID VARYING IN ESTRADIOL CONTENT AFFECTS CUMULUS CELL EXPANSION WITHOUT AFFECTING SUBSEQUENT EMBRYO DEVELOPMENT IN VITRO.

    PubMed

    Harl, A W; Larimore, E L; Al Naib, A; Wooldridge, L K; Ealy, A D; Perry, G A; Rhoads, M L

    2016-01-01

    The objective of this work was to determine how characteristics of bovine follicle fluid (FF; especially oestradiol content) affect cumulus cell expansion and oocyte competence. In the first study, FF was collected from abattoir-derived ovaries and pooled separately for large follicles (≥10mm) and small follicles (≤3mm). A portion of the FF from each category was charcoal stripped. These 4 types of FF were then used as the primary ingredient (75% vol/vol) in oocyte maturation media. A separate control group lacking FF but containing BSA was included to monitor potential impacts of protein on outcomes (control; without FF). Some of the cumulus-oocyte complexes (COC; n=250) were matured in individual drops for analysis of cumulus expansion (photographed and measured at 0 and 21h of maturation). Other COC (n=770) were matured in groups of 12 to 25 in the previously described media, and then subjected to IVF procedures. Cleavage rates were recorded on Day 3, and blastocyst rates were recorded on Day 8 post-fertilization. Cumulus cell expansion was greatest when COC were matured in medium containing FF from large follicles, wherein it even exceeded the controls (P<0.02). Maturation in FF from small follicles resulted in cumulus expansion that was intermediate between large and control. Maturation in charcoal-stripped FF severely restricted cumulus cell expansion (P<0.05) compared with those matured in untreated FF. Despite the observed improvement in cumulus cell expansion, COC that had been matured in media containing FF were less likely to cleave (P<0.05) and also less likely to develop to the blastocyst stage (P<0.01) than those matured in control medium. Cleavage and blastocyst rates did not differ among any of the maturation media containing FF. In the second study, oestrous cycles of 9 crossbred cows were synchronized and FF samples were collected 36 to 42h after prostaglandin F2α injection. Samples from individual cows were categorized as having high

  12. In vitro embryo production in wood bison (Bison bison athabascae) using in vivo matured cumulus-oocyte complexes.

    PubMed

    Cervantes, Miriam P; Palomino, J Manuel; Anzar, Muhammad; Mapletoft, Reuben J; Mastromonaco, Gabriela F; Adams, Gregg P

    2017-02-01

    Experiments were conducted in wood bison to determine the effect of additional maturation time on embryo development of in vivo matured oocytes. In experiment 1, cumulus-oocyte complexes (COC) were collected 30 hours after hCG treatment in superstimulated wood bison, and expanded COC were fertilized immediately or after 4 hours of additional in vitro maturation. Embryo development was assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). No difference in cleavage rate was detected (55.3% vs. 60.5%, P = 0.82), but the Day 8 blastocyst rate was higher after an additional 4 hours of in vitro maturation time (44.7 vs. 18.4%, P = 0.03). In experiment 2, COC were collected at either 30 hours or 34 hours after hCG treatment. Expanded COC from the 30 hours group were fertilized after 4 hours of in vitro maturation, whereas those from the 34 hours group were fertilized immediately. A higher cleavage rate (74.3 vs. 57.0%) and blastocyst rate (54.1 vs. 37.2%) were found in the 34 hours group versus the 30 hours group (P < 0.05). In conclusion, an additional short period of in vitro maturation, or an extended period of in vivo maturation are beneficial for in vitro embryo production in wood bison. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  13. Disruption of fibroblast growth factor receptor signaling in bovine cumulus-oocyte complexes during in vitro maturation reduces subsequent embryonic development.

    PubMed

    Zhang, K; Ealy, A D

    2012-05-01

    Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of ≥ 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 μM) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no time- or FSH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components of bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages of oocyte maturation.

  14. The effect of poly(ADP-ribosyl)ation inhibition on the porcine cumulus-oocyte complex during in vitro maturation.

    PubMed

    Kim, Duk Hyoun; Lee, Hye Ran; Kim, Min Gyeong; Lee, Jun Sung; Jin, Su Jin; Lee, Hoon Taek

    2017-01-29

    Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.

  15. Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

    PubMed

    Accogli, Gianluca; Douet, Cécile; Ambruosi, Barbara; Martino, Nicola Antonio; Uranio, Manuel Filioli; Deleuze, Stefan; Dell'Aquila, Maria Elena; Desantis, Salvatore; Goudet, Ghylène

    2014-12-01

    Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. © 2014 Wiley Periodicals, Inc.

  16. Metabolic differences in bovine cumulus-oocyte complexes matured in vitro in the presence or absence of follicle-stimulating hormone and bone morphogenetic protein 15.

    PubMed

    Sutton-McDowall, Melanie L; Mottershead, David G; Gardner, David K; Gilchrist, Robert B; Thompson, Jeremy G

    2012-10-01

    Bidirectional communication between cumulus cells and the oocyte is necessary to achieve oocyte developmental competence. The aim of the present study was to examine the effects of recombinant human bone morphogenetic protein 15 (rhBMP15) and follicle-stimulating hormone (FSH) supplementation on bovine cumulus-oocyte complex (COC) metabolism during maturation. Bovine COCs were matured in the presence of absence of FSH, rhBMP15, or both for 23 h. The addition of FSH and rhBMP15 increased blastocyst development (without rhBMP15 and FSH, 28.4% ± 7.4%; with FSH and rhBMP15, 51.5% ± 5.4%; P < 0.05). Glucose uptake and lactate production was significantly increased by greater than 2-fold with FSH (P < 0.05), whereas rhBM15 supplementation did not increase these levels. rhBMP15 supplementation (regardless of FSH) significantly decreased ADP levels in COCs, leading to an increase in ATP:ADP ratios (P < 0.05). Indicators of mitochondrial activity and cellular REDOX, oxidized flavin adenine dinucleotide (FAD(++)) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), levels within the oocyte of COCs were significantly higher with rhBMP15 alone, whereas the presence of FSH diminished the rhBMP15 effect. Regardless of treatment, no changes in REDOX state (FAD(++):NAD(P)H). The significant increase in FAD(++) and NAD(P)H in COCs with rhBMP15 was mediated via cumulus cells, because no differences were found in denuded oocytes cultured in the presence or absence of FSH, rhBMP15, or both. The present study demonstrates that a principal metabolic consequence of FSH supplementation of COCs is to alter the glycolytic rate of cumulus cells, whereas that of rhBMP15 is to regulate oxidative phosphorylation in the oocyte, even though it acts via cumulus cells. These effects are tempered when FSH and rhBMP15 are present together but, nonetheless, yield the best oocyte developmental competence.

  17. Effect of vitrification on meiotic maturation, mitochondrial distribution and glutathione synthesis in immature silver fox cumulus oocyte complexes.

    PubMed

    Cao, Xinyan; Li, Jingchun; Xue, Hailong; Wang, Shiyong; Zhao, Weigang; Du, Zhanyu; Yang, Yifeng; Yue, Zhigang

    2017-03-15

    The present study was designed to investigate the effects of vitrifying oocytes obtained from silver foxes on nuclear maturation, mitochondrial distribution and glutathione (GSH) synthesis after in vitro culture for 72 h. Immature oocytes were randomly divided into three groups: (1) fresh GV (germinal vesicle) oocytes (Control group), (2) exposure to the equilibration and vitrification solution but without being plunged into liquid nitrogen (exposed group), and (3) vitrification by the cryoloop method (vitrified-warmed group). The number of survival oocytes was not decreased by either being exposed to the cryoprotectant or being vitrified-warmed compared with the control group (P > 0.05). After IVM, the percentage of resumption of meiosis for vitrified-warmed oocytes (41.9%) was significantly lower than in the control (81.2%) and exposed (79.1%) groups (P < 0.05). However, the proportion of oocytes reaching the metaphase II (MII) stage was similar among the different groups (11.4%, 9.3% and 5.2%, respectively, P > 0.05). The translocation of active mitochondria during fox oocyte maturation was revealed using MitoTracker Red staining and confocal laser microscopy. For fresh oocytes at the GV stage, active mitochondria were distributed around the entire cortex with small granulations and various-sized cavities (no MitoTracker signals). After IVM, the mitochondria formed large granulations and clumps throughout the cytoplasm. Vitrification significantly decreased the proportion of MII oocytes with normal mitochondrial distribution compared with the control and exposed groups (35.4%, 71.9% and 59.2%, respectively, P < 0.05). Similarly, the GSH content was significantly lower in vitrified-warmed oocytes compared with the control and exposed oocytes after IVM (3.4, 5.7 and 4.7 pM/oocyte, respectively, P < 0.05). However, no significant difference was observed between the cryoprotectant exposed and control groups with regard to the normal mitochondrial

  18. Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex

    PubMed Central

    1978-01-01

    Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junction are formed on the oocyte surface by cumulus cell processes that transverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells. In summary, cell communication is a characterisitc feature of the cumulus-oocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation. PMID:670298

  19. Cumulus expansion, nuclear maturation and connexin 43, cyclooxygenase-2 and FSH receptor mRNA expression in equine cumulus-oocyte complexes cultured in vitro in the presence of FSH and precursors for hyaluronic acid synthesis

    PubMed Central

    Dell'Aquila, Maria Elena; Caillaud, Maud; Maritato, Filippo; Martoriati, Alain; Gérard, Nadine; Aiudi, Giulio; Minoia, Paolo; Goudet, Ghylène

    2004-01-01

    The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was increased with 1 microgram/ml eFSH, and was the highest with 20% FCS. It was not influenced by precursors for hyaluronic acid synthesis or glutamine. The expression of transcripts related to cumulus expansion was analyzed in equine cumulus cells before maturation, and after in vivo and in vitro maturation, by using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Connexin 43, cyclooxygenase-2 (COX-2) and FSH receptor (FSHr) mRNA were detected in equine cumulus cells before and after maturation. Their level did not vary during in vivo or in vitro maturation and was influenced neither by FSH nor by precursors for hyaluronic acid synthesis. Results indicate that previously reported regulation of connexin 43 and COX-2 proteins during equine COC maturation may involve post-transcriptional mechanisms. PMID:15212696

  20. Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: effect of denuded oocytes on cumulus expansion and oocyte maturation.

    PubMed

    Appeltant, R; Somfai, T; Nakai, M; Bodó, S; Maes, D; Kikuchi, K; Van Soom, A

    2015-03-01

    The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture

  1. Fatty acid composition of porcine cumulus oocyte complexes (COC) during maturation: effect of the lipid modulators trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) and forskolin.

    PubMed

    Prates, E G; Alves, S P; Marques, C C; Baptista, M C; Horta, A E M; Bessa, R J B; Pereira, R M

    2013-05-01

    The effect of maturation and of two lipid modulators supplementation along in vitro maturation (IVM) on fatty acid (FA) and dimethylacetal (DMA) composition of porcine cumulus oocyte complexes (COC) were studied. Abattoir-derived immature COC were analyzed for FA and DMA or submitted to IVM as follows: control group; t10,c12 CLA group, t10,c12 CLA supplementation for 44 h; Forskolin group, forskolin supplementation during the initial 2 h; t10,c12 CLA + forskolin group, t10,c12 CLA for 44 h and forskolin for just 2h. Each experimental group had five replicates. FA analysis of oocytes, cumulus cells (CC), follicular fluid, and culture media were performed by gas-liquid chromatography. Oocytes and their CC had different FA composition. Oocytes were richer in saturated FA (SFA) preferentially maintaining their FA profile during maturation. Mature CC had the highest polyunsaturated FA (PUFA) content. Five individual and total SFA, and monounsaturated FA (MUFA), notably oleic acid (c9-18:1), percentages were lower (P ≤ 0.023) in mature than in immature CC. t10,c12 CLA was accumulated by COC from t10,c12 CLA and t10,c12 CLA + forskolin groups, mostly in CC where MUFA and an eicosatrienoic isomer decreased (P ≤ 0.043). Nevertheless, PUFA or FA and DMA total content were not affected. Arachidonic acid was reduced in t10,c12 CLA + forskolin CC and hexadecanal-DMA-16:0 in t10,c12 CLA CC. Forskolin alone increased (P ≤ 0.043) c9-18:1 in oocytes. In conclusion, maturation process clearly changed porcine COC FA and DMA profiles, mostly of CC, also more susceptible to modifications induced by t10,c12 CLA. This possibility of manipulating COC lipid composition during IVM could be used to improve oocyte quality/cryopreservation efficiency.

  2. The nuclear and developmental competence of cumulus-oocyte complexes is enhanced by three-dimensional coculture with conspecific denuded oocytes during in vitro maturation in the domestic cat model.

    PubMed

    Morselli, M G; Luvoni, G C; Comizzoli, P

    2017-04-01

    The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes (CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes (COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required. © 2016 Blackwell Verlag GmbH.

  3. Expression and localization of urokinase-type plasminogen activator receptor in bovine cumulus-oocyte complexes.

    PubMed

    García, Daniela C; Miceli, Dora C; Rizo, Gabriela; García, Elina V; Valdecantos, Pablo A; Roldán-Olarte, Mariela

    2016-04-01

    Urokinase-type plasminogen activator (uPA) is a serine protease involved in extracellular matrix remodeling through plasmin generation. uPA usually binds to its receptor, uPAR, which is anchored to the plasma membrane through a glycosylphosphatidylinositol anchor. uPA/uPAR binding increases proteolytic activity in the neighborhood of the cells containing uPAR and activates intracellular signaling pathways involved in extracellular matrix remodeling, cell migration and proliferation. The aim of this work was to study the expression of uPA, uPAR and plasminogen activator inhibitor-1 (PAI-1) in immature and in vitro matured bovine cumulus-oocyte complexes (COCs). uPA is only expressed in the cumulus cells of immature and in vitro matured COCs, while uPAR and PAI-1 are expressed in both the cumulus cells and the immature and in vitro matured oocytes. In addition, uPAR protein was localized by confocal microscopy in the plasma membrane of oocytes and cumulus cells of immature COCs. Results from this research led us to hypothesize that the uPA/uPAR interaction could cause the local production of uPA-mediated plasmin over oocyte and cumulus cell surface; plasmin formation could also be regulated by PAI-1.

  4. Influence of epidermal growth factor and insulin-like growth factor 1 on nuclear maturation and fertilization of buffalo cumulus oocyte complexes in serum free media and their subsequent development in vitro.

    PubMed

    Purohit, G N; Brady, M S; Sharma, S S

    2005-07-01

    The in vitro maturation, fertilization and development of Indian water buffalo (Bubalus sp.) cumulus oocyte complexes (COCs) to blastocysts were studied during culture, either in serum free tissue culture medium 199 (TCM 199) or Waymouth MB (WM). Based on different supplements added to these media, the experimental groups included: (a) no supplement (control); (b) hormones (FSH, LH and oestradiol) (c) Epidermal growth factor (EGF); (d) IGF-1; and (e) EGF + IGF-1. Experiments were conducted to note three end points: (1) nuclear maturation 24 h after culture (eight replicates); (2) fertilization 24 h after insemination (10 replicates); (3) development to blastocysts (nine replicates). The oocytes were cultured in groups of up to five per drop. Using a two-way (5 x 2) factorial model with interactions, the results were compared using generalized linear models with binomial errors and the logit link function. In experiment 1, the proportion of oocytes reaching metaphase II was higher for all the supplement treatments than the control treatment (t = 3.68, p < 0.0001). The proportion of oocytes reaching metaphase II was 74.7, 63.2, 64.7 and 81% with hormone (chi2 = 17.23, p < 0.0001), EGF (chi2 = 7.07, p = 0.007), IGF-1 (chi2 = 19.21, p = 0.002) and EGF + IGF-1 (chi2 = 33.04, p < 0.0001) supplementation, respectively, compared to 46.6% in the control (no supplement) group. Media did not have an effect on outcome. In experiment 2, the proportion of oocytes fertilized was significantly higher with hormones (31.0%, chi2 = 12.5, p = 0.0004), IGF-1 (35.7%, chi2 = 20.53, p < 0.0001), and the EGF + IGF-1 combination (49.7%, chi2 = 51.35, p < 0.0001) compared to control (16.2%). No significant effect of media was seen. In experiment 3, the proportion of oocytes that cleaved at 48 h after culturing was significantly higher for all supplement treatments compared to control. IGF-1 supplementation was the only treatment that did not produce a significantly higher rate of progression

  5. Dual effects of hydrogen sulfide donor on meiosis and cumulus expansion of porcine cumulus-oocyte complexes.

    PubMed

    Nevoral, Jan; Petr, Jaroslav; Gelaude, Armance; Bodart, Jean-Francois; Kucerova-Chrpova, Veronika; Sedmikova, Marketa; Krejcova, Tereza; Kolbabova, Tereza; Dvorakova, Marketa; Vyskocilova, Alena; Weingartova, Ivona; Krivohlavkova, Lenka; Zalmanova, Tereza; Jilek, Frantisek

    2014-01-01

    Hydrogen sulfide (H2S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H2S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H2S donor, Na2S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H2S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H2S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H2S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.

  6. Effect of eicosapentaenoic acid on bovine cumulus-oocyte complex in vitro.

    PubMed

    Nikoloff, Noelia; Pascua, Ana M; Anchordoquy, Juan M; Anchordoquy, Juan P; Sirini, Matías A; Seoane, Analia; Furnus, Cecilia C

    2017-05-01

    The aim of the present study was to investigate the effects of eicosapentaenoic acid (EPA) supplementation during in vitro maturation (IVM) of bovine oocytes. The concentrations tested in all experiments were 1 nM, 1 μM, and 1 mM EPA. The effect of EPA was evaluated on cumulus-oocyte complexes (COC) by oocyte maturation (cumulus expansion area and oocyte nuclear maturation), genotoxicity [single cell gel electrophoresis (SCGE)], and cytotoxicity (apoptosis, viability, and MTT assays) end points. The maturation parameters were affected by exposure of COC to different EPA concentrations in the IVM medium. Cumulus expansion area increased in the presence of 1 nM EPA (P < 0.05) whereas addition of 1 nM EPA (P < 0.05) decreased cumulus expansion after 24 h of IVM. Moreover, the maturation rate significantly decreased when 1 mM of EPA was assayed (P < 0.001). EPA at 1 nM induced genotoxic and cytotoxic effects on bovine cumulus cells (CC) and primary DNA lesions (P < 0.001). A significant increase in the frequency of apoptotic (P < 0.01) and necrotic (P < 0.001) cells was observed after 24 h of treatment with 1 nM, 1 μM, and 1 mM EPA. Mitochondrial activity was altered with 1 mM EPA (P < 0.001). We inferred that optimal oocyte quality was partially dependent on the presence of adequate EPA concentrations; EPA could be beneficial to improve oocyte quality in the maturation process, because low concentration tested (1 nM EPA) improved cumulus expansion. © 2017 International Federation for Cell Biology.

  7. Time course of the meiotic arrest in sheep cumulus-oocyte complexes treated with roscovitine.

    PubMed

    Crocomo, Letícia Ferrari; Marques Filho, Wolff Camargo; Ackermann, Camila Louise; Paschoal, Daniela Martins; Guastali, Midyan Daroz; Dias Maziero, Rosiára Rosária; Sudano, Mateus José; Landim-Alvarenga, Fernanda da Cruz; Bicudo, Sony Dimas

    2016-04-01

    Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

  8. Ejaculated Mouse Sperm Enter Cumulus-Oocyte Complexes More Efficiently In Vitro than Epididymal Sperm

    PubMed Central

    Suarez, Susan S.

    2015-01-01

    The mouse is an established and popular animal model for studying reproductive biology. Epididymal mouse sperm, which lack exposure to secretions of male accessory glands and do not precisely represent ejaculated sperm for the study of sperm functions, have been almost exclusively used in studies. We compared ejaculated and epididymal sperm in an in vitro fertilization setting to examine whether ejaculated sperm enter cumulus-oocyte complexes more efficiently. In order to prepare sperm for fertilization, they were incubated under capacitating conditions. At the outset of incubation, ejaculated sperm stuck to the glass surfaces of slides and the incidences of sticking decreased with time; whereas, very few epididymal sperm stuck to glass at any time point, indicating differences in surface charge. At the end of the capacitating incubation, when sperm were added to cumulus-oocyte complexes, the form of flagellar movement differed dramatically; specifically, ejaculated sperm predominantly exhibited increased bending on one side of the flagellum (a process termed pro-hook hyperactivation), while epididymal sperm equally exhibited increased bending on one or the other side of the flagellum (pro-hook or anti-hook hyperactivation). This indicates that accessory sex gland secretions might have modified Ca2+ signaling activities in sperm, because the two forms of hyperactivation are reported to be triggered by different Ca2+ signaling patterns. Lastly, over time, more ejaculated than epididymal sperm entered the cumulus oocyte complexes. We concluded that modification of sperm by male accessory gland secretions affects the behavior of ejaculated sperm, possibly providing them with an advantage over epididymal sperm for reaching the eggs in vivo. PMID:25996155

  9. Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

    PubMed

    Crocomo, L F; Marques Filho, W C; Ulian, C M V; Branchini, N S; Silva, D T; Ackermann, C L; Landim-Alvarenga, F C; Bicudo, S D

    2015-06-01

    Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression. © 2015 Blackwell Verlag GmbH.

  10. Transition Metal Chelator Induces Progesterone Production in Mouse Cumulus-Oocyte Complexes and Corpora Lutea.

    PubMed

    Tian, X; Anthony, K; Diaz, Francisco J

    2017-04-01

    Progesterone production is upregulated in granulosa cells (cumulus and mural) after the LH surge, but the intra-follicular mechanisms regulating this transition are not completely known. Recent findings show that the transition metal chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN), impairs ovarian function. In this study, we provide evidence that chelating transition metals, including zinc, enhances progesterone production. The findings show that TPEN (transition metal chelator) increases abundance of Cyp11a1 and Star messenger RNA (mRNA) between 8- and 20-fold and progesterone production more than 3-fold in cultured cumulus-oocyte complexes (COC). Feeding a zinc-deficient diet for 10 days, but not 3 days, increased Star, Hsd3b, and prostaglandin F2 alpha receptor (Ptgfr) mRNA ~2.5-fold, suggesting that the effect of TPEN is through modulation of zinc availability. Progesterone from cumulus cells promotes oocyte developmental potential. Blocking progesterone production with epostane during maturation reduced subsequent blastocyst formation from 89 % in control to 18 % in epostane-treated complexes, but supplementation with progesterone restored blastocyst developmental potential to 94 %. Feeding a zinc-deficient diet for 5 days before ovulation did not affect the number of CL, STAR protein, or serum progesterone. However, incubating luteal tissue with TPEN increased abundance of Star, Hsd3b, and Ptgfr mRNA 2-3-fold and increased progesterone production 3-fold. TPEN is known to abolish SMAD2/3 signaling in cumulus cells. However, treatment of COC with the SMAD2/3 phosphorylation inhibitor, SB421542, did not by itself induce steroidogenic transcripts but did potentiate EGF-induced Star mRNA expression. Collectively, the results show that depletion of transition metals with TPEN acutely enhances progesterone biosynthesis in COC and luteal tissue.

  11. Daily differential expression of melatonin-related genes and clock genes in rat cumulus-oocyte complex: changes after pinealectomy.

    PubMed

    Coelho, L A; Peres, R; Amaral, F G; Reiter, R J; Cipolla-Neto, J

    2015-05-01

    This study investigated the maturational stage (immature and mature ovaries) differences of mRNA expression of melatonin-forming enzymes (Aanat and Asmt), melatonin membrane receptors (Mt1 and Mt2) and putative nuclear (Rorα) receptors, and clock genes (Clock, Bmal1, Per1, Per2, Cry1, Cry2) in cumulus-oocyte complexes (COC) from weaning Wistar rats. We also examined the effects of pinealectomy and of melatonin pharmacological replacement on the daily expression of these genes in COC. qRT-PCR analysis revealed that in oocytes, the mRNA expression of Asmt, Mt2, Clock, Bmal1, Per2, and Cry1 were higher (P < 0.05) in immature ovaries than in the mature ones. In cumulus cells, the same pattern of mRNA expression for Asmt, Aanat, Rorα, Clock, Per1, Cry1, and Cry2 genes was observed. In oocytes, pinealectomy altered the daily mRNA expression profiles of Asmt, Mt1, Mt2, Clock, Per1, Cry1, and Cry2 genes. In cumulus cells, removal of the pineal altered the mRNA expression profiles of Mt1, Mt2, Rorα, Aanat, Asmt, Clock, Bmal1, Per2, Cry1, and Cry2 genes. Melatonin treatment partially or completely re-established the daily mRNA expression profiles of most genes studied. The mRNA expression of melatonin-related genes and clock genes in rat COC varies with the maturational stage of the meiotic cellular cycle in addition to the hour of the day. This suggests that melatonin might act differentially in accordance with the maturational stage of cumulus/oocyte complex. In addition, it seems that circulating pineal melatonin is very important in the design of the daily profile of mRNA expression of COC clock genes and genes related to melatonin synthesis and action. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Absence of seasonal changes in FSHR gene expression in the cat cumulus-oocyte complex in vivo and in vitro.

    PubMed

    Hobbs, Rebecca J; Howard, JoGayle; Wildt, David E; Comizzoli, Pierre

    2012-07-01

    Domestic cat oocytes are seasonally sensitive to FSH. Compared with those collected during the breeding season, oocytes from the nonbreeding (NB) season require more FSH during in vitro maturation to achieve comparable developmental competence. This study tested the hypothesis that this seasonal variation was due to altered expression of FSH receptors (FSHR) and/or FSH-induced genes. Relative expression levels of FSHR mRNA and FSH-enhanced gene estrogen receptor β (ESR2) were measured by qPCR in whole ovaries and immature cumulus-oocyte complexes (COCs) isolated from cat ovaries during the natural breeding vs NB seasons. Expression levels of FSH-induced genes prostaglandin-endoperoxide synthase 2 (PTGS2), early growth response protein-1 (EGR1), and epidermal growth factor receptor (EGFR) were examined in mature COCs from both seasons that were a) recovered in vivo or b) matured in vitro with conventional (1 μg/ml) or high (10 μg/ml) FSH concentrations. Overall, FSHR mRNA levels were lower in whole ovaries during the NB compared with breeding season but were similar in immature COCs, whereas ESR2 levels did not differ in either group between intervals. We observed changes in PTGS2, EGR1, and EGFR mRNA expression patterns across maturation in COCs within but not between the two seasons. The lack of seasonal differentiation in FSH-related genes was not consistent with the decreased developmental capacity of oocytes fertilized during the NB season. These findings reveal that the seasonal decrease in cat oocyte sensitivity to FSH occurs both in vivo and in vitro. Furthermore, this decline is unrelated to changes in expression of FSHR mRNA or mRNA of FSH-induced genes in COCs from antral follicles.

  13. Morphology and location of attached follicular cumulus-oocyte complexes in horses, cattle and llamas.

    PubMed

    Del Campo, M R; Del Campo, C H; Mapletoft, R J; Ginther, O J

    1995-02-01

    Morphology and location of the attached cumulus-oocyte complex (COC) were studied in slaughter-house ovaries in horses (49 follicles, 9 to 44 mm), cattle (68 follicles, 6 to 18 mm), and llamas (38 follicles, 3 to 14 mm). The expected point of ovulation was marked, using the ovulation fossa in mares and the center of the projecting follicular surface in cattle and llamas. A follicle was dissected from an ovary, and tissue was removed from the follicle until the COC became visible by transillumination. However, most llama follicles protruded prominently from the ovarian surface so that dissection was not required to locate the COC. The COC was more readily recognized from the external follicular surface in mares and llamas than in cattle, primarily because of a dark oocyte. Compact COC's projected into the antrum with a smooth dome-shape in horses. The COC's in cattle were also dome-shaped but were more irregular and a few contained prominent processes. The mean diameter of the isolated follicle was calculated from 3 planes, except that in llamas the follicles were spherical so that the 3 dimensions were identical. The angle between a straight line connecting the expected ovulation site and the opposite pole and a straight line from the ovulation site to the COC was defined as the COC-location angle. This angle was chosen because it is unaltered by size of a sphere (45 degrees for a COC at the equator). The mean (+/-SEM) COC-location angle differed (P < 0.01) among horses (39.9 +/- 3.3), cattle (50.0 +/- 2.5), and llamas (64.8 +/- 2.1). In mares, the locations of the COC's did not differ from equality between follicular hemispheres, but in cattle and llamas the COC's were located with greater frequency (P < 0.05) in the hemisphere containing the expected ovulation site (cattle, 65%; llamas, 91%).

  14. Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus-oocyte complexes with lissamine green B.

    PubMed

    Dutta, Rahul; Li, Shun; Fischer, Konrad; Kind, Alexander; Flisikowska, Tatiana; Flisikowski, Krzysztof; Rottmann, Oswald; Schnieke, Angelika

    2016-06-01

    We evaluated the usefulness of lissamine green B (LB) staining of cumulus-oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

  15. Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence.

    PubMed

    Sutton-McDowall, Melanie L; Wu, Linda L Y; Purdey, Malcolm; Abell, Andrew D; Goldys, Ewa M; MacMillan, Keith L; Thompson, Jeremy G; Robker, Rebecca L

    2016-01-01

    Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

  16. Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius).

    PubMed

    Wani, N A; Skidmore, J A

    2010-08-01

    In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 microg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 +/- 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 +/- 4.1) and 26-27 h (82.1 +/- 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 +/- 16.3) after GnRH administration. In Experiment 2, a

  17. Relationship between the peripheral concentrations of estradiol-17β (E2) and preovulatory characteristics of cumulus-oocyte complexes (COCs) during superovulation treatment in Japanese Black cows.

    PubMed

    Kitahara, Go; Kamimura, Shunichi; Hamana, Katsumi

    2011-02-01

    The relationship between the peripheral concentrations of estradiol-17β (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (P<0.01). However, there was no difference in the ratio of oocytes showing GVBD between E(2)-84 and E(2)-96 (P=0.73), and the characteristics of the cumulus did not affect the stage of the nucleus in the oocytes in either groups (compact, expanded and nude; P=0.61, 0.81 and 1.00). It was possible that the time until the peak plasma E(2) concentrations after the FSH treatment could become an indicator for the maturation of follicles and oocytes in preovulatory follicles during superovulation treatment in Japanese Black cows.

  18. Experimental evidence for the influence of cumulus-oocyte-complexes on sperm release from the porcine oviductal sperm reservoir.

    PubMed

    Brüssow, Klaus-Peter; Torner, Helmut; Rátky, Jozsef; Manabe, Noboru; Tuchscherer, Armin

    2006-04-01

    In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 +/- 0.2 to 3.1 +/- 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.

  19. An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

    PubMed

    Sánchez, F; Lolicato, F; Romero, S; De Vos, M; Van Ranst, H; Verheyen, G; Anckaert, E; Smitz, J E J

    2017-10-01

    Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM

  20. A new approach for the oocyte genotoxicity assay: adaptation of comet assay on mouse cumulus-oocyte complexes.

    PubMed

    Greco, F; Perrin, J; Auffan, M; Tassistro, V; Orsière, T; Courbiere, B

    2015-07-01

    Conventional genotoxicity tests are technically difficult to apply to oocytes, and results obtained on somatic cells cannot be extrapolated to gametes. We have previously described a comet assay (original-CA) on denuded mouse oocytes, but, in vivo, oocytes are not isolated from their surrounding follicular cells. Our objective was to develop a comet assay on cumulus-oocyte complexes (COC-CA) for a more physiological approach to study the genotoxicity of environmental factors on oocytes. For COC-CA, whole COC were exposed directly to exogenous agents after ovulation and removal from oviducts. Three conditions were studied: a negative control group, and two positive control groups, one of which was exposed to hydrogen peroxide (H2O2) and the other group was incubated with cerium dioxide nanoparticles (CeO2 NPs). With both tests, DNA damage was significant in the presence of both H2O2 and CeO2 NPs compared with the negative control. COC-CA offers an interesting tool for assaying the genotoxicity of environmental agents towards germinal cells. Furthermore, COC-CA is less time-consuming and simplifies the protocol of the original-CA, because COC-CA is easier to perform without the washing-out procedure.

  1. Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks.

    PubMed

    Sun, Ming-Hong; Zheng, Jie; Xie, Feng-Yun; Shen, Wei; Yin, Shen; Ma, Jun-Yu

    2015-01-01

    During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

  2. Hampered cumulus expansion of porcine cumulus-oocyte complexes by excessive presence of alpha2 -macroglobulin is likely mediated via inhibition of zinc-dependent metalloproteases.

    PubMed

    Appeltant, Ruth; Beek, Josine; Maes, Dominiek; Bijttebier, Jo; Van Steendam, Katleen; Nauwynck, Hans; Van Soom, Ann

    2017-09-01

    In vitro maturation (IVM) in serum causes hampered expansion of porcine cumulus-oocyte complexes (COCs) due to excessive alpha2 -macroglobulin (A2M). This study investigated two hypotheses that could explain the effect of A2M: (i) binding of epidermal growth factor (EGF) to A2M, followed by its decreased availability; and (ii) inhibition of zinc-dependent metalloproteases. Cumulus expansion was evaluated based on the diameter of the COCs, the proportion of COCs participating in a floating cloud and the proportion of COCs with loss of cumulus cells. The first hypothesis of decreased EGF availability was tested by increasing the EGF concentration (20 and 50 ng/mL vs. 10 ng/mL), but was not confirmed because cumulus expansion did not improve. To verify the second hypothesis of inhibited zinc-dependent metalloproteases, the effect of tissue inhibitor of metalloproteases-3 (TIMP-3) on cumulus expansion during IVM with and without A2M was investigated. To immuno-neutralize A2M, serum was pre-incubated with A2M antibodies. Impaired cumulus expansion because of TIMP-3 could only be observed during IVM in 10% of serum with A2M antibodies. No effect of TIMP-3 was observed in medium without A2M antibodies. These results indicate that A2M and TIMP-3 share a common target, a zinc-dependent metalloprotease. Future research is directed toward the identification of the protease involved. © 2017 Japanese Society of Animal Science.

  3. Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus-oocytes complexes derived from small follicles.

    PubMed

    Matsunaga, R; Funahashi, H

    2017-03-30

    The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3-6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1-2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus-oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF-derived oocytes than MF-derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co-culture of SF-derived COCs with MF-derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase-II stage. Furthermore, the ooplasmic diameter of SF-derived COCs during IVM was increased to the similar size of MF-derived those in the presence of MF-derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co-culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF-derived CCMs. In conclusion, we demonstrate that supplementation with MF-derived CCMs improves the ooplasmic diameter and meiotic competence of SF-derived oocytes.

  4. Follistatin Rescues Blastocyst Development of Poor Quality Porcine Cumulus-Oocyte Complexes by Delaying Meiotic Resumption With Decreased cGMP.

    PubMed

    Lee, Bo Myeong; Chun, Ju Lan; Lee, Ji Hye; Kim, Eun Young; Park, Kang-Sun; Lee, Jin-Hee; Daigneault, Bradford W; Smith, George W; Kim, Keun Jung; Chang, Kyu-Tae; Lee, Sang-Rae; Kim, Sun-Uk; Choi, Seon-A; Lee, Kyung-Bon; Kim, Min Kyu

    2017-01-01

    Mammalian oocytes resume maturation when removed from follicles and cultured in vitro. During folliculogenesis, oocytes are bathed in follicular fluid (FF), which provides an important and specialized microenvironment for oocyte competence. Follistatin (FST) is one component of FF that may play a role in oocyte maturation and embryo development. This study was conducted to examine possible effects of FST on porcine oocyte competence and embryo development. Exogenous FST in oocyte maturation medium for 22 or 44 hours did not improve nuclear maturation and had no effect on good quality cumulus-oocyte complexes (COCs). However, FST improved blastocyst rates in embryos derived from oocytes with less than 2 layers of cumulus. Follistatin treatment of the poor quality COC group increased transcript levels for genes indicative of oocyte quality. Transcript levels were also altered for cumulus expansion-related genes in response to FST when measured during the germinal vesicle breakdown stage. Interestingly, high-quality oocytes remained at germinal vesicle stage much longer than low-quality oocytes, FST treatment induced temporary blockage of spontaneous meiotic resumption when added during culture of both good and poor quality COCs, and levels of cyclic guanosine monophosphate (cGMP) were higher in FST-treated versus untreated groups for both good and poor quality oocytes. In conclusion, FST treatment of porcine oocytes during in vitro maturation can rescue competency of poor quality oocytes to develop to blastocyst stage following in vitro fertilization. Beneficial effects of addition of FST to culture medium may be mediated by inhibiting degradation of cGMP and temporarily delaying nuclear maturation.

  5. Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.

    PubMed

    Ohtake, Jun; Sakurai, Masahiro; Hoshino, Yumi; Tanemura, Kentaro; Sato, Eimei

    2015-03-01

    We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.

  6. Effects of environmental enrichment on reproductive performance and quantity and morphology of cumulus-oocyte complexes obtained from Rattus norvegicus.

    PubMed

    Fisch, Joana; Oliveira, Iáskara Vieira de; Fank, Juliana; Paim, Lia Mara Gomes; Zandoná, Marília Remuzzi; Lopes, Eliana Franco; Mello, Fernanda Bastos de; Oliveira, Alexandre Tavares Duarte de

    2017-05-01

    Several researchers have observed that environmental enrichment (EE) can be effective in reducing stressful conditions and abnormal behavior and may provide better reproductive performance in rodents. In this context, this study aimed to evaluate the reproductive performance of Wistar rats reared in three different housing conditions. Animals were separated into breeding pairs, one pair per cage and pairs randomly assigned to three experimental groups (ten couples per group): control group were provided cages without any environmental enrichment; PVC group with PVC pipe; and cardboard roll group with a commercially available cardboard tube. To compare the reproductive performance of the three groups, the following were evaluated: number of pups/litter; number of litters; parturition interval; occurrence of cannibalism; weight gain of offspring; as well as the quantity and quality of cumulus-oocyte complexes (COCs) obtained after superovulation of the females born from the first, second and last pregnancy in all groups. Moreover, the plasma level of corticosterone in breeding animals was measured. A total of 60 male rats randomly selected from the first- and last-born litters (20 males from each group) were first tested in an elevated plus-maze (EPM) and on the following day, were tested in an open field test (OFT). Significant differences were found in the number and morphological classification of COCs. In the control group, the number of oocytes in grade 4 (unusual shapes and very heterogeneous ooplasm, presenting no layers of surrounding cumulus cells [13]) presented statistically higher rates (225/2535, 8.9%) compared to the other groups, as well as the number of competent oocytes was higher in the enriched groups (p = 0.001). Moreover, we find that the males of cardboard roll group differed significantly in weight gain compared to PVC group (p = 0.008). In addition to this, we did not detect occurrence of cannibalism in this group. Our findings suggest

  7. Individual bovine in vitro embryo production and cumulus cell transcriptomic analysis to distinguish cumulus-oocyte complexes with high or low developmental potential.

    PubMed

    Bunel, A; Jorssen, E P; Merckx, E; Leroy, J L; Bols, P E; Sirard, M A

    2015-01-15

    Studying cumulus cell (CC) transcriptome is of great interest as it could provide a noninvasive method to assess oocyte quality. In cattle, the search for quality markers has not been done with cumulus-oocyte complexes (COCs) cultured individually from maturation to blastocyst stage. Here, differences between high- and low-potential COCs were examined by transcriptomic analysis of CC biopsies obtained from COCs of 2 to 6 mm follicles (n = 249; eight replicates) before individual in vitro maturation, fertilization, and culture until Day 8 after fertilization. Each COC was individually tracked and categorized based on his fate: embryo at blastocyst stage (CC-Blast) or embryo arrested at 2- to 8-cell stage (CC-2-8-cells). Average blastocyst rates were 27.7% for individual culture and 31.2% for group control (not significantly different). For transcriptomic analysis, five cumulus biopsies per replicate were pooled for each fate. Three CC replicates underwent transcriptomic analysis using RNA microarray assay. Some clear differences in gene expression between the CC-Blast and the CC-2-8-cell groups were identified. Considering a 1.5-fold change (P < 0.05), 68 genes were differentially expressed between the CC-Blast and CC-2-8-cells. Quantitative reverse transcription-polymerase chain reaction validations were performed for 12 selected genes: six upregulated genes for each COC fate. Higher expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) (lipid metabolism), Chloride intracellular channel 3 (CLIC3), Keratin 8 (KRT8), and Lumican (LUM) (molecular transport) was observed in CC-2-8-cells (P < 0.05). The CC-Blast fate analysis revealed a significantly higher expression of Glycine amidinotransferase (L-arginine:glycine amidinotransferase) (GATM) (posttranslational modification, amino acid metabolism, and free radical scavenging). This newly identified set of genes could provide new markers to distinguish COCs associated with good quality embryos from COCs

  8. Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.

    PubMed

    Corbett, Hannah E; Dubé, Chantal D; Slow, Sandy; Lever, Michael; Trasler, Jacquetta M; Baltz, Jay M

    2014-04-01

    Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.

  9. Synchrotron X-ray diffraction to detect glass or ice formation in the vitrified bovine cumulus-oocyte complexes and morulae.

    PubMed

    Anzar, Muhammad; Grochulski, Pawel; Bonnet, Brennan

    2014-01-01

    Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (-171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification.

  10. Synchrotron X-Ray Diffraction to Detect Glass or Ice Formation in the Vitrified Bovine Cumulus-Oocyte Complexes and Morulae

    PubMed Central

    Anzar, Muhammad; Grochulski, Pawel; Bonnet, Brennan

    2014-01-01

    Vitrification of bovine cumulus-oocyte complexes (COCs) is not as successful as bovine embryos, due to oocyte's complex structure and chilling sensitivity. Synchrotron X-ray diffraction (SXRD), a powerful method to study crystal structure and phase changes, was used to detect the glass or ice formation in water, tissue culture medium (TCM)-199, vitrification solution 2 (VS2), and vitrified bovine COCs and morulae. Data revealed Debye's rings and peaks associated with the hexagonal ice crystals at 3.897, 3.635, 3.427, 2.610, 2.241, 1.912 and 1.878 Å in both water and TCM-199, whereas VS2 showed amorphous (glassy) appearance, at 102K (−171°C). An additional peak of sodium phosphate monobasic hydrate (NaH2PO4.H2O) crystals was observed at 2.064 Å in TCM-199 only. All ice and NaH2PO4.H2O peaks were detected in the non-vitrified (control) and vitrified COCs, except two ice peaks (3.145 and 2.655 Å) were absent in the vitrified COCs. The intensities of majority of ice peaks did not differ between the non-vitrified and vitrified COCs. The non-vitrified bovine morulae in TCM-199 demonstrated all ice- and NaH2PO4.H2O-associated Debye's rings and peaks, found in TCM-199 alone. There was no Debye's ring present in the vitrified morulae. In conclusion, SXRD is a powerful method to confirm the vitrifiability of a solution and to detect the glass or ice formation in vitrified cells and tissues. The vitrified bovine COCs exhibited the hexagonal ice crystals instead of glass formation whereas the bovine morulae underwent a typical vitrification. PMID:25536435

  11. Altered Theca and Cumulus Oocyte Complex Gene Expression, Follicular Arrest and Reduced Fertility in Cows with Dominant Follicle Follicular Fluid Androgen Excess

    PubMed Central

    Summers, Adam F.; Pohlmeier, William E.; Sargent, Kevin M.; Cole, Brizett D.; Vinton, Rebecca J.; Kurz, Scott G.; McFee, Renee M.; Cushman, Robert A.; Cupp, Andrea S.; Wood, Jennifer R.

    2014-01-01

    Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development. PMID:25330369

  12. Knockdown of TrkA in cumulus oocyte complexes (COCs) inhibits EGF-induced cumulus expansion by down-regulation of IL-6.

    PubMed

    Wang, Yong; Liang, Ning; Yao, Guidong; Tian, Hui; Zhai, Yiwen; Yin, Yimeng; Sun, Fei

    2014-02-15

    Tyrosine kinase receptor A (TrkA), the high-affinity receptor of nerve growth factor (NGF), is known to play key roles in ovarian follicular development, such as assembly of early follicles and follicular ovulation. However, little is known about the roles of TrkA in cumulus oocyte complex (COC) expansion. In this study, we found that TrkA was abundant in large antral follicles and knockdown of TrkA in COCs attenuated epidermal growth factor (EGF)-induced COC expansion and further decreased the ovulation rate. The effect of TrkA on COC expansion was not mediated through downstream EGF effectors, phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2) or drosophila mothers against decapentaplegic protein (SMAD), or through up-regulation of COC expansion-related transcripts such as prostaglandin-endoperoxide synthase 2 (Ptgs2), hyaluronan synthase 2 (Has2), TNF-induced protein 6 (Tnfaip6) or pentraxin 3 (Ptx3). However, pharmacological blockade of TrkA transducing activity (K252α) in COCs decreased the mRNA expression and protein secretion of interleukin-6 (IL-6), identified from mRNA microarray of K252α-treated COCs. Meanwhile, knockdown of IL-6 attenuated EGF-induced COC expansion. In addition, IL-6 rescued the inhibitory effect of K252α on EGF-induced cumulus expansion. Therefore, IL-6 may act as a new potential cumulus expansion-related transcript, which may be involved in the integration of TrkA and EGF signaling in affecting COC expansion. Here, we provide mechanistic insights into the roles of TrkA in EGF-induced cumulus expansion. Understanding potential cross-points between TrkA and EGF affecting cumulus expansion will help in the discovery of new therapeutic targets in ovulation-related diseases.

  13. Oocyte-dependent activation of MTOR in cumulus cells controls the development and survival of cumulus-oocyte complexes.

    PubMed

    Guo, Jing; Shi, Lanying; Gong, Xuhong; Jiang, Mengjie; Yin, Yaoxue; Zhang, Xiaoyun; Yin, Hong; Li, Hui; Emori, Chihiro; Sugiura, Koji; Eppig, John J; Su, You-Qiang

    2016-08-15

    Communication between oocytes and their companion somatic cells promotes the healthy development of ovarian follicles, which is crucial for producing oocytes that can be fertilized and are competent to support embryogenesis. However, how oocyte-derived signaling regulates these essential processes remains largely undefined. Here, we demonstrate that oocyte-derived paracrine factors, particularly GDF9 and GDF9-BMP15 heterodimer, promote the development and survival of cumulus-cell-oocyte complexes (COCs), partly by suppressing the expression of Ddit4l, a negative regulator of MTOR, and enabling the activation of MTOR signaling in cumulus cells. Cumulus cells expressed less Ddit4l mRNA and protein than mural granulosa cells, which is in striking contrast to the expression of phosphorylated RPS6 (a major downstream effector of MTOR). Knockdown of Ddit4l activated MTOR signaling in cumulus cells, whereas inhibition of MTOR in COCs compromised oocyte developmental competence and cumulus cell survival, with the latter likely to be attributable to specific changes in a subset of transcripts in the transcriptome of COCs. Therefore, oocyte suppression of Ddit4l expression allows for MTOR activation in cumulus cells, and this oocyte-dependent activation of MTOR signaling in cumulus cells controls the development and survival of COCs.

  14. Effect of removing cumulus cells from porcine cumulus-oocyte complexes derived from small and medium follicles during IVM on the apoptotic status and meiotic progression of the oocytes.

    PubMed

    Ferré, Pilar; Bui, Tra Mi Thi; Wakai, Takuya; Funahashi, Hiroaki

    2016-10-15

    The present study was undertaken to examine the apoptotic status and meiotic progression of oocytes from small follicle (SF; 0.5-2 mm in diameter) and medium follicle (MF; 3-6 mm in diameter) when the oocytes were denuded before, during, and after IVM. Cumulus-oocyte complexes (COCs) were collected from SF or MF of prepubertal gilt ovaries. Before or 20 hours after the start of IVM culture, some oocytes were denuded and cultured for IVM. At the end of IVM, apoptotic status and meiotic progression of the oocytes were compared with oocytes matured in the presence of cumulus cells (CCs) by Annexin-V/PI assay and 4',6-Diamidino-2-phenylindole staining. Apoptotic status of the oocytes was only affected by time when the oocytes were denuded. In both oocytes from SF and MF, although the incidence of early and late apoptotic oocytes was significantly higher when the CCs were removed before IVM, the rate was significantly lower when CCs were removed 20 and 44 hours after the start of IVM. The incidence of mature oocytes was significantly affected by both the origin of COCs and time when oocytes were denuded from the COCs. Although the percentage of mature oocytes was higher in MF than SF, maturation rates were significantly higher when oocytes were denuded 20 hours, as compared with 0 and 44 hours after the start of IVM. However, the percentage of mature oocytes with a morphologically normal spindle was significantly higher when oocytes were denuded 44 hours, rather than 22 hours of IVM. In conclusion, removing CCs 20 hours after the start of IVM seems to promote meiotic progression of the oocytes to the metaphase-II stage even when the COCs were collected from SF, although factor(s) from or communication with CCs during IVM may need to obtain a morphologically normal spindle in mature oocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Prophase I arrest of mouse oocytes mediated by natriuretic peptide precursor C requires GJA1 (connexin-43) and GJA4 (connexin-37) gap junctions in the antral follicle and cumulus-oocyte complex.

    PubMed

    Richard, Samantha; Baltz, Jay M

    2014-06-01

    Fully grown germinal vesicle stage mouse oocytes remain arrested in meiotic prophase I until ovulation. This arrest is maintained by cGMP produced in cumulus granulosa cells surrounding the oocyte. Recently, it was found that cGMP production in cumulus cells depends on NPR2 guanylate cyclase activated by its ligand natriuretic peptide precursor C (NPPC). It is assumed that cGMP reaches the oocyte through gap junctions that couple cumulus granulosa cells to each other and to the oocyte. Previous work identified two main types of gap junctions in the follicle, connexin-43 gap junctions (GJA1 protein) between granulosa cells and connexin-37 gap junctions (GJA4) between cumulus cells and the oocyte. However, it had not been established that both types are required for meiotic arrest mediated by NPPC/NPR2 signaling. To investigate this, we used connexin mimetic peptides (CMPs) that specifically disrupt gap junction isoforms within cumulus-oocyte complexes (COCs) and isolated antral follicles in culture. We furthermore developed a punctured antral follicle preparation to permit CMP access to the antral cavity in an otherwise intact follicle. CMP directed against connexin-43 (Cx43 CMP) overcame NPPC-mediated meiotic arrest in both isolated COCs and antral follicles. Cx37 CMP, in contrast, had no effect when present in the medium, but released oocyte arrest in the presence of NPPC when microinjected into the perivitelline space near the oocyte surface in COCs. This is consistent with both connexin isoforms being required for meiotic arrest and with the reported localization of connexin-43 throughout the cumulus cells and connexin-37 at the oocyte surface. © 2014 by the Society for the Study of Reproduction, Inc.

  16. mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

    PubMed

    Silva, A W B; Bezerra, F T G; Glanzner, W G; Dos Santos, J T; Dau, A M P; Rovani, M T; Ilha, G F; Costa, J J N; Cunha, E V; Donato, M A M; Peixoto, C A; Gonçalves, P B D; Bordignon, V; Silva, J R V

    2017-03-01

    This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

  17. Quantification of cyclin B1 and p34(cdc2) in bovine cumulus-oocyte complexes and expression mapping of genes involved in the cell cycle by complementary DNA macroarrays.

    PubMed

    Robert, Claude; Hue, Isabelle; McGraw, Serge; Gagné, Dominic; Sirard, Marc-André

    2002-11-01

    Although high amounts of cyclin B1 mRNA are present in bovine oocytes arrested at the germinal vesicle (GV) stage, the protein is not detectable. Furthermore, there is a depletion of the stored cyclin B1 mRNA in the oocyte as follicular growth progresses. To assess the effect of follicular growth on the accumulation of M-phase promoting factor (MPF) components, mRNA and protein levels of cyclin B1 and p34(cdc2) were measured in GV oocytes collected from diverse follicle size groups (<2 mm, 3-5 mm, and >6 mm). Because oocytes collected from very small follicles have high levels of cyclin B1 mRNA, the onset of its accumulation in the oocytes was evaluated by in situ hybridization of fetal ovaries. Also, a comparative expression map of cell cycle-related genes expressed in the oocyte and cumulus cells was established using nylon-based cDNA arrays, which allowed the detection of 35 different genes transcribed mostly in oocytes. Both components of the pre-MPF complex were expressed at the mRNA level in GV oocytes, whereas p34(cdc2) was the only pre-MPF protein detected at that stage, thus indicating that meiosis resumption in bovine oocytes is differentially regulated as compared with other mammals, and meiosis resumption seems to be regulated by the translation of cyclin B1 mRNA.

  18. Impaired maturation, fertilization, and embryonic development of porcine oocytes following exposure to an environmentally relevant organochlorine mixture.

    PubMed

    Campagna, C; Sirard, M A; Ayotte, P; Bailey, J L

    2001-08-01

    The reproductive health risks related to exposure to persistent organic pollutants in the environment remain controversial. This debate is partly because most studies have investigated only one or two chemicals at a time, whereas populations are exposed to a large spectrum of persistent chemicals in their environment. Using the pig as a toxicological model, we hypothesized that exposing immature cumulus-oocyte complexes to an organochlorine mixture during in vitro maturation (IVM) would adversely affect oocyte maturation, fertilization, and subsequent embryo development. This organochlorine mixture mimics that which contaminates the Arctic marine food chain. Cumulus-oocyte complexes were cultured in IVM medium containing increasing concentrations of the organochlorine mixture, similar to that found in women of highly exposed populations. Organochlorines reduced the quality of cumulus expansion and the viability of cumulus cells in a dose-response manner. The proportion of apoptotic cumulus cells also increased due to organochlorine exposure. Half of the oocytes were fixed after insemination, and the remainders were cultured for 8 days. Concentrations of organochlorines did not affect the rates of oocyte degeneration, sperm penetration, and development to morula. However, incidence of incompletely matured oocytes increased and polyspermy rate decreased, both in a dose-response manner with increasing organochlorine concentrations. Blastocyst formation and number of cells per blastocyst declined with organochlorine concentration. Exposing porcine cumulus-oocyte complexes to an environmentally pertinent organochlorine mixture during IVM disturbs oocyte development, supporting recent concerns that such pollutants harm reproductive health in humans and other mammalian species.

  19. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

  20. Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

    PubMed

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L; Ritter, Lesley J; White, Melissa A; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/- FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/- FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.

  1. Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

    PubMed

    Liu, Shuzhen; Jiang, Ligang; Zhong, Tao; Kong, Shuhui; Zheng, Rongbin; Kong, Fengyun; Zhang, Cong; Zhang, Lei; An, Liguo

    2015-01-01

    Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus-oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus-oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

  2. MicroRNA expression profile in bovine cumulus-oocyte complexes during late oogenesis

    USDA-ARS?s Scientific Manuscript database

    During late oogenesis, the mammalian oocyte synthesizes and stores mRNA necessary to guide the early stages of embryo development prior to the activation of embryonic transcription. The oocyte also contains many post-transcriptional regulatory mechanisms that coordinate mRNA stability and translati...

  3. In vitro maturation and artificial activation of donkey oocytes.

    PubMed

    Zhao, Gaoping; Wu, Kaifeng; Cui, Liang; Zhao, Lixia; Liu, Yiyi; Tan, Xiuwen; Zhou, Huanmin

    2011-09-01

    Three media were evaluated for their ability to support in vitro maturation of donkey (Equus asinus) oocytes and their development after parthenogenetic activation. The basal medium for Medium 1 (M1) and Medium 2 (M2) was M199 and DMEM/F12 respectively, whereas, Medium 3 (M3) consisted of equal parts (v/v) of M199 and DMEM/F12. All three media were supplemented with 10% (v/v) fetal calf serum, 0.01 units/mL porcine FSH, 0.01 units/mL equine LH, 200 ng/mL insulin-like growth factor 1(IGF-I), 10 μl/mL insulin-transferrin-selenium (ITS), 0.1 mg/mL taurine, 0.1 mg/mL L-cysteine, 0.05 mg/mL L-glutamine, 0.11 mg/mL sodium pyruvate, and 25 mg/mL gentamycin. There were no significant differences among the three maturation media for oocyte maturation. Maturation rate of donkey oocytes in M1 was 53% for compact (Cp) cumulus-oocyte complexes and 75% for expanded (Ex) cumulus-oocyte complexes; in M2 these were 55 and 77%, respectively; and in M3, 58 and 75%. The percentage of cleaved parthenotes and 4- or 8-cell embryos were not significantly different for oocytes matured in the various media (61 and 24% for M1; 66 and 32% for M2; and 67 and 33% for M3). Oocytes matured in M3 tended to yield a higher rate of advanced embryo development (morula) than oocytes matured in M1 (22 vs 9%; P = 0.07). In conclusion, donkey oocytes were matured and parthenogenetically activated in vitro, using methods similar to those used in the horse.

  4. Influence of co-culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus-enclosed porcine oocytes in a defined system.

    PubMed

    Appeltant, Ruth; Somfai, Tamás; Kikuchi, Kazuhiro; Maes, Dominiek; Van Soom, Ann

    2016-04-01

    Co-culture of cumulus-oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte-secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β-mercaptoethanol. Cumulus-oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co-culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus-enclosed porcine oocytes in a defined system.

  5. Effect of sericin supplementation in maturation medium on cumulus cell expansion, oocyte nuclear maturation, and subsequent embryo development in Sanjabi ewes during the breeding season.

    PubMed

    Aghaz, F; Hajarian, H; Shabankareh, H Karami; Abdolmohammadi, A

    2015-12-01

    The purpose of this study was to evaluate the effect of sericin with different concentrations (0% [control], 0.1%, 0.5%, 1.0%, and 2.5%) added to the IVM medium on cumulus cell expansion, oocyte nuclear maturation, and subsequent embryo development in Sanjabi ewes during the breeding season. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body extrusion. After IVF with fresh ram semen, presumptive zygotes were cultured 8 days in potassium simplex optimization medium supplemented by amino acids, and the percentages developing to the two-cell and blastocyst stages were measured as the indicators of early embryonic developmental competence. More cumulus-oocyte complexes matured with 0.5% sericin underwent germinal vesicle breakdown and reached metaphase II stage compared with the control cumulus-oocyte complexes matured without sericin (P ≤ 0.05). The present findings indicated that supplementation with 0.5% sericin during the maturation culture may improve the nuclear maturation and the cumulus cell expansion. Furthermore, the percentage of blastocysts obtained from 0.5% and 0.1% sericin (37.8 ± 1.76% and 34.8 ± 1.09%, respectively) was higher (P ≤ 0.05) than that of the control medium (29.60 ± 1.67%). However, addition of 1% and 2.5% of sericin to the IVM medium oocytes had a negative effect on nuclear maturation and cumulus cell expansion. Furthermore, the percentage of cleavage and blastocyst rate was significantly lower in the 1% and 2.5% sericin groups than in the control group. These findings showed that supplementation of IVM medium with 0.5% sericin may improve the meiotic competence of oocytes and early embryonic development in Sanjabi ewes during the breeding season.

  6. Exposure to cadmium during in vitro maturation at environmental nanomolar levels impairs oocyte fertilization through oxidative damage: A large animal model study.

    PubMed

    Martino, N A; Marzano, G; Mangiacotti, M; Miedico, O; Sardanelli, A M; Gnoni, A; Lacalandra, G M; Chiaravalle, A E; Ciani, E; Bogliolo, L; Minervini, F; Pizzi, F; Dell'Aquila, M E

    2017-02-07

    Cadmium is a highly toxic heavy metal with negative effects on oocyte fertilization. The aim of this study was to analyse whether cadmium-induced impairment of fertilization is caused by mitochondria dysfunction and oxidative stress in the cumulus-oocyte complex (COC). Preliminarily, 19 trace element levels were measured in ovaries from juvenile and adult ewes and age-related cadmium ovarian bioaccumulation at nanomolar concentrations was found. COCs from juvenile and adult ewes, exposed during in vitro maturation to 1nM or 100nM CdCl2, and subjected to in vitro fertilization showed significantly lower fertilization rates in exposed COCs compared with controls. In vitro matured exposed and control COCs underwent confocal microscopy analysis of mitochondria activity and reactive oxygen species (ROS) levels and lipid peroxidation (LPO) assay at cumulus cell and oocyte level. In both age groups, cadmium at nanomolar concentrations induced cumulus-oocyte mitochondria over-activity and oxidative damage which were related to impaired oocyte fertilization.

  7. Caffeine supplementation during IVM improves frequencies of nuclear maturation and preimplantation development of dromedary camel oocytes following IVF.

    PubMed

    Fathi, Mohamed; Seida, Adel A; Sobhy, Refaat R; Darwish, Gamal M; Badr, Magdy R; Moawad, Adel R

    2014-06-01

    Caffeine supplementation during oocyte IVM has been reported to improve preimplantation embryo development and the quality of in vitro-produced blastocysts in a range of species; but no studies have been done in camels. The present study investigated the effect of caffeine supplementation during dromedary camel oocyte IVM on nuclear maturation rates, IVF events, and subsequent preimplantation development. Cumulus-oocyte complexes obtained at slaughter were matured in vitro in caffeine supplemented medium either for 30 hours (caffeine 30 hours) or in the medium without caffeine supplement for 24 hours and then transferred to freshly prepared IVM medium supplemented with 10 mM caffeine for another 6 hours (caffeine 6 hours). Cumulus-oocyte complexes matured for 30 hours in the medium without caffeine supplement were used as a control. Matured oocytes were fertilized in vitro by epididymal spermatozoa of mature male camels collected from a local slaughterhouse. Eighteen hours after insemination, presumptive zygotes were cultured in modified KSOMaa medium for 7 days. Maturation and fertilization rates were significantly higher in the caffeine 6-hour group compared with the control group (P < 0.05), whereas IVM of oocytes in caffeine-supplemented medium for 30 hours did not affect these parameters (P > 0.05). Interestingly, IVM of oocytes in caffeine supplemented medium for 6 hours significantly (P < 0.05) increased the frequencies of blastocyst development by more than two-fold when compared with control (27.78% vs. 11.76%). In conclusion, culturing dromedary camel oocytes in maturation medium without caffeine for 24 hours and then in the medium supplemented with 10 mM caffeine for 6 hours during 30-hour IVM can significantly improve frequencies of nuclear maturation, fertilization rate, and subsequent preimplantation development.

  8. The effect of cumulus cells on domestic cat (Felis catus) oocytes during in vitro maturation and fertilization.

    PubMed

    Sowińska, N; Frankowska, K; Filipczyk, A; Adamaszek, A; Nalik, K; Fic, K; Pietsch-Fulbiszewska, A

    2017-04-01

    The aim of this study was to evaluate the effect of co-culture of denuded oocytes with cumulus cells (CC) or cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) and in vitro fertilization (IVF). Immature oocytes were collected from ovaries of domestic cats following a routine ovariectomy. Oocytes were matured in vitro for 24 hr within four groups: (i) denuded oocytes (DO), (ii) DO co-cultured with CC, (iii) DO co-cultured with COC and (iv) COC as a control group. In further experiments, COCs were matured in vitro for 24 hr, and then, oocytes were randomly divided into four groups as previously described and fertilized in vitro. Embryos were cultured for up to 7 days. At the end of each experiment, oocytes/embryos were stained with Hoechst 33342 solution and observed under an inverted fluorescence microscope. The results of oocyte maturation showed that their meiotic competence decreased significantly in all experimental groups, compared to the control group. The maturation rates were approximately 45%, 24%, 43% and 76% in experiment 1, and 21%, 14%, 33% and 50% in experiment 2 in groups (i), (ii), (iii) and (iv), respectively. Examination of in vitro fertilization revealed that embryos developed up to the morula stage in all experimental groups. DO and oocytes cultured with COC during fertilization showed a lower cleavage rate-36% and 25% as opposed to those co-cultured with loose CC and the control group-43% and 42%, respectively. Results of this study indicate that cumulus cells connected with an oocyte into a cumulus-oocyte complex are irreplaceable for the maturation of domestic cat oocyte, but that the addition of loose CC may be beneficial for IVF. © 2016 Blackwell Verlag GmbH.

  9. Effect of sericin supplementation during in vitro maturation on the maturation, fertilization and development of porcine oocytes.

    PubMed

    Do, L T K; Namula, Z; Luu, V V; Sato, Y; Taniguchi, M; Isobe, T; Kikuchi, K; Otoi, T

    2014-04-01

    This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA-fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.

  10. Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization.

    PubMed

    Saini, N; Singh, M K; Shah, S M; Singh, K P; Kaushik, R; Manik, R S; Singla, S K; Palta, P; Chauhan, M S

    2015-12-01

    In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus-oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus-oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus-oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.

  11. Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration.

    PubMed

    Yoshimura, Y; Nakamura, Y; Oda, T; Ando, M; Ubukata, Y; Karube, M; Koyama, N; Yamada, H

    1992-08-01

    The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.

  12. Role of PTGS2-generated PGE2 during gonadotrophin-induced bovine oocyte maturation and cumulus cell expansion.

    PubMed

    Marei, Waleed F; Abayasekara, D Robert E; Wathes, D Claire; Fouladi-Nashta, Ali A

    2014-03-01

    Prostaglandin E2 (PGE2) is an autocrine/paracrine factor which mediates gonadotrophin (Gn) stimulation of cumulus expansion and oocyte maturation in rodents. Its role in bovine oocyte maturation is less characterized. This study detected PTGS2 (COX2) and PGE synthases (PTGES1, PTGES2 and PTGES3) in bovine cumulus-oocyte complexes (COC). Only PTGS2 and PTGES1 expression changed during maturation. In Gn-free media, no cumulus expansion and ∼45% nuclear maturation was achieved, while Gn-induced maturation showed full cumulus expansion (score 3) and ∼87% maturation. PGE2 supplementation without Gn induced mild cumulus expansion (score 0.5-1) but increased nuclear maturation to levels similar to those obtained with Gn alone. In the presence of Gn, exogenous PGE2 did not affect expansion or nuclear maturation and subsequent embryo development. Treatment with PTGS2 selective inhibitor (NS398), PTGS2-specific siRNA or PTGER2-receptor antagonist (AH6809) resulted in ∼20-25% reduction in nuclear maturation. NS398 and AH6809 did not affect cumulus expansion. Most oocytes not reaching metaphase of second meiosis (MII) following NS398, AH6809 and PTGS2-specific siRNA treatments were at MI. After longer maturation, NS398-treated oocytes had normal MII rate and uncompromised embryo development. PGE2 has a limited role in cumulus expansion in bovine COC but is important for the timing of Gn-induced nuclear maturation. We confirmed that genes involved in the synthesis of prostaglandin E2 (PGE2) are expressed by cumulus-oocyte complexes (or eggs) of cows and that PGE2 is synthesized during oocyte maturation in the presence of gonadotrophin hormones. When we inhibited synthesis of PGE2 or blocked its receptors, oocyte maturation, but not cumulus expansion, was compromised. Further investigation showed that oocyte maturation is delayed but not arrested when PGE2 synthesis is inhibited. On the other hand, addition of exogenous PGE2 induced a high maturation rate and mild cumulus

  13. Birth after human chorionic gonadotropin-primed oocyte in vitro maturation and fertilization with testicular sperm in a normo-ovulatory patient

    PubMed Central

    González-Ortega, Claudia; Piña-Aguilar, Raul Eduardo; Cancino-Villareal, Patricia; Gutiérrez-Gutiérrez, Antonio Martin

    2016-01-01

    In this report, we present a case of in vitro maturation (IVM) with surgical retrieved testicular sperm in a normo-ovulatory female. Human chorionic gonadotropin-primed IVM, testicular biopsy for sperm retrieval and intracytoplasmic sperm injection with fresh sperm were performed. Fourteen cumulus-oocyte complexes were obtained in germinal vesicle or metaphase I stage, eight oocytes reached metaphase II, seven presumptive zygotes were obtained, and three cleavage stages embryos in day 2 were transferred producing a singleton pregnancy. A single healthy newborn was obtained. Our results suggest that IVM may be an alternative for in vitro fertilization in normo-ovulatory women even if surgical retrieval of sperm is needed. Further research is required to depict contributing factors to the success of IVM in indications different from polycystic ovaries syndrome and the role of male gamete. PMID:27803591

  14. Effect of FSH and LH hormones on oocyte maturation of buffalo and gene expression analysis of their receptors and Cx43 in maturing oocytes.

    PubMed

    Pandey, Alok; Gupta, S C; Gupta, Neelam

    2010-08-01

    Follicle stimulating hormone (FSH) and luteinizing hormone (LH) are commonly added to maturation media to improve cumulus expansion known to be a predictor of oocyte maturation. Therefore, effects of various concentrations of FSH (1000 ng/ml), LH (1000 ng/ml) and FSH + LH (1000 ng/ml each) in comparison with control (without FSH + LH) cultured oocytes were investigated. FSH and LH (1000 ng/ml each) induced significantly more cumulus expansion and polar body numbers, as compared with control and treatments of 1000 ng/ml FSH and 1000 ng/ml LH alone. Expression of FSH receptor (r), LHr and Cx43 mRNAs was determined by real-time PCR in cumulus-oocyte complexes (COCs) and denuded oocytes at different maturation times. Expression of all three genes was higher in COCs compared with denuded oocytes, confirming the importance of cumulus cells in oocyte maturation. FSHr and connexin 43 (Cx43) mRNA abundance in both COCs and denuded oocytes was highest at 0-6 h of maturation and decreased subsequently. However, LHr mRNA abundance increased from 6 h up to 24 h of maturation. The study concluded that FSH, LH receptors and Cx43 gene expression regulation is an index related to oocyte maturation.

  15. Effect of melatonin on maturation capacity and fertilization of Nili-Ravi buffalo (Bubalus bubalis) oocytes

    PubMed Central

    Nagina, G.; Asima, A.; Nemat, U.; Shamim, A.

    2016-01-01

    This study evaluated the effect of melatonin supplementation of in vitro maturation media on in vitro maturation (IVM) and in vitro fertilization (IVF) rate of buffalo oocytes. Cumulus oocytes complexes (COCs) were aspirated from follicles of 2-8 mm diameter. In experiment I, COCs were matured in IVM medium supplemented with 0 (control), 250, 500, and 1000 μM melatonin for 22-24 hours in CO2 incubator at 38.5°C with 5% CO2 and at 95% relative humidity. The maturation rate did not differ in media supplemented with melatonin at 250 μM, 500 μM, 1000 μM and control (0 μM). In experiment II, the matured oocytes were fertilized in 50 μl droplets of Tyrode’s Albumin Lactate Pyruvate (TALP) medium having 10 ug/ml heparin for sperm (2 million/ml) capacitation. The fertilization droplets were then kept for incubation at 5% CO2, 39°C and at 95% relative humidity for 18 hours. The fertilization rate was assessed by sperm penetration and pronuclear formation. Fertilization rate was improved when maturation medium was supplemented with 250 μM melatonin compared to control. In conclusion, melatonin supplementation to serum free maturation media at 250 μM improved the fertilization rate of buffalo oocytes. PMID:27540514

  16. The importance of manganese in the cytoplasmic maturation of cattle oocytes: blastocyst production improvement regardless of cumulus cells presence during in vitro maturation.

    PubMed

    Anchordoquy, Juan Patricio; Anchordoquy, Juan Mateo; Sirini, Matias Angel; Testa, Juan Alberto; Peral-García, Pilar; Furnus, Cecilia Cristina

    2016-02-01

    Adequate dietary intake of manganese (Mn) is required for normal reproductive performance in cattle. This study was carried out to investigate the effect of Mn during in vitro maturation of bovine cumulus-oocyte complexes (COC) on apoptosis of cumulus cells, cumulus expansion, and superoxide dismutase (SOD) activity in the COC. The role of cumulus cells on Mn transport and subsequent embryo development was also evaluated. Early apoptosis decreased in cumulus cells matured with Mn compared with medium alone. Cumulus expansion did not show differences in COC matured with or without Mn supplementation. SOD activity was higher in COC matured with 6 ng/ml Mn than with 0 ng/ml Mn. Cleavage rates were higher in COC and denuded oocytes co-cultured with cumulus cells, either with or without Mn added to in vitro maturation (IVM) medium. Regardless of the presence of cumulus cells during IVM, the blastocyst rates were higher when 6 ng/ml Mn was supplemented into IVM medium compared with growth in medium alone. Blastocyst quality was enhanced when COC were matured in medium with Mn supplementation. The results of the present study indicated that Mn supplementation to IVM medium enhanced the 'health' of COC, and improved subsequent embryo development and embryo quality.

  17. Effect of perfluorooctane sulfonate on viability, maturation and gap junctional intercellular communication of porcine oocytes in vitro.

    PubMed

    Domínguez, A; Salazar, Z; Arenas, E; Betancourt, M; Ducolomb, Y; González-Márquez, H; Casas, E; Teteltitla, M; Bonilla, E

    2016-09-01

    Perfluorooctane sulfonate (PFOS) is a broadly used man-made surfactant whose long half-life has led to bioaccumulation. This perfluorinated compound is ubiquitous in human body fluids. PFOS concentrations as high as 26μM in plasma have been reported in occupationally exposed populations, and high levels of PFOS in human follicular fluid have been associated with subfertility. However, the effect of PFOS on the maturation of oocytes in mammals has not been reported to date. The aim of this study was to determine the effects of PFOS during oocyte maturation. Results indicate that PFOS inhibits oocyte viability (Lethal Concentration50=32μM) and maturation (inhibition of maturation50=22μM) at physiologically relevant concentrations. In order to evaluate the mechanisms of oocyte maturation inhibition by PFOS, gap junctional intercellular communication (GJIC) between oocytes and granulosa cells was assessed. GJIC between granulosa cells and the oocyte was significantly affected during the first 8h of maturation. However, the inhibitory effect of PFOS on GJIC was not due to an alteration on the expression of connexin genes Cx43, Cx45 and Cx60. These findings suggest that occupationally exposed populations could be at risk, and that PFOS might affect oocyte maturation by interfering the GJIC in the cumulus-oocyte complexes during the first hours of maturation. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix*

    PubMed Central

    Briggs, David C.; Birchenough, Holly L.; Ali, Tariq; Rugg, Marilyn S.; Waltho, Jon P.; Ievoli, Elena; Jowitt, Thomas A.; Enghild, Jan J.; Richter, Ralf P.; Salustri, Antonietta; Milner, Caroline M.; Day, Anthony J.

    2015-01-01

    The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix. PMID:26468290

  19. Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation.

    PubMed

    Yerushalmi, G M; Salmon-Divon, M; Yung, Y; Maman, E; Kedem, A; Ophir, L; Elemento, O; Coticchio, G; Dal Canto, M; Mignini Renzinu, M; Fadini, R; Hourvitz, A

    2014-08-01

    Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mammalian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment.

  20. Oocyte-secreted factors in oocyte maturation media enhance subsequent development of bovine cloned embryos.

    PubMed

    Su, Jianmin; Wang, Yongsheng; Zhang, Lei; Wang, Bo; Liu, Jun; Luo, Yan; Guo, Zekun; Quan, Fusheng; Zhang, Yong

    2014-04-01

    Successful in vitro maturation (IVM) and oocyte quality both affect the subsequent development of cloned embryos derived from somatic-cell nuclear transfer (SCNT). Developmental competence is usually lower in oocytes matured in vitro compared with those that matured in vivo, possibly due to insufficient levels of oocyte-secreted factors (OSFs) and disrupted oocyte-cumulus communication. This study investigated the effects of OSFs secreted by denuded oocytes (DOs) during IVM on the subsequent developmental competence of cloned bovine embryos. Cumulus-oocyte complexes (COCs) from antral follicles of slaughtered-cow ovaries collected from an abattoir were divided into four groups: COCs co-cultured with and without DOs in maturation media used for SCNT, as well as COCs co-cultured with and without DOs in maturation media used for in vitro fertilization (IVF). Based on the developmental competence and embryo quality of bovine embryos generated from these four groups, we found that co-culturing the COCs with DOs enhanced the in vitro development of IVF and cloned bovine embryos, and potentially generated more high-quality cloned blastocysts that possessed locus-specific histone modifications at levels similar to in vitro-fertilized embryos. These results strongly suggest that co-culturing COCs with DOs enhances subsequent developmental competence of cloned bovine embryo. © 2014 Wiley Periodicals, Inc.

  1. In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid

    PubMed Central

    AGUNG, Budiyanto; OTOI, Takeshige; FUCHIMOTO, Dai-ichiro; SENBON, Shoichiro; ONISHI, Akira; NAGAI, Takashi

    2013-01-01

    Abstract This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 106 cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium. PMID:23428620

  2. Estimation of the optimal timing of fertilization for embryo development of in vitro-matured bovine oocytes based on the times of nuclear maturation and sperm penetration.

    PubMed

    Koyama, Keisuke; Kang, Sung-Sik; Huang, Weiping; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2014-05-01

    The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14-22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12-30 hr, sperm penetration was examined after 4-18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12-30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x - 0.297x(2), P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr.

  3. Estimation of the Optimal Timing of Fertilization for Embryo Development of In Vitro-Matured Bovine Oocytes Based on the Times of Nuclear Maturation and Sperm Penetration

    PubMed Central

    KOYAMA, Keisuke; KANG, Sung-Sik; HUANG, Weiping; YANAGAWA, Yojiro; TAKAHASHI, Yoshiyuki; NAGANO, Masashi

    2014-01-01

    ABSTRACT The objective of this research was to estimate the optimal timing for fertilization to achieve proper embryonic development of in vitro-matured bovine oocytes. First, cumulus-oocyte complexes were subjected to in vitro maturation (IVM) for 14–22 hr. The timing when 50% of oocytes reached metaphase II stage was estimated to be 17.5 hr after IVM start. Next, using oocytes subjected to IVM for 12–30 hr, sperm penetration was examined after 4–18 hr of in vitro fertilization (IVF). A significant negative correlation between IVM duration and the timing when 50% of oocytes were penetrated by sperm after IVF start was observed (P<0.01). Finally, oocytes subjected to 12–30 hr of IVM were inseminated and cultured for 6 days to examine embryonic development. In the group with 22 hr of IVM, the percentages of cleaved embryos and blastocysts were the highest values in all groups. According to the regression equation describing the time from nuclear maturation to sperm penetration (x) and the percentage of blastocysts (y) (y=7.23x − 0.297x2, P<0.01), the blastocyst rate peaked when sperm penetration occurred at 12.2 hr after achieving nuclear maturation. In conclusion, under the present IVM/IVF conditions, it was estimated that oocytes acquired their highest developmental competence at about 30 hr after IVM start, and thus, the optimal IVM duration was calculated to be about 21 hr. PMID:24430663

  4. Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation.

    PubMed

    Okudaira, Yuichi; Wakai, Takuya; Funahashi, Hiroaki

    2017-02-23

    The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.

  5. Capturing Complexity through Maturity Modelling

    ERIC Educational Resources Information Center

    Underwood, Jean; Dillon, Gayle

    2004-01-01

    The impact of information and communication technologies (ICT) on the process and products of education is difficult to assess for a number of reasons. In brief, education is a complex system of interrelationships, of checks and balances. This context is not a neutral backdrop on which teaching and learning are played out. Rather, it may help, or…

  6. Capturing Complexity through Maturity Modelling

    ERIC Educational Resources Information Center

    Underwood, Jean; Dillon, Gayle

    2004-01-01

    The impact of information and communication technologies (ICT) on the process and products of education is difficult to assess for a number of reasons. In brief, education is a complex system of interrelationships, of checks and balances. This context is not a neutral backdrop on which teaching and learning are played out. Rather, it may help, or…

  7. Production of lion (Panthera leo) blastocysts after in vitro maturation of oocytes and intracytoplasmic sperm injection.

    PubMed

    Fernandez-Gonzalez, Lorena; Hribal, Romy; Stagegaard, Julia; Zahmel, Jennifer; Jewgenow, Katarina

    2015-04-01

    Assisted reproductive techniques are becoming widely applied to the breeding of endangered species, but establishing reliable protocols for the production of embryos in vitro is challenging because of the scarcity of sample material. In our study, we applied an assisted reproductive technique protocol for IVM and intracytoplasmic sperm injection (ICSI), developed in the domestic cat, to oocytes retrieved from ovaries of four 2-year-old lionesses (Panthera leo) eight hours postmortem. In total, 68 cumulus-oocyte complexes of good quality were randomly distributed and cultured for 32 to 34 hours in two different maturation culture media, consisting of Medium 199 with Earle's salts, 3 mg/mL BSA, 0.1 mg/mL cysteine, 1.4 mg/mL sodium pyruvate, 0.6 mg/mL sodium lactate, 0.15 mg/mL l-glutamine, and 0.055 mg/mL gentamicin. Hormonal supplementation of IVM_1 was 0.02 IU/mL FSH and 0.05 IU/mL LH; IVM_2 consisted of 1.64 IU/mL FSH, 1.06 IU/mL LH, and 1 μg/mL 17ß-estradiol. Differences in hormonal supplementation did not produce significant differences in oocyte maturation rates, which were 39.4% in IVM_1 and 34.3% in IVM_2. Matured oocytes were microinjected with homologous frozen-thawed spermatozoa, and subsequent cleavage rates were 30.8% and 58.3%, respectively. Half of the embryos derived from oocytes matured in IVM_1 developed into blastocysts, whereas only 28.6% of embryos from oocytes matured in IVM_2 reached the blastocyst stage. Morula stages were present from Day 6 onward, and blastocyst stages from Day 9 on, indicating a slower developmental speed in comparison with domestic cats. This is the first report of in vitro-produced blastocysts using ICSI in the lion, and the results report that IVM and ICSI can be successfully performed with cumulus-oocyte complexes retrieved from ovaries after eight hours of shipping, obtaining competent embryos in culture.

  8. The influence of powdered coconut water (ACP-318®) in in vitro maturation of canine oocytes.

    PubMed

    Silva, A E F; Cavalcante, L F; Rodrigues, B A; Rodrigues, J L

    2010-12-01

    The objective of this study was to determine the influence of powdered coconut water (ACP-318(®)) diluted in high glucose (11.0 mM) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Cumulus oocyte complexes (COCs) (n = 632) were randomly allocated into three experimental groups named as group 1 (control group), group 2 (5% powdered coconut water) and group 3 (10% powdered coconut water). The percentage of meiotic resumption (MR) (GVBD to MII) was 39.1% (81/207), 50.2% (108/215) and 46.6% (98/210) for groups 1, 2 and 3 respectively (p < 0.05). There were no differences in MR rates among groups 2 and 3. The medium with ACP-318(®) slightly enhanced the nuclear maturation of canine oocytes when a comparison was established with rates of maturation exhibited by oocytes in the experimental group 1 without ACP-318(®) (p < 0.05). The results suggest that oocytes' nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water. Further investigation must be performed for a better understanding of powdered coconut water influence in cellular events during IVM of dog oocytes.

  9. MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes.

    PubMed

    Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Grázia, João Gabriel V; Santos, Fábio N; Arnold, Daniel R; Basso, Andrea C; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Sá Rosa-E-Silva, Ana Carolina J

    2017-04-01

    The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

  10. MicroRNA expression profile in bovine cumulus–oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes

    USDA-ARS?s Scientific Manuscript database

    The expression of microRNAs (miRs) in bovine cumulus-oocyte complexes (COCs) during late oogenesis was profiled to determine the potential for regulation of maternal mRNAs by this class of small RNAs. A cDNA cloning and sequencing strategy resulted in 1812 putative miR sequences, representing 72 di...

  11. Beneficial Effects of Melatonin on the In Vitro Maturation of Sheep Oocytes and Its Relation to Melatonin Receptors.

    PubMed

    Tian, Xiuzhi; Wang, Feng; Zhang, Lu; He, Changjiu; Ji, Pengyun; Wang, Jing; Zhang, Zhenzhen; Lv, Dongying; Abulizi, Wusiman; Wang, Xuguang; Lian, Zhengxing; Liu, Guoshi

    2017-04-17

    (1) Background: The binding sites of melatonin, as a multifunctional molecule, have been identified in human, porcine, and bovine samples. However, the binding sites and mechanisms of melatonin have not been reported in sheep; (2) Methods: Cumulus-oocyte complexes (COCs) were cultured in TCM-199 supplemented with melatonin at concentrations of 0, 10(-3), 10(-5), 10(-7), 10(-9), and 10(-11) M. Melatonin receptors (MT1 and MT2) were evaluated via immunofluorescence and Western blot. The effects of melatonin on cumulus cell expansion, nuclear maturation, embryo development, and related gene (GDF9, DNMT1, PTX3, HAS2, and EGFR) expression were investigated. The level of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were evaluated in oocytes and cumulus, respectively; (3) Results: Both MT1 and MT2 were expressed in oocytes, cumulus cells, and granulosa cells. Melatonin with a concentration of 10(-7) M significantly enhanced the rates of nuclear maturation, cumulus cells expansion, cleavage, and blastocyst. Melatonin enhanced the expression of BMP15 in oocytes and of PTX3, HAS2, and EGFR in cumulus cells. Melatonin decreased the cAMP level of oocytes but enhanced the cGMP level in oocytes and cumulus cells; (4) Conclusion: The higher presence of MT1 in GV cumulus cells and the beneficial effects of melatonin indicated that its roles in regulating sheep oocyte maturation may be mediated mainly by the MT1 receptor.

  12. Analyses of apoptosis and DNA damage in bovine cumulus cells after in vitro maturation with different copper concentrations: consequences on early embryo development.

    PubMed

    Rosa, D E; Anchordoquy, J M; Anchordoquy, J P; Sirini, M A; Testa, J A; Mattioli, G A; Furnus, C C

    2016-12-01

    The aim of this study was to investigate the influence of copper (Cu) during in vitro maturation (IVM) on apoptosis and DNA integrity of cumulus cells (CC); and oocyte viability. Also, the role of CC in the transport of Cu during IVM was evaluated on oocyte developmental capacity. Damage of DNA was higher in CC matured without Cu (0 µg/dl Cu, P < 0.01) with respect to cells treated with Cu for cumulus-oocyte complexes (COCs) exposed to 0, 20, 40, or 60 µg/dl Cu). The percentage of apoptotic cells was higher in CC matured without Cu than in CC matured with Cu. Cumulus expansion and viability of CC did not show differences in COC treated with 0, 20, 40, or 60 µg/dl Cu during IVM. After in vitro fertilization (IVF), cleavage rates were higher in COC and DO + CC (denuded oocytes + CC) with or without Cu than in DO. Independently of CC presence (COC, DO + CC or DO) the blastocyst rates were higher when 60 µg/dl Cu was added to IVM medium compared to medium alone. These results indicate that Cu supplementation to IVM medium: (i) decreased DNA damage and apoptosis in CC; (ii) did not modify oocyte viability and cumulus expansion; and (iii) improved subsequent embryo development up to blastocyst stage regardless of CC presence during IVM.

  13. In vivo and in vitro maturation of oocytes collected from superstimulated wood bison (Bison bison athabascae) during the anovulatory and ovulatory seasons.

    PubMed

    Cervantes, Miriam P; Palomino, J Manuel; Anzar, Muhammad; Mapletoft, Reuben J; Adams, Gregg P

    2016-10-01

    Experiments were done to compare the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison. In Experiment 1 (anovulatory season), follicular ablation was done to synchronize follicle wave emergence among bison on Day -1, and FSH was given on Days 0 and 2. Bison were then assigned to 5 groups (n=5/group) in which COC were collected by transvaginal follicle aspiration on Day 4 and either fixed immediately with no maturation (control), matured in vitro for 24 or 30h, or collected on Day 5 after in vivo maturation for 24 or 30h (i.e., after hCG treatment). In Experiment 2 (ovulatory season), bison were treated as described for Experiment 1, but PGF2α (cloprostenol) was given to control the luteal phase on Days -9 and 3. In both experiments, cumulus cell expansion was more extensive following in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30h groups. Nuclear maturation occurred more rapidly in vitro; 60-70% of oocytes were at the MII stage 24h after in vitro maturation while only 25-27% of oocytes had reached the MII stage after 24h of in vivo maturation. In conclusion, nuclear maturation occurred more rapidly during in vitro vs. in vivo maturation, but was associated with less cumulus expansion than in vivo maturation. In vivo oocyte maturation was more complete at 30 vs. 24h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes.

  14. In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up-derived oocytes have different kinetics and requirements regarding maturation media.

    PubMed

    Souza-Fabjan, Joanna Maria G; Locatelli, Yann; Duffard, Nicolas; Corbin, Emilie; Touzé, Jean-Luc; Perreau, Christine; Beckers, Jean François; Freitas, Vicente José F; Mermillod, Pascal

    2014-05-01

    A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the

  15. Heat stress and antioxidant enzyme activity in bubaline ( Bubalus bubalis) oocytes during in vitro maturation

    NASA Astrophysics Data System (ADS)

    Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

    2016-09-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly ( P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly ( P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

  16. Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation.

    PubMed

    Waiz, Syma Ashraf; Raies-Ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T Sridhar; Chauhan, M S; Upadhyay, R C

    2016-09-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

  17. Chemical manipulation of glucose metabolism in porcine oocytes: effects on nuclear and cytoplasmic maturation in vitro.

    PubMed

    Herrick, Jason R; Brad, Amber M; Krisher, Rebecca L

    2006-02-01

    The objectives of this study were to manipulate metabolism of glucose through glycolysis and the pentose phosphate pathway (PPP) in porcine oocytes during in vitro maturation, and determine the effects of this manipulation on meiotic progression, intracellular glutathione (GSX) concentrations and embryonic development. Cumulus-oocyte complexes isolated from abattoir ovaries were matured (40-44 h) in Purdue Porcine Medium for maturation alone (control) or supplemented with pyrroline-5 carboxylate (PC, 0.1 microM; PPP stimulator), diphenyleneiodonium (DPI, 0.1 microM; PPP inhibitor), dinitrophenol (DNP, 10 microM; glycolytic stimulator), hexametaphosphate (HMP, 100 microM; glycolytic inhibitor), PC + HMP or DNP + DPI. At the conclusion of in vitro maturation, cumulus cells were removed and oocytes were randomly allocated for analysis of GSX, metabolism and nuclear maturation, or in vitro fertilization and embryo culture. Both DPI and DNP + DPI decreased (P < or = 0.05) the activity of glycolysis and the PPP, increased (P < or = 0.05) the percentage of immature oocytes, and decreased (P < or = 0.05) the proportion of mature oocytes compared with control oocytes and oocytes from the other treatments. Embryonic development (cleavage and blastocyst stage) and the intracellular content of GSX were also decreased (P < or = 0.05) following exposure to DPI or DNP + DPI compared with control oocytes and oocytes from the other treatments. Oocyte metabolism, nuclear maturation, GSX content and embryonic development were unaffected (P > 0.05) following exposure to PC, DNP, HMP or PC + HMP. Our results suggest that metabolism of glucose through the PPP and/or glycolysis plays a key role in the control of nuclear and cytoplasmic maturation of porcine oocytes in vitro.

  18. Effect of In Vitro Maturation Technique and Alpha Lipoic Acid Supplementation on Oocyte Maturation Rate: Focus on Oxidative Status of Oocytes

    PubMed Central

    Zavareh, Saeed; Karimi, Isaac; Salehnia, Mojdeh; Rahnama, Ali

    2016-01-01

    Background Therapeutic potential of in vitro maturation (IVM) in infertility is growing with great promise. Although significant progress is obtained in recent years, existing IVM protocols are far from favorable results. The first aim of this study was to investigate whether two step IVM manner change reactive oxygen species (ROS) and total anti- oxidant capacity (TAC) levels. The second aim was to find the effect of alpha lipoic acid (ALA) supplementation on oocyte maturation rate and on ROS/TAC levels during IVM. Materials and Methods In this experimental study, mouse germinal vesicle (GV) oocytes divided into cumulus denuded oocytes (DOs) and cumulus oocyte complexes (COCs) groups. GVs were matured in vitro in the presence or absence of ALA only for 18 hours (control) or with pre-culture of forskolin plus cilostamide for an additional 18 hours. Matured oocytes obtained following 18 and 36 hours based on experimental design. In parallel, the ROS and TAC levels were measured at different time (0, 18 and 36 hours) by 2',7'-dichlorodihydrofluorescein (DCFH) probe and ferric reducing/antioxidant power (FRAP) assay, respectively. Results Maturation rate of COCs was significantly higher than DOs in control group (P<0.05), while there was no significant difference between COCs and DOs when were pre-cultured with forskolin plus cilostamide. ROS and TAC levels was increased and decreased respectively in DOs after 18 hours while in COCs did not change at 18 hours and showed a significant increase and decrease respectively at 36 hours (P<0.05). ROS and TAC levels in the presence of ALA were significantly decreased and increased respectively after 36 hours (P<0.05) whereas, maturation rates of COCs and DOs were similar to their corresponding control groups. Conclusion ALA decreased ROS and increased TAC but could not affect maturation rate of both COCs and DOs in one or two step IVM manner. PMID:26985332

  19. Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess

    USDA-ARS?s Scientific Manuscript database

    To date, animal models with naturally occurring androgen excess have not been identified. Serendipitously, we discovered two subpopulations of cows with dramatically different follicular fluid androgen concentrations in dominant follicles within our research herd. In the cow, androstenedione is the...

  20. MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells.

    PubMed

    Pan, Bo; Toms, Derek; Shen, Wei; Li, Julang

    2015-03-15

    We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion (HAS2, PTGS2) and oocyte maturation (CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zona pellucida 3 (ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

  1. Effect of alpha-tocopherol and ascorbic acid on bovine oocyte in vitro maturation.

    PubMed

    Dalvit, G; Llanes, S P; Descalzo, A; Insani, M; Beconi, M; Cetica, P

    2005-04-01

    In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.

  2. Involvement of the serine protease inhibitor, SERPINE2, and the urokinase plasminogen activator in cumulus expansion and oocyte maturation.

    PubMed

    Lu, Chung-Hao; Lee, Robert Kuo-Kuang; Hwu, Yuh-Ming; Lin, Ming-Huei; Yeh, Ling-Yu; Chen, Ying-Jie; Lin, Shau-Ping; Li, Sheng-Hsiang

    2013-01-01

    The serpin peptidase inhibitor, clade E, member 2 (SERPINE2) inhibits urokinase-type plasminogen activator (PLAU) and tissue-type plasminogen activator. Higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. The objective of this study was to evaluate whether high SERPINE2 levels in cumulus cells are associated with oocyte immaturity. Using the mouse cumulus-oocyte complex as an experimental model, the effects of elimination and overexpression of SERPINE2 in cumulus cells on cumulus expansion and oocyte maturation were assayed by in vitro maturation. Serpine2 and PLAU transcripts were the most highly expressed serpins and plasminogen activators, respectively. Their expression was coordinately regulated in cumulus cells during gonadotropin-induced oocyte maturation. Silencing of Serpine2 expression using small interfering RNAs or blockage of SERPINE2 protein using a specific antibody had no effect on oocyte maturation. However, overexpression of Serpine2 or exogenous supplementation with high levels of SERPINE2 impaired cumulus expansion and oocyte maturation, probably by decreasing hyaluronan synthase 2 (Has2) and versican (Vcan) mRNA expression. Amiloride, a specific PLAU inhibitor, also suppressed these processes. PLAU supplementation of the oocyte in vitro maturation medium caused earlier and more extensive expansion of cumulus cells and oocyte maturation that may be mediated by increased Has2 mRNA expression. However, these effects were neutralized by coincubation with SERPINE2 or amiloride and PLAU. In conclusion, SERPINE2 and PLAU are involved in cumulus expansion and oocyte maturation. High SERPINE2 levels impair these processes, probably by decreasing cumulus matrix gene expression as well as reducing cumulus hyaluronan contents and inhibiting PLAU activity. These findings may explain why cumulus cells surrounding immature human oocytes express high SERPINE2 levels.

  3. Age and anti-Müllerian hormone levels predict the success of in vitro maturation of cat oocytes.

    PubMed

    Snoeck, F; Sarrazin, S; Wydooghe, E; Van Soom, A

    2016-11-15

    Up to date, in vitro maturation (IVM) rates of oocytes are highly variable between individual cats. This study was carried out to investigate the predictive value of age and anti-Müllerian hormone (AMH) concentration in relation to capacity for IVM of cat oocytes. Ovaries were collected from 33 cats, which were divided into three age groups: (i) 0-3 months (pre-pubertal); (ii) 3-12 months (peripubertal); and (iii) older than 12 months (pubertal). The cumulus-oocyte complexes (COCs) were matured and subsequently stained to check nuclear maturation status, and blood was taken for AMH analysis. Increasing age was significantly associated with decreasing AMH levels, and mean AMH levels differed significantly between all age categories: group 1: mean AMH 18.71 μg/L; group 2: mean AMH 9.27 μg/L; and group 3: mean AMH 4.13 μg/L. Moreover, the probability of maturation was more likely in groups 2 and 3 compared to group 1. Between categories 2 and 3, no significant difference in maturation probability was found (p = .31). Finally, the probability of oocyte maturation decreased significantly with increasing AMH levels. In age group 2, oocytes with a higher AMH level were less likely to mature. In age groups 1 and 3, no significant association between the AMH level and the proportion of maturated COC was found. We can conclude that if a higher probability of nuclear maturation is required, it is preferable to use cats with lower AMH levels and older than 3 months of age to improve cat IVM.

  4. Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development.

    PubMed

    Takada, L; Junior, A Martins; Mingoti, G Z; Balieiro, J C C; Cipolla-Neto, J; Coelho, L A

    2012-02-01

    The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5μg/ml FSH and 5.0μg/ml LH (FSH-LH); 10(-9)M melatonin (MEL) or FSH-LH+MEL (FSH-LH-MEL). After 24h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6±2.4) than in FSH-LH-MEL (28.0±2.4) and FSH-LH (17.8±2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro.

  5. In vitro acute exposure to DEHP affects oocyte meiotic maturation, energy and oxidative stress parameters in a large animal model.

    PubMed

    Ambruosi, Barbara; Uranio, Manuel Filioli; Sardanelli, Anna Maria; Pocar, Paola; Martino, Nicola Antonio; Paternoster, Maria Stefania; Amati, Francesca; Dell'Aquila, Maria Elena

    2011-01-01

    Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos

  6. Optimal concentration of hyaluronan and plant protein in different culture systems for in vitro maturation of bovine oocytes.

    PubMed

    Opiela, Jolanta; Latasiewicz, Ewa; Smorag, Zdzisław

    2012-12-01

    With a view to search for optimal concentration of hyaluronan (HA) and plant protein (PP) in different culture systems for in vitro maturation of bovine oocytes, cumulus-oocyte complexes (COCs) were matured in vitro in 2 culture systems (first co-cultured with granulose cells and estrus calf serum (ECS) in 2 mL volume, second without co-culture where ECS was replaced by exogenous hormones and BSA or PP in 100 microL dose under mineral oil). Seven types of media were used; 3 in first system and 4 in second system. To evaluate HA and PP effect on oocytes after in vitro culture an estimation of meiosis stage and a level of DNA fragmentation was performed by TUNEL staining. The highest meiotic maturation (84%) was observed in oocytes cultured in medium enriched with ECS in co-culture with granulose cells (1st system). The lowest meiotic maturation was noted in medium with addition of BSA (43%). The addition of HA in the medium enriched with BSA significantly increased the rate of matured oocytes (67%) and also didn't affect the chromatin quality of individual oocytes. The addition of HA to the culture medium supplemented with a PP decreased the rate of matured oocytes to 54% but no statistical differences were noted. The results of the present study showed that HA supplementation didn't have a detrimental impact on oocyte chromatin integrity and improved bovine oocytes' meiotic maturation in medium supplemented only with BSA without co-culture of granulose cells.

  7. Impact of gonadotropins on oocyte maturation, fertilisation and developmental competence in vitro.

    PubMed

    Wang, Xuemei; Tsai, Tony; Qiao, Jie; Zhang, Zhan; Feng, Huai L

    2014-06-01

    The aim of the present study was to evaluate the dose-dependent effects of gonadotropins, either singly (Bravelle (B), Luveris (L), Menupur (M), Repronex (R), Gonal-F (G), Follism (F) and Norvarel (N)) or in combination (Menupur+Bravelle; Repronext+Bravelle; and Bravelle+Norvarel), on rates of oocyte maturation, fertilisation and early embryo development in vitro in an animal model. Bovine cumulus-oocyte complexes (COCs) were purchased commercially and cultured in TCM-199 with 10% fetal bovine serum supplemented with varying concentrations of gonadotropin (0, 5, 10, 20, 40IU or United States Pharmacopoeia (USP) mL-1) for 24 and 48h according to current IVF clinical stimulation protocols. All gonadotropins enhanced oocyte maturation in vitro in a dose-dependent manner. Individually, Gonal-F (Merck KGaA, Darmstadt, Germany), Follism (Merck Co, Whitehouse Station, NJ, USA) and Repronext (Ferring, Parsippany, NJ, USA) promoted oocyte maturation; in combination, they effectively enhanced COC expansion and increased the maturation competence of MII oocytes. However, high concentrations of gonadotropins may result in maturation arrest. Specific combinations of gonadotropins may change the rate of early embryonic development (8-16-cells) and morula-blastocyst formation. These data provide support for the responsiveness of bovine oocytes to gonadotropins in vitro and the need to consider variations in the relative concentrations and ratio of combinations (FSH/LH or human chorionic gonadotropin) for optimisation of oocyte developmental competence. The results of the present study could be applied to therapeutic clinical stimulation protocols and help improve IVF success rates.

  8. Increasing the cAMP concentration during in vitro maturation of pig oocytes improves cumulus maturation and subsequent fertilization in vitro.

    PubMed

    Appeltant, R; Beek, J; Vandenberghe, L; Maes, D; Van Soom, A

    2015-02-01

    Porcine IVF faces various problems such as incomplete cytoplasmic maturation of the oocyte and polyspermy. Previous studies proved the importance of cAMP in regulating nuclear and cytoplasmic maturation of oocytes. This study investigated the effect of the cAMP-modulating agents 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cAMP sodium salt (dbcAMP) on several parameters during in vitro production of porcine embryos. First, we wanted to see if oocyte collection in IBMX could meiotically arrest oocytes and, as such, improve synchronization of nuclear and cytoplasmic maturation. To this end, cumulus-oocyte complexes (COCs) were collected from gilts in HEPES-buffered Tyrode balanced salt solution medium with 0.5-mM IBMX or without IBMX. At the end of oocyte collection, the effect of IBMX on chromatin configuration was evaluated. However, no differences could be observed in nuclear configuration between IBMX- and IBMX+ oocytes (P > 0.05). Second, we added dbcAMP during IVM to improve cytoplasmic maturation and evaluated cumulus expansion (lack of adhesion), a disintegrin and metalloproteinase with thrombospondin-like repeats (ADAMTS-1) levels in cumulus cells, fertilization, and blastocyst rates. Cumulus-oocyte complexes were matured in modified North Carolina State University medium 37 with or without 1-mM dbcAMP. Frozen-thawed, epididymal, boar spermatozoa were used for IVF. After IVF, presumed zygotes were cultured for 7 days in North Carolina State University medium 23. Penetration rate decreased in dbcAMP+ (57.3%) compared with dbcAMP- (67.8%), but the polyspermy rate also decreased (43.3% vs. 53.4%, respectively) leading to an increased normal fertilization rate (56.7% vs. 46.6%, respectively; P < 0.05). Only 7.2% of the COCs showed adhesion in dbcAMP+ which was lower than 15.7% in dbcAMP- (P < 0.05) probably because of an upregulation of the ADAMTS-1 protein by dbcAMP. When the adherent oocytes were removed during maturation, no difference could be

  9. Effect of the addition of insulin-transferrin-selenium and/or L-ascorbic acid to the in vitro maturation of prepubertal bovine oocytes on cytoplasmic maturation and embryo development.

    PubMed

    Córdova, B; Morató, R; Izquierdo, D; Paramio, T; Mogas, T

    2010-11-01

    This study examines the effects of adding insulin-transferrin-selenium (ITS) and/or L-ascorbic acid (ASC) to a conventional medium for maturing prepubertal calf oocytes on chromosome organization, cortical granule (CG) distribution, and embryo development to the blastocyst stage. Cumulus-oocyte complexes (COCs) were matured in medium TCM 199 containing PVA and EGF (control), and supplemented with ITS and/or ASC for 12 or 24 h at 38.5 °C in a 5% CO(2) atmosphere. Calf oocytes matured with ITS + ASC or ASC for 12 h showed significantly higher percentages of peripherally distributed CG (83.3% and 86.2% respectively) than control oocytes (71.4%) or those matured with ITS alone (71.4%). No effects on chromosome organization were detected. Conversely, 24 h of supplementation did not affect CG distribution patterns, while the addition of ASC gave rise to significantly higher percentages of oocytes showing a normal alignment of their chromosomes (72.9%) compared to controls (58.7%). At 48 hpi, similar cleavage rates were observed among treatments regardless of the treatment time. However, the presence of ITS + ASC for 12 h rendered significantly higher blastocyst rates than those recorded in the remaining groups. Supplementation for 24 h with ITS or ITS + ASC had no significant effects on the percentage of blastocysts obtained, while the presence of ASC significantly reduced the proportions of embryos developing to the blastocyst stage. Our data suggest that ITS plus L-ascorbic acid supplementation during the first 12 h of in vitro maturation improves cytoplasm maturation and the developmental competence of embryos produced from prepubertal calf oocytes.

  10. In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development.

    PubMed

    Arias-Álvarez, M; García-García, R M; López-Tello, J; Rebollar, P G; Gutiérrez-Adán, A; Lorenzo, P L

    2016-09-28

    In vivo-matured cumulus-oocyte complexes are valuable models in which to assess potential biomarkers of rabbit oocyte quality that contribute to enhanced IVM systems. In the present study we compared some gene markers of oocytes and cumulus cells (CCs) from immature, in vivo-matured and IVM oocytes. Moreover, apoptosis in CCs, nuclear maturation, mitochondrial reallocation and the developmental potential of oocytes after IVF were assessed. In relation to cumulus expansion, gene expression of gap junction protein, alpha 1, 43 kDa (Gja1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) was significantly lower in CCs after in vivo maturation than IVM. In addition, there were differences in gene expression after in vivo maturation versus IVM in both oocytes and CCs for genes related to cell cycle regulation and apoptosis (V-Akt murine thymoma viral oncogene homologue 1 (Akt1), tumour protein 53 (Tp53), caspase 3, apoptosis-related cysteine protease (Casp3)), oxidative response (superoxide dismutase 2, mitochondrial (Sod2)) and metabolism (glucose-6-phosphate dehydrogenase (G6pd), glyceraldehyde-3-phosphate dehydrogenase (Gapdh)). In vivo-matured CCs had a lower apoptosis rate than IVM and immature CCs. Meiotic progression, mitochondrial migration to the periphery and developmental competence were higher for in vivo-matured than IVM oocytes. In conclusion, differences in oocyte developmental capacity after IVM or in vivo maturation are accompanied by significant changes in transcript abundance in oocytes and their surrounding CCs, meiotic rate, mitochondrial distribution and apoptotic index. Some of the genes investigated, such as Gja1, could be potential biomarkers for oocyte developmental competence in the rabbit model, helping improve in vitro culture systems in these species.

  11. Bovine OPU-derived oocytes can be matured in vitro for 16-28 h with similar developmental capacity.

    PubMed

    Merton, J S; de Roos, A P W; Koenen, E P C; Roelen, B A J; Vos, P L A M; Mullaart, E; Knijn, H M

    2012-12-01

    The aim of this study was to determine the optimal maturation culture period of ovum pick up (OPU)-derived cumulus oocytes complexes (COCs) in relation to their developmental capacity. Embryo production, embryo cryotolerance, post-transfer embryonic survival and calf characteristics such as gestation length, birthweight and sex ratio were investigated. This retrospective study covers the analyses of ovum pick up -in vitro production and calving results from a commercial programme that took place between March 1994 and September 2004. Donors were both heifers (of which approximately 90% pregnant) and cows (of which approximately 10% pregnant). Embryo production analyses were based on 7800 OPU sessions conducted from January 1995 until January 1999. Analyses of calving rate were based on 13 468 embryo transfers performed during January 1995 until May 2002. Analyses on calf characteristics were based on 2162 calves born between March 1994 and September 2004. The in vitro maturation culture period ranged from 16 to 28 h. The mean production rate of transferable embryos was 16.5% (1.2 embryos per OPU session). Length of maturation culture period did not affect the production of transferable embryos. Mean calving rate was 40.9% and 38.7% for fresh and frozen/thawed embryos, respectively. Calving rate was not affected by the maturation culture period. Mean birthweight, gestation length and proportion of male calves were 46 kg, 281.9 days and 52.8%, respectively. Maturation culture period did not affect these variables. In conclusion, this study shows that the in vitro maturation culture period within the range of 16-28 h does not affect in vitro embryo production, embryo cryotolerance, post-transfer embryonic survival and calf characteristics, suggesting that all COC batches collected by OPU on the same day, can be fertilized in one IVF session without a significant loss in the production from oocyte to calf.

  12. Influence of manganese on apoptosis and glutathione content of cumulus cells during in vitro maturation in bovine oocytes.

    PubMed

    Anchordoquy, Juan Patricio; Anchordoquy, Juan Mateo; Picco, Sebastián J; Sirini, Matías A; Errecalde, Ana Lía; Furnus, Cecilia C

    2014-02-01

    We have investigated the effect of different Mn concentrations on (1) DNA integrity of cumulus cells by olive tail moment (OTM); (2) cumulus cells apoptosis by Annexin V staining assay; (3) intracellular total glutathione (GSH-GSSG) content; and (4) oocyte nuclear maturation and embryo cleavage after in vitro fertilisation (IVF). For this purpose, 0 (control), 2 (Mn1), 5 (Mn2) and 6 ng/mL (Mn3) Mn concentrations were added to IVM medium. Comet assay analysed by OTM was significantly higher in cumulus cells arising from COCs matured without Mn (control, P < 0.01) respect to cumulus cells obtained from COCs matured with Mn (control: 5.18 ± 2.3; Mn1: 2.93 ± 2.2; Mn2: 2.63 ± 2.4; Mn3: 2.92 ± 2.4). The frequency of apoptotic cells was higher in the control group (control: 6.63 ± 0.59; Mn1: 5.05 ± 0.5; Mn2: 4.61 ± 0.49; Mn3: 3.33 ± 0.42). Intracellular concentration of GSH-GSSG increased in oocytes and cumulus cells matured in the presence of Mn (P < 0.01). There were no differences in percentages of nuclear maturation when Mn was added to IVM medium at any concentration, but at 6 ng/mL Mn a higher cleavage rate was observed respect to the control group (P < 0.05). In conclusion, deficiency in Mn concentration during in vitro maturation increased the damage in the DNA molecule and the frequency of apoptotic cumulus cells. However, the addition of an adequate Mn concentration (6 ng/mL Mn) to IVM medium improved the health of cumulus-oocyte complexes and produced more cleaved embryos 48 h after IVF.

  13. MicroRNA-224 delays oocyte maturation through targeting Ptx3 in cumulus cells.

    PubMed

    Li, Xiufang; Wang, Huidan; Sheng, Yan; Wang, Zhongqing

    2017-02-01

    MicroRNAs (miRNAs) have been improved to regulate oocyte development in a cell- or stage-specific manner. In this study, we aimed to clarify microRNA-224's (miR-224) role in cumulus cells (CCs), to find out whether a change level of miR-224 in CCs could influence the maturation of oocyte. We found that overexpression of miR-224 of CCs led to the impairment of cell expansion, along with a decrease in the gene expression associated with cell expansion and maturation of oocyte. The increased expression of miR-224 in CC interrupted oocyte cell cycle at the GV stage. The GDF9, BMP15 and ZP3 of the oocytes were also down-regulated. The following in vitro fertilization had yielded a lower number of oocytes from cumulus-oocyte complexes (COCs) overexpressing miR-224 when reaching the blastocyst stage. The suppressive effect of miR-224 in the maturation of COC is validated by the miR-224 knockdown model, where the expansion of cumulus cell was increased and oocyte was developed to MII stage. In addition, the expression of aromatase in CCs was down-regulated by miR-224, resulting in a decreased level of estradiol (E2). A further investigation found that miR-224 down-regulated the expression of protein and mRNA of Ptx3 by targeting its 3'UTR. Our study revealed that miR-224 regulates the gene expression and function of CCs, which influences the maturation of oocyte, at least in part, via targeting Ptx3.

  14. Fatty acid metabolism during maturation affects glucose uptake and is essential to oocyte competence.

    PubMed

    Paczkowski, M; Schoolcraft, W B; Krisher, R L

    2014-10-01

    Fatty acid β-oxidation (FAO) is essential for oocyte maturation in mice. The objective of this study was to determine the effect of etomoxir (a FAO inhibitor; 100 μM), carnitine (1 mM), and palmitic acid (1 or 100 μM) during maturation on metabolism and gene expression of the oocyte and cumulus cells, and subsequent embryo development in the mouse. Carnitine significantly increased embryo development, while there was a decrease in development following maturation with 100 μM palmitic acid or etomoxir (P<0.05) treatment. Glucose consumption per cumulus-oocyte complex (COC) was decreased after treatment with carnitine and increased following etomoxir treatment (P<0.05). Intracellular oocyte lipid content was decreased after carnitine or etomoxir exposure (P<0.05). Abundance of Slc2a1 (Glut1) was increased after etomoxir treatment in the oocyte and cumulus cells (P<0.05), suggesting stimulation of glucose transport and potentially the glycolytic pathway for energy production when FAO is inhibited. Abundance of carnitine palmitoyltransferase 2 (Cpt2) tended to increase in oocytes (P=0.1) after treatment with 100 μM palmitic acid and in cumulus cells after exposure to 1 μM palmitic acid (P=0.07). Combined with carnitine, 1 μM palmitic acid increased the abundance of Acsl3 (P<0.05) and Cpt2 tended to increase (P=0.07) in cumulus cells, suggesting FAO was increased during maturation in response to stimulators and fatty acids. In conclusion, fatty acid and glucose metabolism are related to the mouse COC, as inhibition of FAO increases glucose consumption. Stimulation of FAO decreases glucose consumption and lipid stores, positively affecting subsequent embryo development, while an overabundance of fatty acid or reduced FAO negatively affects oocyte quality.

  15. A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.

    PubMed

    Ledda, S; Idda, A; Kelly, J; Ariu, F; Bogliolo, L; Bebbere, D

    2016-04-01

    The aim of this work was to develop a microbioreactor using liquid marble (LM) as a novel system for oocyte in vitro maturation (IVM) in small volumes. Cumulus-oocyte complexes (COCs) obtained from slaughterhouse sheep ovaries were in vitro matured in a LM system prepared by placing a drop (30 μl containing 10 COCs) suspended in TCM 199 supplemented with 10 % (v/v) oestrus sheep serum (OSS) and 0.1 IU FSH and LH onto a polytetrafluoroethylene (PTFE) particle bed (LM group). As a control group (CTRL group), COCs were in vitro matured in standard volume and conditions (600 μl of IVM medium in a four-well dish). After 24-h culture at 38.5 °C in 5 % CO2 in air, COCs were released from LM and the following parameters were evaluated: (a) percentage of MII oocytes, (b) oocyte developmental competence following in vitro fertilization (IVF) or parthenogenetic activation (PA) and embryo culture for 8 days in synthetic oviductal fluid (SOF) medium at 38.5 °C in 5 % O2, 5 % CO2, and 90 % N2. The results indicated similar percentage of MII oocytes in LM and CTRL groups (88.0 vs. 92.0 %). No differences were observed in blastocyst rate after IVF (LM 47.5 % vs. CTRL 50.2 %, P=0.637) or PA (LM 44.4 % vs. CTRL 48.3 %, P=0.426). The results indicate that LM microbioreactor is a viable technique that provides a suitable microenvironment to induce oocyte in vitro maturation.

  16. IGF-I slightly improves nuclear maturation and cleavage rate of bovine oocytes exposed to acute heat shock in vitro.

    PubMed

    Meiyu, Qi; Liu, Di; Roth, Zvi

    2015-08-01

    An in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus-oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.

  17. Combined epidermal growth factor and hyaluronic acid supplementation of in vitro maturation medium and its impact on bovine oocyte proteome and competence.

    PubMed

    Ríos, G L; Buschiazzo, J; Mucci, N C; Kaiser, G G; Cesari, A; Alberio, R H

    2015-03-15

    The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 μg/mL HA, and 100-μM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P < 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P < 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P < 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive

  18. Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture.

    PubMed

    Saadeldin, Islam M; Koo, Ok Jae; Kang, Jung Taek; Kwon, Dae Kee; Park, Sol Ji; Kim, Su Jin; Moon, Joon Ho; Oh, Hyun Ju; Jang, Goo; Lee, Byeong Chun

    2012-01-01

    Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

  19. Addition of recombinant human growth hormone to in vitro maturation medium of bovine oocytes.

    PubMed

    Pozzobon, S E; Lagares, M A; Brum, D S; Leivas, F G; Rubin, M I B

    2005-02-01

    The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.

  20. Expression of hyaluronan synthases and corresponding hyaluronan receptors is differentially regulated during oocyte maturation in cattle.

    PubMed

    Schoenfelder, Martin; Einspanier, Ralf

    2003-07-01

    In response to the gonadotropin surge, the compact cumulus-oocyte complex (COC) undergoes expansion by synthesis of the mucopolysaccharide hyaluronan (HA) accompanying oocyte maturation. The objective of the present study was to quantify mRNA transcripts of the HA synthase (HAS) 1, HAS2, and HAS3 and the HA-receptors CD44 and RHAMM (receptor for HA-mediated motility). Additionally, we determined the histological localization of HA and its receptor, CD44, in maturing bovine COCs and cultured granulosa cells (GCs). Full-length transcript of bovine HAS2 and a part of the bovine RHAMM sequence has been made available. Real-time reverse transcriptase-polymerase chain reaction was used for individual mRNA expressions of bovine COCs in comparison to follicular GC gonadotropin treatment. Localization of CD44 and HA were done by immunohistochemistry and biotinylated HA-binding protein, respectively. Gonadotropins caused a rapid, 120-fold increase of HAS2 mRNA, whereas a delayed, 2-fold up-regulation of HAS3 mRNA was observed. The HAS1 transcripts were barely detected. Expression of CD44 mRNA greatly increased during in vitro maturation of COCs, indicating an important role when compared to an unchanged, steady-state RHAMM expression. As a consequence, HA was locally enriched after COC expansion, but only limited change was observed in the GCs. In cultured GCs, HAS2 expression was stimulated through FSH application, followed by the effective treatments of FSH+LH and LH. Treatment with LH induced the highest increase of the CD44 receptor, followed by FSH and FSH+LH treatments. These results suggest that HAS2 is mainly responsible for rapid HA synthesis in bovine COCs and GCs. In bovine COCs, the transcriptional up-regulation of both HAS2 and the receptor CD44 appear to be important prerequisites for initiating HA-mediated effects during final oocyte development and sperm-egg interaction.

  1. Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes.

    PubMed

    Yoon, Junchul David; Jeon, Yubyeol; Cai, Lian; Hwang, Seon-Ung; Kim, Eunhye; Lee, Eunsong; Kim, Dae Y; Hyun, Sang-Hwan

    2015-01-15

    In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbecco's modified Eagle's medium for 48 hours with varying numbers of CSCs (0.0, 2.5 × 10(4), 5.0 × 10(4), and 10.0 × 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 μL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 × 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 × 10(4) and 5.0 × 10(4) CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 × 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 × 10(4) and 5.0 × 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 × 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 × 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that

  2. Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

    PubMed

    Hosoe, Misa; Yoshida, Nao; Hashiyada, Yutaka; Teramoto, Hidetoshi; Takahashi, Toru; Niimura, Sueo

    2014-01-01

    Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

  3. Bone morphogenetic protein 1 is expressed in porcine ovarian follicles and promotes oocyte maturation and early embryonic development

    PubMed Central

    LEI, Xiaocan; CUI, Kuiqing; CAI, Xiaoyan; REN, Yanping; LIU, Qingyou; SHI, Deshun

    2016-01-01

    In the present study, we tried to determine whether bone morphogenetic protein 1 (BMP1) plays a role in ovarian follicular development and early embryo development. We systematically investigated the expression and influence of BMP1 during porcine follicle and early embryonic development. Immunohistochemistry demonstrated that the BMP1 protein is expressed in granular cells and oocytes during follicular development, from primary to pre-ovulatory follicles, including atretic follicles and the corpus luteum. The mRNA expression of BMP1 significantly increased as the porcine follicles grew. Immunofluorescence analysis indicated that BMP1 was expressed in cumulus-oocyte complexes (COCs), oocytes and porcine embryos during early in vitro culture. qPCR and western blot analysis showed that the expression of BMP1 was significantly up-regulated in mature porcine oocytes and COCs compared to immature oocytes and COCs. BMP1 is expressed in early porcine embryos, and its expression reaches a peak at the 8-cell stage. To determine the effect of BMP1 on the maturation of oocytes and the development of early embryos, various concentrations of BMP1 recombinant protein or antibody were added to the in vitro culture media, respectively. BMP1 significantly affected the porcine oocyte maturation rate, the cleavage rate and the blastocyst development rate of embryos cultured in vitro in a positive way, as well as the blastocyst cell number. In conclusion, BMP1 is expressed throughout porcine ovarian follicle development and early embryogenesis, and it promotes oocyte maturation and the developmental ability of embryos during early in vitro culture. PMID:27890905

  4. Recombinant human follicle-stimulating hormone and transforming growth factor-alpha enhance in vitro maturation of porcine oocytes.

    PubMed

    Mito, Tomomi; Yoshioka, Koji; Noguchi, Michiko; Yamashita, Shoko; Hoshi, Hiroyoshi

    2013-07-01

    The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1 IU/ml FSH in the presence or absence of 10 ng/ml TGF-α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01-0.1 IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF-α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05-0.1 IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF-α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF-α-induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF-α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion-related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF-α upregulated the expression of HAS2 mRNA after 20 hr culture and HAPLN1 mRNA after 44 hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF-α alone. These results suggest that FSH and TGF-α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways.

  5. Sericin Accelerates the Production of Hyaluronan and Decreases the Incidence of Polyspermy Fertilization in Bovine Oocytes During In Vitro Maturation

    PubMed Central

    HOSOE, Misa; YOSHIDA, Nao; HASHIYADA, Yutaka; TERAMOTO, Hidetoshi; TAKAHASHI, Toru; NIIMURA, Sueo

    2014-01-01

    Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes. PMID:24748396

  6. In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr).

    PubMed

    Berlinguer, F; González, R; Succu, S; del Olmo, A; Garde, J J; Espeso, G; Gomendio, M; Ledda, S; Roldan, E R S

    2008-02-01

    The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.

  7. Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes.

    PubMed

    Asada, M; Tetsuka, M; Ishikawa, H; Ohsumi, S; Fukui, Y

    2001-09-01

    The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA

  8. Improving in vitro maturation and pregnancy outcome in cattle using a novel oocyte shipping and maturation system not requiring a CO₂ gas phase.

    PubMed

    Barceló-Fimbres, M; Campos-Chillón, L F; Mtango, N R; Altermatt, J; Bonilla, L; Koppang, R; Verstegen, J P

    2015-07-01

    The present work evaluated the benefit of a novel shipping and maturation medium (SMM) not requiring a CO2 gas for maturation and subsequent embryonic development of slaughterhouse and ovum pickup (OPU) bovine cumulus-oocyte complexes (COCs). Four experiments were conducted. In experiment 1, COCs were maturated for 18 hours in SMM and then incubated for 6 hours in, or 24 hours in a conventional system (control). Experiment 2 compared maturation for 24 hours in SMM versus 24 hours in the control. Experiment 3 compared three different incubation temperatures (37 °C, 38 °C, and 38.5 °C) for COCs maturation in SMM. In experiment 4, COCs obtained from 166 OPU sessions (representing two dairy and two beef breeds) in two locations (Wisconsin and California) were matured in SMM or control and evaluated relative to embryo production and pregnancy rates. Frozen semen was used for all experiments. The results for experiment 1 showed that the blastocyst rate and total embryo production rate (TE, Day-7 morulae plus all blastocysts) were higher for SMM than those in the control. However, no differences were observed for cleavage rate or blastocyst stage. In experiment 2, the blastocyst rate and TE were higher for SMM than those in the control; however, there was no difference for cleavage rate, total cell number, blastocyst stage. In experiment 3, the cleavage rate was similar, but the blastocyst rate and TE were greater for 38.5 °C than those for 38.0 °C and 37.5 °C. For experiment 4, Wisconsin OPU-derived COCs had a greater cleavage rate, blastocyst rate, TE, and blastocyst stage for SMM versus control. There were no breed effects. For the California trial, OPU-derived COCs matured in SMM had similar cleavage and pregnancy rates at Day 35 but greater blastocyst rates and transferred embryos per session than the control, which resulted in 2.2 more pregnancies per OPU session. Holstein COCs had superior embryonic development but similar pregnancy compared with Jersey. We

  9. Effects of gonadotropins on in vitro maturation and of electrical stimulation on parthenogenesis of canine oocytes.

    PubMed

    Kim, B S; Lee, S R; Hyun, B H; Shin, M J; Yoo, D H; Lee, S; Park, Y S; Ha, J H; Ryoo, Z Y

    2010-02-01

    The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 mus with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 mus without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.

  10. "Bone Development" Is an Ontology Group Upregulated in Porcine Oocytes Before In Vitro Maturation: A Microarray Approach.

    PubMed

    Budna, Joanna; Bryja, Artur; Celichowski, Piotr; Kranc, Wiesława; Ciesiółka, Sylwia; Borys, Sylwia; Rybska, Marta; Kolecka-Bednarczyk, Agata; Jeseta, Michal; Bukowska, Dorota; Antosik, Paweł; Brüssow, Klaus P; Bruska, Małgorzata; Nowicki, Michał; Zabel, Maciej; Kempisty, Bartosz

    2017-08-01

    Mammalian cumulus-oocyte complexes (COCs) reach full developmental capability during folliculogenesis and oogenesis. It is well recognized that only gametes achieving MII stage after in vivo or in vitro maturation (IVM) are successfully fertilized by a single spermatozoon. Although the process of oocyte nuclear and/or cytoplasmic maturation in pigs is well determined, there exist many differences that promote these processes in vivo and in vitro. Therefore, this study aimed to investigate the differences in RNA expression profiles between porcine oocytes before and after IVM using microarray and real-time quantitative polymerase chain reaction (RT-qPCR) assays. Experiments were performed on oocytes isolated from 55 pubertal crossbred Landrace gilts. The oocytes were analyzed both before and after IVM and only Brilliant Cresyl Blue (BCB)-positive gametes were used for subsequent microarray analysis (Affymetrix) and RT-qPCR analysis. The microarray assay, which measures expression of 12,258 transcripts, revealed 419 differentially expressed transcripts in porcine oocytes, from which 379 were downregulated and 40 were upregulated before IVM compared to those analyzed after IVM. After DAVID analysis, we found eight different transcripts, including IHH, BMP1, WWTR1, CHRDL1, KLF10, EIF2AK3, MMP14, and STC1. Their expression is related to the "bone development" ontology group and was further subjected to hierarchical clusterization. Using RT-qPCR analysis, we confirmed the results of the microarray assay, showing increased expression of the eight genes in oocytes before IVM compared to oocytes after maturation in vitro. It has been suggested that "bone development" belongs to one ontological group involving genes substantially upregulated in porcine oocytes before IVM. We suggest that the gamete mRNA expression profile before IVM may comprise stored transcripts, which are templates for protein biosynthesis following fertilization. We also hypothesize that these mRNAs may

  11. Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

    PubMed

    Romero-Aguirregomezcorta, Jon; Santa, Ángela Patricia; García-Vázquez, Francisco Alberto; Coy, Pilar; Matás, Carmen

    2014-01-01

    Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

  12. Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica).

    PubMed

    Gjørret, J O; Crichton, E G; Loskutoff, N M; Armstrong, D L; Hyttel, P

    2002-09-01

    The application of assisted reproduction techniques to wild cats has been stalled by a lack of basic knowledge of the reproductive biology in these species. In this study, the ultrastructure of Siberian tiger (Panthera tigris altaica) cumulus-oocyte-complexes (COCs), as well as in vitro produced (IVP) zygotes and embryos were investigated, to estimate the normality of the manipulated reproduction processes. Adult female tigers were subjected to a purified porcine pFSH/pLH stimulation treatment followed by oocyte aspiration. According to morphological appearance at the stereomicroscopical level, COCs were classified as mature, immature, or degenerated, and then allocated into the following groups: presumptively immature COCs, which were in vitro matured (IVM-group) before fixation; presumptively mature COCs, which were either fixed after retrieval (pre-IVF-group), following in vitro insemination (IVF-group) or following in vitro insemination and subsequent in vitro culture (IVC-group). All specimens were processed for light and transmission electron microscopy (TEM). Both the IVM- and pre-IVF-group included oocytes in meiotic stages ranging from prophase I to metaphase II, and some prophase I oocytes in the IVM-group were apparently in their growth phase. The IVF-group presented features of presumptive normal fertilization, but aberrations such as polynucleation were also noted. The IVC-group included cleavage stage embryos of which, however, many were polynucleated. In conclusion, the procedures used for stimulation, aspiration, and classification of COCs resulted in retrieval of a heterogeneous population of oocytes which, following IVF and IVC, displayed a high rate of developmental deviations. Copyright 2002 Wiley-Liss, Inc.

  13. Protective effects of antioxidants on linoleic acid-treated bovine oocytes during maturation and subsequent embryo development.

    PubMed

    Khalil, Wael A; Marei, Waleed F A; Khalid, Muhammad

    2013-07-15

    Linoleic acid (LA; n-6, 18:2) is the most abundant polyunsaturated fatty acid in the ovarian follicular fluid and is known to inhibit oocyte maturation and its subsequent development. In the present study, we investigated how its effects on cumulus cell expansion, oocyte nuclear maturation, and blastocyst development are altered by supplementation of the media with vitamin E (VE; 100 μM) and glutathione peroxidase (GPx; 1 μM) either alone or in combination, and whether it has any effect on the mRNA expression of GPx1, GPx4, or superoxide dismutase (SOD2) in the bovine cumulus oocyte complexes (COCs). LA supplementation of the culture media significantly (P ≤ 0.05) reduced the percentage of COCs exhibiting full cumulus cell expansion and the percentage of oocytes reaching metaphase II stage, and lowered the blastocyst rate compared with controls. And these inhibitory effects were associated with a reduction in the relative mRNA expression of GPx1 and SOD2 but not of GPx4 compared with controls. However, VE and GPx, both alone and in combination, completely abrogated the inhibitory effects of LA on nuclear maturation of oocytes and blastocyst rate but failed to do so for cumulus cell expansion. In conclusion, these data suggest that the detrimental effects of LA on oocyte developmental competence are mediated, at least in part, by a reduction in GPx1 and SOD2 mRNA expression. Moreover, VE and GPx may provide protection to most of the inhibitory effects of LA.

  14. Effect of different manganese concentrations during in vitro maturation of bovine oocytes on DNA integrity of cumulus cells and subsequent embryo development.

    PubMed

    Anchordoquy, J P; Anchordoquy, J M; Sirini, M A; Mattioli, G; Picco, S J; Furnus, C C

    2013-12-01

    Manganese (Mn) is a trace element present in forages and cereals, and its concentration depends on soil status. Manganese deficiency in cattle, goats and ewes not only impairs oestrous cycle but reduces calf birth weight. The achievement of the first oestrus is delayed, and more attempts are necessary to obtain a successful conception. This study was conducted to investigate the effect of the availability of supplemental Mn during IVM on DNA damage of cumulus cells and total glutathione (GSH) content in oocytes and cumulus cells. The effect of supplementary Mn during IVM on subsequent embryo development was also studied. The results reported here indicate (i) DNA damage in cumulus cells decreased with 0, 2, 5 and 6 ng/ml Mn supplementation during IVM (p < 0.05). (ii) Intracellular GSH-GSSG content increased (p < 0.01) with different Mn concentrations in oocytes and cumulus cells. Also, cumulus cell number per cumulus oocyte-complexes (COC) did not differ either before or after IVM. (iii) Addition of Mn to maturation medium resulted in similar cleavage rates (p > 0.05) at 0, 2, 5 and 6 ng/ml Mn. However, subsequent embryo development to blastocyst stage was significantly higher (p < 0.01) in oocytes matured with 5 and 6 ng/ml Mn. (iv) There was also an increase (p < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 5 and 6 ng/ml respect to zero Mn (IVM alone) and 2 ng/ml Mn. This study provides evidence that optimal embryo development to the blastocyst stage was partially dependent on the presence of Mn during IVM. Moreover, the availability of Mn during oocyte maturation ensures 'normal' intracellular GSH content in COCs and protects DNA integrity of cumulus cells.

  15. Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules.

    PubMed

    Vireque, A A; Camargo, L S A; Serapião, R V; Rosa E Silva, A A M; Watanabe, Y F; Ferreira, E M; Navarro, P A A S; Martins, W P; Ferriani, R A

    2009-03-01

    In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured in TCM plus FSH and 10% estrous cow serum. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P<0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless, there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. Similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis.

  16. Successful piglet production in a chemically defined system for in-vitro production of porcine embryos: dibutyryl cyclic amp and epidermal growth factor-family peptides support in-vitro maturation of oocytes in the absence of gonadotropins.

    PubMed

    Akaki, Yuka; Yoshioka, Koji; Noguchi, Michiko; Hoshi, Hiroyoshi; Funahashi, Hiroaki

    2009-08-01

    To induce meiotic resumption of porcine oocytes, it is thought to be necessary to expose the cumulus-oocyte complexes (COCs) to gonadotropins during in-vitro maturation (IVM). However, the detailed mechanism of meiotic resumption by gonadotropins is still unknown, and successful piglet production has not been reported by using oocytes matured in gonadotropin-free media and fertilized in vitro. The present study was undertaken to examine the combinational effects of epidermal growth factor (EGF)-family members and dibutyryl cyclic AMP (cAMP) in a chemically defined medium on IVM of porcine oocytes and the developmental competence following in vitro fertilization (IVF). The basic IVM medium was a chemically defined medium, modified porcine oocyte medium (mPOM). Supplementation of the IVM medium with 10 or 1000 ng/ml EGF, amphiregulin and betacellulin during the whole IVM period, except for 10 ng/ml amphiregulin, increased the percentage of oocytes maturing to the metaphase-II stage. When COCs were exposed to both dibutyryl cAMP and EGF-family members during the first 20-h of IVM and then culture was continued in the absence of EGF-family members and dibutyryl cAMP, the incidence of metaphase-II oocytes was significantly increased and was not different from that of oocytes cultured in a standard IVM system with gonadotropins. The developmental competence of the oocytes to the blastocyst stage following IVF was no different from that of control oocytes matured with gonadotropins. When these blastocysts were transferred into the uterine horn of three recipients, all of gilts became pregnant and delivered a total of 11 piglets. These observations indicate that supplementation of a chemically defined maturation medium with EGF-family members and dibutyryl cAMP during the first 20 h of IVM can support well the meiotic progress and developmental competence of porcine oocytes.

  17. Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes.

    PubMed

    Wani, N A; Nowshari, M A

    2005-07-01

    Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro

  18. Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence.

    PubMed

    Naruse, Kenji; Iga, Kosuke; Shimizu, Manabu; Takenouchi, Naoki; Akagi, Satoshi; Somfai, Tamas; Hirao, Yuji

    2012-01-01

    In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 μM of milrinone, the enucleation rate was significantly improved by 100 μM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 μM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 μM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.

  19. Activation of 5' adenosine monophosphate-activated protein kinase blocks cumulus cell expansion through inhibition of protein synthesis during in vitro maturation in Swine.

    PubMed

    Santiquet, Nicolas; Sasseville, Maxime; Laforest, Martin; Guillemette, Christine; Gilchrist, Robert B; Richard, François J

    2014-08-01

    The serine/threonine kinase 5' adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein known as a metabolic switch, is involved in oocyte nuclear maturation in mice, cattle, and swine. The present study analyzed AMPK activation in cumulus cell expansion during in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COC). 5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) is a well-known activator of AMPK. It inhibited oocyte meiotic resumption in COC. Moreover, cumulus cell expansion did not occur in the presence of AICAR, demonstrating its marked impact on cumulus cells. Activation of AMPK was supported by AICAR-mediated phosphorylation of alpha AMPK subunits. Furthermore, the presence of AICAR increased glucose uptake, a classical response to activation of this metabolic switch in response to depleted cellular energy levels. Neither nuclear maturation nor cumulus expansion was reversed by glucosamine, an alternative substrate in hyaluronic acid synthesis, through the hexosamine biosynthetic pathway, which ruled out possible depletion of substrates. Both increased gap junction communication and phosphodiesterase activity in COC are dependent on protein synthesis during the initial hours of IVM; however, both were inhibited in the presence of AICAR, which supports the finding that activation of AMPK by AICAR mediated inhibition of protein synthesis. Moreover, this protein synthesis inhibition was equivalent to that of the well-known protein synthesis inhibitor cycloheximide, as observed on cumulus expansion and protein concentration. Finally, the phosphorylation level of selected kinases was investigated. The pattern of raptor phosphorylation is supportive of activation of AMPK-mediated inhibition of protein synthesis. In conclusion, AICAR-mediated AMPK activation in porcine COC inhibited cumulus cell expansion and protein synthesis. These results bring new considerations to the importance of this kinase in ovarian

  20. In vitro fertilisation of in vivo matured porcine oocytes obtained from prepuberal gilts at different time intervals after hCG injection.

    PubMed

    Rátky, J; Rath, D; Brüssow, K P

    2003-01-01

    The goal of the present study was to find out the best interval after hCG injection in PMSG primed prepuberal gilts for retrieval of in vivo matured oocytes for in vitro fertilisation (IVF). Altogether 66 gilts were superovulated with 1500 IU PMSG and 500 IU hCG 72 h later. Ovum pick up was performed endoscopically 24, 28, 32 or 36 h after hCG and a total of 869 cumulus-oocyte-complexes (COCs) were aspirated from 1400 follicles. COCs were tested for quality, and an aliquot was immediately fixed and stained to determine meiotic configuration. The remaining COCs were fertilised in vitro using frozen-thawed epididymal semen. Quality and developmental stage of embryos were tested after IVF, and the number of nuclei was counted. At 24 to 32 h after hCG only few oocytes have entered the second meiotic cycle (18 to 25% vs. 58% at 36 h, p < 0.05). The overall cleavage rate was significantly influenced by insufficient maturation rate at the early collection times (14% at 24 h vs. 49% at 36 h). Additionally, when oocytes were collected 24 to 32 h vs. 36 h the cleavage rate based on mature oocytes was lower (26 vs. 62%, p < 0.05). Once embryonic development has been initiated, the further in vitro development to blastocyst stages did not differ between groups. However, the number of cells was lower at collection times 24 to 32 h as compared to 36 h after hCG (12 to 15 cells vs. 22 cells, p < 0.05). The results indicate that the time of COC collection affects the in vitro developmental competence up to the blastocyst stage and should not be performed earlier than 36 h after hCG treatment.

  1. Effect of recombinant human follicle-stimulating hormone and luteinizing hormone on in vitro maturation of porcine oocytes evaluated by the subsequent in vitro development of embryos obtained by in vitro fertilization, intracytoplasmic sperm injection, or parthenogenetic activation.

    PubMed

    Silvestre, M A; Alfonso, J; García-Mengual, E; Salvador, I; Duque, C C; Molina, I

    2007-05-01

    The aim of this work was to study the effect of recombinant human (rh) FSH and LH on in vitro maturation of pig oocytes compared with a conventional hormonal supplement based on equine (PMSG) and human chorionic gonadotropins (hCG), as evaluated by the developmental ability of 3 types of pig embryos obtained by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or artificial activation (ATA). In Exp. 1, one cumulus-oocyte complex group (A group) was supplemented with rh-FSH and rh-LH (0.1 IU/mL each), and the other group (B group) was supplemented with PMSG and hCG (10 IU/mL each). No differences in nuclear maturation between the A and B groups were observed (68.5 vs. 71.4%, respectively). No differences were detected between hormonal treatments in the rates of cleavage or blastocyst formation of ATA, IVF, and ICSI embryos. Total cell number of the embryos was not significantly different in any experimental group (A: 31.1, 28.5, and 19.8 vs. B: 25.2, 25.5, and 20.6 for ATA, IVF, and ICSI embryos, respectively). In Exp. 2, the effects of different concentrations of rh-FSH and rh-LH (0.5, 0.1, or 0.05 IU/mL) in maturation medium on nuclear maturation and in vitro development of embryos obtained by IVF were studied. No effect of different hormonal concentrations on blastocyst formation rates was observed (8.5, 13.0, and 5.7%, respectively). Blastocyst cell number was not different in any experimental group. In conclusion, the results obtained here permit us to substitute PMSG and hCG with rh-FSH and rh-LH and to produce pig embryos obtained by IVF, ICSI, or ATA.

  2. Effect of leptin during in vitro maturation of prepubertal calf oocytes: embryonic development and relative mRNA abundances of genes involved in apoptosis and oocyte competence.

    PubMed

    Córdova, Bladimir; Morató, Roser; de Frutos, Celia; Bermejo-Álvarez, Pablo; Paramio, Teresa; Gutiérrez-Adán, Alfonso; Mogas, Teresa

    2011-12-01

    During the in vitro maturation of adult bovine oocytes, leptin has beneficial effects on blastocyst development, apoptosis and transcription levels of developmentally important genes. The present study analyzes the differential effects of leptin on prepubertal bovine oocytes and cumulus cells. Effects were determined of leptin treatment during oocyte maturation on their developmental capacity after fertilization (Exp. 1), incidence of apoptosis in cumulus oocyte complexes (COCs) (Exp. 2) or on relative mRNA abundances of genes in cumulus cells and oocytes (Exp. 3). COCs were matured in serum-free medium containing 1 mg/mL polyvinyl alcohol and 0, 10, 100, or 1000 ng/mL leptin (L0, L10, L100, and L1000, respectively), or in medium supplemented with 10% fetal calf serum (FCS) as a positive control. Addition of leptin during oocyte maturation had no effect on cleavage rates after fertilization (FCS, 68.6%; L0, 62.9%; L10, 66.9%; L100, 63.4%; L1000, 60.9%). Similarly, no significant differences in blastocyst rates were observed when oocytes were matured in the presence of L0 (8.4%), L10 (9.3%), L100 (6.7%), L1000 (8.2%), compared to control FCS (9.4%). In Experiment 2, maturation in the presence of 1000 ng/mL of leptin increased the proportion of TUNEL-positive cumulus cell (6.9%) with respect to those matured in the presence of FCS (4.96%), but not at the lower leptin doses. When relative mRNA abundances were examined for seven genes by qRT-PCR, five (TP53, BAX, DNMT3A, PGTS2 and LEPR) showed differences among groups. LEPR expression was significantly higher in the oocytes matured with FCS compared with the other groups and in those matured with PVA (L0) without leptin compared with the three groups of oocytes matured in the presence of leptin. In conclusion, the addition of leptin to the in vitro maturation medium used for prepubertal bovine oocytes does not increase the development potential of the oocytes or reduce the percentage of apoptosis in cumulus cells

  3. Stoichiometry and structure of a lantibiotic maturation complex

    PubMed Central

    Reiners, Jens; Abts, André; Clemens, Rebecca; Smits, Sander H. J.; Schmitt, Lutz

    2017-01-01

    Lantibiotics are ribosomally synthesized antimicrobial peptides secreted by mainly Gram-positive bacteria. Class 1 lantibiotics mature via two modification steps introduced by a modification LanBC complex. For the lantibiotic nisin, the dehydratase NisB catalyzes the dehydration of serine and threonine residues in the so-called core peptide. Second, five (methyl)-lanthionine rings are introduced in a regio- and stereospecific manner by the cyclase NisC. Here, we characterized the assembly of the NisBC complex in vitro, which is only formed in the presence of the substrate. The complex is composed of a NisB dimer, a monomer of NisC and one prenisin molecule. Interestingly, the presence of the last lanthionine ring prevented complex formation. This stoichiometry was verified by small-angle X-ray scattering measurements, which revealed the first structural glimpse of a LanBC complex in solution. PMID:28169337

  4. In Vitro Acute Exposure to DEHP Affects Oocyte Meiotic Maturation, Energy and Oxidative Stress Parameters in a Large Animal Model

    PubMed Central

    Sardanelli, Anna Maria; Pocar, Paola; Martino, Nicola Antonio; Paternoster, Maria Stefania; Amati, Francesca; Dell'Aquila, Maria Elena

    2011-01-01

    Phthalates are ubiquitous environmental contaminants because of their use in plastics and other common consumer products. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and it impairs fertility by acting as an endocrine disruptor. The aim of the present study was to analyze the effects of in vitro acute exposure to DEHP on oocyte maturation, energy and oxidative status in the horse, a large animal model. Cumulus cell (CC) apoptosis and oxidative status were also investigated. Cumulus-oocyte complexes from the ovaries of slaughtered mares were cultured in vitro in presence of 0.12, 12 and 1200 µM DEHP. After in vitro maturation (IVM), CCs were removed and evaluated for apoptosis (cytological assessment and TUNEL) and intracellular reactive oxygen species (ROS) levels. Oocytes were evaluated for nuclear chromatin configuration. Matured (Metaphase II stage; MII) oocytes were further evaluated for cytoplasmic energy and oxidative parameters. DEHP significantly inhibited oocyte maturation when added at low doses (0.12 µM; P<0.05). This effect was related to increased CC apoptosis (P<0.001) and reduced ROS levels (P<0.0001). At higher doses (12 and 1200 µM), DEHP induced apoptosis (P<0.0001) and ROS increase (P<0.0001) in CCs without affecting oocyte maturation. In DEHP-exposed MII oocytes, mitochondrial distribution patterns, apparent energy status (MitoTracker fluorescence intensity), intracellular ROS localization and levels, mt/ROS colocalization and total SOD activity did not vary, whereas increased ATP content (P<0.05), possibly of glycolytic origin, was found. Co-treatment with N-Acetyl-Cysteine reversed apoptosis and efficiently scavenged excessive ROS in DEHP-treated CCs without enhancing oocyte maturation. In conclusion, acute in vitro exposure to DEHP inhibits equine oocyte maturation without altering ooplasmic energy and oxidative stress parameters in matured oocytes which retain the potential to be fertilized and develop into embryos

  5. Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation.

    PubMed

    Yokoo, Masaki; Tientha, Paisan; Kimura, Naoko; Niwa, Koji; Sato, Eimei; Rodriguez-Martinez, Heriberto

    2002-11-01

    Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

  6. The exosome complex establishes a barricade to erythroid maturation

    PubMed Central

    McIver, Skye C.; Kang, Yoon-A; DeVilbiss, Andrew W.; O’Driscoll, Chelsea A.; Ouellette, Jonathan N.; Pope, Nathaniel J.; Camprecios, Genis; Chang, Chan-Jung; Yang, David; Bouhassira, Eric E.; Ghaffari, Saghi

    2014-01-01

    Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1–mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program. PMID:25115889

  7. Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation.

    PubMed

    Guignot, F; Bezard, J; Palmer, E

    1999-10-01

    In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and

  8. In vitro maturation of dromedary (Camelus dromedarius) oocytes: effect of different protein supplementations and epidermal growth factor*.

    PubMed

    Wani, Na; Wernery, U

    2010-10-01

    The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4-well culture plates containing 500 μl of the maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO(2) in air for 32-36 h. The basic maturation medium consisted of TCM-199 supplemented with 0.1 mg/ml L-glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto-orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase-I, anaphase-I (A-I), metaphase-II (M-II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M-II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M-II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any

  9. Effect of soybean phosphatidylcholine on lipid profile of bovine oocytes matured in vitro.

    PubMed

    Pitangui-Molina, Caroline P; Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Santos, Vanessa G; Ferreira, Christina R; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Rosa-E-Silva, Ana Carolina J S

    2017-04-01

    The phospholipid (PL) composition of embryo and oocyte membranes affects thermal phase behavior and several physicochemical properties such as fluidity and permeability. The characterization of PL profiles and the development of suitable in vitro maturation (IVM) protocols, that are able to modify membrane's composition, may result in significant improvements in oocyte developmental potential and cryotolerance. Using soybean phosphatidylcholine (PC) as a model supplement, we evaluated the effect of PL supplementation during IVM on bovine cumulus-oocyte-complex (COC). Substantial changes in the lipid profiles of oocyte membrane were observed and associated with pre-implantation data. The propensity of the PC supplement to become soluble in the maturation medium and/or diffuse into mineral oil was also assessed. Oocytes were matured in TCM without supplementation, i.e. control, (n=922) or supplemented with 50 or 100μM PC (n=994). The maturation media and mineral oil pre- and post- IVM, along with control and PC-treated oocytes were then analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and the lipid profiles were compared via principal component analysis (PCA). Soybean PCs are bioavailable and stable in IVM medium; further, PCs did not diffuse to the mineral oil, which also remained unaltered by the metabolism of treated oocytes. PC supplementation at 100μM resulted in substantially greater relative abundances of polyunsatured PL, namely PC (32:1), PC (34:2), PC (36:6), PC (36:4), and PC (38:6), in oocyte membrane. These differences indicated that short-term exposure to the PC supplement could indeed modify the lipid composition of IVM-oocytes in a dose-dependent manner. Membrane incorporation of polyunsaturated molecular species of PC was favored, and does so without compromising the viability of the subsequent embryo in regards to cleavage, blastocyst development and hatching rate. The reported approach will allow for the

  10. Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: optimization of protocols.

    PubMed

    Wani, N A

    2008-03-15

    Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P<0.01) of oocytes was activated in ionomycine/6-DMAP and ionomycine/roscovitine groups when compared with ethanol/6-DMAP, ethanol/roscovitine and in vitro fertilized groups. However, there was no difference (P>0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P<0.01) in ionomycine/6-DMAP and ionomycine/roscovitine when compared with ethanol/6-DMAP and ethanol/roscovitine treated oocytes. A higher proportion of oocytes reached blastocyst stage when they were exposed to ionomycine for 3 min but they were not significantly different from the others (P>0.05). The proportion of blastocysts

  11. Optimum gas atmosphere for in vitro maturation and in vitro fertilization of bovine oocytes.

    PubMed

    Pinyopummintr, T; Bavister, B D

    1995-09-01

    The objective of this study was to determine optimal gas atmosphere conditions for in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes. In Experiment 1, groups of 10 to 12 cumulus-oocyte complexes (COCs) were matured (24 h) and fertilized (18 h) under 1) 5% CO(2), 5% O(2;) 2) 5% CO(2), 10% O(2) or 3) 5% CO(2), 20% 0(2.) The COCs were cultured in 50 microl drops of maturation medium (TCM-199 + 10% bovine calf serum + oLH, oFSH and estrogen) or fertilization medium (TALP + swim-up separated spermatozoa +1 microg/ml heparin sulfate) under a layer of 10 ml paraffin oil at 39 degrees C with saturated humidity. Half of the oocytes in each drop were assigned randomly for maturation scoring and the remainder were inseminated. Reduced atmospheric O(2) drastically decreased proportions of oocytes reaching MII (71.4, 26.9 and 9.3% with 20, 10 and 5% O(2), respectively; P < 0.05). The percentages of total fertilization in 10 and 20% O(2) were similar and considerably higher than in 5% O(2) (80.3, 87.0 and 53.1%, respectively; P < 0.05). In addition, the percentage of polyspermy markedly increased when IVF was conducted in reduced O(2) (26.6 and 28.8% in 5 and 10% O(2) vs 15.4% in 20% O(2;) P < 0.05). Experiment 2 was similar to Experiment 1 except that CO(2) was the variable: 1) 2.5% CO(2) in air, 2) 5% CO(2) in air and 3) 10% CO(2) in air. The proportion of MII oocytes did not differ across treatments (64.9, 68.9 and 61.9%, respectively; P > 0.05). Although the percentages of total fertilization among treatments were not different (75.4, 80.9 and 76.1%, respectively), the proportion of normal fertilization was significantly reduced in 10% C0(2) (55.1%) when compared with that of either 2.5% CO(2) (62.7%) or 5% CO(2) (68.7%; P < .05). This study indicates that low O(2) is detrimental for IVM/IVF of bovine oocytes and that optimal atmospheric conditions are either 2.5 or 5% CO(2) and 20% O(2).

  12. Quality and developmental ability of dromedary (Camelus dromedarius) embryos obtained by IVM/IVF, in vivo matured/IVF or in vivo matured/fertilized oocytes.

    PubMed

    Khatir, H; Anouassi, A; Tibary, A

    2007-06-01

    The effect of source of cumulus-oocytes-complexes (COCs), maturation and fertilization conditions on developmental competence of dromedary embryos was examined. Thirty-six adult females were superovulated with equine Chorionic Gonadotropin (eCG) injection (3500 IU, IM) and divided in three groups of 12 females each. Group 1 provided 138 COC's collected from follicles >or= 5 mm 10 days after stimulation prior hCG treatment and matured in vitro for 30 h. Group 2 provided 120 in vivo matured oocytes which were aspirated from their follicles 20 h after hCG (3000 IU, IV) given on day 10 follow eCG injection. Group 3 provided 65 in vivo matured/fertilized oocytes. Females in Group 3 received hCG on day 10 following eCG treatment and then were mated 24 h later. Fertilized oocytes were collected from the oviducts of females 48-h post-mating. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of COCs. All cultures were performed in three replicates (n = 3) at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. Nuclear maturation rate for Groups 1 and 2 was determined by epifluorescence microscopy in a sample of COCs (n = 30) denuded, fixed and stained with Hoechst 33342. To study the viability of obtained embryos, hatched blastocysts from each group were transferred to recipients followed by pregnancy diagnosis using ultrasonography at 15, 60 and 90 days. The percentage of COCs reaching metaphase II (MII) after 30 h of maturation was slightly but not significantly higher for in vivo matured oocytes (28/30; 93%) than those in vitro matured (25/30; 84%). The total rate of cleavage (2 cells to blastocyst stage) was not different for the three groups. However, significantly (p < 0.05) more blastocyst and hatched blastocysts were obtained from in vivo matured and in vivo fertilized oocytes (Group 3; 52% and 73%) than from in vitro

  13. The effect of conspecific ampulla oviductal epithelial cells during in vitro maturation on oocyte developmental competence and maturation-promoting factor (MPF) activity in sheep.

    PubMed

    Dadashpour Davachi, Navid; Kohram, Hamid; Zare Shahneh, Ahmad; Zhandi, Mahdi; Goudarzi, Abbas; Fallahi, Roozbeh; Masoudi, Reza; Yousefi, Ali Reza; Bartlewski, Pawel M

    2017-01-15

    The acquisition of fertilization ability by oocytes is one of the prerequisites for successful in vitro embryo production. In the present study, we examined the influence of conspecific ampulla oviductal epithelial cells incubated with cumulus-oocyte complexes (COCs) throughout the IVM phase on the developmental competence and maturation-promoting factor (MPF) activity of sheep oocytes. There were six experimental groups in this study, namely four groups with and two groups without oviductal epithelial cells added to IVM media: adult COCs matured in vitro with the ampulla oviductal epithelial cells obtained from adult (AAE; G1) or prepubertal donors (prepubertal sheep ampulla oviductal epithelial cells [PAE]; G4), COCs obtained from prepubertal animals cocultured with AAE (G2) or PAE (G3), and adult (C1) and prepubertal sheep COCs (C2) matured without oviductal epithelial cells. Coincubation of oocytes retrieved from both adult and sexually immature donors with AAE (G1 and G2) resulted in significantly improved rates of metaphase-II (M-II) attainment (G1: 85.1 ± 2.0 and G2: 40.2 ± 1.3) and blastocyst formation (G1: 42.2 ± 1.1 and G2: 21.2 ± 1.0) as well as blastocyst development (total cell count; G1: 130.3 ± 7.8, G2: 70.2 ± 3.5) compared with their respective controls (C1: 94.3 ± 4.1 and C2: 49.7 ± 2.0). Prior to IVM, the activity of MPF was greater (P < 0.05) for oocytes obtained from ewes (G1, G4, and C1) compared with those from ewe lambs (G2, G3, and C2). The greatest increment in MPF activity was recorded in G2 (MPF activity before IVM/MPF activity after IVM = 3.62) followed by C2 and G3 (2.22 and 2.20, respectively), and then all remaining groups of oocytes (C1: 1.89, G1: 1.87, and G4: 1.86). In summary, coincubation with AAE during the 24-hour IVM had a positive impact on ovine oocyte competence and ensuing in vitro embryo production efficiency. A significant increase in MPF activity following IVM of G2 oocytes could be responsible, at

  14. Brain Maturation Changes Characterized by Algorithmic Complexity (Lempel and Ziv Complexity)

    NASA Astrophysics Data System (ADS)

    Fernández, J. G.; Larrondo, H. A.; Figliola, A.; Serrano, E.; Rostas, J. A. P.; Hunter, M.; Rosso, O. A.

    2007-05-01

    Recent experimental results suggest that basal electroencephalogram (EEG)changes reflect the widespread functional evolution in neuronal circuits, occurring in chicken brain during the "synapse maturation" period, between 3 and 8 weeks' posthatch. In present work a quantitative analysis based on the Algorithmic Complexity (Lempel and Ziv Complexity) is performed. It is shown that this complexity presents a peak at week 2 posthatch 2, and a tendency to stabilize its values after the week 5 posthatch.

  15. Females with reduced fertility have excess androstenedione in follicular fluid, altered theca gene expression and increased VEGFA164b, maternal effect, and microRNA processing mRNA levels in cumulus-oocyte complexes

    USDA-ARS?s Scientific Manuscript database

    Ovarian dysfunction contributes significantly to female infertility. However, the intrinsic and exogenous factors that result in abnormal ovarian function are poorly defined. Thus, we have established a cow model of fertility to identify mechanisms regulating follicular growth, steroidogenesis and o...

  16. In vivo effect of interleukin-1beta and interleukin-1RA on oocyte cytoplasmic maturation, ovulation, and early embryonic development in the mare

    PubMed Central

    Caillaud, Maud; Duchamp, Guy; Gérard, Nadine

    2005-01-01

    A growing body of evidence suggests that the interleukin-1 system is involved in periovulatory events. Previous work from our lab demonstrated that in the mare, interleukin-1beta (IL-1beta) increases the ovulatory rate of metaphase II oocytes. The present study was conducted to analyze in vivo the effect of IL-1 on oocyte cytoplasmic maturation, ovulation and pregnancy rate. In the present work, IL-1beta (experiment 1, n = 13; experiment 2, n = 25) and interleukin-1RA (IL-1RA; experiment 1, n = 25) were injected intrafollicularly by using the transvaginal ultrasound-guided injection method. Injections were performed on cyclic mares when the diameter of the growing dominant follicle reached 30–34 mm. In experiment 1, mares were inseminated the day of the treatment and all the other day until ovulation. The time of ovulation was determined and a pregnancy diagnosis was performed 14 days after ovulation of the injected follicle. In experiment 2, the cumulus-oocyte complex from each injected follicle was collected by transvaginal ultrasound-guided aspiration 38 h after the intrafollicular injection. Oocyte nuclear stage and oocyte cytoplasmic maturation were assessed by analyzing chromatin configuration, cortical granules migration and mitochondria distribution under a confocal microscope. The results from experiment 1 confirm that an intrafollicular injection of 1 microgram IL-1beta induces ovulation in the mare whereas IL-1RA has no effect at the dose used in the present study. Furthemore, we demonstrated, that in our experimental conditions, IL-1beta and IL-1RA induced a decrease in embryo development. Experiment 2 leads us to observe that IL-1beta is unable to induce cortical granules migration and remodelling of mitochondria, that commonly occurs during oocyte maturation, whereas it acts on nuclear maturation. This result may explain the decrease in embryo development we observed after IL-1beta intrafollicular injection. In conclusion, the present study tends to

  17. Effect of leptin supplementation during in vitro oocyte maturation and embryo culture on bovine embryo development and gene expression patterns.

    PubMed

    Arias-Alvarez, M; Bermejo-Alvarez, P; Gutierrez-Adan, A; Rizos, D; Lorenzo, P L; Lonergan, P

    2011-03-15

    Leptin is a metabolic hormone related to body condition and nutritional status that influences fertility in assisted reproductive technologies modulating oocyte and embryo quality. The aim of the present study was to establish the effect of various leptin concentrations (0, 10, 100 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on bovine embryo development and quality in terms of gene expression. The relative mRNA abundance of the genes encoding solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1), Bcl-2-associated X protein (BAX), placenta-specific 8 (PLAC8), aldo-keto reductase family 1 member B1 (AKR1B1) and leptin receptor (LEPR) were determined on Day 7 blastocysts by qRT-PCR. Cleavage rate (P < 0.005) and blastocyst yield (P = 0.05) was significantly lower when cumulus-oocyte complexes (COCs) were matured with 100 ng/mL leptin compared to 0 or 10 ng/mL leptin. No significant effect of different concentrations of leptin added during IVC on blastocyst yield was observed. The presence of 100 ng/mL leptin in both IVM and IVC further decreased cleavage rate (P < 0.005) and blastocyst yield compared to the control group without leptin (P = 0.05) and those supplemented with 10 ng/mL leptin or FCS (P < 0.005). There was no evidence of any leptin-induced difference in the relative transcript abundance of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts. Expression of AKR1B1 was significantly lower in blastocysts from COCs matured with 100 ng/mL leptin compared to those matured with 0 or 10 ng/mL leptin (P < 0.005). LEPR expression was up regulated when leptin concentration was increased from 0 ng/mL during IVM to 10 ng/mL during IVC, but it was down-regulated in the opposite situation (P < 0.005). In conclusion, high leptin concentrations possibly related to obesity seem to be more detrimental rather than the absence of this hormone for preimplantation embryo survival; this effect is independent of LEPR gene

  18. Effect of recombinant bovine somatotropin (rbST) on cytoplasmic maturation of bovine oocytes and their developmental competence in vitro.

    PubMed

    Kuzmina, Tatjana I; Alm, Hannelore; Denisenko, Vitaly; Tuchscherer, Armin; Kanitz, Wilhelm; Torner, Helmut

    2007-04-01

    The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2). The culture medium of the treatment groups was modified by supplementation of the control medium with 10 ng/ml rbST (CM 1A and CM 2A), 10(6)/ml granulosa cells (CM 1B and CM 2B), or 10 ng/ml rbST plus 10(6)/ml granulosa cells (CM 1C and CM 2C). No differences were observed in the percentages of oocytes reaching metaphase II between the groups. However, the proportion of blastocysts was highest in treatment groups CM 1C and CM 2C (P<0.05). The type of serum did not alter the positive effect of rbST on the developmental competence of embryos. The fluorescence intensity of metabolically active mitochondria measured by intensity per oocyte (Em 570) after MitoTracker CMTM Ros Orange labeling was significantly increased in oocytes matured in the presence of 10 ng/ml rbST and granulosa cells (309.21 vs. 119.97 microA; P<0.01). In parallel, the concentration of [Ca(2+)](is) in oocytes, determined using fluorophore chlortetracycline, was significantly decreased (0.85 +/- 0.02 vs. 0.97 +/- 0.03 AU; P<0.05). Based on these results, we concluded that rbST, in interaction with granulosa cells stimulates the oxidative activity of ooplasmic mitochondria and decreases the content of [Ca(2+)](is) in oocytes. These facts support the hypothesis that somatotropin influences the developmental competence of bovine oocytes during maturation in vitro, and this effect can be modulated by granulosa

  19. Metabolic Maturation of Auditory Neurones in the Superior Olivary Complex

    PubMed Central

    Trattner, Barbara; Gravot, Céline Marie; Grothe, Benedikt; Kunz, Lars

    2013-01-01

    Neuronal activity is energetically costly, but despite its importance, energy production and consumption have been studied in only a few neurone types. Neuroenergetics is of special importance in auditory brainstem nuclei, where neurones exhibit various biophysical adaptations for extraordinary temporal precision and show particularly high firing rates. We have studied the development of energy metabolism in three principal nuclei of the superior olivary complex (SOC) involved in precise binaural processing in the Mongolian gerbil (Meriones unguiculatus). We used immunohistochemistry to quantify metabolic markers for energy consumption (Na+/K+-ATPase) and production (mitochondria, cytochrome c oxidase activity and glucose transporter 3 (GLUT3)). In addition, we calculated neuronal ATP consumption for different postnatal ages (P0–90) based upon published electrophysiological and morphological data. Our calculations relate neuronal processes to the regeneration of Na+ gradients perturbed by neuronal firing, and thus to ATP consumption by Na+/K+-ATPase. The developmental changes of calculated energy consumption closely resemble those of metabolic markers. Both increase before and after hearing onset occurring at P12–13 and reach a plateau thereafter. The increase in Na+/K+-ATPase and mitochondria precedes the rise in GLUT3 levels and is already substantial before hearing onset, whilst GLUT3 levels are scarcely detectable at this age. Based on these findings we assume that auditory inputs crucially contribute to metabolic maturation. In one nucleus, the medial nucleus of the trapezoid body (MNTB), the initial rise in marker levels and calculated ATP consumption occurs distinctly earlier than in the other nuclei investigated, and is almost completed by hearing onset. Our study shows that the mathematical model used is applicable to brainstem neurones. Energy consumption varies markedly between SOC nuclei with their different neuronal properties. Especially for the

  20. Effect of growth hormone releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) on in vitro bovine oocyte maturation.

    PubMed

    Beker, A R; Izadyar, F; Colenbrander, B; Bevers, M M

    2000-06-01

    The aim of this study was to investigate the effects of growth hormone releasing hormone (GHRH) and the structural-related peptide vasoactive intestinal peptide (VIP) on nuclear maturation, cortical granule distribution and cumulus expansion of bovine oocytes. Bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of either 100 ng/mL bovine GHRH or 100 ng/mL porcine VIP. The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage was assessed after 16 or 24 h of incubation using DAPI staining. Cortical granule distribution was assessed after 24 h of incubation using FITC-PNA staining. To assess the effects of GHRH and VIP on cumulus expansion, COCs were incubated for 24 h under the conditions described above. In addition, 0.05 IU/mL recombinant human FSH was added to GHRH and VIP groups. Cultures without GHRH/VIP/FSH or with only FSH served as negative and positive controls, respectively. At 16 h neither GHRH (42.9%) nor VIP (38.5%) influenced the percentage of MII stage oocytes compared with their respective controls (44.2 and 40.8%). At 24 h there also was no difference in the percentage of MII oocytes between GHRH (77.0%), VIP (75.3%) and their respective controls (76.0 and 72%). There was no significant cumulus expansion in the GHRH or VIP group, while FSH induced significant cumulus expansion compared with the control groups, which were not inhibited by GHRH or VIP. Distribution of cortical granules was negatively affected by GHRH and VIP. The percentage of oocytes showing more or less evenly dispersed cortical granules in the cortical cytoplasm aligning the oolemma (Type 3) was lower in the GHRH (2.7%) and VIP (7.8%) groups than in the control group (15.9%). In conclusion, GHRH and VIP have no effect on nuclear maturation or cumulus expansion of bovine COCs but retard cytoplasmic maturation, as reflected by delayed cortical granule migration.

  1. The Role of Teacher Stress, Cognitive Complexity, and Career Maturity in Teacher Socialization.

    ERIC Educational Resources Information Center

    Franz, John B.; Dembo, Myron H.

    A research study investigated the relationship of stress in teachers' work environment to teachers' level of cognitive complexity (level of thinking) and their career maturity, and the relationship of stress, cognitive complexity, and career maturity to teaching experience. Participants were teaching elementary school in an urban environment: 23…

  2. Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes.

    PubMed

    Hermansson, Ulrika; Axnér, Eva; Holst, Bodil Ström

    2007-10-01

    Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes. Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20 degrees C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO2 in air at 38 degrees C and 100% humidity in the presence of 5 x 106 fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM). Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger. In conclusion, under the conditions of the present study, immature oocytes recovered from

  3. Cumulus cell expansion and first polar body extrusion during in vitro oocyte maturation in relation to morphological and morphometric characteristics of the dromedary camel ovary.

    PubMed

    Mesbah, F; Kafi, M; Nili, H

    2016-12-01

    The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6-hr interval) in Hams-F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4-year-old and >4-year-old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2-4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4-year-old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2-6 mm follicles are cultivable. The most slaughterhouse-derived COCs retrieved from 2-6 mm follicles of non-pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24-30 hr; the optimal time for CC expansion is 24-30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.

  4. Degrees of maturity: the complex structure and biology of flaviviruses.

    PubMed

    Pierson, Theodore C; Diamond, Michael S

    2012-04-01

    Flaviviruses are small enveloped virions that enter target cells in a pH-dependent fashion. Virus attachment, entry, and membrane fusion are orchestrated by the envelope (E) and pre-membrane (prM) proteins, the two structural proteins displayed on the surface of virions. Flaviviruses assemble as an immature non-infectious form onto which prM and E form trimeric spikes. During egress from infected cells, flaviviruses undergo dramatic structural changes characterized by the formation of a herringbone arrangement of E proteins that lie flat against the surface of the virion and cleavage of the prM protein by the cellular protease furin. The result is a relatively smooth, infectious mature virion. This dynamic process is now understood in structural detail at the atomic level. However, recent studies indicate that many of the virions released from cells share structural features of both immature and mature virus particles. These mosaic partially mature virions are infectious and interact uniquely with target cells and the host immune response. Here, we will discuss recent advances in our understanding of the biology and significance of partially mature flaviviruses.

  5. Syntactic Maturity: The Complex Sentence in Intermediate Spanish.

    ERIC Educational Resources Information Center

    Garrott, Carl L.

    This paper begins with a literature review of research on syntactic maturity, defined as the developmental stages from one- and two-word utterances to the hierarchical structures of adult speech, and seeks to answer questions in the context of past and current research in this area. It attempts to study some of the ramifications of the movement…

  6. 176 REGULATORY ROLE OF miR-20a DURING BOVINE OOCYTE MATURATION.

    PubMed

    Andreas, E; Salilew-Wondim, D; Rings, F; Held, E; Hoelker, M; Neuhoff, C; Tholen, E; Schellander, K; Tesfaye, D

    2016-01-01

    The role of microRNA in oocyte maturation is mostly associated with optimal turnover of the accumulated maternal transcripts during their growth to allow maturation. MiR-20a is a member of the miR-17-92 cluster, which has been found to be differentially expressed in bovine granulosa cells derived from preovulatory dominant and subordinate follicles. Our recent study showed that miR-20a is involved in the regulation of granulosa cell proliferation, differentiation, and progesterone synthesis by targeting PTEN and BMPR2 genes. Here, we aimed to investigate the role of miR-20a in the bovine oocyte maturation processes. For this, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2-8mm in diameter) and cultured in groups of 50 in 400µL of maturation media (TCM-199 media supplemented with 12% oestrus cow serum and 10µg/ml Follitropin®) at 39°C in a humidified atmosphere with 5% (vol/vol) CO2 in the air for 22h. The cumulus cells and oocytes before (germinal vesicle) and after maturation (metaphase II) were mechanically separated in 0.1% hyaluronidase (in TCM-199 media). To study whether the presence of cumulus cells or oocyte has an impact on the miR-20a expression, we cultured oocytectomized cumulus cells and oocytes with and without their companion cells. Moreover, COC were co-cultured with miR-20a mimic, inhibitor, or corresponding controls to investigate the role of this miRNA in oocyte maturation. The total RNA from cumulus cells and oocytes was extracted using miRNeasy® mini kit (Qiagen GmbH, Hilden, Germany). Total RNA from respective samples was reverse transcribed for mRNA and microRNA expression analysis. Quantitative expression analysis was performed using StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) and subsequent data were analysed using a comparative cycle threshold method. The progesterone released in the spent media was measured using progesterone enzyme-linked immunosorbent assay kit (ENZO Life Sciences

  7. Mediastinal mature teratoma with complex rupture into the lung, bronchus and skin: a case report.

    PubMed

    Serraj, Mounia; Lakranbi, Marouane; Ghalimi, Jamal; Ouadnouni, Yassine; Smahi, Mohamed

    2013-06-01

    Mature teratoma is the most common primary germ cell tumor in the mediastinum. On rare occasions, cystic teratomas rupture in adjacent structures, such as pleural space, pericardium, lung or tracheobronchial tree. We present a case of a mediastinal mature cystic teratoma in 16-year-old female with complex rupture into the lung, bronchus and skin. Mature mediastinal teratoma fistulized to the skin has not been previously described.

  8. Effect of protein synthesis inhibition before or during in vitro maturation on subsequent development of bovine oocytes.

    PubMed

    Lonergan, P; Fair, T; Khatir, H; Cesaroni, G; Mermillod, P

    1998-08-01

    The overall objective of this study was to assess the effect of maintaining meiotic arrest in bovine oocytes in vitro on developmental competence. In Experiment 1 the effect of inhibition of meiotic resumption using cycloheximide (CX), on subsequent was examined. Immature cumulus oocyte complexes (COCs, n = 804) were cultured in the absence (24 h) or presence of CX for 6, 12, 18 or 24 h. The control was inseminated 24 h later, while CX-treated oocytes were cultured for a further 24 h before insemination. In Experiment 2 the effect of exposing the oocyte (n = 1239) during meiotic arrest to putative stimulatory substances (pFSH and FCS) was examined. In Experiment 3, to study the importance of protein synthesis during maturation, synthesis was blocked for a 6-h period at various times (6, 12, 18 h) after start of culture (n = 1117). In Experiment 1, there was no difference in cleavage rate between treatments. However, the percentage of 5 to 8 cell embryos at 72 h post insemination was significantly lower after CX treatment (64 vs 42 to 51%; P < 0.05). This was reflected in a lower rate of blastocysts at Day 6 (9 to 15 vs 31%, P < 0.002). While the blastocyst rate at Day 8 was lower in CX-treated oocytes, the effect was only significant when CX was present for longer than 12 h. A marked decrease in development was noted following inhibition for 18 h or more compared with the control (17 to 19 vs 40%; P < 0.0002). In Experiment 2, addition of either FSH or FCS to oocytes in the presence of CX had no effect on any of the parameters studied, even though there was a positive effect in control oocytes. In Experiment 3, treatment with CX after the oocytes had matured for varying periods resulted in decreased blastocyst rates at Days 6 and 8 of culture. The most significant drop in development occurred when oocytes were cultured for 12 h before exposure to CX (15 vs 40%; P < 0.0001). In conclusion, CX-blocked oocytes retained their developmental competence, although final

  9. Toxicity evaluation of ethanol treatment during in vitro maturation of porcine oocytes and subsequent embryonic development following parthenogenetic activation and in vitro fertilization.

    PubMed

    Lee, Sanghoon; Kim, Eunhye; Hyun, Sang-Hwan

    2014-11-01

    Ethanol is frequently used as a solvent in several techniques for in vitro production (IVP). It is also used for the parthenogenetic activation (PA) of oocytes. Although a number of studies have suggested that ethanol has detrimental effects on fibroblasts and neuronal cells, little attention has been paid to the effects of ethanol on porcine oocytes. Thus, the aim of this study was to evaluate the effects of the addition of ethanol to in vitro maturation (IVM) medium. We investigated the effects of ethanol (0, 1 and 3%) on the following parameters: nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic development following PA and in vitro fertilization (IVF). After 44 h of IVM, the 3% group showed a significant (P<0.05) decrease in nuclear maturation (34.0%) compared with the control group (70.3%). The 1 and 3% groups exhibited a significant (P<0.05) decrease in GSH levels and an increase in ROS levels compared with the control group. Compared with the control group, the 3% group had significantly (P<0.05) lower cleavage rates following PA (51.6 vs. 86.9%) and IVF (53.2 vs. 70.6%), as well as lower blastocyst formation rates and decreased total cell numbers following PA (11.3% and 31.8 vs. 53.6% and 65.4, respectively) and IVF (4.1% and 22.0 vs. 36.1% and 70.3, respectively). We evaluated the mRNA expression levels of DNA repair‑related and apoptosis‑related genes in the cumulus oocyte complexes (COCs). The 1% ethanol group showed significantly (P<0.05) higher mRNA expression levels of poly(ADP‑ribose) polymerase‑1 (PARP‑1), Bax, Bak and caspase‑3, and the 3% ethanol group had significantly (P<0.05) increased PARP‑1, Bax and caspase‑3 mRNA expression levels compared with the control group. Our results suggest that treatment with >1% ethanol during IVM exerts a toxic effect on the developmental potential of PA and IVF porcine embryos by decreasing the intracellular GSH level, thereby

  10. A long-lived mesoscale convective complex. II - Evolution and structure of the mature complex

    NASA Technical Reports Server (NTRS)

    Wetzel, P. J.; Cotton, W. R.; Mcanelly, R. L.

    1983-01-01

    The present investigation is concerned with an eight-day episode, during which a series of mesoscale convective complexes (MCC) developed and moved across the country, producing heavy rain and some flooding over an extensive region. An overview of the considered period from August 3 to August 10, 1977 is presented, and the evolution of the August 4 storm is examined. The structure of the mature MCC is discussed, taking into account the August 4-5 storm, a comparative case involving the August 3-4 storm, and an evaluation of the observed phenomena. It is concluded that MCCs are basically tropical in nature and that their dynamics are dominated by buoyant accelerations. It was found that the MCCs developed a warm-core, divergent anticyclonic flow pattern in the upper troposphere which was not present prior to the development of convection. A similar structure is observed in tropical cloud clusters.

  11. Influence of Grape Maturity on Complex Carbohydrate Composition of Red Sparkling Wines.

    PubMed

    Martínez-Lapuente, Leticia; Apolinar-Valiente, Rafael; Guadalupe, Zenaida; Ayestarán, Belén; Pérez-Magariño, Silvia; Williams, Pascale; Doco, Thierry

    2016-06-22

    This paper studied how grape maturity affected complex carbohydrate composition during red sparkling wine making and wine aging. Grape ripening stage (premature and mature grapes) showed a significant impact on the content, composition, and evolution of polysaccharides and oligosaccharides of sparkling wines. Polysaccharides rich in arabinose and galactose, mannoproteins, rhamnogalacturonans II, and oligosaccharides in base wines increased with maturity. For both maturity stages, polysaccharides rich in arabinose and galactose, and the glucuronic acid glycosyl residue of the oligosaccharides were the major carbohydrates detected in all vinification stages. The total glycosyl content of oligosaccharides decreased during the whole period of aging on yeast lees. The reduction of polysaccharides rich in arabinose and galactose and rhamnogalacturonans type II during the aging was more pronounced in mature samples. To our knowledge, this is the first report of the polysaccharide and oligosaccharide composition of red sparkling wines.

  12. Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development.

    PubMed

    Ortiz-Escribano, N; Smits, K; Piepers, S; Van den Abbeel, E; Woelders, H; Van Soom, A

    2016-07-15

    This study was designed to determine the role of cumulus cells during vitrification of bovine oocytes. Mature cumulus-oocyte complexes (COCs) with many layers of cumulus cells, corona radiata oocytes (CRs), with a few layers of cumulus cells, and denuded oocytes (DOs) without cumulus cells were vitrified in 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5-M sucrose. Oocytes that survived the vitrification process were fertilized. Denuded oocytes were fertilized with or without supplementation of intact COCs (DOsCOCs). First, survival and embryo development rates were studied. Higher survival rates were obtained for DOs and DOsCOCs (94% and 95%, respectively) compared with COCs (82.7%, P < 0.05). Corona radiata oocytes showed similar survival rates when compared with DOs. The cleavage and blastocyst rates of vitrified DOs were compromised because cumulus cells were not present during the fertilization (34% and 2.7%, respectively). However, the situation could be reverted when DOs were supplemented with intact COCs (DOsCOCs; 62.7% and 12.7%, respectively, P < 0.05). Vitrified CRs showed similar cleavage and blastocyst rate (49.3% and 7.7%, respectively) compared with COCs (54.8% and 4.9%, respectively). In the second experiment, the penetration rate was analyzed. Removing cumulus cells before fertilization reduced the fertilization of vitrified DOs compared with COCs (24.3% vs. 52.8%, P < 0.05). The supplementation of DOs with intact COCs (DOsCOCs) improved the fertilization rate though (49.6%, P < 0.05). No differences in the fertilization rate were found between CRs and COCs. In the third experiment, parthenogenetic activation was examined. Interestingly, the CRs group showed higher cleavage and blastocyst rates (76.8% and 29.6%, respectively) than the COCs (39.1% and 7.5%, respectively, P < 0.05). Furthermore, oocytes from vitrified CRs had the same odds to become a blastocyst as fresh oocytes (1.1 vs. 1.5, respectively). In conclusion, our data

  13. Service quality and maturity of health care organizations through the lens of Complexity Leadership Theory.

    PubMed

    Horvat, Ana; Filipovic, Jovan

    2017-07-17

    This research focuses on Complexity Leadership Theory and the relationship between leadership-examined through the lens of Complexity Leadership Theory-and organizational maturity as an indicator of the performance of health organizations. The research adopts a perspective that conceptualizes organizations as complex adaptive systems and draws upon a survey of opinion of 189 managers working in Serbian health organizations. As the results indicate a dependency between functions of leadership and levels of the maturity of health organizations, we propose a model that connects the two. The study broadens our understanding of the implications of complexity thinking and its reflection on leadership functions and overall organizational performance. The correlations between leadership functions and maturity could have practical applications in policy processing, thus improving the quality of outcomes and the overall level of service quality. © 2017 John Wiley & Sons, Ltd.

  14. Maturation of embryonic tissues in a lymph node: a new approach for bioengineering complex organs.

    PubMed

    Francipane, Maria Giovanna; Lagasse, Eric

    2014-01-01

    Given our recent finding that the lymph node (LN) can serve as an in vivo factory to generate complex structures like liver, pancreas, and thymus, we investigated whether LN could also support early development and maturation from several mid-embryonic (E14.5/15.5) mouse tissues including brain, thymus, lung, stomach, and intestine. Here we observed brain maturation in LN by showing the emergence of astrocytes with well-developed branching processes. Thymus maturation in LN was monitored by changes in host immune cells. Finally, newly terminally differentiated mucus-producing cells were identified in ectopic tissues generated by transplantation of lung, stomach and intestine in LN. Thus, we speculate the LN offers a unique approach to study the intrinsic and extrinsic differentiation potential of cells and tissues during early development, and provides a new site for bioengineering complex body parts.

  15. The first dromedary (Camelus dromedarius) offspring obtained from in vitro matured, in vitro fertilized and in vitro cultured abattoir-derived oocytes.

    PubMed

    Khatir, Hadj; Anouassi, AbdelHaq

    2006-06-01

    Dromedary offspring have never been produced fully in vitro. We have previously demonstrated that embryos obtained by culture in semi-defined medium (mKSOMaa) have better in vitro development ability than those cultured with oviductal epithelial cells. The aim of the present experiment was to study the pregnancy rate after embryo transfer of in vitro-produced (IVP) dromedary embryos cultured in semi-defined modified medium (mKSOMaa). IVM/IVF procedures were conducted on six hundred and sixty four (664) cumulus oocytes complexes (COCs) aspirated from ovaries collected at a local slaughterhouse and cultured in vitro (38.5 degrees C; 5% CO2, and maximum humidity >95%). Maturation was completed by incubation in TCM-199 medium supplemented with 10% heat-treated Fetal Calf Serum (FCS), 10 ng/mL EGF, 1 microg/mL FSH, 1 microg/mL E2 and 500 microM cysteamine for 30 h. In vitro fertilization was performed using fresh semen (0.5 x 10(6) spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 38.5 degrees C, 5% CO2 and 90% N2 with maximum humidity (>95%). All IVC steps were done in seven replicates. The cleavage rate (two cells to blastocyst stage) was 64% (425/664) and the percentage of oocytes reaching the blastocyst stage was 23% (155/664). The hatching rate of blastocyst obtained after culture was 46% (71/155). Good quality hatched blastocysts (n = 66) were transferred individually to synchronized recipients. Pregnancy rates, determined by ultrasonography at 15, 60 and 90 days after embryo transfer (ET), were 38%, 32% and 27%, respectively. Out of 18 pregnant females 5 aborted between the fifth and seventh month of pregnancy and 13 females (20%) remained pregnant. After 385 days of pregnancy, the first healthy and normal male-dromedary offspring produced fully in vitro was born at a birth weight of 38 kg. More dromedary calves (n = 4) were born later on. The remaining pregnant females (n = 8) are due to calf within the next months. In

  16. Thermoprotective effect of insulin-like growth factor 1 on in vitro matured bovine oocyte exposed to heat shock.

    PubMed

    Rodrigues, Thaís A; Ispada, Jessica; Risolia, Pedro H B; Rodrigues, Mariana T; Lima, Rafaela S; Assumpção, Mayra E O A; Visintin, José A; Paula-Lopes, Fabíola F

    2016-11-01

    The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 °C for 22 hours) and heat shock (41 °C for 14 hours followed by 38.5 °C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 °C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented

  17. [Evoked motor activity and maturation sequence of striate complex structures during chick embryogenesis].

    PubMed

    Gevorgian, E G; Bogdanov, O V; Medvedeva, M V

    1977-01-01

    Morphological and functional maturation of different structures of the striatal complex takes place heterochronously, as revealed in studies on motor activity of 17--21-day chick embryos evoked by electrical stimulation of these structures. Phylogenetically more ancient structures, i. e. archi- and paleostriatum, are the first to be involved into regulation of the motor activity. These structures together with the structures of the midbrain and cerebellum are considered as "the primary" step of regulatory mechanisms which develop during functional maturation of the motor analyser. Neostriatal mechanisms operate from the 19th day of incubation, whereas hyperstriatal ones--only to the day of hatching.

  18. 141 BOVINE EMBRYO DEVELOPMENT RATES ARE AFFECTED WHEN OOCYTES ARE MATURED IN DIFFERENT VIALS CONTAINING HEPES/BICARBONATE BUFFERED MEDIUM.

    PubMed

    Hashem, N; Secher, J O; Pryor, J H; Long, C R; Looney, C R; Avery, B; Hyttel, P; Stroebech, L

    2016-01-01

    Laboratory ware for the in vitro-produced embryos is generally made from embryo-tested plastic instead of glass. The quality of the plastic is crucial for the outcome because plastic is often toxic to gametes (Nijs et al. 2009 Fertil. Steril. 92, 527-535). In addition, gas molecules permeate through the plastic at a rate that depends on a variety of factors, such as diffusion coefficient and thickness of the plastic. In an incubator with appropriate concentration of CO2 and vented culture vessels, the gas permeability of the plastic is not important. When oocytes are transported outside a controlled atmosphere, gas permeability, toxicity, and oocyte cumulus cell CO2 metabolism could perturb the outcome. Medium containing bicarbonate buffer increases pH outside of a controlled atmosphere within minutes, whereas medium buffered with HEPES maintains suitable pH for hours. Previously, we tested that gas permeability differs among plastic vials and glass vials with no cumulus-oocyte complexes (COC) by measuring pH after 2, 5, and 24h at the same temperature. The objective of this study was to compare pH post-maturation, blastocyst development rates on Day 8 post-IVF (Day 0=IVF) between 2 different 1.2-mL polypropylene cryovials (A: VWR DK, 479-1219; B: Sigma-Aldrich, St. Louis, MO, USA, CLS430289), glass vial (VWR DK, NSCAC4015-96), and 4-well plate (4WP) as control (Thermo Fisher Scientific, Waltham, MA, USA, 144444). A total of 1135 abattoir-derived COC in Exp. 1 and 133 in Exp. 2 were divided equally between the treatments (20-25 COC per vessel). Vials/4WP contained 0.8/0.5mL of BO-IVM HEPES, a HEPES/bicarbonate medium (IVF Bioscience; BO-HEPES-IVM, UK). Maturation lasted 22 to 24h at 38.8°C in an incubator with either a humidified atmosphere of 5.5% CO2 in air (Exp. 1) or with no CO2 contact (Exp. 2). In Exp. 1, oocyte vials were matured without a vial lid while in Exp. 2 vial lids were closed. Statistical analysis was performed with chi-square and mean±SD. In Exp

  19. Effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas.

    PubMed

    Ruiz, Jaime; Paulo Santayana, R; José Mendoza, M; Leandra Landeo, J; Huamán, Elizabeth; Ticllacuri, Flamel; Fidel Mujica, L; Silva, Mauricio; Ratto, Marcelo H

    2017-06-01

    We evaluated the effect of in vitro maturation time, sperm selection and oxygen tension on alpaca embryo development. In Experiment I, Cumulus Oocyte- Complexes (COCs) were obtained from abattoir ovaries and in vitro matured in TCM-199 for 24 (n = 217), 28 (215), or 32 h (223) at 38.5 °C, high humidity and 5% CO2 in air. Oocytes from 24 (n = 392), 28 (n = 456) or 32 (n = 368) h groups were in vitro fertilized with epididymal sperm and cultured in SOFaa at 38.5 °C, high humidity and 5% CO2, 5% O2 and 90% N2 for 7 days. Embryo development was evaluated on Day 2, 5 and Day 7 of in vitro culture (Day 0 = in vitro fertilization). In Experiment II, a 2 by 2-factorial design was used to determine the effect of sperm selection (Swim-up vs Percoll) and oxygen tension (20% vs 5%) during embryo culture and their interaction on embryo development. COCs were in vitro matured for 32 h at 38.5 °C and 5% CO2 in air and then in vitro inseminated with epididymal sperm processed by swim-up or Percoll. Zygotes were cultured in SOFaa + cumulus cells at 38.5 °C under 20 or 5% of O2 tension and high humidity for 7 days. A total of 235, 235, 253 and 240 oocytes were assigned to: swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, groups respectively. The proportion of oocytes reaching MII stage was highest after 32 h of in vitro maturation (P < 0.05). Blastocyst rate (29.1 ± 2.7%) was also highest for COCs matured for 32 h (Exp I). In Experiment II, Blastocysts rate (26.03 ± 4.7; 27.7 ± 4.3; 29.7 ± 3.8 and 27.6 ± 4.2% for swim-up+20 O2, swim-up+5 O2 or Percoll+20 O2, Percoll+5 O2, respectively) was not affected by sperm selection method (P = 0.8), oxygen tension (P = 0.9) or their interaction (P = 0.5). Copyright © 2017 Elsevier Inc. All rights reserved.

  20. New Insights into the Complex Relationship between Weight and Maturity of Burgundy Truffles (Tuber aestivum)

    PubMed Central

    Büntgen, Ulf; Bagi, István; Fekete, Oszkár; Molinier, Virginie; Peter, Martina; Splivallo, Richard; Vahdatzadeh, Maryam; Richard, Franck; Murat, Claude; Tegel, Willy; Stobbe, Ulrich; Martínez-Peña, Fernando; Sproll, Ludger; Hülsmann, Lisa; Nievergelt, Daniel; Meier, Barbara; Egli, Simon

    2017-01-01

    Despite an increasing demand for Burgundy truffles (Tuber aestivum), gaps remain in our understanding of the fungus’ overall lifecycle and ecology. Here, we compile evidence from three independent surveys in Hungary and Switzerland. First, we measured the weight and maturity of 2,656 T. aestivum fruit bodies from a three-day harvest in August 2014 in a highly productive orchard in Hungary. All specimens ranging between 2 and 755 g were almost evenly distributed through five maturation classes. Then, we measured the weight and maturity of another 4,795 T. aestivum fruit bodies harvested on four occasions between June and October 2015 in the same truffière. Again, different maturation stages occurred at varying fruit body size and during the entire fruiting season. Finally, the predominantly unrelated weight and maturity of 81 T. aestivum fruit bodies from four fruiting seasons between 2010 and 2013 in Switzerland confirmed the Hungarian results. The spatiotemporal coexistence of 7,532 small-ripe and large-unripe T. aestivum, which accumulate to ~182 kg, differs from species-specific associations between the size and ripeness that have been reported for other mushrooms. Although size-independent truffle maturation stages may possibly relate to the perpetual belowground environment, the role of mycelial connectivity, soil property, microclimatology, as well as other abiotic factors and a combination thereof, is still unclear. Despite its massive sample size and proof of concept, this study, together with existing literature, suggests consideration of a wider ecological and biogeographical range, as well as the complex symbiotic fungus-host interaction, to further illuminate the hidden development of belowground truffle fruit bodies. PMID:28125633

  1. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes

    PubMed Central

    1996-01-01

    We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal- associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step. PMID:8601581

  2. Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

    PubMed

    Canosa, S; Adriaenssens, T; Coucke, W; Dalmasso, P; Revelli, A; Benedetto, C; Smitz, J

    2017-05-01

    Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in

  3. The LGI1-ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function.

    PubMed

    Lovero, Kathryn L; Fukata, Yuko; Granger, Adam J; Fukata, Masaki; Nicoll, Roger A

    2015-07-28

    Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

  4. From metamorphosis to maturity in complex life cycles: equal performance of different juvenile life history pathways.

    PubMed

    Schmidt, Benedikt R; Hödl, Walter; Schaub, Michael

    2012-03-01

    Performance in one stage of a complex life cycle may affect performance in the subsequent stage. Animals that start a new stage at a smaller size than conspecifics may either always remain smaller or they may be able to "catch up" through plasticity, usually elevated growth rates. We study how size at and date of metamorphosis affected subsequent performance in the terrestrial juvenile stage and lifetime fitness of spadefoot toads (Pelobates fuscus). We analyzed capture-recapture data of > 3000 individuals sampled during nine years with mark-recapture models to estimate first-year juvenile survival probabilities and age-specific first-time breeding probabilities of toads, followed by model selection to assess whether these probabilities were correlated with size at and date of metamorphosis. Males attained maturity after two years, whereas females reached maturity 2-4 years after metamorphosis. Age at maturity was weakly correlated with metamorphic traits. In both sexes, first-year juvenile survival depended positively on date of metamorphosis and, in males, also negatively on size at metamorphosis. In males, toads that metamorphosed early at a small size had the highest probability to reach maturity. However, because very few toadlets metamorphosed early, the vast majority of male metamorphs had a very similar probability to reach maturity. A matrix projection model constructed for females showed that different juvenile life history pathways resulted in similar lifetime fitness. We found that the effects of date of and size at metamorphosis on different juvenile traits cancelled each other out such that toads that were small or large at metamorphosis had equal performance. Because the costs and benefits of juvenile life history pathways may also depend on population fluctuations, ample phenotypic variation in life history traits may be maintained.

  5. Scrapie Affects the Maturation Cycle and Immune Complex Trapping by Follicular Dendritic Cells in Mice

    PubMed Central

    McGovern, Gillian; Mabbott, Neil; Jeffrey, Martin

    2009-01-01

    Transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurological disorders of man and animals, characterised by abnormal disease-associated prion protein (PrPd) accumulations in the brain and lymphoreticular system (LRS). Prior to neuroinvasion, TSE agents often accumulate to high levels within the LRS, apparently without affecting immune function. However, our analysis of scrapie-affected sheep shows that PrPd accumulations within the LRS are associated with morphological changes to follicular dendritic cells (FDCs) and tingible body macrophages (TBMs). Here we examined FDCs and TBMs in the mesenteric lymph nodes (MLNs) of scrapie-affected mice by light and electron microscopy. In MLNs from uninfected mice, FDCs could be morphologically categorised into immature, mature and regressing forms. However, in scrapie-affected MLNs this maturation cycle was adversely affected. FDCs characteristically trap and retain immune complexes on their surfaces, which they display to B-lymphocytes. In scrapie-affected MLNs, some FDCs were found where areas of normal and abnormal immune complex retention occurred side by side. The latter co-localised with PrPd plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrPd accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrPd. Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrPd accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function. PMID:19997557

  6. Longitudinal sleep EEG trajectories indicate complex patterns of adolescent brain maturation.

    PubMed

    Feinberg, Irwin; Campbell, Ian G

    2013-02-15

    New longitudinal sleep data spanning ages 6-10 yr are presented and combined with previous data to analyze maturational trajectories of delta and theta EEG across ages 6-18 yr in non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. NREM delta power (DP) increased from age 6 to age 8 yr and then declined. Its highest rate of decline occurred between ages 12 and 16.5 yr. We attribute the delta EEG trajectories to changes in synaptic density. Whatever their neuronal underpinnings, these age curves can guide research into the molecular-genetic mechanisms that underlie adolescent brain development. The DP trajectories in NREM and REM sleep differed strikingly. DP in REM did not initially increase but declined steadily from age 6 to age 16 yr. We hypothesize that the DP decline in REM reflects maturation of the same brain arousal systems that eliminate delta waves in waking EEG. Whereas the DP age curves differed in NREM and REM sleep, theta age curves were similar in both, roughly paralleling the age trajectory of REM DP. The different maturational curves for NREM delta and theta indicate that they serve different brain functions despite having similar within-sleep dynamics and responses to sleep loss. Period-amplitude analysis of NREM and REM delta waveforms revealed that the age trends in DP were driven more by changes in wave amplitude rather than incidence. These data further document the powerful and complex link between sleep and brain maturation. Understanding this relationship would shed light on both brain development and the function of sleep.

  7. ASSEMBLY, MATURATION, AND TRAFFICKING OF THE γ-SECRETASE COMPLEX IN ALZHEIMER’S DISEASE

    PubMed Central

    Dries, Daniel R.; Yu, Gang

    2008-01-01

    In this review, we discuss the biology of γ-secretase, an enigmatic enzyme complex that is responsible for the generation of the amyloid-β peptide that constitutes the amyloid plaques of Alzheimer’s disease. We begin with a brief review on the processing of the amyloid precursor protein and a brief discussion on the family of enzymes involved in regulated intramembrane proteolysis, of which γ-secretase is a member. We then identify the four major components of the γ-secretase complex – presenilin, nicastrin, Aph-1, and Pen-2 – with a focus on the identification of each and the role that each plays in the maturation and activity of the complex. We also discuss two new proteins that may play a role in modulating the assembly and activity of the γ-secretase complex. Next, we summarize the known subcellular locations of each γ-secretase component and the sites of γ-secretase activity, as defined by the production of Aβ. Finally, we close by synthesizing all of the included topics into an overarching model for the assembly and trafficking of the γ-secretase complex, which serves as a launching point for further questions into the biology and function of γ-secretase in Alzheimer’s disease. PMID:18393798

  8. Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics1[OPEN

    PubMed Central

    2017-01-01

    The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes. PMID:28400496

  9. Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus

    PubMed Central

    Yau, Suk-Yu; Li, Ang; Tong, Jian-Bin; Bostrom, Crystal; Christie, Brian R.; Lee, Tatia M.C.; So, Kwok-Fai

    2016-01-01

    Background: Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. Objective: We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. Methods: We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. Results: Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. Conclusion: These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons. PMID:27567758

  10. Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus.

    PubMed

    Yau, Suk-Yu; Li, Ang; Tong, Jian-Bin; Bostrom, Crystal; Christie, Brian R; Lee, Tatia M C; So, Kwok-Fai

    2016-09-21

    Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons.

  11. NMDA receptors are selectively partitioned into complexes and supercomplexes during synapse maturation

    PubMed Central

    Frank, René A. W.; Komiyama, Noboru H.; Ryan, Tomás J.; Zhu, Fei; O'Dell, Thomas J.; Grant, Seth G. N.

    2016-01-01

    How neuronal proteomes self-organize is poorly understood because of their inherent molecular and cellular complexity. Here, focusing on mammalian synapses we use blue-native PAGE and ‘gene-tagging' of GluN1 to report the first biochemical purification of endogenous NMDA receptors (NMDARs) directly from adult mouse brain. We show that NMDARs partition between two discrete populations of receptor complexes and ∼1.5 MDa supercomplexes. We tested the assembly mechanism with six mouse mutants, which indicates a tripartite requirement of GluN2B, PSD93 and PSD95 gate the incorporation of receptors into ∼1.5 MDa supercomplexes, independent of either canonical PDZ-ligands or GluN2A. Supporting the essential role of GluN2B, quantitative gene-tagging revealed a fourfold molar excess of GluN2B over GluN2A in adult forebrain. NMDAR supercomplexes are assembled late in postnatal development and triggered by synapse maturation involving epigenetic and activity-dependent mechanisms. Finally, screening the quaternary organization of 60 native proteins identified numerous discrete supercomplexes that populate the mammalian synapse. PMID:27117477

  12. Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics.

    PubMed

    Merton, J S; Knijn, H M; Flapper, H; Dotinga, F; Roelen, B A J; Vos, P L A M; Mullaart, E

    2013-09-01

    Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13

  13. The effects of elevated body temperature on the complexity of the diaphragm EMG signals during maturation.

    PubMed

    Akkurt, David; Akay, Yasemin M; Akay, Metin

    2009-04-01

    significant. These results furthermore suggest that during maturation, the output of the central pattern generator becomes less complex probably because the elevated body temperature reduces the neural activity and alters the behavior of the central respiratory controller, making it more susceptible to sudden infant death syndrome (SIDS).

  14. Patterns of (3H) thymidine incorporation differ in immature rats and mature, cycling rats

    SciTech Connect

    Hirshfield, A.N.

    1986-02-01

    By the time follicular development has progressed to the preovulatory stage, granulosa cells abutting the basement membrane no longer incorporate (3H) thymidine (3H-TdR). The purpose of this experiment was to determine when, during the course of follicular growth, cell proliferation in these mural granulosa cells ceases. Autoradiographs were prepared following continuous 3H-TdR infusion in vivo, or incubation with 3H-TdR in vitro. In cycling rats, the concentration of silver grains over mural regions of the granulosa layer was lower than over antral regions of most follicles with greater than 1000 cells in the largest cross section (LCS). This centripetal labeling pattern became more striking as follicular size increased. By proestrus, only the cells of the discus proligerus (cumulus and the portion of the follicular wall supporting the cumulus oocyte complex) continued to incorporate 3H-TdR. In contrast to cycling rats, centripetal labeling patterns were not seen in ovaries of prepubertal rats, even in follicles of the same size. The difference in follicular growth patterns between these two types of animals suggests an influence of cyclic gonadotropin surges on the control of granulosa cell proliferation.

  15. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation.

    PubMed

    Kim, Sungsu; Wairkar, Yogesh P; Daniels, Richard W; DiAntonio, Aaron

    2010-03-08

    Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.

  16. Brain maturation is delayed in infants with complex congenital heart defects

    PubMed Central

    Licht, Daniel J.; Shera, David M.; Clancy, Robert R.; Wernovsky, Gil; Montenegro, Lisa M.; Nicolson, Susan C.; Zimmerman, Robert A.; Spray, Thomas L.; Gaynor, J. William; Vossough, Arastoo

    2009-01-01

    Objective Small head circumferences and white matter injury in the form of periventricular leukomalacia have been observed in populations of infants with severe forms of congenital heart defects. This study tests the hypothesis that congenital heart defects delay in utero structural brain development. Methods Full-term infants with hypoplastic left heart syndrome or transposition of the great arteries were prospectively evaluated with preoperative brain magnetic resonance imaging. Patients with independent risk factors for abnormal brain development (shock, end-organ injury, or intrauterine growth retardation) were excluded. Outcome measures included head circumferences and the total maturation score on magnetic resonance imaging. Total maturation score is a previously validated semiquantitative anatomic scoring system used to assess whole brain maturity. The total maturation score evaluates 4 parameters of maturity: (1) myelination, (2) cortical infolding, (3) involution of glial cell migration bands, and (4) presence of germinal matrix tissue. Results The study cohort included 29 neonates with hypoplastic left heart syndrome and 13 neonates with transposition of the great arteries at a mean gestational age of 38.9 ± 1.1 weeks. Mean head circumference was 1 standard deviation below normal. The mean total maturation score for the cohort was 10.15 ± 0.94, significantly lower than reported normative data in infants without congenital heart defects, corresponding to a delay of 1 month in structural brain development. Conclusion Before surgery, term infants with hypoplastic left heart syndrome and transposition of the great arteries have brains that are smaller and structurally less mature than expected. This delay in brain development may foster susceptibility to periventricular leukomalacia in the preoperative, intraoperative, and postoperative periods. PMID:19258059

  17. The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

    SciTech Connect

    Li, Long; Lok, Shee-Mei; Yu, I-Mei; Zhang, Ying; Kuhn, Richard J.; Chen, Jue; Rossmann, Michael G.

    2008-09-17

    Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

  18. The Arp2/3 Complex Is Essential for Distinct Stages of Spine Synapse Maturation, Including Synapse Unsilencing

    PubMed Central

    Spence, Erin F.; Kanak, Daniel J.; Carlson, Benjamin R.

    2016-01-01

    Dendritic filopodia are actin-rich structures that are thought to contribute to early spine synapse formation; however, the actin regulatory proteins important for early synaptogenesis are poorly defined. Using organotypic hippocampal slice cultures and primary neuron hippocampal cultures from Arp2/3 conditional knock-out mice, we analyze the roles of the Arp2/3 complex, an actin regulator that creates branched actin networks, and demonstrate it is essential for distinct stages of both structural and functional maturation of excitatory spine synapses. Our data show that initially the Arp2/3 complex inhibits the formation of dendritic filopodia but that later during development, the Arp2/3 complex drives the morphological maturation from filopodia to typical spine morphology. Furthermore, we demonstrate that although the Arp2/3 complex is not required for key spine maturation steps, such as presynaptic contact and recruitment of MAGUK (membrane-associated guanylate kinase) scaffolding proteins or NMDA receptors, it is necessary for the recruitment of AMPA receptors. This latter process, also known as synapse unsilencing, is a final and essential step in the neurodevelopment of excitatory postsynaptic synaptogenesis, setting the stage for neuronal interconnectivity. These findings provide the first evidence that the Arp2/3 complex is directly involved in functional maturation of dendritic spines during the developmental period of spinogenesis. SIGNIFICANCE STATEMENT Excitatory spine synapse formation (spinogenesis) is a poorly understood yet pivotal period of neurodevelopment that occurs within 2–3 weeks after birth. Neurodevelopmental disorders such as intellectual disability and autism are characterized by abnormal spine structure, which may arise from abnormal excitatory synaptogenesis. The initial stage of spinogenesis is thought to begin with the emergence of actin-rich dendritic filopodia that initiate contact with presynaptic axonal boutons. However, it

  19. The Arp2/3 Complex Is Essential for Distinct Stages of Spine Synapse Maturation, Including Synapse Unsilencing.

    PubMed

    Spence, Erin F; Kanak, Daniel J; Carlson, Benjamin R; Soderling, Scott H

    2016-09-14

    Dendritic filopodia are actin-rich structures that are thought to contribute to early spine synapse formation; however, the actin regulatory proteins important for early synaptogenesis are poorly defined. Using organotypic hippocampal slice cultures and primary neuron hippocampal cultures from Arp2/3 conditional knock-out mice, we analyze the roles of the Arp2/3 complex, an actin regulator that creates branched actin networks, and demonstrate it is essential for distinct stages of both structural and functional maturation of excitatory spine synapses. Our data show that initially the Arp2/3 complex inhibits the formation of dendritic filopodia but that later during development, the Arp2/3 complex drives the morphological maturation from filopodia to typical spine morphology. Furthermore, we demonstrate that although the Arp2/3 complex is not required for key spine maturation steps, such as presynaptic contact and recruitment of MAGUK (membrane-associated guanylate kinase) scaffolding proteins or NMDA receptors, it is necessary for the recruitment of AMPA receptors. This latter process, also known as synapse unsilencing, is a final and essential step in the neurodevelopment of excitatory postsynaptic synaptogenesis, setting the stage for neuronal interconnectivity. These findings provide the first evidence that the Arp2/3 complex is directly involved in functional maturation of dendritic spines during the developmental period of spinogenesis. Excitatory spine synapse formation (spinogenesis) is a poorly understood yet pivotal period of neurodevelopment that occurs within 2-3 weeks after birth. Neurodevelopmental disorders such as intellectual disability and autism are characterized by abnormal spine structure, which may arise from abnormal excitatory synaptogenesis. The initial stage of spinogenesis is thought to begin with the emergence of actin-rich dendritic filopodia that initiate contact with presynaptic axonal boutons. However, it remains enigmatic how actin

  20. Bovine non-competent oocytes (BCB-) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development.

    PubMed

    Salviano, M B; Collares, F J F; Becker, B S; Rodrigues, B A; Rodrigues, J L

    2016-04-01

    Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus-oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 - 60%) and embryonic development rates to the blastocyst stage (10/48 - 21%) as the results obtained with the BCB control group oocytes (45/77 - 58% and 08/45 - 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB- oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB- (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB-; and (4) BCB+ matured in same IVM medium drop as (5) BCB- at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage

  1. Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes

    SciTech Connect

    Takai, Hiroyuki; Xie, Yihu; de Lange, Titia; Pavletich, Nikola P.

    2010-09-20

    We reported previously that the stability of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs) depends on their interaction with Tel2, the ortholog of yeast Tel2 and Caenorhabditis elegans Clk-2. Here we provide evidence that Tel2 acts with Hsp90 in the maturation of PIKK complexes. Quantitative immunoblotting showed that the abundance of Tel2 is low compared with the PIKKs, and Tel2 preferentially bound newly synthesized ATM, ATR, mTOR, and DNA-PKcs. Tel2 complexes contained, in addition to Tti1-Tti2, the Hsp90 chaperone, and inhibition of Hsp90 interfered with the interaction of Tel2 with the PIKKs. Analysis of in vivo labeled nascent protein complexes showed that Tel2 and Hsp90 mediate the formation of the mTOR TORC1 and TORC2 complexes and the association of ATR with ATRIP. The structure of yeast Tel2, reported here, shows that Tel2 consists of HEAT-like helical repeats that assemble into two separate {alpha}-solenoids. Through mutagenesis, we identify a surface patch of conserved residues involved in binding to the Tti1-Tti2 complex in vitro. In vivo, mutation of this conserved patch affects cell growth, levels of PIKKs, and ATM/ATR-mediated checkpoint signaling, highlighting the importance of Tti1-Tti2 binding to the function of Tel2. Taken together, our data suggest that the Tel2-Tti1-Tti2 complex is a PIKK-specific cochaperone for Hsp90.

  2. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

    PubMed

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H; van Wijk, Klaas J

    2015-11-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

  3. Comparison of different fertilisation media for an in vitro maturation?fertilisation?culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production.

    PubMed

    Ferré, Luis B; Bogliotti, Yanina; Chitwood, James L; Fresno, Cristóbal; Ortega, Hugo H; Kjelland, Michael E; Ross, Pablo J

    2015-05-13

    High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P < 0.05) in cleavage rate (M199 = 57%, SOF = 71% and TALP = 72%), blastocyst formation (M199 = 9%, SOF = 20% and TALP = 19%), proportion of Grade 1 blastocysts (M199 = 15%, SOF = 52% and TALP = 51%), proportion of Grade 3 blastocysts (M199 = 58%, SOF = 21% and TALP = 20%) and hatching rates (M199 = 29%, SOF = 60% and TALP = 65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

  4. The role of canopy structural complexity in wood net primary production of a maturing northern deciduous forest.

    PubMed

    Hardiman, Brady S; Bohrer, Gil; Gough, Christopher M; Vogel, Christoph S; Curtisi, Peter S

    2011-09-01

    The even-aged northern hardwood forests of the Upper Great Lakes Region are undergoing an ecological transition during which structural and biotic complexity is increasing. Early-successional aspen (Populus spp.) and birch (Betula papyrifera) are senescing at an accelerating rate and are being replaced by middle-successional species including northern red oak (Quercus rubra), red maple (Acer rubrum), and white pine (Pinus strobus). Canopy structural complexity may increase due to forest age, canopy disturbances, and changing species diversity. More structurally complex canopies may enhance carbon (C) sequestration in old forests. We hypothesize that these biotic and structural alterations will result in increased structural complexity of the maturing canopy with implications for forest C uptake. At the University of Michigan Biological Station (UMBS), we combined a decade of observations of net primary productivity (NPP), leaf area index (LAI), site index, canopy tree-species diversity, and stand age with canopy structure measurements made with portable canopy lidar (PCL) in 30 forested plots. We then evaluated the relative impact of stand characteristics on productivity through succession using data collected over a nine-year period. We found that effects of canopy structural complexity on wood NPP (NPPw) were similar in magnitude to the effects of total leaf area and site quality. Furthermore, our results suggest that the effect of stand age on NPPw is mediated primarily through its effect on canopy structural complexity. Stand-level diversity of canopy-tree species was not significantly related to either canopy structure or NPPw. We conclude that increasing canopy structural complexity provides a mechanism for the potential maintenance of productivity in aging forests.

  5. Effects of in-vitro or in-vivo matured ooplasm and spindle-chromosome complex on the development of spindle-transferred oocytes.

    PubMed

    Ding, Chenhui; Li, Tao; Zeng, Yanhong; Hong, Pingping; Xu, Yanwen; Zhou, Canquan

    2014-12-01

    To study the effects of in-vitro matured ooplasm and spindle-chromosome complex (SCC) on the development of spindle-transferred oocytes, reciprocal spindle transfer was conducted between in-vivo and in-vitro matured oocytes. The reconstructed oocytes were divided into four groups according to their different ooplasm sources and SCC, artificially activated and cultured to the blastocyst stage. Oocyte survival, activation and embryo development after spindle transfer manipulation were compared between groups. Survival, activation, and cleavage rates of reconstructed oocytes after spindle transfer manipulation did not differ significantly among the four groups. The eight-cell stage embryo formation rates on day 3 and the blastocyst formation rate on day 6 were not significantly different between the in-vitro and in-vivo matured SCC groups when they were transplanted into in-vivo matured ooplasm. The rate of eight-cell stage embryo formation with in-vitro matured ooplasm was significantly lower (P < 0.05) than that of embryos with in-vivo matured ooplasm, and none of the embryos developed to the blastocyst stage. Therefore, SCC matured in vitro effectively supported the in-vitro development of reconstructed oocytes. Ooplasm matured in vitro, however, could not support the development of reconstructed oocytes, and may not be an appropriate source of ooplasm donation for spindle transfer.

  6. Nicotine and elevated body temperature reduce the complexity of the genioglossus and diaphragm EMG signals in rats during early maturation

    NASA Astrophysics Data System (ADS)

    Akkurt, David; Akay, Yasemin M.; Akay, Metin

    2009-10-01

    In this paper, we examined the effect of nicotine exposure and increased body temperature on the complexity (dynamics) of the genioglossus muscle (EMGg) and the diaphragm muscle (EMGdia) to explore the effects of nicotine and hyperthermia. Nonlinear dynamical analysis of the EMGdia and EMGg signals was performed using the approximate entropy method on 15 (7 saline- and 8 nicotine-treated) juvenile rats (P25-P35) and 19 (11 saline- and 8 nicotine-treated) young adult rats (P36-P44). The mean complexity values were calculated over the ten consecutive breaths using the approximate entropy method during mild elevated body temperature (38 °C) and severe elevated body temperature (39-40 °C) in two groups. In the first (nicotine) group, rats were treated with single injections of nicotine enough to produce brain levels of nicotine similar to those achieved in human smokers (2.5 (mg kg-1)/day) until the recording day. In the second (control) group, rats were treated with injections of saline, beginning at postnatal 5 days until the recording day. Our results show that warming the rat by 2-3 °C and nicotine exposure significantly decreased the complexity of the EMGdia and EMGg for the juvenile age group. This reduction in the complexity of the EMGdia and EMGg for the nicotine group was much greater than the normal during elevated body temperatures. We speculate that the generalized depressive effects of nicotine exposure and elevated body temperature on the respiratory neural firing rate and the behavior of the central respiratory network could be responsible for the drastic decrease in the complexity of the EMGdia and EMGg signals, the outputs of the respiratory neural network during early maturation.

  7. Tukau Field: Finding new oil in matured and complex field after 20 years of production

    SciTech Connect

    Shariff, M.D.; Ridza, M.; Majid, P.

    1996-12-31

    The Tukau Field is located some 30 km offshore Sarawak, Malaysia. in water depth of about 160 ft. The field, discovered by TK-2 in 1966 found 235 ft net oil sand and 16 ft wet gas sand. After further seismic data acquisition and interpretation, six (6) appraisal wells were drilled from 1973 to 1975 before the field could be commercially developed. The Tukau structure is a structurally complex feature formed as a domal anticlinal uplift, located along the Tukau I Bakau / Baram trend. It is dissected at the shallow level by normal synthetic and antithetic faults. These fault system divide the field into seven (7) fault blocks. The major hydrocarbon accumulations are between 2400 ftss and 7500 ftss and the main prospective sequence consists of fine to very fine grained sand of the upper cycle V of late Miocene age and deposited in a deltaic, fluviomarine, coastal to near shore environment. Development drilling commenced in 1975 with a total of 23 wells. To date a total of nine (9) rounds of development activities were carried out resulting in 55 wells being drilled and nine (9) well jackets installed. In 1975, based on the seismic and well data. the field is estimated to contain some 300 MMSTB of oil. Following subsequent field reviews Incorporating some 50 odd well data and seismic reinterpretation in 1987. the field STOIIP increased to 500 MMST. 3D seismic was acquired in 1992 and field review carried out In 1995 resulted In some development potential and appraisal / exploration opportunities. The appraisal well drilled in October 1995, increased the field STOIIP by some 50 MMSTB. Preliminary evaluation based on geological, engineering and economic information indicated that Tukau field will be further developed with additional well jacket and this will boost the field production by about 50%.

  8. COMMUNICATION: The effects of elevated body temperature on the complexity of the diaphragm EMG signals during maturation

    NASA Astrophysics Data System (ADS)

    Akkurt, David; Akay, Yasemin M.; Akay, Metin

    2009-04-01

    suggest that during maturation, the output of the central pattern generator becomes less complex probably because the elevated body temperature reduces the neural activity and alters the behavior of the central respiratory controller, making it more susceptible to sudden infant death syndrome (SIDS).

  9. Restricted occurrence of Locusta migratoria ovary maturing parsin in the brain-corpora cardiaca complex of various insect species.

    PubMed

    Richard, O; Tamarelle, M; Geoffre, S; Girardie, J

    1994-09-01

    Ovary maturing parsin (OMP) is a gonadotrophic molecule previously isolated from the neurosecretory lobes of the corpora cardiaca of Locusta migratoria (acridian Orthoptera). A polyclonal antiserum directed against the two biologically active domains of the L. migratoria (Lom) OMP was used to investigate the occurrence of Lom OMP-like substances in brain-corpora cardiaca complexes of other insect species. Using immunohistochemistry, specimens of 40 different insect species belonging to 13 insect orders were tested. The Lom OMP-like substance was strictly limited to specimens of insect species belonging to the Acridae. It occurred in non-basophilic cells of the pars intercerebralis that project to the corpora cardiaca, as in Locusta. Although the antiserum only detected Lom OMP-like material in the Acridae, it is possible that related molecules exist in other insects. The antiserum may be very specific for domains of the Lom OMP molecule that have not been highly conserved during evolution or possibly these domains are not accessible to the antiserum in other insects.

  10. Virus maturation.

    PubMed

    Delgui, Laura R; Rodríguez, José F

    2013-01-01

    The formation of infectious virus particles is a highly complex process involving a series of sophisticated molecular events. In most cases, the assembly of virus structural elements results in the formation of immature virus particles unable to initiate a productive infection. Accordingly, for most viruses the final stage of the assembly pathway entails a set of structural transitions and/or biochemical modifications that transform inert precursor particles into fully infectious agents. In this chapter, we review the most relevant maturation mechanisms involved in the generation of infectious virions for a wide variety of viruses.

  11. Vaccinia Mature Virus Fusion Regulator A26 Protein Binds to A16 and G9 Proteins of the Viral Entry Fusion Complex and Dissociates from Mature Virions at Low pH

    PubMed Central

    Chang, Shu-Jung; Shih, Ao-Chun; Tang, Yin-Liang

    2012-01-01

    Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422–8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive. PMID:22278246

  12. Vaccinia mature virus fusion regulator A26 protein binds to A16 and G9 proteins of the viral entry fusion complex and dissociates from mature virions at low pH.

    PubMed

    Chang, Shu-Jung; Shih, Ao-Chun; Tang, Yin-Liang; Chang, Wen

    2012-04-01

    Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.

  13. Should what we know about neurobehavioral development, complex congenital heart disease, and brain maturation affect the timing of corrective cardiac surgery?

    PubMed

    DiNardo, James A

    2011-07-01

    Despite remarkable improvements in perioperative care, adverse neurobehavioral outcomes following neonatal and infant cardiac surgery are commonplace and are associated with substantial morbidity. It is becoming increasingly clear that complex congenital heart disease is associated with both abnormalities in neuroanatomic development and a delay in fetal brain maturation. Substantial cerebral ischemic/hypoxic injury has been detected in neonates with complex congenital heart disease both prior to and following corrective cardiac surgery. The brain of the neonate with complex congenital heart disease appears to be uniquely vulnerable to the types of ischemic/hypoxic injury associated with perioperative care. It remains to be determined whether delaying surgical correction to allow for brain maturation will be associated with improvements in neurobehavioral outcomes.

  14. An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

    PubMed Central

    Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

    2017-01-01

    The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

  15. Förster Resonance Energy Transfer (FRET) as a Tool for Dissecting the Molecular Mechanisms for Maturation of the Shigella Type III Secretion Needle Tip Complex

    PubMed Central

    Dickenson, Nicholas E.; Picking, William D.

    2012-01-01

    Förster resonance energy transfer (FRET) provides a powerful tool for monitoring intermolecular interactions and a sensitive technique for studying Å-level protein conformational changes. One system that has particularly benefited from the sensitivity and diversity of FRET measurements is the maturation of the Shigella type III secretion apparatus (T3SA) needle tip complex. The Shigella T3SA delivers effector proteins into intestinal cells to promote bacterial invasion and spread. The T3SA is comprised of a basal body that spans the bacterial envelope and a needle with an exposed tip complex that matures in response to environmental stimuli. FRET measurements demonstrated bile salt binding by the nascent needle tip protein IpaD and also mapped resulting structural changes which led to the recruitment of the translocator IpaB. At the needle tip IpaB acts as a sensor for host cell contact but prior to secretion, it is stored as a heterodimeric complex with the chaperone IpgC. FRET analyses showed that chaperone binding to IpaB’s N-terminal domain causes a conformational change in the latter. These FRET analyses, with other biophysical methods, have been central to understanding T3SA maturation and will be highlighted, focusing on the details of the FRET measurements and the relevance to this particular system. PMID:23203116

  16. An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

    PubMed

    Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P

    2017-06-01

    The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. © 2017 Kaur et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. Attempts at in vitro fertilization and culture of in vitro matured oocytes in sei ( Balaenoptera borealis) and Bryde's ( B. edeni) whales.

    PubMed

    Bhuiyan, M M U; Suzuki, Y; Watanabe, H; Matsuoka, K; Fujise, Y; Ishikawa, H; Ohsumi, S; Fukui, Y

    2009-02-01

    The cumulus-oocyte-complexes (COCs) recovery rates with respect to reproductive status per sei (Balaenoptera borealis) and Bryde's (B. edeni) whales were determined in Experiment 1. The number of COCs recovered ranged from 16.0 to 30.6 and from 6.7 to 26.8 per sei and Bryde's whales, respectively. The effects of COCs grades and protein supplementation in embryo culture medium on development of in vitro fertilized (IVF) embryos were evaluated in sei and Bryde's whales in Experiment 2. The COCs were classified into either Grade A (COCs with five or more layers of compact cumulus cells) or Grade B (COCs with less than five layers of compact or expanded cumulus cells) before being cultured for IVM. The cleavage (12.0 to 19.5%), 4-cell (8.0 to 12.0%) and 8-cell (4.0 to 8.0%) formation rates in sei whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either fetal whale serum (FWS)- or bovine serum albumin (BSA)-supplemented medium. The cleavage (4.0 to 14.8%), 4-cell (0.0 to 7.5%) and 8-cell (0.0 to 2.6%) formation rates in Bryde's whales did not vary significantly between embryos derived from either grade A or B oocytes and between embryos cultured in either FWS- or BSA-supplemented medium. The grade B oocytes cultured in FWS-supplemented medium developed to morula stage (1.1%) in sei whales. In conclusion, the present study indicates that IVF in sei whales is possible to achieve cleaved embryos developing to morula stage. This is the first in vitro embryo production attempt in sei and Bryde's whales.

  18. Ovarian tissue cryopreservation in female-to-male transgender people: insights into ovarian histology and physiology after prolonged androgen treatment.

    PubMed

    De Roo, Chloë; Lierman, Sylvie; Tilleman, Kelly; Peynshaert, Karen; Braeckmans, Kevin; Caanen, Mirte; Lambalk, Cornelius B; Weyers, Steven; T'Sjoen, Guy; Cornelissen, Ria; De Sutter, Petra

    2017-03-21

    Female-to-male transgender people (trans men) are faced with the risk of losing their reproductive potential owing to gender-affirming hormone treatment and genital reconstructive surgery. This observational, prospective cohort study investigates the effect of prolonged androgen therapy on their ovarian histology and fertility preservation perspectives. Hormone serum levels, ovarian histology and cumulus-oocyte complexes (COC) of 40 transsexual men were analysed at the moment of hysterectomy with bilateral oophorectomy in the context of genital reconstructive surgery after testosterone treatment (58.18 ± 26.57 weeks). In the cortex, most follicles were primordial (68.52% total follicle count) compared with 20.26% intermediate and 10.74%primary follicles. Few secondary follicles (0.46%) and a single antral follicle were found in the sections analysed. In total, 1313 COC were retrieved from the medulla of 35 patients (37.51 ± 33.58 COC per patient). Anti-Müllerian hormone serum levels were significantly correlated with number of COC (Rs 0.787, P < 0.001). After 48 h in-vitro maturation, 34.30% metaphase II oocytes were obtained, with 87.10% having a normal spindle structure. In conclusion, the cortical follicle distribution in trans men, after more than a year of testosterone treatment, seems to be surprisingly normal. This work confirms the presence and in-vitro maturation potential of cumulus-oocyte complexes.

  19. Transcription factor complex formation and chromatin fine structure alterations at the murine c-fms (CSF-1 receptor) locus during maturation of myeloid precursor cells

    PubMed Central

    Tagoh, Hiromi; Himes, Roy; Clarke, Deborah; Leenen, Pieter J.M.; Riggs, Arthur D.; Hume, David; Bonifer, Constanze

    2002-01-01

    Expression of the gene for the macrophage colony stimulating factor receptor (CSF-1R), c-fms, has been viewed as a hallmark of the commitment of multipotent precursor cells to macrophages. Lineage-restricted expression of the gene is controlled by conserved elements in the proximal promoter and within the first intron. To investigate the developmental regulation of c-fms at the level of chromatin structure, we developed an in vitro system to examine the maturation of multipotent myeloid precursor cells into mature macrophages. The dynamics of chromatin fine structure alterations and transcription factor occupancy at the c-fms promoter and intronic enhancer was examined by in vivo DMS and UV-footprinting. We show that the c-fms gene is already transcribed at low levels in early myeloid precursors on which no CSF-1R surface expression can be detected. At this stage of myelopoiesis, the formation of transcription factor complexes on the promoter was complete. By contrast, occupancy of the enhancer was acutely regulated during macrophage differentiation. Our data show that cell-intrinsic differentiation decisions at the c-fms locus precede the appearance of c-fms on the cell surface. They also suggest that complex lineage-specific enhancers such as the c-fms intronic enhancer regulate local chromatin structure through the coordinated assembly and disassembly of distinct transcription factor complexes. PMID:12101129

  20. A protective role of cumulus cells after short-term exposure of rat cumulus cell-oocyte complexes to lifestyle or environmental contaminants.

    PubMed

    Campen, Kelly A; McNatty, Kenneth P; Pitman, Janet L

    2017-04-01

    Ovarian follicular fluid provides a potential reservoir for exogenous compounds that may adversely affect oocyte quality. This study examined the effects of common lifestyle and environmental contaminants, namely bisphenol-A (BPA), caffeine, 3,4-methylenedioxymethamphetamine (MDMA), nicotine and Δ(9)-tetrahydrocannabinol (THC) on gap junction genes (Gja1, Gja4) and proteins (GJA1), glucose metabolism genes (Gfpt1, Pfkp) and oocyte growth factor genes (Bmp15, Gdf9), as well as gap junction transfer rate, in rat cumulus-oocyte complexes (COCs). In vitro exposure to MDMA and THC accelerated the timing of meiotic resumption and all contaminants altered either gap junction gene expression (BPA, caffeine, MDMA and THC) or transfer rate (BPA and nicotine). In vitro exposure of COCs to MDMA also altered glucose metabolism genes. Overall, oocyte-derived genes were largely unaffected following exposure to any contaminant. In summary, the impact of short-term exposure to lifestyle and environmental contaminants on oocyte function may be diminished due to protective properties of cumulus cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples.

    PubMed

    Bienvenut, Willy Vincent; Scarpelli, Jean-Pierre; Dumestier, Johan; Meinnel, Thierry; Giglione, Carmela

    2017-03-20

    Characterization of mature protein N-termini by large scale proteomics is challenging. This is especially true for proteins undergoing cleavage of transit peptides when they are targeted to specific organelles, such as mitochondria or chloroplast. Protein neo-N-termini can be located up to 100-150 amino acids downstream from the initiator methionine and are not easily predictable. Although some bioinformatics tools are available, they usually require extensive manual validation to identify the exact N-terminal position. The situation becomes even more complex when post-translational modifications take place at the neo-N-terminus. Although N-terminal acetylation occurs mostly in the cytosol, it is also observed in some organelles such as chloroplast. To date, no bioinformatics tool is available to define mature protein starting positions, the associated N-terminus acetylation status and/or yield for each proteoform. In this context, we have developed the EnCOUNTer tool (i) to score all characterized peptides using discriminating parameters to identify bona fide mature protein N-termini and (ii) to determine the N-terminus acetylation yield of the most reliable ones. Based on large scale proteomics analyses using the SILProNAQ methodology, tandem mass spectrometry favoured the characterization of thousands of peptides. Data processing using the EnCOUNTer tool provided an efficient and rapid way to extract the most reliable mature protein N-termini. Selected peptides were subjected to N-terminus acetylation yield determination. In an A. thaliana cell lysate, 1232 distinct proteotypic N-termini were characterized of which 648 were located at the predicted protein N-terminus (position 1/2) and 584 were located further downstream (starting at position > 2). A large number of these N-termini were associated with various well-defined maturation processes occurring on organelle-targeted proteins (mitochondria, chloroplast and peroxisome), secreted proteins or membrane

  2. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability1[OPEN

    PubMed Central

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H.; van Wijk, Klaas J.

    2015-01-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. PMID:26371235

  3. Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

    PubMed

    Chen, Wei; Bao, Yige; Chen, Xuerong; Burton, Jeremy; Gong, Xueli; Gu, Dongqing; Mi, Youjun; Bao, Lang

    2016-04-01

    Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance.

  4. Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica).

    PubMed

    Crichton, Elizabeth G; Bedows, Elliott; Miller-Lindholm, Amanda K; Baldwin, David M; Armstrong, Douglas L; Graham, Laura H; Ford, J Joe; Gjorret, Jakob O; Hyttel, Poul; Pope, C Earle; Vajta, Gabor; Loskutoff, Naida M

    2003-01-01

    A comparison of the amino acid sequences demonstrated that Siberian tiger gonadotropins are more homologous with those of porcine than any other commercially available preparation. The present study measured the efficacy of repeated ovarian stimulation with purified porcine gonadotropins on the follicular, hormonal, and immunogenic responses in Siberian tigers as well as on the ability of oocytes retrieved by laparoscopic follicular aspiration to fertilize and cleave in vitro. Controlled rate and vitrification cryopreservation methods were also compared for their ability to support ongoing cleavage following thawing of presumptive 2- to 4-cell tiger embryos generated in vitro. Vitrification supported continued embryonic cleavage in vitro while controlled rate freezing did not. Stereological microscopy indicated an excellent ovarian response with the recovery of quality cumulus-oocyte complexes that apparently fertilized and cleaved in vitro. However, ultrastructural and physiological examination revealed abnormal and unnatural responses such as the failure of some cumulus-oocyte complexes to reach maturity and progestagen levels to approach normalcy. At the same time, analyses of blood for antibodies failed to detect an immune reaction to these foreign gonadotropins in an assay that tested positive for the chorionic gonadotropin-stimulated domestic cat. Together, these observations suggest that porcine gonadotropins may be effective for the ovarian stimulation of tigers but that some modifications to administration protocols are needed to produce a more natural response.

  5. Cortical maturation of long latency auditory evoked potentials in hearing children: the complex P1-N1-P2-N2.

    PubMed

    Silva, Liliane Aparecida Fagundes; Magliaro, Fernanda Cristina Leite; Carvalho, Ana Claudia Martinho de; Matas, Carla Gentile

    2017-09-04

    The purpose of this study was to monitor the emergence and changes to the components of the Long Latency Auditory Evoked Potentials (LLAEP) in normal hearing children. This longitudinal study included children of both genders: seven aged between 10 and 35 months, and eight children between 37 and 63 months. The electrophysiological hearing evaluation consisted of analysis of LLAEP obtained in a sound field generated with loudspeakers positioned at an azimuth of 90°, through which the syllable /ba/ was played at an intensity of 70 dB HL. Each child underwent an initial evaluation followed by two re-evaluations three and nine months later. The emergence of LLAEP components across the nine-month follow-up period was observed. P1 and N2 were the most common components in children of this age range. There was no statistically significant difference regarding the occurrence of P1, N1, P2, and N2 components amongst younger and older children. Regarding latency values, the greatest changes overtime were observed in the P1 component for younger children and in the N2 component for older children. Only the P1 component significantly differed between the groups, with the highest latency values observed in younger children. LLAEP maturation occurs gradually and the emergence of complex components appears to be related more to the maturation of the central auditory nervous system than to chronological age.

  6. The Effects of Strategic Communication Principles on C2 Agility, Complexity/Uncertainty, and C2 Maturity

    DTIC Science & Technology

    2014-06-01

    TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Turkish Army Staff College,Harp Akademisi Lojmanlari,4.Levent...the complexity of entity--individual, system , organization, or collectives (Alberts, 58). The capability of entity is also another factor that...communication systems , to include the characteristics of various media channels and the intentions, capabilities and efforts of other influencers within and

  7. Single-event multilevel acute total correction of complex equinocavovarus deformity in skeletally mature patients with spastic cerebral palsy hemiparesis.

    PubMed

    Bishay, Sherif N G

    2013-01-01

    Complex multiplanar ankle/foot deformity as equinocavovarus is a common problem in patients with spastic cerebral palsy hemiparesis. The data from 30 consecutive patients (30 feet), treated between March 2009 and March 2010, with equinocavovarus and toe clawing secondary to spastic cerebral palsy hemiparesis, aged 16 to 18 years, were analyzed clinically and radiographically. All the patients had received conservative physiotherapy treatment and ankle/foot orthoses before undergoing combined soft tissue and bony surgical procedures performed in a single session to correct the complex toe clawing, cavus, varus, and equinus deformities. Preoperative measurements of certain foot angles were compared with their corresponding postoperative values. A grading system for evaluation of the results using a point scoring system was used to accurately evaluate both the clinical and the radiographic results after an average follow-up period of 2.5 years. Of the 30 patients (30 feet), 18 (60%) had excellent, 9 (30%) good, 3 (10%) fair, and 0 had poor outcomes. Neither vascular problems nor nonunion occurred. Significant improvement was seen postoperatively (p < .0333). Neither staged surgical procedures nor gradual distraction techniques using external fixators are ideal modalities to correct complex ankle/foot equinocavovarus deformity in patients with spastic cerebral palsy. Single-event, multilevel surgery with complete soft tissue and bony correction appears to be the treatment of choice in such cases. It shortens the treatment period and avoids patient dissatisfaction associated with multiple procedures, without major complications.

  8. Dynamic rupture propagation on geometrically complex fault with along-strike variation of fault maturity: insights from the 2014 Northern Nagano earthquake

    NASA Astrophysics Data System (ADS)

    Ando, Ryosuke; Imanishi, Kazutoshi; Panayotopoulos, Yannis; Kobayashi, Tomokazu

    2017-09-01

    Understanding the effect of the complex fault geometry on the dynamic rupture process and discriminating it from the complexity originating from the rheological properties of faults, is an essential subject in earthquake science. The 2014 Northern Nagano earthquake, which occurred near the end zone of a major active fault system, provided unique observations that enabled us to investigate both the detailed geometrical fault structure and surface deformation patterns as well as the temporal sequence led up from a prominent foreshock activity. We first develop a geometrical fault model with a substantial variation along strike, and a model for the regional stress field, which is well constrained by the observations. This significant along-strike variation in fault geometry probably reflects the difference of fault maturity at the end zone of the complex fault system. We used this model in order to conduct a set of dynamic rupture simulations using the highly efficient spatiotemporal boundary integral equation method. Based on our simulations, we show that the observed surface deformation can be reasonably explained as the effect of the non-planar fault geometry with a number of branch faults and bends.[Figure not available: see fulltext.

  9. Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex

    PubMed Central

    Dean, Anudariya B.; Mitchell, David R.

    2015-01-01

    Axonemal dyneins are multisubunit enzymes that must be preassembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules, where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport, and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors—ODA5, ODA8, and ODA10—show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10. Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immuno–electron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number 1, which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules. PMID:26310446

  10. Comparative proteomics of tuber induction, development and maturation reveal the complexity of tuberization process in potato (Solanum tuberosum L.).

    PubMed

    Agrawal, Lalit; Chakraborty, Subhra; Jaiswal, Dinesh Kumar; Gupta, Sonika; Datta, Asis; Chakraborty, Niranjan

    2008-09-01

    Tuberization in potato ( Solanum tuberosum L.) is a developmental process that serves a double function, as a storage organ and as a vegetative propagation system. It is a multistep, complex process and the underlying mechanisms governing these overlapping steps are not fully understood. To understand the molecular basis of tuberization in potato, a comparative proteomic approach has been applied to monitor differentially expressed proteins at different development stages using two-dimensional gel electrophoresis (2-DE). The differentially displayed proteomes revealed 219 protein spots that change their intensities more than 2.5-fold. The LC-ES-MS/MS analyses led to the identification of 97 differentially regulated proteins that include predicted and novel tuber-specific proteins. Nonhierarchical clustering revealed coexpression patterns of functionally similar proteins. The expression of reactive oxygen species catabolizing enzymes, viz., superoxide dismutase, ascorbate peroxidase and catalase, were induced by more than 2-fold indicating their possible role during the developmental transition from stolons into tubers. We demonstrate that nearly 100 proteins, some presumably associated with tuber cell differentiation, regulate diverse functions like protein biogenesis and storage, bioenergy and metabolism, and cell defense and rescue impinge on the complexity of tuber development in potato.

  11. Effects of maturation and acidosis on the chaos-like complexity of the neural respiratory output in the isolated brainstem of the tadpole, Rana esculenta

    PubMed Central

    Samara, Ziyad; Fiamma, Marie-Noëlle; Bautin, Nathalie; Ranohavimparany, Anja; Le Coz, Patrick; Golmard, Jean-Louis; Darré, Pierre; Zelter, Marc; Poon, Chi-Sang; Similowski, Thomas

    2011-01-01

    Human ventilation at rest exhibits mathematical chaos-like complexity that can be described as long-term unpredictability mediated (in whole or in part) by some low-dimensional nonlinear deterministic process. Although various physiological and pathological situations can affect respiratory complexity, the underlying mechanisms remain incompletely elucidated. If such chaos-like complexity is an intrinsic property of central respiratory generators, it should appear or increase when these structures mature or are stimulated. To test this hypothesis, we employed the isolated tadpole brainstem model [Rana (Pelophylax) esculenta] and recorded the neural respiratory output (buccal and lung rhythms) of pre- (n = 8) and postmetamorphic tadpoles (n = 8), at physiologic (7.8) and acidic pH (7.4). We analyzed the root mean square of the cranial nerve V or VII neurograms. Development and acidosis had no effect on buccal period. Lung frequency increased with development (P < 0.0001). It also increased with acidosis, but in postmetamorphic tadpoles only (P < 0.05). The noise-titration technique evidenced low-dimensional nonlinearities in all the postmetamorphic brainstems, at both pH. Chaos-like complexity, assessed through the noise limit, increased from pH 7.8 to pH 7.4 (P < 0.01). In contrast, linear models best fitted the ventilatory rhythm in all but one of the premetamorphic preparations at pH 7.8 (P < 0.005 vs. postmetamorphic) and in four at pH 7.4 (not significant vs. postmetamorphic). Therefore, in a lower vertebrate model, the brainstem respiratory central rhythm generator accounts for ventilatory chaos-like complexity, especially in the postmetamorphic stage and at low pH. According to the ventilatory generators homology theory, this may also be the case in mammals. PMID:21325645

  12. Caspase-8 tyrosine-380 phosphorylation inhibits CD95 DISC function by preventing procaspase-8 maturation and cycling within the complex

    PubMed Central

    Powley, I R; Hughes, M A; Cain, K; MacFarlane, M

    2016-01-01

    Caspase-8 is a key initiator of apoptotic cell death where it functions as the apical protease in death receptor-mediated apoptosis triggered via the death-inducing signalling complex (DISC). However, the observation that caspase-8 is upregulated in many common tumour types led to the discovery of alternative non-apoptotic, pro-survival functions, many of which are contingent on phosphorylation of a tyrosine residue (Y380) found in the linker region between the two catalytic domains of the enzyme. Furthermore, Src-mediated Y380 phosphorylation leads to increased resistance to CD95-induced apoptosis; however, the mechanism underlying this impaired response to extrinsic apoptotic stimuli has not been identified. Consequently, we have employed a number of model systems to further dissect this protective mechanism. First, using an in vitro DISC model together with recombinant procaspase-8 variants, we show that Y380 phosphorylation inhibits procaspase-8 activation at the CD95 DISC, thereby preventing downstream activation of the caspase cascade. Second, we validated this finding in a cellular context using transfected neuroblastoma cell lines deficient in caspase-8. Reconstitution of these lines with phosphomimetic-caspase-8 results in increased resistance to CD95-mediated apoptosis and enhanced cell migration. When the in vitro DISC is assembled in the presence of cell lysate, caspase-8 Y380 phosphorylation attenuates DISC activity by inhibiting procaspase-8 autoproteolytic activity but not recruitment or homodimerization of caspase-8 within the complex. Once incorporated into the DISC, phosphorylated caspase-8 is unable to be released from the complex; this inhibits further cycling and release of active catalytic subunits into the cytoplasm, thus resulting in increased apoptotic resistance. Taken together, our novel findings expand our understanding of the key mechanisms underlying the anti-apoptotic functions of caspase-8 which may act as a critical block to

  13. Increase of learning abilities and maturation of the vertical lobe complex during postembryonic development in the cuttlefish, Sepia.

    PubMed

    Dickel, L; Chichery, M P; Chichery, R

    2001-09-01

    When shown prawns in a glass tube, cuttlefish quickly learn to inhibit their predatory behavior. By using a visual learning paradigm, we studied training and retention performances of cuttlefish aged from 8 to 90 days. We found an improvement in the acquisition of learning abilities during the first 2 months of life as well as an increase of 24-hr retention performance between 30 and 90 days of age. Using morphometric measurements of different lobes of the central nervous system, we correlated the emergence of these learning abilities with the postembryonic development of related nervous structures. Our results show that only the growth of the superior frontal and vertical lobes appears to be significantly correlated with the improvement of learning and long-term retention performances. Thus, as found in earlier data collected in Octopus, the vertical lobe complex of the cuttlefish seems to be involved in these learning processes. Copyright 2001 John Wiley & Sons, Inc.

  14. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis.

    PubMed

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J Julian; Gartner, Anton

    2016-03-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis.

  15. The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis

    PubMed Central

    Hong, Ye; Sonneville, Remi; Agostinho, Ana; Meier, Bettina; Wang, Bin; Blow, J. Julian; Gartner, Anton

    2016-01-01

    Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Here, using Caenorhabditis elegans we show that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, we found that the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. Our results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis. PMID:27010650

  16. Mature area of new frontier Northeast British Columbia reveals new high potential in the structurally complex region of the western Canada sedimentary basin

    SciTech Connect

    Hutton, A.N.; Varsek, J.L. )

    1993-09-01

    Northeast British Columbia has been extensively explored since the early fifties. Evaluated as a part of the Western Canada sedimentary basin, with a passive undeformed basement and only Laramide deformation, the basin must be considered mature. Recent work involving detailed stratigraphic analysis, extensive field observations, potential field data analysis, and deep crustal reflection seismic work lead to an exciting new view of this portion of the Western Canada basin. Situated over the middle Proterozoic continental margin, a complexly deformed and deeply truncated foreland forms a ramp against highly magnetic crystalline rocks. Dipping features within the Proterozoic intersect the Phanerozoic, producing local structure and uplift which has had a profound influence on Devonian reef paleogeography. A major contractional episode created previously unrecognized Devonian to Carboniferous folds and this discovery has led to a complete reinterpretation of the structural style and deformational history of the area. The structural complexity of the basin is increased by right lateral strike-slip faulting. This system is highlighted in the subsurface by a series of faults fanning out across the basin with displacements of up to 20 km in the Proterozoic foreland and accommodation continuing until the Cretaceous. Strike-slip deformation has generated an embayment in the Proterozoic continental margin and within the Phanerozoic shelf, indicating the occurrence of several previously unrecognized prospective shelf to basin transitions. The complex interplay of structure and paleogeography results in a series of play opportunities that could result in new giant discoveries.

  17. Escherichia coli harboring a natural IncF conjugative F plasmid develops complex mature biofilms by stimulating synthesis of colanic acid and Curli.

    PubMed

    May, Thithiwat; Okabe, Satoshi

    2008-11-01

    It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F(+) cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.

  18. Phagosome maturation: aging gracefully.

    PubMed Central

    Vieira, Otilia V; Botelho, Roberto J; Grinstein, Sergio

    2002-01-01

    Foreign particles and apoptotic bodies are eliminated from the body by phagocytic leucocytes. The initial stage of the elimination process is the internalization of the particles into a plasma membrane-derived vacuole known as the phagosome. Such nascent phagosomes, however, lack the ability to kill pathogens or to degrade the ingested targets. These properties are acquired during the course of phagosomal maturation, a complex sequence of reactions that result in drastic remodelling of the phagosomal membrane and contents. The determinants and consequences of the fusion and fission reactions that underlie phagosomal maturation are the topic of this review. PMID:12061891

  19. Characterization of mature maize (Zea mays L.) root system architecture and complexity in a diverse set of Ex-PVP inbreds and hybrids.

    PubMed

    Hauck, Andrew L; Novais, Joana; Grift, Tony E; Bohn, Martin O

    2015-01-01

    The mature root system is a vital plant organ, which is critical to plant performance. Commercial maize (Zea mays L.) breeding has resulted in a steady increase in plant performance over time, along with noticeable changes in above ground vegetative traits, but the corresponding changes in the root system are not presently known. In this study, roughly 2500 core root systems from field trials of a set of 10 diverse elite inbreds formerly protected by Plant Variety Protection plus B73 and Mo17 and the 66 diallel intercrosses among them were evaluated for root traits using high throughput image-based phenotyping. Overall root architecture was modeled by root angle (RA) and stem diameter (SD), while root complexity, the amount of root branching, was quantified using fractal analysis to obtain values for fractal dimension (FD) and fractal abundance (FA). For each trait, per se line effects were highly significant and the most important contributor to trait performance. Mid-parent heterosis and specific combining ability was also highly significant for FD, FA, and RA, while none of the traits showed significant general combining ability. The interaction between the environment and the additive line effect was also significant for all traits. Within the inbred and hybrid generations, FD and FA were highly correlated (rp ≥ 0.74), SD was moderately correlated to FD and FA (0.69 ≥ rp ≥ 0.48), while the correlation between RA and other traits was low (0.13 ≥ rp ≥ -0.40). Inbreds with contrasting effects on complexity and architecture traits were observed, suggesting that root complexity and architecture traits are inherited independently. A more comprehensive understanding of the maize root system and the way it interacts with the environment will be useful for defining adaptation to nutrient acquisition and tolerance to stress from drought and high plant densities, critical factors in the yield gains of modern hybrids.

  20. CcmI Subunit of CcmFHI Heme Ligation Complex Functions as an Apocytochrome c Chaperone during c-Type Cytochrome Maturation*

    PubMed Central

    Verissimo, Andreia F.; Yang, Honghui; Wu, Xiaomin; Sanders, Carsten; Daldal, Fevzi

    2011-01-01

    Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c2 as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c2 but not holocytochrome c2. Mainly the C-terminal portions of both CcmI and apocytochrome c2 mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c2, like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c2, but this interaction requires a free thiol group at apocytochrome c2 heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae. PMID:21956106

  1. The Signal Peptide of the Junín Arenavirus Envelope Glycoprotein Is Myristoylated and Forms an Essential Subunit of the Mature G1-G2 Complex

    PubMed Central

    York, Joanne; Romanowski, Victor; Lu, Min; Nunberg, Jack H.

    2004-01-01

    Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking. PMID:15367645

  2. In vitro maturation of oocytes.

    PubMed

    Smith, G D

    2001-10-01

    In vitro maturation (IVM) of human oocytes is an emerging assisted reproductive technology with great promise. To be successful, this process must entail both nuclear and cytoplasmic maturation. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. Although nuclear maturation during human oocyte IVM progresses normally, cytoplasmic maturation is significantly lacking, as exemplified by poor embryonic developmental competence and pregnancy rates. Advances made in immature oocyte isolation and oocyte and embryo culture conditions have increased the clinical feasibility of IVM. However, in order to achieve acceptable birth rates, future studies should focus on characterization and regulation of oocyte cytoplasmic maturation, and how oocyte-derived factors influence zygotic genome activation and embryonic developmental competence.

  3. Inoculation with a psychrotrophic-thermophilic complex microbial agent accelerates onset and promotes maturity of dairy manure-rice straw composting under cold climate conditions.

    PubMed

    Gou, Changlong; Wang, Yuqiong; Zhang, Xiqing; Lou, Yujie; Gao, Yunhang

    2017-06-20

    The objective was to determine the effects of psychrotrophic-thermophilic complex microbial agent (PTCMA) comprised of a psychrotrophic bacterium consortium (PBC) and a thermophilic cellulolytic fungi consortium (TCFC), on composting in a cold climate. Mixtures of dairy manure and rice straw were inoculated with PTCMA, PBC, TCFC and sterile water (control) and composted at an initial ambient temperatures of -2 to 5°C. In compost piles inoculated with PBC or PTCMA, temperatures reached the thermophilic phase (>55°C) faster (8-11d) than piles inoculated with TCFC or control. Furthermore, compost inoculated with TCFC or PTCMA had greater decreases in total organic carbon and carbon-to-nitrogen ratios, as well as significant increases in total nitrogen, degradation of cellulose and lignin and germination index than PBC inoculation or Control compost. Consequently, inoculation with both (i.e. PTCMA) accelerated the onset and promoted maturity of composting under cold-climate conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. The regulatory gamma subunit SNF4b of the sucrose non-fermenting-related kinase complex is involved in longevity and stachyose accumulation during maturation of Medicago truncatula seeds.

    PubMed

    Rosnoblet, Claire; Aubry, Catherine; Leprince, Olivier; Vu, Benoit Ly; Rogniaux, Hélène; Buitink, Julia

    2007-07-01

    The sucrose non-fermenting-related kinase complex (SnRK1) is a heterotrimeric complex that plays a central role in metabolic adaptation to nutritional or environmental stresses. Here we investigate the role of a regulatory gamma-subunit of the complex, MtSNF4b, in Medicago truncatula seeds. Western blot indicated that MtSNF4b accumulated during seed filling, whereas it disappeared during imbibition of mature seeds. Gel filtration chromatography suggested that MtSNF4b assembled into a complex (450-600 kDa) at the onset of maturation drying, and dissociated during subsequent imbibition. Drying of desiccation-tolerant radicles led to a reassembly of the complex, in contrast to sensitive tissues. Silencing of MtSNF4b using a RNA interference (RNAi) approach resulted in a phenotype with reduced seed longevity, evident from the reduction in both germination percentage and seedling vigour in aged RNAi MtSNF4b seeds compared with the wild-type seeds. In parallel to the assembly of the complex, seeds of the RNAi MtSNF4b lines showed impaired accumulation of raffinose family oligosaccharides compared with control seeds. In mature seeds, the amount of stachyose was reduced by 50-80%, whereas the sucrose content was 60% higher. During imbibition, the differences in non-reducing sugar compared with the control disappeared in parallel to the disassembly of the complex. No difference was observed in dry weight or reserve accumulation such as proteins, lipids and starch. These data suggest that the regulatory gamma-subunit MtSNF4b confers a specific and temporal function to SnRK1 complexes in seeds, improving seed longevity and affecting the non-reducing sugar content at later stages of seed maturation.

  5. Sexual maturation and administration of 17β-estradiol and testosterone induce complex gene expression changes in skin and increase resistance of Atlantic salmon to ectoparasite salmon louse.

    PubMed

    Krasnov, Aleksei; Wesmajervi Breiland, Mette S; Hatlen, Bjarne; Afanasyev, Sergey; Skugor, Stanko

    2015-02-01

    The crustacean ectoparasitic salmon louse (Lepeophtheirus salmonis) is a major problem of Atlantic salmon aquaculture in the Northern hemisphere. Host-pathogen interactions in this system are highly complex. Resistance to the parasite involves variations in genetic background, nutrition, properties of skin, and status of the endocrine and immune systems. This study addressed the relationship between sex hormones and lice infection. Field observation revealed a sharp reduction of lice prevalence during sexual maturation with no difference between male and female fish. To determine if higher resistance against lice was related to sex hormones, post-smolt salmon were administered control feed and feeds containing 17β-estradiol (20 mg/kg) and testosterone (25 mg/kg) during a 3-week pre-challenge period. After challenge with lice, counts were reduced 2-fold and 1.5-fold in fish that received 17β-estradiol and testosterone, respectively. Gene expression analyses were performed from skin of salmon collected in the field trial and from the controlled lab experiment at three time points (end of feeding-before challenge, 3 days post challenge (dpc) and 16 dpc) using oligonucleotide microarray and qPCR. Differential expression was observed in genes associated with diverse biological processes. Both studies revealed similar changes of several antibacterial acute phase proteins; of note was induction of cathelicidin and down-regulation of a defensin gene. Treatment with hormones revealed their ability to modulate T helper cell (Th)-mediated immunity in skin. Enhanced protection achieved by 17β-estradiol administration might in part be due to the skewing of Th responses away from the prototypic anti-parasitic Th2 immunity and towards the more effective Th1 responses. Multiple genes involved in wound healing, differentiation and remodelling of skin tissue were stimulated during maturation but suppressed with sex hormones. Such opposite regulation suggested that these processes

  6. Genetical and biotechnological methods of utilization of female reproductive potential in mammals.

    PubMed

    Katska-Ksiazkiewicz, Lucyna; Lechniak-Cieślak, Dorota; Korwin-Kossakowska, Agnieszka; Alm, Hannelore; Ryńska, Bozenna; Warzych, Ewelina; Sosnowski, Jarosław; Sender, Grazyna

    2006-01-01

    The investigations included: 1/ Establishment of culture systems that would maintain the three-dimensional structure of bovine intact early antral follicles (EAF) or isolated cumulus-oocyte-granulosa complexes (COCGs) and increase the resulting portion of cumulus-oocyte complexes (COCs) with normal morphology for subsequent in vitro maturation (IVM) and in vitro fertilization (IVF), 2/ Quality assessment of IVM bovine oocytes and resulting day-8 blastocysts produced in TCM199 medium supplemented with fetal bovine serum (FBS), fatty acid free bovine serum albumin (fafBSA) or polyvinylpyrrolidone (PVP40), 3/ Testing the polymorphism of the genes: retinol binding protein (RBP4), epidermal growth factor (EGF), prostaglandin-endoperoxide synthase 2 (PTGS2), insulin-like growth factor 2 (IGF2), amphiregulin (AREG) and prolactin (PRL), and their effects on reproductive traits in swine. Isolated COCGs created in culture follicle-like structures and their oocytes achieved meiotic competence and matured to metaphase II at a higher rate than did oocytes from smaller diameter follicles which were cultured intact. The proportion of COCs with normal morphology significantly increased when isolated COCGs were embedded in microdrops of collagen gel or cultured on inserts covered with gel rather than in large gel volumes. No significant effect of maturation media composition on meiotic spindle morphology and the rate of apoptotic bovine oocytes was observed. Among day-8 embryos derived from oocytes matured with PVP40 a reduced blastocyst rate and elevated apoptotic index were found, whereas total cell count was not affected. Gene expression study also revealed a decrease in relative abundance for IGF2 and its receptor (IGF2R), and heat shock protein 70 (Hsp70) genes in PVP40 group and a significant elevation in fafBSA derived embryos. The significant effect of reproduction traits of swine (litter size and litter weight) was found for RBP4, EGF, IGF2 and AREG genes. A new

  7. Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

    PubMed

    Sand, Michael; Skrygan, Marina; Georgas, Dimitrios; Arenz, Christoph; Gambichler, Thilo; Sand, Daniel; Altmeyer, Peter; Bechara, Falk G

    2012-11-01

    The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA-induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute-1 (AGO1), argonaute-2 (AGO2), as well as double-stranded RNA-binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P < 0.05). There was no significant difference in the TARBP2 expression levels between groups (P > 0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer.

  8. Timing of maturation of a Neoproterozoic oceanic arc during Pan-African Orogeny: the Asmlil complex (Anti-Atlas, South Morocco)

    NASA Astrophysics Data System (ADS)

    Triantafyllou, Antoine; Berger, Julien; Baele, Jean-Marc; Bruguier, Olivier; Diot, Hervé; Ennih, Nasser; Plissart, Gaëlle; Monnier, Christophe; Watlet, Arnaud; Vandycke, Sara

    2016-04-01

    produced by partial melting of a REE-depleted gabbronorite with cpx + garnet-rich residue, as typically observed in the basal crustal part of paleo-arc sections (e.g. Talkeetna, Kohistan arcs). Field observations, geochemical signatures, P-T estimates and new geochronological data allow to track the timing of Asmlil arc maturation. Combining our results to the entire Pan-African suture (Sirwa and Bou Azzer inliers), geochronological data clearly show that three distinct flare-ups give the tempo of arc magmatism during Pan-African Orogeny. First event is the early construction of the 755-745 My oceanic arc marked by intermediate volcanic to subvolcanic massifs. Second event occurred around 700 My and results from mafic products intruding previous arc. A last event also dated at 660-650 My in the Sirwa window marks the emplacement of hot hornblenditic arc-magmas into older arc massifs during the tectonic extrusion of the arc section. This late event is also related to intense melt production at different level of the arc contributing to differentiation of the whole arc complex. We thus interpreted the Asmlil complex as the final composite product of successive magmatic pulses during arc life cycle which can be compared to relatively long-lived and paced active arc systems (e.g. Aleutian, IBM arcs).

  9. Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies.

    PubMed

    An, Ran; Wang, Chunmin; Turek, John; Machaty, Zoltan; Nolte, David D

    2015-03-01

    The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates.

  10. Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies

    PubMed Central

    An, Ran; Wang, Chunmin; Turek, John; Machaty, Zoltan; Nolte, David D.

    2015-01-01

    The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates. PMID:25798318

  11. Frequency analysis of cytotoxic T lymphocyte precursors in chimeric mice. Evidence for intrathymic maturation of clonally distinct self- major histocompatibility complex- and allo-major histocompatiblilty complex-restricted virus-specific T cells

    PubMed Central

    1981-01-01

    To study whether the thymic major histocompatibility complex (MHC) imposes a constraint on the receptor repertoire of maturating cytotoxic T lymphocyte (CTL) precursors, the restriction phenotypes of virus- specific CTL of MHC-compatible and of MHC-incompatible thymus- and bone marrow-grafted (A X B)F1 chimeric mice were compared. Dependent on the mode of in vitro sensitization, thymocytes or splenocytes of both types of chimeric mice generated Sendai virus-specific, self-MHC-or allo-MHC- restricted CTL. By applying the limiting-dilution technique, the CTL- precursor (CTL-P) frequencies of self-MHC-restricted and allo-MHC- restricted virus-specific T cells as well as of alloreactive T cells were determined. The data obtained revealed that independent of MHC differences between thymus and bone marrow, the frequencies of self-MHC- restricted and allo-MHC-restricted CTL-P were comparable, and in the same older of magnitude as those previously determined in conventionally reared mice. Self-MHC-restricted, virus-specific CTL-P were in a three- to fivefold excess over allo-MHC-restricted CTL-P. A segregation analysis revealed that clonally distinct CTL-P give rise to either self-restricted or allo-MHC-restricted, virus-specific CTL. Both sets were found not only in the spleen, but also in the thymus of chimeric mice, formally demonstrating the intrathymic differentiation pathway of self-MHC as well of allo-MHC-restricted CTL-P. These data reveal no major constraint of the thymic MHC on the capacity of T cells to recognize viral antigens either in the context of self-MHC or of allogeneic MHC products. PMID:6265587

  12. Effect of convective transport in porous media on the conditions of organic matter maturation and generation of hydrocarbons in trap rocks complexes

    NASA Astrophysics Data System (ADS)

    Yurie Khachay, Professor; Mindubaev, Mansur

    2016-04-01

    One of the main problems of the study of the intrusion thermal effects on the maturation of the organic matter is to estimate the volume, intensity, thermal effects of the intrusion and its redistribution in porous media by convection. A numerical algorithm for solving the problem of the developed convection in two-dimensional and three-dimensional models of the porous medium depending on the incline angle is developed. It is defined that the convective stability in the medium decreases with increasing incline angle. It was found that depending on the incline angle the structure of convection from many cells for a flat horizontal layer changes and it transfers to more elongated structures along the layer. It is shown that depending on the incline angles, invading sill and imbedding volume of the porous medium it can be realized either stationary or non-stationary convection that provides a principal different thermal conditions of hydrocarbons maturation in the motherboard porous medium. We give numerical examples of the influence of the incline angle on the flow structure inside the porous inclusion. By the stationary convection the volume of the boundary layers between the convective sells increases. That can lead to increasing of the part of motherboard rocks that are outer the temperature conditions of oil catalysis and as a consequence to the overestimation of the deposits.

  13. Bovine oocytes show a higher tolerance to heat shock in the warm compared with the cold season of the year.

    PubMed

    Maya-Soriano, M J; López-Gatius, F; Andreu-Vázquez, C; López-Béjar, M

    2013-01-15

    Heat stress is especially harmful for bovine ovarian follicle development and oocyte competence. In this study, we assessed the effects of heat shock on oocyte maturation in oocytes collected during the cold (February-March; n = 114) or warm (May-June; n = 116) periods of the year. In both cases, cumulus-oocyte complexes were matured under control (38 °C) and heat shock conditions (41.5 °C, 18-21 h of maturation). For each oocyte, nuclear stage, cortical granule distribution and steroidogenic activity of cumulus cells were evaluated. Based on the odds ratio, heat-shocked oocytes were 26.83 times more likely to show an anomalous metaphase II morphology. When matured under heat shock conditions, oocytes obtained in both seasons were similarly affected in terms of nuclear maturation, whereas a seasonal effect was observed on cytoplasmic maturation. For oocytes collected during the cold season, the likelihood to show an anomalous maturation was 25.96 times higher when exposed to the heat treatment than when matured under control conditions. By contrast, oocytes collected during the warm season matured under control or heat shock did not show significant risk of showing an anomalous cytoplasmic maturation. Our findings indicate an increased rate of premature oocytes in response to heat shock as well as a higher tolerance to this stress of oocytes harvested in the warm season compared with those collected in the colder period.

  14. Natriuretic peptides stimulate oocyte meiotic resumption in bovine.

    PubMed

    De Cesaro, Matheus P; Macedo, Mariana P; Santos, Joabel T; Rosa, Paulo R A; Ludke, Charles A; Rissi, Vitor B; Gasperin, Bernardo G; Gonçalves, Paulo B D

    2015-08-01

    The aim of the present study was to evaluate the expression of mRNA encoding natriuretic peptides (NPs) and their receptors in the cumulus-oocyte complex in cattle, a monovular mammalian species, and also to investigate the role of NPs in oocyte meiotic resumption in vitro. mRNA was observed for the NP precursor type-A (NPPA), type-C (NPPC), NP receptor-1 (NPR-1), receptor-2 (NPR-2) and receptor-3 (NPR-3) in bovine cumulus cells, and NPR-2 mRNA was observed in oocytes. These results are different from those obtained in mouse and pig models. The effects of NPPA, NP precursor type-B (NPPB) and NPPC on the resumption of arrested meiosis maintained by forskolin were studied at three different doses (10, 100 and 1000nM) with a 12h culture system. The germinal vesicle breakdown rates were greater (P≤0.05) in oocytes that were cultured in the presence of one or a combination of NPs (from 44% to 73%) than the negative control (from 24% to 27%). Additionally, it was demonstrated that the concentration of cyclic guanosine 3',5'-monophosphate (cGMP) is increased by NPPA and NPPC in oocytes and cumulus cells after 3h of in vitro maturation. However, in both groups, the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) in the oocyte did not increase between 3 and 6h of culture, even when forskolin was used. In summary, we observed the presence of mRNA for NPs and their receptors in the bovine cumulus-oocyte complex and demonstrated that, in vitro, NPPA, NPPB and NPPC stimulate oocyte meiotic resumption in a monovular species.

  15. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  16. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  17. The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.

    PubMed

    San Francisco, Brian; Sutherland, Molly C; Kranz, Robert G

    2014-03-01

    Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches haem covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of holocytochromes c by CcmFH and CcmG, a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH-only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I, with a cleaved signal sequence and two covalent bonds to haem. We engineered increased cytochrome c production by CcmFGH-only, with yields approaching those from the full system I. Three conserved histidines in CcmF (TM-His1, TM-His2 and P-His1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH. Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis. © 2014 John Wiley & Sons Ltd.

  18. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    PubMed

    Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo; Schuck, Peter; Louis, John M; Pierson, Jason; Rao, Prashant; Subramaniam, Sriram; Gustchina, Alla; Clore, G Marius; Wlodawer, Alexander

    2013-01-01

    A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design.

  19. An active Mitochondrial Complex II Present in Mature Seeds Contains an Embryo-Specific Iron-Sulfur Subunit Regulated by ABA and bZIP53 and Is Involved in Germination and Seedling Establishment.

    PubMed

    Restovic, Franko; Espinoza-Corral, Roberto; Gómez, Isabel; Vicente-Carbajosa, Jesús; Jordana, Xavier

    2017-01-01

    Complex II (succinate dehydrogenase) is an essential mitochondrial enzyme involved in both the tricarboxylic acid cycle and the respiratory chain. In Arabidopsis thaliana, its iron-sulfur subunit (SDH2) is encoded by three genes, one of them (SDH2.3) being specifically expressed during seed maturation in the embryo. Here we show that seed SDH2.3 expression is regulated by abscisic acid (ABA) and we define the promoter region (-114 to +49) possessing all the cis-elements necessary and sufficient for high expression in seeds. This region includes between -114 and -32 three ABRE (ABA-responsive) elements and one RY-enhancer like element, and we demonstrate that these elements, although necessary, are not sufficient for seed expression, our results supporting a role for the region encoding the 5' untranslated region (+1 to +49). The SDH2.3 promoter is activated in leaf protoplasts by heterodimers between the basic leucine zipper transcription factors bZIP53 (group S1) and bZIP10 (group C) acting through the ABRE elements, and by the B3 domain transcription factor ABA insensitive 3 (ABI3). The in vivo role of bZIP53 is further supported by decreased SDH2.3 expression in a knockdown bzip53 mutant. By using the protein synthesis inhibitor cycloheximide and sdh2 mutants we have been able to conclusively show that complex II is already present in mature embryos before imbibition, and contains mainly SDH2.3 as iron-sulfur subunit. This complex plays a role during seed germination sensu-stricto since we have previously shown that seeds lacking SDH2.3 show retarded germination and now we demonstrate that low concentrations of thenoyltrifluoroacetone, a complex II inhibitor, also delay germination. Furthermore, complex II inhibitors completely block hypocotyl elongation in the dark and seedling establishment in the light, highlighting an essential role of complex II in the acquisition of photosynthetic competence and the transition from heterotrophy to autotrophy.

  20. An active Mitochondrial Complex II Present in Mature Seeds Contains an Embryo-Specific Iron–Sulfur Subunit Regulated by ABA and bZIP53 and Is Involved in Germination and Seedling Establishment

    PubMed Central

    Restovic, Franko; Espinoza-Corral, Roberto; Gómez, Isabel; Vicente-Carbajosa, Jesús; Jordana, Xavier

    2017-01-01

    Complex II (succinate dehydrogenase) is an essential mitochondrial enzyme involved in both the tricarboxylic acid cycle and the respiratory chain. In Arabidopsis thaliana, its iron–sulfur subunit (SDH2) is encoded by three genes, one of them (SDH2.3) being specifically expressed during seed maturation in the embryo. Here we show that seed SDH2.3 expression is regulated by abscisic acid (ABA) and we define the promoter region (-114 to +49) possessing all the cis-elements necessary and sufficient for high expression in seeds. This region includes between -114 and -32 three ABRE (ABA-responsive) elements and one RY-enhancer like element, and we demonstrate that these elements, although necessary, are not sufficient for seed expression, our results supporting a role for the region encoding the 5’ untranslated region (+1 to +49). The SDH2.3 promoter is activated in leaf protoplasts by heterodimers between the basic leucine zipper transcription factors bZIP53 (group S1) and bZIP10 (group C) acting through the ABRE elements, and by the B3 domain transcription factor ABA insensitive 3 (ABI3). The in vivo role of bZIP53 is further supported by decreased SDH2.3 expression in a knockdown bzip53 mutant. By using the protein synthesis inhibitor cycloheximide and sdh2 mutants we have been able to conclusively show that complex II is already present in mature embryos before imbibition, and contains mainly SDH2.3 as iron–sulfur subunit. This complex plays a role during seed germination sensu-stricto since we have previously shown that seeds lacking SDH2.3 show retarded germination and now we demonstrate that low concentrations of thenoyltrifluoroacetone, a complex II inhibitor, also delay germination. Furthermore, complex II inhibitors completely block hypocotyl elongation in the dark and seedling establishment in the light, highlighting an essential role of complex II in the acquisition of photosynthetic competence and the transition from heterotrophy to autotrophy. PMID

  1. Health- and disease-associated species clusters in complex natural biofilms determine the innate immune response in oral epithelial cells during biofilm maturation.

    PubMed

    Langfeldt, Daniela; Neulinger, Sven C; Stiesch, Meike; Stumpp, Nico; Bang, Corinna; Schmitz, Ruth A; Eberhard, Jörg

    2014-11-01

    The aim of the present study was to verify our hypothesis concerning the differential induction of various antimicrobial and immunomodulatory responses in oral epithelial cells by diverse bacterial species clusters. For this purpose, oral biofilms between 1 and 14 days of maturation (36 volunteers) were co-incubated with gingival epithelial cells. Subsequently, human β-defensin (hBD)-2, hBD-3, LL-37, interleukin (IL)-1β, IL-6, IL-8 and IL-10 mRNA expression profiles were quantified by quantitative reverse transcription PCR. The correlation between bacterial species and the host innate immune response was determined by relating these results to existing 16S rRNA phylogenetic analysis by amplicon sequencing (Langfeldt et al. 2014. PLoS One 9: e87449). Data were analysed by multiple factor analysis. Transcription of hBD-2 and hBD-3 was significantly associated with the abundance of species of the Prevotella cluster and the absence of species of the Streptococcus cluster. IL-1β, -6, -8 and -10 mRNA syntheses were significant correlated with Leptotrichia species [Leptotrichia 302H02 (0.448, P < 0.0001), Leptotrichia nbw822e09c1 (0.214, P = 0.008) and Leptotrichia wadei (0.218, P = 0.007)] of the Prevotella cluster. In the third dimension IL-10 and members of the Prevotella cluster were negatively correlated, whereas hBD-3 and IL-1β, IL-6 and IL-8 were positive correlated to axis 3, like members of the Proteobacteria cluster. In conclusion, distinct species of health- and disease-associated bacterial clusters induce antibacterial or immunomodulatory reactions in oral epithelial cells during early stages of bacteria-host interactions.

  2. Removal of both Ycf48 and Psb27 in Synechocystis sp. PCC 6803 disrupts Photosystem II assembly and alters Q(A)(-) oxidation in the mature complex.

    PubMed

    Jackson, Simon A; Hervey, John R D; Dale, Asher J; Eaton-Rye, Julian J

    2014-10-16

    The Photosystem II (PS II) assembly factors Psb27 and Ycf48 are transiently associated with PS II during its biogenesis and repair pathways. We investigated the function of these proteins by constructing knockout mutants in Synechocystis sp. PCC 6803. In ΔYcf48 cells, PS II electron transfer and stable oxygen evolution were perturbed. Additionally, Psb27 was required for photoautotrophic growth of cells lacking Ycf48 and assembly beyond the RC47 assembly complex in ΔYcf48:ΔPsb27 cells was impeded. Our results suggest the RC47 complex formed in ΔYcf48 cells is defective and that this deficiency is exacerbated if CP43 binds in the absence of Psb27. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure.

    PubMed

    Kim, Jin Hong; Gripon, Philippe; Bouezzedine, Fidaa; Jeong, Mun Sik; Chi, Seung-Wook; Ryu, Seong-Eon; Hong, Hyo Jeong

    2015-01-16

    To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)-specific preS1 humanized antibody (HzKR127), we further humanized it through specificity-determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy-chain complementarity-determining regions (CDR), on the basis of the crystal structure of the antigen-antibody complex. HzKR127-3.2 exhibited 2.5-fold higher affinity and enhanced virus-neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus-neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127-3.2 may be a good candidate for HBV immunoprophylaxis.

  4. Complexity.

    PubMed

    Gómez-Hernández, J Jaime

    2006-01-01

    It is difficult to define complexity in modeling. Complexity is often associated with uncertainty since modeling uncertainty is an intrinsically difficult task. However, modeling uncertainty does not require, necessarily, complex models, in the sense of a model requiring an unmanageable number of degrees of freedom to characterize the aquifer. The relationship between complexity, uncertainty, heterogeneity, and stochastic modeling is not simple. Aquifer models should be able to quantify the uncertainty of their predictions, which can be done using stochastic models that produce heterogeneous realizations of aquifer parameters. This is the type of complexity addressed in this article.

  5. The Asymmetry in the Mature Amino-Terminus of ClpP Facilitates a Local Symmetry Match in ClpAP and ClpXP Complexes

    SciTech Connect

    Bewley,M.; Graziano, V.; Griffin, K.; Flanagan, J.

    2006-01-01

    ClpP is a self-compartmentalized proteolytic assembly comprised of two, stacked, heptameric rings that, when associated with its cognate hexameric ATPase (ClpA or ClpX), form the ClpAP and ClpXP ATP-dependent protease, respectively. The symmetry mismatch is an absolute feature of this large energy-dependent protease and also of the proteasome, which shares a similar barrel-shaped architecture, but how it is accommodated within the complex has yet to be understood, despite recent structural investigations, due in part to the conformational lability of the N-termini. We present the structures of Escherichia coli ClpP to 1.9 Angstroms and an inactive variant that provide some clues for how this might be achieved. In the wild type protein, the highly conserved N-terminal 20 residues can be grouped into two major structural classes. In the first, a loop formed by residues 10-15 protrudes out of the central access channel extending {approx}12-15 Angstroms from the surface of the oligomer resulting in the closing of the access channel observed in one ring. Similar loops are implied to be exclusively observed in human ClpP and a variant of ClpP from Streptococcus pneumoniae. In the other ring, a second class of loop is visible in the structure of wt ClpP from E. coli that forms closer to residue 16 and faces toward the interior of the molecule creating an open conformation of the access channel. In both classes, residues 18-20 provide a conserved interaction surface. In the inactive variant, a third class of N-terminal conformation is observed, which arises from a conformational change in the position of F17. We have performed a detailed functional analysis on each of the first 20 amino acid residues of ClpP. Residues that extend beyond the plane of the molecule (10-15) have a lesser effect on ATPase interaction than those lining the pore (1-7 and 16-20). Based upon our structure-function analysis, we present a model to explain the widely disparate effects of individual

  6. Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

    PubMed

    Rodrigues-Cunha, Maria Carolina; Mesquita, Lígia G; Bressan, Fabiana; Collado, Maite Del; Balieiro, Júlio C C; Schwarz, Kátia R L; de Castro, Fernanda C; Watanabe, Osnir Y; Watanabe, Yeda F; de Alencar Coelho, Lia; Leal, Cláudia L V

    2016-10-15

    Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P < 0.05) compared with the other groups (51.6%-69.1%, P > 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P < 0.05) whereas Cu,ZnSOD transcripts were more abundant in melatonin group (10(-6)M; P < 0.05). Nuclear fragmentation in cumulus cells was highest in controls (37.4%/10,000 cells; P < 0.05) and lower in FSH and 10(-6)M melatonin (29.4% and 25.6%/10,000 cells, respectively). Reactive oxygen species levels were lower in oocytes matured with 10(-6)M melatonin than in control and FSH groups (P < 0.05). Embryo development from oocytes matured only with melatonin was similar to those matured in complete medium (P > 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support

  7. Antisense inhibition of the 2-oxoglutarate dehydrogenase complex in tomato demonstrates its importance for plant respiration and during leaf senescence and fruit maturation.

    PubMed

    Araújo, Wagner L; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Nunes-Nesi, Adriano; Fernie, Alisdair R

    2012-06-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon-nitrogen interactions.

  8. Antisense Inhibition of the 2-Oxoglutarate Dehydrogenase Complex in Tomato Demonstrates Its Importance for Plant Respiration and during Leaf Senescence and Fruit Maturation[W][OA

    PubMed Central

    Araújo, Wagner L.; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Nunes-Nesi, Adriano; Fernie, Alisdair R.

    2012-01-01

    Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon–nitrogen interactions. PMID:22751214

  9. [Adolescent brain maturation].

    PubMed

    Holzer, L; Halfon, O; Thoua, V

    2011-05-01

    Recent progress in neuroscience has yielded major findings regarding brain maturation during adolescence. Unlike the body, which reaches adult size and morphology during this period, the adolescent brain is still maturing. The prefrontal cortex appears to be an important locus of maturational change subserving executive functions that may regulate emotional and motivational issues. The recent expansion of the adolescent period has increased the lag between the onset of emotional and motivational changes activated by puberty and the completion of cognitive development-the maturation of self-regulatory capacities and skills that are continuing to develop long after puberty has occurred. This "disconnect" predicts risk for a broad set of behavioral and emotional problems. Adolescence is a critical period for high-level cognitive functions such as socialization that rely on maturation of the prefrontal cortex. Intervention during the period of adolescent brain development provides opportunities and requires an interdisciplinary approach.

  10. Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4–HLA-DR1 complex

    PubMed Central

    Wang, Xin Xiang; Li, Yili; Yin, Yiyuan; Mo, Min; Wang, Qian; Gao, Wei; Wang, Lili; Mariuzza, Roy A.

    2011-01-01

    Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4–MHC class II interaction is its exceptionally low affinity, which ranges from KD = ∼200 μM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell–cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with KD = 8.8 μM, compared with >400 μM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and β2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development. PMID:21900604

  11. Increased expression of pentraxin 3 after in vivo and in vitro stimulation with gonadotropins in porcine oocyte-cumulus complexes and granulosa cells.

    PubMed

    Nagyova, E; Kalous, J; Nemcova, L

    2016-07-01

    It has been previously shown that multimeric pentraxin 3 (PTX3) is a key component of the cumulus oophorus extracellular matrix (ECM) in mice. In response to the ovulatory LH surge, the cumulus cells assemble a unique ECM that envelopes the oocyte and cumulus cell complex. Importantly, cumuli from PTX3(-/-) mice were defective in their ECM organization and their fertility was impaired. It has been demonstrated that tumor necrosis factor alpha-induced protein 6 catalyzes the formation of heavy chains of (inter-alpha-trypsin inhibitor) -hyaluronan complexes and these are then cross-linked via PTX3. This process is tightly regulated and requires the proteins to meet/interact in the correct order. Finally, in this way, the above-listed proteins form the cumulus oophorus ECM. We investigated whether PTX3 is expressed in the porcine preovulatory follicle. Porcine oocyte-cumulus complexes (OCC) and mural granulosa cells (MGC) from gilts were obtained either after stimulation in vivo with eCG/hCG (4, 8, 16, 24, and 32 h) or culture in vitro (4, 24, and 44 h) in FSH/LH-supplemented medium. The methods performed were real-time reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunostaining. The expression of PTX3 transcripts was significantly increased 24 h after either in vivo hCG stimulation or in vitro FSH/LH treatment in both OCC and MGC. Western blot analysis with PTX3 antibody revealed that not only matrix extracts from in vivo-stimulated gilts contain high levels of PTX3 protein but also matrix extracts of FSH/LH-stimulated OCC cultured in medium supplemented either with follicular fluid or with porcine serum. The localization of PTX3 in the cumulus oocyte complex was confirmed by immunostaining. In conclusion, PTX3 is produced by porcine OCC and MGC both in vivo and in vitro with gonadotropin stimuli inducing cumulus expansion.

  12. Does supplementation of in-vitro culture medium with melatonin improve IVF outcome in PCOS?

    PubMed

    Kim, Mi Kyoung; Park, Eun A; Kim, Hyung Joon; Choi, Won Yun; Cho, Jung Hyun; Lee, Woo Sik; Cha, Kwang Yul; Kim, You Shin; Lee, Dong Ryul; Yoon, Tae Ki

    2013-01-01

    Human pre-ovulatory follicular fluid (FF) contains a higher concentration of melatonin than serum. The aim of this study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcomes of an in-vitro maturation (IVM) IVF-embryo transfer programme for patients with polycystic ovarian syndrome (PCOS). Melatonin concentrations in the culture media of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and the clinical outcomes after using IVM media with or without melatonin were analysed. In the culture media of GC or COC, melatonin concentrations gradually increased. When human chorionic gonadotrophin priming protocols were used, implantation rates in the melatonin-supplemented group were higher than those of the non-supplemented control group (P<0.05). Pregnancy rates were also higher, although not significantly. The findings suggest that the addition of melatonin to IVM media may improve the cytoplasmic maturation of human immature oocytes and subsequent clinical outcomes. It is speculated that follicular melatonin may be released from luteinizing GC during late folliculogenesis and that melatonin supplementation may be used to improve the clinical outcomes of IVM IVF-embryo transfer. Melatonin is primarily produced by the pineal gland and regulates a variety of important central and peripheral actions related to circadian rhythms and reproduction. Interestingly, human pre-ovulatory follicular fluid contains a higher concentration of melatonin than serum. However, in contrast to animal studies, the direct role of melatonin on oocyte maturation in the human system has not yet been investigated. So, the aim of the study was to evaluate the effect of melatonin supplementation of culture medium on the clinical outcome of an in-vitro maturation (IVM) IVF-embryo transfer programme for PCOS patients. The melatonin concentrations in culture medium of granulosa cells (GC) or cumulus-oocyte-complexes (COC) were measured and

  13. Toward Teacher Maturity.

    ERIC Educational Resources Information Center

    Pickle, Judy

    1985-01-01

    The essence of teacher maturity can be synthesized into personal, professional, and process domains. Although overlapping, these categories add a multidimensional approach to the search for what is good in teaching and provide a model for professional development. (MT)

  14. Knockout of the gamma subunit of the AP-1 adaptor complex in the human parasite Trypanosoma cruzi impairs infectivity and differentiation and prevents the maturation and targeting of the major protease cruzipain.

    PubMed

    Moreira, Claudia Maria do Nascimento; Batista, Cassiano Martin; Fernandes, Jessica Chimenes; Kessler, Rafael Luis; Soares, Maurilio José; Fragoso, Stenio Perdigão

    2017-01-01

    The AP-1 Adaptor Complex assists clathrin-coated vesicle assembly in the trans-Golgi network (TGN) of eukaryotic cells. However, the role of AP-1 in the protozoan Trypanosoma cruzi-the Chagas disease parasite-has not been addressed. Here, we studied the function and localization of AP-1 in different T. cruzi life cycle forms, by generating a gene knockout of the large AP-1 subunit gamma adaptin (TcAP1-γ), and raising a monoclonal antibody against TcAP1-γ. Co-localization with a Golgi marker and with the clathrin light chain showed that TcAP1-γ is located in the Golgi, and it may interact with clathrin in vivo, at the TGN. Epimastigote (insect form) parasites lacking TcAP1-γ (TcγKO) have reduced proliferation and differentiation into infective metacyclic trypomastigotes (compared with wild-type parasites). TcγKO parasites have also displayed significantly reduced infectivity towards mammalian cells. Importantly, TcAP1-γ knockout impaired maturation and transport to lysosome-related organelles (reservosomes) of a key cargo-the major cysteine protease cruzipain, which is important for parasite nutrition, differentiation and infection. In conclusion, the defective processing and transport of cruzipain upon AP-1 ablation may underlie the phenotype of TcγKO parasites.

  15. In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals.

    PubMed

    Abdelkhalek, A E; Gabr, Sh A; Khalil, W A; Shamiah, Sh M; Pan, L; Qin, G; Farouk, M H

    2017-03-28

    Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

  16. Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos.

    PubMed

    Su, Jianmin; Hu, Guangdong; Wang, Yongsheng; Liang, Dong; Gao, Mingqing; Sun, Hongzheng; Zhang, Yong

    2014-08-01

    Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.

  17. Structure, function and dynamics in adenovirus maturation

    DOE PAGES

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  18. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  19. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  20. Structure, function and dynamics in adenovirus maturation.

    PubMed

    Mangel, Walter F; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a "molecular sled" to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed.

  1. Effects of non-esterified fatty acids on bovine granulosa cells and developmental potential of oocytes in vitro.

    PubMed

    Jorritsma, R; César, M L; Hermans, J T; Kruitwagen, C L J J; Vos, P L A M; Kruip, T A M

    2004-04-01

    High yielding dairy cows experience a negative energy balance (NEB) shortly after parturition, which is accompanied by high concentrations of non-esterified fatty acids (NEFA) in blood up to approximately 3 weeks post partum. We hypothesized that the elevated plasma NEFA concentration causes lower fertility by exerting negative effects on granulosa cells and oocytes in the ovary, leading to less viable embryos and insufficient corpora lutea. In two series of experiments, we studied the effects of a realistic NEFA (C18:1) concentration on both the proliferation and the progesterone production of follicular granulosa cells in vitro (part I) and on maturation, fertilization and developmental potential of oocytes (part II). For part I, granulosa cells were added to 4 groups of dishes with four different media and cultured for nine consecutive days. After a preculture period of 42h, the presence of NEFA had a negative effect on the proliferation of granulosa cells. No effect of NEFA on the amount of progesterone production per cell was observed. For part II, a total of 1804 cumulus-oocyte-complexes were collected from slaughterhouse ovaries. Using a subgroup of 690 COC, maturation medium with NEFA caused a delay in maturation. Using another 1114 COC, fertilization, cleavage, and embryonic development after maturation in presence of NEFA were significantly reduced. We concluded that the presence of NEFA in follicular fluid and blood of post partum cows may reduce fertility due to hampered embryonic development and subnormal CL function.

  2. Preservation of primordial follicles from lions by slow freezing and xenotransplantation of ovarian cortex into an immunodeficient mouse.

    PubMed

    Wiedemann, C; Hribal, R; Ringleb, J; Bertelsen, M F; Rasmusen, K; Andersen, C Y; Kristensen, S G; Jewgenow, K

    2012-12-01

    Assisted reproductive technology (ART) is considered an important tool in the conservation of endangered species, but often the most limiting factor of ART is the availability of mature oocytes. The aim of the present study was to investigate the feasibility of preserving female germ cells from ovaries of female lions (Panthera leo). Good quality cumulus-oocyte complexes (COCs) were isolated and subjected to in vitro maturation (IVM). In addition, ovarian cortex was obtained and cut into pieces for culture and cryopreservation by slow freezing. The survival of ovarian follicles was assessed by histology. Frozen-thawed samples of ovarian cortex samples were xenotransplanted under the skin of ovariectomized immunodeficient mouse for 28 days. Overall, 178 intact COCs were obtained from 13 lions, but only 28.1% were matured in vitro indicating insufficient IVM conditions. In contrast, almost all follicles within the ovarian cortex survived culture when the original sample was from a young healthy lion collected immediately after euthanasia. Within the xenotransplants, the number of primordial follicles decreased after 28 days by 20%, but the relation between primordial and growing follicles changed in favour of follicular growth. Female gamete rescue from valuable felids may be performed by slow freeze cryopreservation of ovarian cortex. Although the IVM protocol for lions is not yet optimized, mature oocytes may be obtained after long-term xenotransplantation and IVM and could potentially represent one way of salvage of endangered felid species in the future.

  3. Maturation in Larch 1

    PubMed Central

    Greenwood, Michael S.; Hopper, Catherine A.; Hutchison, Keith W.

    1989-01-01

    The time course of maturation in eastern larch (Larix laricina [Du Roi] K. Koch) was examined by grafting scions from trees of different ages onto 2-year-old root stock and following scion development for several years. Height, diameter, foliar chlorophyll content, and rooting ability of scion-derived cuttings all varied linearly as a function of log10 age. Chlorophyll content (milligrams per gram of dry weight) increased while height, diameter, and ability to root decreased with age (P < 0.01). The tendency toward orthotropic growth and branch formation per centimeter of main stem decreased abruptly between age 1 and 5 years (P < 0.01). Total chlorophyll content of both long and short shoot foliage increased by 30 to 50% with increasing age, but the chlorophyll a/b ratio did not change. Also, juvenile long shoot needles were significantly longer than mature (P < 0.01). Surprisingly, the juvenile scions produced more total strobili over two successive years, but the mature scions produced a significantly higher proportion of male strobili (P < 0.001 year 1; P < 0.02 year 2). The age-related changes in foliar traits were not associated with changes in DNA methylation between juvenile and mature scions. Using HPLC, we found that 20% of foliar DNA cytosine residues were methylated in both scion types. Images Figure 1 PMID:16666785

  4. Jealousy and Moral Maturity.

    ERIC Educational Resources Information Center

    Mathes, Eugene W.; Deuger, Donna J.

    Jealousy may be perceived as either good or bad depending upon the moral maturity of the individual. To investigate this conclusion, a study was conducted testing two hypothesis: a positive relationship exists between conventional moral reasoning (reference to norms and laws) and the endorsement and level of jealousy; and a negative relationship…

  5. Mature Students Studying Mathematics.

    ERIC Educational Resources Information Center

    Hirst, Keith

    1999-01-01

    Discusses mature students in the single subject area of mathematics in a single institution and makes comparisons with traditional universities. Reviews some features of the age distribution, entry qualifications, degree-class distribution, non-completion rates and gender distribution. (Author/ASK)

  6. Brain maturation and epilepsy.

    PubMed

    Dulac, Olivier; Milh, Mathieu; Holmes, Gregory L

    2013-01-01

    At full term, both glutamate and gamma-amino-butyric acid (GABA) are excitatory; cortical synapses are beginning to appear, there is little myelin in the cerebral hemispheres, and long tracts hardly start to develop. Neonatal myoclonic encephalopathy can result from premature activation of N-methyl-D-aspartate (NMDA) transmission. Benign neonatal seizures and migrating partial seizures in infancy could involve excessive or premature excitability of deep cortical layers. Benign rolandic epilepsy and continuous spike waves in slow sleep are consistent with an excess of both excitatory and inhibitory cortical synapses. West and Lennox-Gastaut syndromes express age-related diffuse cortical hyperexcitability, the pattern depending on the age of occurrence; synchronization of spikes is becoming possible with maturation of the myelin. Idiopathic generalized epilepsy is itself modulated by maturation that causes frontal hyperexcitability generating myoclonic-astatic seizures, between the ages of infantile and juvenile myoclonic epilepsies. Physiological delay of hippocampo-neocortical pathways maturation could account for the delayed occurrence of mesial temporal epilepsy following infantile damage, whereas premature maturation could contribute to fronto-temporal damage characteristic of fever-induced epileptic encephalopathy in school-age children, a dramatic school-age epileptic encephalopathy.

  7. An unexpected twist in viral capsid maturation

    SciTech Connect

    Gertsman, Ilya; Gan, Lu; Guttman, Miklos; Lee, Kelly; Speir, Jeffrey A.; Duda, Robert L.; Hendrix, Roger W.; Komives, Elizabeth A.; Johnson, John E.

    2009-04-14

    Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 {angstrom} resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 {angstrom}. A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and -sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol{sup -1} of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic

  8. CHEMERIN (RARRES2) decreases in vitro granulosa cell steroidogenesis and blocks oocyte meiotic progression in bovine species.

    PubMed

    Reverchon, Maxime; Bertoldo, Michael J; Ramé, Christelle; Froment, Pascal; Dupont, Joëlle

    2014-05-01

    CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation. © 2014 by the Society for the Study of Reproduction, Inc.

  9. Complex odontoma.

    PubMed

    Preetha, A; Balikai, Bharati S; Sujatha, D; Pai, Anuradha; Ganapathy, K S

    2010-01-01

    Odontomas are hamartomatous lesions or malformations composed of mature enamel, dentin, and pulp. They may be compound or complex, depending on the extent of morphodifferentiation or their resemblance to normal teeth. The etiology of odontoma is unknown, although several theories have been proposed. This article describes a case of a large infected complex odontoma in the residual mandibular ridge, resulting in considerable mandibular expansion.

  10. Delayed visual maturation.

    PubMed Central

    Cole, G F; Hungerford, J; Jones, R B

    1984-01-01

    Sixteen blind babies who were considered to be showing the characteristics of delayed visual maturation were studied prospectively. The diagnosis was made on clinical grounds, and the criteria for this are discussed. All of these infants developed visual responses between 4 and 6 months of age and had normal or near normal visual acuities by 1 year of age. Long term follow up, however, has shown neurological abnormalities in some of these children. PMID:6200080

  11. Calcium ion currents mediating oocyte maturation events

    PubMed Central

    Tosti, Elisabetta

    2006-01-01

    During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. PMID:16684344

  12. Source rock maturation, San Juan sag

    SciTech Connect

    Gries, R.R.; Clayton, J.L.

    1989-09-01

    Kinetic modeling for thermal histories was simulated for seven wells in the San Juan sag honoring measured geochemical data. Wells in the area of Del Norte field (Sec. 9, T40N, R5E), where minor production has been established from an igneous sill reservoir, show that the Mancos Shale source rocks are in the mature oil generation window as a combined result of high regional heat flow and burial by approximately 2,700 m of Oligocene volcanic rocks. Maturation was relatively recent for this area and insignificant during Laramide subsidence. In the vicinity of Gramps field (Sec. 24, T33N, R2E) on the southwest flank of the San Juan sag, these same source rocks are exposed due to erosion of the volcanic cover but appear to have undergone a similar maturation history. At the north and south margins of the sag, two wells (Champlin 34A-13, Sec. 13, T35N, R4.5E; and Champlin 24A-1, Sec. 1, T44N, R5E) were analyzed and revealed that although the regional heat flow was probably similar to other wells, the depth of burial was insufficient to cause maturation (except where intruded by thick igneous sills that caused localized maturation). The Meridian Oil 23-17 South Fork well (Sec. 17, T39N, R4E) was drilled in a deeper part of the San Juan sag, and source rocks were intruded by numerous igneous sills creating a complex maturation history that includes overmature rocks in the lowermost Mancos Shale, possible CO{sub 2} generation from the calcareous Niobrara Member of the Mancos Shale, and mature source rocks in the upper Mancos Shale.

  13. Ultrastructure of in vitro Matured Human Oocytes.

    PubMed

    Shahedi, Abbas; Khalili, Mohammad Ali; Soleimani, Mehrdad; Morshedizad, Shekoufeh

    2013-12-01

    Approximately 20% of recovered oocytes are immature and discarded in intracytoplasmic sperm injection (ICSI) procedures. These oocytes represent a potential resource for both clinical and basic science application. The aim of this study was to evaluate the ultrastructure architecture of in vitro matured human oocytes using transmission electron microscopy (TEM). A total of 204 immature oocytes from infertile patients who underwent ICSI cycles were included in this prospective study. Immature oocytes were divided into two groups: (i) GV oocytes (n = 101); and (ii) MI oocytes (n = 103). Supernumerary fresh in vivo matured oocytes (n = 10) were used as control. The rates of maturations were 61.38% for GV and 73.78% for MI oocytes in IVM medium (P = 0.07). However, the rate of oocyte arrest was significant between groups (P <0 .05). Ultrastructurally; in vitro and in vivo matured oocytes appeared round, with a homogeneous cytoplasm, an intact oolemma and an intact zona pellucida. However, immature oocytes indicated numerous large mitochondria-vesicle complexes (M-VC). Ultrastructural changes of M-VC in IVM groups emphasize the need for further research in order to refine culture conditions and improve the implantation rate of in-vitro matured oocytes.

  14. Evaluation of equine oocyte developmental competence using polarized light microscopy.

    PubMed

    Bertero, A; Ritrovato, F; Evangelista, F; Stabile, V; Fortina, R; Ricci, A; Revelli, A; Vincenti, L; Nervo, T

    2017-06-01

    The purpose of this study was to observe in vitro-matured equine oocytes with an objective computerized technique that involves the use of a polarized light microscope (PLM) in addition to the subjective morphological evaluation obtained using a classic light microscope (LM). Equine cumulus-oocyte complexes (COCs, n = 922) were subjected to different in vitro maturation times (24, 36 or 45 h), however, only 36-h matured oocytes were analyzed using CLM. The 36-h matured oocytes that reached maturity were parthenogenetically activated to evaluate the quality and meiotic competence. Average maturation percentages per session in groups 1, 2 and 3 (24-, 36- and 45-h matured oocytes respectively) were 29.31 ± 13.85, 47.01 ± 9.90 and 36.62 ± 5.28%, whereas the average percentages of immature oocytes per session were 28.78 ± 20.17, 7.83 ± 5.51 and 22.36 ± 8.39% respectively. The zona pellucida (ZP) birefringent properties were estimated and correlated with activation outcome. ZP thickness and retardance of the inner layer of the zona pellucida (IL-ZP) were significantly increased in immature oocytes compared with mature oocytes (P < 0.001 and P < 0.01 respectively). The comparison between parthenogenetically activated and non-activated oocytes showed a significant increase in the area and thickness of the IL-ZP in parthenogenetically activated oocytes (P < 0.01). These results show that the 36-h in vitro maturation (IVM) protocol allowed equine oocytes to reach maturity, and PLM observation of ZP can be used to distinguish mature and immature oocytes as well as activated and non-activated oocytes. © 2017 Society for Reproduction and Fertility.

  15. Oxygen-regulated gene expression in murine cumulus cells.

    PubMed

    Kind, Karen L; Tam, Kimberley K Y; Banwell, Kelly M; Gauld, Ashley D; Russell, Darryl L; Macpherson, Anne M; Brown, Hannah M; Frank, Laura A; Peet, Daniel J; Thompson, Jeremy G

    2015-01-01

    Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

  16. The Reproductive Toxicity of CdSe/ZnS Quantum Dots on the in vivo Ovarian Function and in vitro Fertilization

    PubMed Central

    Xu, Gaixia; Lin, Guimiao; Lin, Suxia; Wu, Na; Deng, Yueyue; Feng, Gang; Chen, Qiang; Qu, Junle; Chen, Danni; Chen, Siping; Niu, Hanben; Mei, Shujiang; Yong, Ken-Tye; Wang, Xiaomei

    2016-01-01

    Despite the usefulness of quantum dots (QDs) in biomedicine and optoelectronics, their toxicity risks remain a major obstacle for clinical usages. Hence, we studied the reproductive toxicity of CdSe/ZnS QDs on two aspects, (i) in vivo ovarian functions and (ii) in vitro fertilization process. The body weight, estrous cycles, biodistribution of QDs, and oocyte maturation are evaluated on female mice treated with QDs. The mRNA level of the follicle-stimulating hormone receptor (FSHr) and luteinizing hormone receptor (LHr) in ovaries are assayed. Then, the matured cumulus-oocyte-complexes are harvested to co-culture with in vitro capacitated sperms, and the in vitro fertilization is performed. The result revealed that QDs are found in the ovaries, but no changes are detected on the behavior and estrous cycle on the female mice. The mRNA downregulations of FSHr and LHr are observed and the number of matured oocytes has shown a significant decrease when the QDs dosage was above 1.0 pmol/day. Additionally, we found the presence of QDs has reduced the in vitro fertilization success rate. This study highly suggests that the exposure of CdSe/ZnS QDs to female mice can cause adverse effects to the ovary functions and such QDs may have limited applications in clinical usage. PMID:27876896

  17. Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations.

    PubMed

    Van Hoeck, V; Rizos, D; Gutierrez-Adan, A; Pintelon, I; Jorssen, E; Dufort, I; Sirard, M A; Verlaet, A; Hermans, N; Bols, P E J; Leroy, J L M R

    2015-01-01

    Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus-oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (=control); (2) elevated (75 µM) stearic acid (SA) concentrations (=HIGH SA); and (3) elevated (425 µM) NEFA concentrations (=HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.

  18. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  19. Capability Maturity Model for Software,

    DTIC Science & Technology

    1991-08-01

    This paper provides a technical overview of the Capability Maturity Model for Software and reflects the most current version. Specifically, this...paper, in combination with the Key Practices of the Capability Maturity Model , is intended to help software organizations use the CMM as a guide to improve the maturity of their software process.

  20. Behaviour of cytoplasmic organelles and cytoskeleton during oocyte maturation.

    PubMed

    Mao, Luna; Lou, Hangying; Lou, Yiyun; Wang, Ning; Jin, Fan

    2014-03-01

    Assisted reproduction technology (ART) has become an attractive option for infertility treatment and holds tremendous promise. However, at present, there is still room for improvement in its success rates. Oocyte maturation is a process by which the oocyte becomes competent for fertilization and subsequent embryo development. To better understand the mechanism underlying oocyte maturation and for the future improvement of assisted reproduction technology, this review focuses on the complex processes of cytoplasmic organelles and the dynamic alterations of the cytoskeleton that occur during oocyte maturation. Ovarian stimulation and in-vitro maturation are the major techniques used in assisted reproduction technology and their influence on the organelles of oocytes is also discussed. Since the first birth by assisted reproduction treatment was achieved in 1978, numerous techniques involved in assisted reproduction have been developed and have become attractive options for infertility treatment. However, the unsatisfactory success rate remains as a main challenge. Oocyte maturation is a process by which the oocyte becomes competent for fertilization and subsequent embryo development. Oocyte maturation includes both nuclear and cytoplasmic maturation. Nuclear maturation primarily involves chromosomal segregation, which has been well studied, whereas cytoplasmic maturation involves a series of complicated processes, and there are still many parts of this process that remain controversial. Ovarian stimulation and in-vitro maturation (IVM) are the major techniques of assisted reproduction. The effect of ovarian stimulation or IVM on the behaviour of cell organelles of the oocyte has been postulated as the reason for the reduced developmental potential of in-vitro-produced embryos. To further understanding of the mechanism of oocyte maturation and future improvement of assisted reproduction treatment, the complex events of cytoplasmic organelles and the cytoskeleton that

  1. Hydrocarbon maturation in thrust belts: Thermal considerations

    NASA Astrophysics Data System (ADS)

    Furlong, Kevin P.; Edman, Janell D.

    Sedimentary strata within thrust belts experience transient thermal histories which perturb the maturation paths of organic material contained within the rocks. Calculation of the thermal history, including perturbations which occur with overthrusting, for a particular sequence of tectonic events, allows us to evaluate the timing of maturation reactions and the remaining generative potential in the source rock during the evolution of the geologic terrain. In addition, thermal-maturation indicators can be used to constrain tectonic models for a region and eliminate nonviable geologic interpretations. We have utilized a numerical model to evaluate the thermal response to burial, erosion and thrusting. This model allows us to specify reasonably complex (and geologically reasonable) tectonic histories, including time varying erosion and sedimentation, syn-thrusting erosion, and multiple thrusting events. Such complexities are not easily incorporated in analytic thermal models of thrust belt evolution. In case studies of the western overthrust belt of Wyoming, thermal modeling of geologic histories provides insight into maturation processes, timing and geometries of thrust sheets, and pre-thrusting tectonism. In particular, the timing of thrust sheet motion in the Wyoming portion of the thrust belt appears to be younger than normally thought. Much of the thin skinned thrusting evaluated here appears (from a thermal perspective) to be of similar age to Laramide thrusting in the region.

  2. Dissociation of motor maturation.

    PubMed

    DiMario, Francis J

    2003-06-01

    We prospectively acquired clinical data regarding the presentation, evaluation, and developmental progress of all patients identified with dissociated motor maturation to define their clinical outcomes. Children (N = 8) referred for evaluation of suspected cerebral palsy because of delayed sitting or walking and identified to have dissociated motor maturation were followed with serial clinical examination. All displayed the characteristic "sitting on air" posture while held in vertical suspension and had otherwise normal developmental assessments. This posture is composed of the hips held in flexion and abduction with the knees extended and feet plantar or dorsiflexed. Three children were initially evaluated at 10 months of age owing to absence of sitting and five other children were evaluated at a mean of 14 months (range 12-19 months) owing to inability to stand. Follow-up evaluations were conducted over a mean of 10.5 months (range 5-34 months). Five children were born prematurely at 34 to 36 weeks gestation. Denver Developmental Screening Test and general and neurologic examinations were normal except to note hypotonia in six children and the "sitting on air" posture in all of the children. Four children have older siblings or parents who "walked late" (after 15 months). On average, the children attained sitting by 8 months (range 7-10 months). One child did not crawl prior to independent walking, two children scooted rather than crawled, and five children crawled at an average of 13.5 months (range 10-16 months). All children cruised by a mean of 18 months (range 16-21.5 months) and attained independent walking by 20.1 months (range 18-25 months). Neuroimaging and serum creatine kinase enzyme testing were normal in two children who were tested. These eight children conform to the syndrome of dissociated motor maturation. The "sitting on air" posture serves as a diagnostic sign and anticipated excellent prognosis, but follow-up is required to ensure a normal

  3. Maturation in Larch 1

    PubMed Central

    Hutchison, Keith W.; Sherman, Christopher D.; Weber, Jill; Smith, Sandra Schiller; Singer, Patricia B.; Greenwood, Michael S.

    1990-01-01

    The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO2 per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS

  4. Pitch perception prior to cortical maturation

    NASA Astrophysics Data System (ADS)

    Lau, Bonnie K.

    Pitch perception plays an important role in many complex auditory tasks including speech perception, music perception, and sound source segregation. Because of the protracted and extensive development of the human auditory cortex, pitch perception might be expected to mature, at least over the first few months of life. This dissertation investigates complex pitch perception in 3-month-olds, 7-month-olds and adults -- time points when the organization of the auditory pathway is distinctly different. Using an observer-based psychophysical procedure, a series of four studies were conducted to determine whether infants (1) discriminate the pitch of harmonic complex tones, (2) discriminate the pitch of unresolved harmonics, (3) discriminate the pitch of missing fundamental melodies, and (4) have comparable sensitivity to pitch and spectral changes as adult listeners. The stimuli used in these studies were harmonic complex tones, with energy missing at the fundamental frequency. Infants at both three and seven months of age discriminated the pitch of missing fundamental complexes composed of resolved and unresolved harmonics as well as missing fundamental melodies, demonstrating perception of complex pitch by three months of age. More surprisingly, infants in both age groups had lower pitch and spectral discrimination thresholds than adult listeners. Furthermore, no differences in performance on any of the tasks presented were observed between infants at three and seven months of age. These results suggest that subcortical processing is not only sufficient to support pitch perception prior to cortical maturation, but provides adult-like sensitivity to pitch by three months.

  5. Correlation between dental maturity and cervical vertebral maturity.

    PubMed

    Chen, Jianwei; Hu, Haikun; Guo, Jing; Liu, Zeping; Liu, Renkai; Li, Fan; Zou, Shujuan

    2010-12-01

    The aim of this study was to investigate the association between dental and skeletal maturity. Digital panoramic radiographs and lateral skull cephalograms of 302 patients (134 boys and 168 girls, ranging from 8 to 16 years of age) were examined. Dental maturity was assessed by calcification stages of the mandibular canines, first and second premolars, and second molars, whereas skeletal maturity was estimated by the cervical vertebral maturation (CVM) stages. The Spearman rank-order correlation coefficient was used to measure the association between CVM stage and dental calcification stage of individual teeth. The mean chronologic age of girls was significantly lower than that of boys in each CVM stage. The Spearman rank-order correlation coefficients between dental maturity and cervical vertebral maturity ranged from 0.391 to 0.582 for girls and from 0.464 to 0.496 for boys (P < 0.05). In girls, the mandibular second molar had the highest and the canine the lowest correlation. In boys, the canine had the highest and the first premolar the lowest correlation. Tooth calcification stage was significantly correlated with cervical vertebral maturation stage. The development of the mandibular second molar in females and that of the mandibular canine in males had the strongest correlations with cervical vertebral maturity. Therefore, it is practical to consider the relationship between dental and skeletal maturity when planning orthodontic treatment. Copyright © 2010 Mosby, Inc. All rights reserved.

  6. CFD - Mature Technology?

    NASA Technical Reports Server (NTRS)

    Kwak, Dochan

    2005-01-01

    Over the past 30 years, numerical methods and simulation tools for fluid dynamic problems have advanced as a new discipline, namely, computational fluid dynamics (CFD). Although a wide spectrum of flow regimes are encountered in many areas of science and engineering, simulation of compressible flow has been the major driver for developing computational algorithms and tools. This is probably due to a large demand for predicting the aerodynamic performance characteristics of flight vehicles, such as commercial, military, and space vehicles. As flow analysis is required to be more accurate and computationally efficient for both commercial and mission-oriented applications (such as those encountered in meteorology, aerospace vehicle development, general fluid engineering and biofluid analysis) CFD tools for engineering become increasingly important for predicting safety, performance and cost. This paper presents the author's perspective on the maturity of CFD, especially from an aerospace engineering point of view.

  7. Mechanisms of Hierarchical Cortical Maturation

    PubMed Central

    Chomiak, Taylor; Hu, Bin

    2017-01-01

    Cortical information processing is structurally and functionally organized into hierarchical pathways, with primary sensory cortical regions providing modality specific information and associative cortical regions playing a more integrative role. Historically, there has been debate as to whether primary cortical regions mature earlier than associative cortical regions, or whether both primary and associative cortical regions mature simultaneously. Identifying whether primary and associative cortical regions mature hierarchically or simultaneously will not only deepen our understanding of the mechanisms that regulate brain maturation, but it will also provide fundamental insight into aspects of adolescent behavior, learning, neurodevelopmental disorders and computational models of neural processing. This mini-review article summarizes the current evidence supporting the sequential and hierarchical nature of cortical maturation, and then proposes a new cellular model underlying this process. Finally, unresolved issues associated with hierarchical cortical maturation are also addressed. PMID:28959187

  8. Polyadenylated tail length variation pattern in ultra-rapid vitrified bovine oocytes

    PubMed Central

    Dutta, D. J.; Raj, Himangshu; Dev, and Hiramoni

    2016-01-01

    Aim: Thecurrent study aims at investigating the polyadenylated (poly[A]) tail length of morphologically high and low competent oocytes at different developmental stages. Furthermore, effect of ultra-rapid vitrification on the poly(A) tail length was studied. Materials and Methods: Fresh bovine cumulus oocyte complexes from abattoir originated ovaries were graded based on morphological characters and matured in vitro. Cryopreservation was done by ultra-rapid vitrification method. mRNA was isolated from different categories of oocyte and subjected to ligation-mediated poly(A) test followed by polymerase chain reaction for determining the poly(A) tail length of β actin, gap junction protein alpha 1 (GJA1), poly(A) polymerase alpha (PAPOLA), and heat shock 70 kDa protein (HSP70) transcripts. Results: GJA1, PAPOLA, and HSP70 showed significantly higher poly(A) in immature oocytes of higher competence irrespective of vitrification effects as compared to mature oocytes of higher competence. Conclusion: mRNA poly(A) tail size increases in developmentally high competent immature bovine oocytes. There was limited effect of ultra-rapid vitrification of bovine oocytes on poly(A). PMID:27847415

  9. Are oocytes from the anestrous bitch competent for meiosis?

    PubMed

    Chastant-Maillard, S; Saint-Dizier, M; Grimard, B; Chebrout, M; Thoumire, S; Reynaud, K

    2012-12-01

    In the bitch, oocyte meiosis resumption takes place in the oviduct. Using oocytes from anestrous bitches, in vitro maturation (IVM) generally gives very poor results. To investigate the contribution of oocyte competence to the low IVM yield, we compared in vivo maturation in an optimal environment with conventional IVM. A total of 418 grade 1 cumulus-oocyte complexes (COCs) from 10 anestrous bitches were transferred into the oviducts of recipient bitches either on Day -1 (n = 3 recipients), Day 0 (n = 2) or on Day +1 (n = 2) relative to ovulation. For each donor bitch, 20 grade 1 COCs were also cultured in vitro. After 72 h of in vivo or IVM, the nuclear stage of oocytes was determined after DNA and tubulin staining. Of the 154 oocytes recovered and examined after intratubal transfer, only 2% reached the metaphase I or II stage and 38.3% were degenerated. Oocytes cultured in vitro displayed a higher metaphase rate (7.6%, n = 170) and lower degeneration rate (12.9%) compared with transferred oocytes (p < 0.001). These results clearly demonstrate that the oocyte competence is the major limiting factor of IVM efficiency in the dog. Mimicking the tubal environment may thus not be sufficient to increase IVM yield in this species.

  10. Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals.

    PubMed

    Prochazka, Radek; Blaha, Milan

    2015-01-01

    In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.

  11. Negative energy balance affects imprint stability in oocytes recovered from postpartum dairy cows.

    PubMed

    O'Doherty, Alan M; O'Gorman, Aoife; al Naib, Abdullah; Brennan, Lorraine; Daly, Edward; Duffy, Pat; Fair, Trudee

    2014-09-01

    Ovarian follicle development in post-partum, high-producing dairy cows, occurs in a compromised endogenous metabolic environment (referred to as negative energy balance, NEB). Key events that occur during oocyte/follicle growth, such as the vital process of genomic imprinting, may be detrimentally affected by this altered ovarian environment. Imprinting is crucial for placental function and regulation of fetal growth, therefore failure to establish and maintain imprints during oocyte growth may contribute to early embryonic loss. Using ovum pick-up (OPU), oocytes and follicular fluid samples were recovered from cows between days 20 and 115 post-calving, encompassing the NEB period. In a complimentary study, cumulus oocyte complexes were in vitro matured under high non-esterified fatty acid (NEFA) concentrations and in the presence of the methyl-donor S-adenosylmethionine (SAM). Pyrosequencing revealed the loss of methylation at several imprinted loci in the OPU derived oocytes. The loss of DNA methylation was observed at the PLAGL1 locus in oocytes, following in vitro maturation (IVM) in the presence of elevated NEFAs and SAM. Finally, metabolomic analysis of postpartum follicular fluid samples revealed significant differences in several branched chain amino acids, with fatty acid profiles bearing similarities to those characteristic of lactating dairy cows. These results provide the first evidence that (1) the postpartum ovarian environment may affect maternal imprint acquisition and (2) elevated NEFAs during IVM can lead to the loss of imprinted gene methylation in bovine oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Estrogen receptors in granulosa cells govern meiotic resumption of pre-ovulatory oocytes in mammals.

    PubMed

    Liu, Wei; Xin, Qiliang; Wang, Xiao; Wang, Sheng; Wang, Huarong; Zhang, Wenqiang; Yang, Ye; Zhang, Yanhao; Zhang, Zhiyuan; Wang, Chao; Xu, Yang; Duan, Enkui; Xia, Guoliang

    2017-03-09

    In mammals, oocytes are arrested at the diplotene stage of meiosis I until the pre-ovulatory luteinizing hormone (LH) surge triggers meiotic resumption through the signals in follicular granulosa cells. In this study, we show that the estradiol (E2)-estrogen receptors (ERs) system in follicular granulosa cells has a dominant role in controlling oocyte meiotic resumption in mammals. We found that the expression of ERs was controlled by gonadotropins under physiological conditions. E2-ERs system was functional in maintaining oocyte meiotic arrest by regulating the expression of natriuretic peptide C and natriuretic peptide receptor 2 (NPPC/NPR2), which was achieved through binding to the promoter regions of Nppc and Npr2 genes directly. In ER knockout mice, meiotic arrest was not sustained by E2 in most cumulus-oocyte complexes in vitro and meiosis resumed precociously in pre-ovulatory follicles in vivo. In human granulosa cells, similar conclusions are reached that ER levels were controlled by gonadotropins and E2-ERs regulated the expression of NPPC/NPR2 levels. In addition, our results revealed that the different regulating patterns of follicle-stimulating hormone and LH on ER levels in vivo versus in vitro determined their distinct actions on oocyte maturation. Taken together, these findings suggest a critical role of E2-ERs system during oocyte meiotic progression and may propose a novel approach for oocyte in vitro maturation treatment in clinical practice.

  13. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  14. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  15. Smart Grid Interoperability Maturity Model

    SciTech Connect

    Widergren, Steven E.; Levinson, Alex; Mater, J.; Drummond, R.

    2010-04-28

    The integration of automation associated with electricity resources (including transmission and distribution automation and demand-side resources operated by end-users) is key to supporting greater efficiencies and incorporating variable renewable resources and electric vehicles into the power system. The integration problems faced by this community are analogous to those faced in the health industry, emergency services, and other complex communities with many stakeholders. To highlight this issue and encourage communication and the development of a smart grid interoperability community, the GridWise Architecture Council (GWAC) created an Interoperability Context-Setting Framework. This "conceptual model" has been helpful to explain the importance of organizational alignment in addition to technical and informational interface specifications for "smart grid" devices and systems. As a next step to building a community sensitive to interoperability, the GWAC is investigating an interoperability maturity model (IMM) based on work done by others to address similar circumstances. The objective is to create a tool or set of tools that encourages a culture of interoperability in this emerging community. The tools would measure status and progress, analyze gaps, and prioritize efforts to improve the situation.

  16. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes surrounded by cumulus cells connected to membrana granulosa.

    PubMed

    van Tol, H T; van Eijk, M J; Mummery, C L; van den Hurk, R; Bevers, M M

    1996-10-01

    Cumulus oocyte complexes (COCs) and cumulus oocyte complexes connected to a piece of the membrane granulosa (COCGs) were isolated from bovine antral follicles with a diameter of 2 to 8 mm. After culture of COCGs without gonadotrophic hormones for 22 hr approximately 50% of the oocytes were still in the germinal vesicle (GV) stage. Histology of the COCGs showed that the pieces of the membrana granulosa were free of thecal cells and parts of the basal membrane. This indicates that the membrana granulosa solely inhibits the progression of meiosis. To investigate the effect of gonadotropins on the resumption of meiosis of oocytes from small and medium sized antral follicles, COCs and COCGs were cultured with or without rec-hFSH or hCG. Addition of 0.05 IU rec-hFSH to the culture medium of COCGs resulted in germinal vesicle breakdown in 97.8% of the oocytes compared to 46% in the control group, and an increase of the diameter of the COCs (479 microns vs. 240 microns in the control group). Addition of 0.05 IU hCG to the culture medium had no effect on nuclear maturation (47.2% GV vs. 48.5% GV in the control group) nor on cumulus expansion (246 microns vs. 240 microns in the control group). RT-PCR on cDNA of the follicular wall, cumulus cells, granulosa cells, COCs, and oocytes revealed that mRNA for FSH receptor was present in all cell types except oocytes. mRNA of the LH receptor was detected exclusively in thecal cells. Nucleotide sequence analysis and alignment of the cloned PCR products showed the presence of two isoforms of the FSH receptor mRNA and two isoforms of the LH receptor mRNA. It is concluded that, in vitro, resumption of meiosis of oocytes, originating from small and medium sized antral follicles and meiotically arrested by the membrana granulosa, is triggered by FSH and not by LH. This is supported by the fact that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of these follicles.

  17. Ovum pick up and in vitro embryo production in cows superstimulated with an individually adapted superstimulation protocol.

    PubMed

    De Roover, R; Genicot, G; Leonard, S; Bols, P; Dessy, F

    2005-03-01

    The aim of this experiment was to apply an ovarian superstimulation protocol prior to ovum pick up (OPU), tailored to the individual donor response, to evaluate its advantages and disadvantages in terms of follicle numbers and diameters, the numbers of retrieved oocytes and day 7 cultured blastocysts. Ten adult non-lactating dairy cows were superstimulated with pFSH and subjected to ovum pick up-in vitro fertilisation (OPU-IVF) 6 times at 2-week intervals. On day 0 of each 2-week period, all follicles >8mm were ablated and an ear implant (Crestar, Intervet, Belgium) was inserted. On day 2, 48 h after follicle ablation the animals were administered six equal doses of pFSH, divided into morning and evening doses for 3 days. On day 7, 48 h following the last pFSH injection, follicle diameters were measured by ultrasound and all follicles were subjected to OPU. All cumulus-oocyte complexes (COC), regardless of their quality, were subjected to in vitro maturation-in vitro fertilisation-in vitro culture (IVM-IVF-IVC). The total dose of pFSH prior to the first OPU session was 300 microg per animal. During the following OPU sessions, the total pFSH dose was either kept unchanged, increased or reduced (+/-50 microg), according to the percentage of follicles of more than 11 mm in diameter, present in the previous session of that particular donor. The mean number of punctured follicles per session was 11.9 +/- 7.7 (mean +/- S.D.), with 16% of follicles exceeding 11 mm. These follicles yielded a mean of 5.6 +/- 4.1 cumulus oocyte complexes (COC), 32% of which had >/=3 layers of cumulus cells (quality 1 and 2). The recovery rate was 47%. Finally, all COC were subjected to IVM-IVF-IVC, which resulted in a mean of 2.0 +/- 2.3 blastocysts on day 7 postinsemination. The subtle changes in pFSH dose influenced the sizes but not the numbers of follicles, the latter parameter was influenced by the individual donor and the OPU session.

  18. The Mature Athlete

    PubMed Central

    McCarthy, Moira M.; Hannafin, Jo A.

    2014-01-01

    Context: Aging changes the biology, healing capacity, and biomechanical function of tendons and ligaments and results in common clinical pathologies that present to orthopedic surgeons, primary care physicians, physical therapists, and athletic trainers. A better understanding of the age-related changes in these connective tissues will allow better patient care. Evidence Acquisition: The PubMed database was searched in December 2012 for English-language articles pertaining to age-related changes in tendons and ligaments. Level of Evidence: Level 5. Results: The mature athlete faces challenges associated with age-dependent changes in the rotator cuff, Achilles tendon, lateral humeral epicondylar tendons, quadriceps tendon, and patellar tendon. The anterior cruciate ligament and the medial collateral ligament are the most studied intra-articular and extra-articular ligaments, and both are associated with age-dependent changes. Conclusion: Tendons and ligaments are highly arranged connective tissue structures that maintain joint motion and joint stability. These structures are subject to vascular and compositional changes with increasing age that alter their mechanotransduction, biology, healing capacity, and biomechanical function. Emerging research into the etiology of age-dependent changes will provide further information to help combat the age-related clinical complications associated with the injuries that occur to tendons and ligaments. PMID:24427441

  19. Career Maturity of Welfare Recipients.

    ERIC Educational Resources Information Center

    Beckman, Carol M.

    To investigate the career maturity of welfare recipients, this thesis examines six independent variables: (1) race; (2) sex; (3) age; (4) level of formal education; (5) general intelligence; and (6) locus of control. Scales taken from the Career Maturity Inventory served as the dependent variables. The sample consisted of 83 welfare recipients who…

  20. Treating mature stands for wildlife

    Treesearch

    William H. Healy; Gary F. Houf

    1989-01-01

    Stands older than 60 years or that are medium to large sawtimber size generally provide good wildlife habitat. Mature trees usually produce abundant mast and provide den sites (see fig. 1 in Note 9.04 Treating Immature Stands). The undergrowth in these stands produces moderate amounts of browse and herbage. Mature stands also provide opportunities for management...

  1. Financial maturity of yellow birch

    Treesearch

    William B. Leak

    1969-01-01

    The methods used to compute financial maturity of yellow birch sawtimber are similar to those used for paper birch sawtimber, except for minor differences in detail. The procedure followed for yellow-birch veneer-log trees was also similar, except that local veneer grades and local veneer-log prices were used as the basis for the financial maturity computations.

  2. Career Education and Career Maturity.

    ERIC Educational Resources Information Center

    Trebilco, Geoffrey R.

    1984-01-01

    Investigated the relationships between career maturity and career curriculum in 38 Melbourne metropolitan secondary schools (N=2280 students) using an Australian adaption of the Career Development Inventory. Results confirmed that schools with career education programs achieved higher gains in student career maturity. (JAC)

  3. Role of COPI in phagosome maturation.

    PubMed

    Botelho, R J; Hackam, D J; Schreiber, A D; Grinstein, S

    2000-05-26

    Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

  4. Decreased expression of tumor necrosis factor-alpha-stimulated gene 6 in cumulus cells of the cyclooxygenase-2 and EP2 null mice.

    PubMed

    Ochsner, Scott A; Russell, Darryl L; Day, Anthony J; Breyer, Richard M; Richards, Joanne S

    2003-03-01

    Ovulation, the release of fertilizable oocytes from mature follicles, involves tissue remodeling and increased prostaglandin (PG) signaling. Cyclooxygenase (COX)-2 is the rate-limiting enzyme during PG synthesis. Female mice null for either COX-2 or the PGE(2) receptor EP2 are infertile, show decreased ovulation, and exhibit abnormal cumulus expansion. Cumulus expansion is the production of a complex extracellular matrix surrounding the cumulus-oocyte complex (COC). Matrix components consist of hyaluronan, proteoglycans, and proteins with hyaluronan binding domains. One such hyaluronan binding protein is TNFalpha-stimulated gene 6 (TSG-6). By various methods, we show induction of TSG-6 and hyaluronan synthase-2 mRNA in ovaries of mice treated with pregnant mare serum gonadotropin and human chorionic gonadotropin. By in situ hybridization, we show that both genes are expressed in periantral mural granulosa cells and cumulus cells of the mouse ovary. Notably, RT-PCR and in situ hybridization show that TSG-6 mRNA but not hyaluronan synthase-2 mRNA expression is selectively reduced in cumulus cells of COX-2 and EP2 null mice. Western analysis further confirms that TSG-6 protein is reduced in isolated COCs but remains covalently associated with inter alpha-trypsin inhibitor in COX-2 null mice. These observations identify TSG-6 as a target of PG action and show that its production in ovulatory follicles is associated with proper formation of the cumulus-derived extracellular matrix.

  5. Extracellular Vesicles from Bovine Follicular Fluid Support Cumulus Expansion.

    PubMed

    Hung, Wei-Ting; Hong, Xioman; Christenson, Lane K; McGinnis, Lynda K

    2015-11-01

    Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation-inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present; however, no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on cumulus-oocyte complex (COC) expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3-5 mm) and large (>9 mm) antral bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy, and Western blot analysis. To test for bioactivity, mouse and bovine COCs were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression.

  6. Extracellular Vesicles from Bovine Follicular Fluid Support Cumulus Expansion1

    PubMed Central

    Hung, Wei-Ting; Hong, Xioman; Christenson, Lane K.; McGinnis, Lynda K.

    2015-01-01

    Expansion of the cumulus complex surrounding the oocyte is critical for ovulation of a fertilizable egg. The ovulation-inducing surge of luteinizing hormone leads to an increased expression of genes such as prostaglandin-endoperoxide synthase 2 (Ptgs2), pentraxin-related protein 3 (Ptx3), and tumor necrosis factor alpha-induced protein 6 (Tnfaip6) that support cumulus expansion. Factors released by mural granulosa and cumulus granulosa cells into the follicular fluid induce paracrine signaling within the follicular compartment. The follicular fluid that separates these distinct granulosa cell types is an enriched fluid containing numerous proteins, nucleic acids, and other macromolecules. Extracellular vesicles (EVs) are also present; however, no physiologically relevant functions of follicular EVs have yet been demonstrated. In our study, the effect of follicular EVs on cumulus-oocyte complex (COC) expansion and relevant gene expression was assayed. Follicular EVs were isolated using ultracentrifugation from follicular fluid of small (3–5 mm) and large (>9 mm) antral bovine follicles, then characterized by nanoparticle tracking analysis, electron microscopy, and Western blot analysis. To test for bioactivity, mouse and bovine COCs were cultured with follicular EVs. Cumulus expansion and Ptgs2, Ptx3, and Tnfaip6 gene expression were measured following COC maturation culture. The results demonstrated that follicular EVs can support both measurable cumulus expansion and increased gene expression. PMID:26423123

  7. The effects of roscovitine on cumulus cell apoptosis and the developmental competence of domestic cat oocytes.

    PubMed

    Sananmuang, T; Techakumphu, M; Tharasanit, T

    2010-01-15

    The developmental competence of cat oocytes matured in vitro is relatively poor when compared with that of in vivo oocytes. The study aimed to investigate the effect of roscovitine on the developmental competence of cat Felis catus oocytes matured in vitro. Cumulus-oocyte complexes (COCs) were classified as Grade I and II to III. Groups of COCs were cultured in 0, 12.5, 25, 50, 100, and 200 microM roscovitine for 24h and were either fixed to assess the stages of nuclear maturation (Experiment 1) or additionally matured in vitro for 24h before fixation (Experiment 2). In Experiment 3, cumulus cells from the COCs treated with roscovitine were examined for apoptosis. Experiment 4 examined the developmental competence of cat oocytes after roscovitine treatment and in vitro fertilization in terms of cleavage and morula and blastocyst formation rates. Roscovitine reversibly arrested cat oocytes at an immature stage in a dose-dependent manner. Roscovitine at 12.5 and 25 microM demonstrated less efficiency compared with that of other doses. However, higher doses of roscovitine induced cumulus cell apoptosis and resulted in a high number of degenerated oocytes after in vitro maturation. Roscovitine at 12.5 and 25 microM were therefore used to evaluate their effect on embryo development. Pretreatment with 12.5 and 25 microM roscovitine prior to in vitro maturation decreased the developmental competence of cat oocytes compared with that of non-roscovitine-treated controls. In conclusion, roscovitine reversibly maintained cat oocytes at the germinal vesicle stage without detrimental effect on nuclear maturation. However, it negatively affected cumulus cell viability and developmental competence.

  8. Feasibility of Optimizing Recovery and Reserves from a Mature and Geological Complex Multiple Turbidite Offshore Calif. Reservoir through the Drilling and Completion of a Trilateral Horizontal Well, Class III

    SciTech Connect

    Pacific Operators Offshore, Inc.

    2001-04-04

    The intent of this project was to increase production and extend the economic life of this mature field through the application of advanced reservoir characterization and drilling technology, demonstrating the efficacy of these technologies to other small operators of aging fields. Two study periods were proposed; the first to include data assimilation and reservoir characterization and the second to drill the demonstration well. The initial study period showed that a single tri-lateral well would not be economically efficient in redevelopment of Carpinteria's multiple deep water turbidite sand reservoirs, and the study was amended to include the drilling of a series of horizontal redrills from existing surplus well bores on Pacific Operators' Platform Hogan.

  9. Predictive Capability Maturity Model (PCMM).

    SciTech Connect

    Swiler, Laura Painton; Knupp, Patrick Michael; Urbina, Angel

    2010-10-01

    Predictive Capability Maturity Model (PCMM) is a communication tool that must include a dicussion of the supporting evidence. PCMM is a tool for managing risk in the use of modeling and simulation. PCMM is in the service of organizing evidence to help tell the modeling and simulation (M&S) story. PCMM table describes what activities within each element are undertaken at each of the levels of maturity. Target levels of maturity can be established based on the intended application. The assessment is to inform what level has been achieved compared to the desired level, to help prioritize the VU activities & to allocate resources.

  10. The AGU Data Management Maturity Model Initiative

    NASA Astrophysics Data System (ADS)

    Bates, J. J.

    2015-12-01

    In September 2014, the AGU Board of Directors approved two initiatives to help the Earth and space sciences community address the growing challenges accompanying the increasing size and complexity of data. These initiatives are: 1) Data Science Credentialing: development of a continuing education and professional certification program to help scientists in their careers and to meet growing responsibilities and requirements around data science; and 2) Data Management Maturity (DMM) Model: development and implementation of a data management maturity model to assess process maturity against best practices, and to identify opportunities in organizational data management processes. Each of these has been organized within AGU as an Editorial Board and both Boards have held kick off meetings. The DMM model Editorial Board will recommend strategies for adapting and deploying a DMM model to the Earth and space sciences create guidance documents to assist in its implementation, and provide input on a pilot appraisal process. This presentation will provide an overview of progress to date in the DMM model Editorial Board and plans for work to be done over the upcoming year.

  11. Epigenetic mechanisms in pubertal brain maturation

    PubMed Central

    Morrison, Kathleen E.; Rodgers, Ali B.; Morgan, Christopher P.; Bale, Tracy L.

    2014-01-01

    Puberty is a critical period of development during which the reemergence of gonadotropin releasing hormone secretion from the hypothalamus triggers a cascade of hormone-dependent processes. Maturation of specific brain regions including the prefrontal cortex occurs during this window, but the complex mechanisms underlying these dynamic changes are not well understood. Particularly, the potential involvement of epigenetics in this programming has been under-examined. The epigenome is known to guide earlier stages of development, and it is similarly poised to regulate vital pubertal-driven brain maturation. Further, as epigenetic machinery is highly environmentally responsive, its involvement may also lend this period of growth to greater vulnerability to external insults, resulting in reprogramming and increased disease risk. Importantly, neuropsychiatric diseases commonly present in individuals during or immediately following puberty, and environmental perturbations including stress may precipitate disease onset by disrupting the normal trajectory of pubertal brain development via epigenetic mechanisms. In this review, we discuss epigenetic processes involved in pubertal brain maturation, the potential points of derailment, and the importance of future studies for understanding this dynamic developmental window and gaining a better understanding of neuropsychiatric disease risk. PMID:24239720

  12. Transcriptional Programs Controlling Perinatal Lung Maturation

    PubMed Central

    Xu, Yan; Wang, Yanhua; Besnard, Valérie; Ikegami, Machiko; Wert, Susan E.; Heffner, Caleb; Murray, Stephen A.; Donahue, Leah Rae; Whitsett, Jeffrey A.

    2012-01-01

    The timing of lung maturation is controlled precisely by complex genetic and cellular programs. Lung immaturity following preterm birth frequently results in Respiratory Distress Syndrome (RDS) and Broncho-Pulmonary Dysplasia (BPD), which are leading causes of mortality and morbidity in preterm infants. Mechanisms synchronizing gestational length and lung maturation remain to be elucidated. In this study, we designed a genome-wide mRNA expression time-course study from E15.5 to Postnatal Day 0 (PN0) using lung RNAs from C57BL/6J (B6) and A/J mice that differ in gestational length by ∼30 hr (B6maturation. We identified both temporal and strain dependent gene expression patterns during lung maturation. For time dependent changes, cell adhesion, vasculature development, and lipid metabolism/transport were major bioprocesses induced during the saccular stage of lung development at E16.5–E17.5. CEBPA, PPARG, VEGFA, CAV1 and CDH1 were found to be key signaling and transcriptional regulators of these processes. Innate defense/immune responses were induced at later gestational ages (E18.5–20.5), STAT1, AP1, and EGFR being important regulators of these responses. Expression of RNAs associated with the cell cycle and chromatin assembly was repressed during prenatal lung maturation and was regulated by FOXM1, PLK1, chromobox, and high mobility group families of transcription factors. Strain dependent lung mRNA expression differences peaked at E18.5. At this time, mRNAs regulating surfactant and innate immunity were more abundantly expressed in lungs of B6 (short gestation) than in A/J (long gestation) mice, while expression of genes involved in chromatin assembly and histone modification were expressed at lower levels in B6 than in A/J mice. The present study systemically mapped key regulators, bioprocesses

  13. Maturation of sugar maple seed

    Treesearch

    Clayton M., Jr. Carl; Albert G., Jr. Snow; Albert G. Snow

    1971-01-01

    The seeds of a sugar maple tree (Acer saccharum Marsh.) do not mature at the same time every year. And different trees mature their seeds at different times. So time of year is not a reliable measure of when seeds are ripe. Better criteria are needed. In recent studies we have found that moisture content and color are the best criteria for judging when sugar maple...

  14. Naturally Engineered Maturation of Cardiomyocytes

    PubMed Central

    Scuderi, Gaetano J.; Butcher, Jonathan

    2017-01-01

    Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This

  15. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the normal...

  16. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the normal...

  17. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the normal...

  18. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure...

  19. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure...

  20. Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients

    PubMed Central

    Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

    2016-01-01

    Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women. PMID:28004769

  1. AMPK: a master energy regulator for gonadal function

    PubMed Central

    Bertoldo, Michael J.; Faure, Melanie; Dupont, Joëlle; Froment, Pascal

    2015-01-01

    From C. elegans to mammals (including humans), nutrition and energy metabolism significantly influence reproduction. At the cellular level, some detectors of energy status indicate whether energy reserves are abundant (obesity), or poor (diet restriction). One of these detectors is AMPK (5′ AMP-activated protein kinase), a protein kinase activated by ATP deficiency but also by several natural substances such as polyphenols or synthetic molecules like metformin, used in the treatment of insulin resistance. AMPK is expressed in muscle and liver, but also in the ovary and testis. This review focuses on the main effects of AMPK identified in gonadal cells. We describe the role of AMPK in gonadal steroidogenesis, in proliferation and survival of somatic gonadal cells and in the maturation of oocytes or spermatozoa. We discuss also the role of AMPK in germ and somatic cell interactions within the cumulus-oocyte complex and in the blood testis barrier. Finally, the interface in the gonad between AMPK and modification of metabolism is reported and discussion about the role of AMPK on fertility, in regards to the treatment of infertility associated with insulin resistance (male obesity, polycystic ovary syndrome). PMID:26236179

  2. The presence of corpus luteum may have a negative impact on in vitro developmental competency of bovine oocytes.

    PubMed

    Hajarian, Hadi; Shahsavari, Mohammad H; Karami-shabankareh, Hamed; Dashtizad, Mojtaba

    2016-03-01

    The aim of the current study was to investigate the effects of the presence or absence of corpus luteum (CL) on in vitro developmental competence of bovine oocytes. In experiment 1, cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and divided according to the presence (CL(+) oocytes) or absence (CL(-) oocytes) of a CL in the ovary. Control oocytes (C group) were obtained from ovaries which were not selected toward the presence or absence of CL. All oocytes were submitted to in vitro maturation, fertilization and culture. In experiment 2, the oocytes from the CL(+) and CL(-) ovaries were divided into grown (BCB(+)) and growing (BCB(-)) categories by means of the brilliant cresyl blue (BCB) test. The oocytes from all groups (CL(+)/BCB(+), CL(-)/BCB(+), CL(+)/BCB(-), CL(-)/BCB(-) and control oocytes) were subjected to in vitro embryo production. In experiment 1, the cleavage and blastocyst rates of CL(-) oocytes were higher than those of CL(+) oocytes (83.9% and 43% vs. 69.3% and 22.5%, respectively). In experiment 2, there was less BCB(+) oocytes (more competent oocytes) in the group of CL(+) oocytes than in the group of CL(-) oocytes. Furthermore, developmental competence of all CL(+) oocytes (CL(+)/BCB(+) and CL(+)/BCB(-)) was lower than that of all CL(-) oocytes (CL(-)/BCB(+) and CL(-)/BCB(-)). Thus, the presence of a corpus luteum in the ovary may have negative effects on developmental competence of ipsilateral oocytes.

  3. The differentiation of mammalian ovarian granulosa cells – living in the shadow of cellular developmental capacity.

    PubMed

    Chachuła, A; Kranc, W; Budna, J; Bryja, A; Ciesiólka, S; Wojtanowicz-Markiewicz, K; Piotrowska, H; Bukowska, D; Krajecki, M; Antosik, P; Brüssow, K P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

    2016-01-01

    The mammalian cumulus-oocyte complex (COCs) promotes oocyte growth and development during long stages of folliculogenesis and oogenesis. Before ovulation, the follicle is formed by a variety of fully differentiated cell populations; cumulus cells (CCs) that tightly surround the female gamete, granulosa cells (GCs) and theca cells (TCs) which build the internal and external mass of the follicular wall. It is well documented that CCs surrounding the oocyte are necessary for resumption of meiosis and full maturation of the gamete. However, the role of the granulosa cells in acquisition of MII stage and/or full fertilization ability is not yet entirely known. In this article, we present an overview of mammalian oocytes and their relationship to the surrounding cumulus and granulosa cells. We also describe the processes of GCs differentiation and developmental capacity. Finally, we describe several markers of mammalian GCs, which could be used for positive identification of isolated cells. The developmental capacity of oocytes and surrounding somatic cells – a “fingerprint” of folliculogenesis and oogenesis.

  4. Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients.

    PubMed

    Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

    2016-12-22

    Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women.

  5. Brilliant cresyl blue staining does not present cytotoxic effects on human luteinized follicular cells, according to gene/protein expression, as well as to cytotoxicity tests.

    PubMed

    Alcoba, Diego Duarte; Schneider, Júlia; Arruda, Letícia; Martiny, Patrícia Borba; Capp, Edison; von Eye Corleta, Helena; Brum, Ilma Simoni

    2017-03-01

    In vitro oocyte maturation presents many advantages and its success is related to the selection of fully grown oocytes. In animal models, staining of cumulus-oocyte complexes (COCs) with Brilliant Cresyl Blue (BCB) is widely used for this selection. However, a lack of information about the safety of BCB makes its applicability in humans questionable. Because granulosa and cumulus cells have a close relationship with the oocyte and taking into account that follicular cells are also exposed to BCB when this pre-selection method is applied, we aimed to evaluate the effects of BCB on human follicular cells exposed to BCB. Cytotoxicity tests (Sulforhodamine B and Neutral Red Uptake) and gene and protein expression of elements related to the cell cycle (BAX, BCL2, TP53 and CDKN1A), as well as to cell death and metabolism (GAPDH), glucose consumption, and estradiol and progesterone secretion, were examined in granulosa and cumulus cells with and without exposure to BCB. Regardless estradiol secretion and glucose consumption, all other evaluations presented similar results between control and treated (BCB) groups, independently of cell type. Therefore, our results demonstrate convincingly that BCB seems to be safe for use in humans and it should encourage future studies to evaluate the development of embryos derived from human oocytes selected by BCB staining. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

  6. Type of gonadotropin used during controlled ovarian stimulation induces differential gene expression in human cumulus cells: A randomized study.

    PubMed

    Cruz, María; Requena, Antonio; Agudo, David; García-Velasco, Juan Antonio

    2017-08-01

    The cumulus-oocyte complex plays a central role in the regulation of folliculogenesis where it is important for the maturation, reprogramming, and fertilization of oocytes. Consequently, cumulus cell gene expression profiling is being explored as a promising method for assessing oocyte competence in the near future. Through DNA microarray technology, we analyzed the potential differences in the gene expression profiles of cumulus cells from preovulatory follicles after controlled ovarian stimulation using different types of gonadotropins. A prospective, randomized study was performed among 90 women participating in an oocyte donation program. Subjects were assigned to receive recombinant follicle-stimulating hormone (FSH), urinary FSH, or human menopausal gonadotropin (hMG). The gene expression profile in cumulus cells was analyzed according the type of gonadotropin received during ovarian stimulation. Furthermore, we also performed a gene ontology analysis to provide structural knowledge. Hierarchical clustering, principal component analysis, and gene enrichment analysis revealed greater differences between the urinary FSH and hMG groups compared to the rest of the pair-wise comparisons; recombinant FSH vs hMG and urinary FSH vs recombinant FSH. Data suggest that controlled ovarian stimulation induces specific gene expression profiles in human cumulus cells depending on the type of gonadotropin used. Registered at clinicaltrials.gov; identifier NCT022437032. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Effect of gonadotrophins, oestradiol and insulin on cumulus expansion of Nili Ravi buffalo oocytes.

    PubMed

    Shahid, Beenish; Jalali, Samina; Khan, Muhamad Ijaz; Shami, Sa

    2015-01-01

    The objective of the present study was to investigate the cumulus expansions of Nili Ravi buffalo oocytes during cultured in TCM-199 supplemented with 2 μg/ml oestradiol (E(2)), 0.05 IU/ml recombinant human follicle stimulating hormone (rhFSH), 2IU/ml human chorionic gonadotrophin (hCG), and 0.12 IU/ml insulin (I). The cumulus oocytes complexes (COCs) were collected from 2-8mm follicles from local abattoir ovaries. Supplementation of medium with single hormones showed significant (P<0.0001) increase in mean diameter of COCs with rhFSH except E(2), hCG and insulin after 24 hours compared to the increase in the mean diameter of COCs matured in TCM-199 without any hormonal supplementation. With rhFSH even at 8th hour, significant increase (P<0.001) in cumulus expansion was observed. In combination of hormones the significant (P<0.0001) cumulus expansion was achieved in E(2)+rhFSH treatment group. The non significant (P>0.05) cumulus expansion was observed in treatment groups viz. E(2)+hCG, E(2)+Insulin, rhFSH+hCG, rhFSH+Insulin, hCG+Insulin, E(2)+rhFSH+hCG and E(2)+rhFSH+hCG+Insulin after 24 hours. In conclusion, supplementation of rhFSH alone and in combination with E(2)in TCM-199 has highly significant effect on cumulus expansion.

  8. Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization.

    PubMed

    Hara, H; Yamane, I; Noto, I; Kagawa, N; Kuwayama, M; Hirabayashi, M; Hochi, S

    2014-11-01

    Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with β-mercaptoethanol (βME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without βME/Cys × fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with βME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.

  9. Expression and cellular distribution of INHA and INHB before and after in vitro cultivation of porcine oocytes isolated from follicles of different size.

    PubMed

    Kempisty, Bartosz; Jackowska, Marta; Woźna, Magdalena; Antosik, Paweł; Piotrowska, Hanna; Zawierucha, Piotr; Bukowska, Dorota; Jaśkowski, Jędrzej M; Nowicki, Michał; Brüssow, Klaus P

    2012-01-01

    Cumulus-oocyte-complexes (COCs) were collected from small (<3 mm), medium (3-5 mm), and large (>5 mm) porcine follicles, and the INHA and INHB expression and cellular localization were studied. Developmentally competent (BCB+) COCs were cultured for 44 h. Samples of mRNA were isolated before and after in vitro maturation (IVM) from oocytes collected from follicles of different size for RQ-PCR assay. The INHA and INHB protein distribution within the oocytes was observed by confocal microscopy. INHA mRNA expression was increased in oocytes from large compared to medium and small follicles before IVM (P < 0.001), and to oocytes of small follicles after IVM (P < 0.001). The INHB expression was not different before IVM, but the IHNB mRNA level was gradually higher in oocytes from large follicles after IVM (P < 0.01). INHA was not differently expressed before IVM; however, in large follicle oocytes the protein was distributed in the peripheral area of the cytoplasm; in oocytes from small follicles it was in the entire cytoplasm. After IVM, INHA was strongly expressed in oocytes from small follicles and distributed particularly in the zona pellucida (ZP). Similarly and both before and after IVM, INHB protein was highly expressed in small follicle oocytes and within the cytoplasm. In summary, INHs can be recognized as a marker of porcine oocyte quality.

  10. Resumption of oocyte meiosis in mammals: on models, meiosis activating sterols, steroids and EGF-like factors.

    PubMed

    Tsafriri, A; Cao, X; Ashkenazi, H; Motola, S; Popliker, M; Pomerantz, S H

    2005-04-29

    De novo synthesis of meiosis activating sterols (MAS) was stimulated by LH- and AY-9944 in rat cultured follicles and cumulus oocyte complexes (COCs), but could not be measured in denuded oocytes. Thus, MAS synthesized by the somatic compartment of the follicle could serve as a signal for the resumption of meiosis. Nevertheless, the delay in germinal vesicle breakdown (GVB) after MAS or AY-9944 stimulation as compared with gonadotropins, obtained by several groups, remains the strongest evidence against the suggested role of MAS as an essential mediator of LH in meiosis resumption. Recently several studies using mammalian COCs in culture have implied that steroids, like in fish and amphibians, serve as signals in mediating the LH/hCG stimulation of meiosis. However, in these studies there was no clear distinction between the requirement for steroids for the acquisition of meiotic competence, oocyte and follicle wellbeing or as a signal for meiotic resumption. Further, some of the authors overlooked earlier studies showing that blocking ovarian or follicular steroidogenesis does not affect GVB, the first step of meiosis resumption. Finally, in vivo and in vitro studies in the rat confirm and extend recent studies showing that locally produced and released EGF-like factors, such as epiregulin, seem to mediate at least part of the LH/hCG actions on oocyte maturation and release of ova at ovulation.

  11. Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro.

    PubMed

    Pomini Pinto, R F; Fontes, P K; Loureiro, B; Sousa Castilho, A C; Sousa Ticianelli, J; Montanari Razza, E; Satrapa, R A; Buratini, J; Moraes Barros, C

    2015-02-01

    Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM-199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

  12. Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop.

    PubMed

    Moawad, Adel R; Zhu, Jie; Choi, Inchul; Amarnath, Dasari; Chen, Wenchao; Campbell, Keith H S

    2013-01-01

    The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus-oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P<0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

  13. Maternal liver damage delays meiotic resumption in bovine oocytes through impairment of signalling cascades originated from low p38MAPK activity in cumulus cells.

    PubMed

    Tanaka, H; Takeo, S; Monji, Y; Kuwayama, T; Iwata, H

    2014-02-01

    The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

  14. Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique.

    PubMed

    Fernandez-Gonzalez, L; Jewgenow, K

    2017-04-01

    Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes (IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen (SN2 ) for ultra-rapid freezing to improve survival rates. Cumulus-oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit(®) Freeze/Thaw (n = 89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato(®) kit (38.7%, n = 137) and IZW protocol (24.5%, n = 143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN2 did not result in any improvement of oocytes' cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN2 (n = 144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato(®) (n = 62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato(®) kit, but the use of SN2 is improving neither maturation nor cleavage percentages when combined with these procedures. © 2016 Blackwell Verlag GmbH.

  15. Developmental competence and embryo quality of small oocytes from pre-pubertal goats cultured in IVM medium supplemented with low level of hormones, insulin-transferrin-selenium and ascorbic acid.

    PubMed

    Hammami, S; Morató, R; Romaguera, R; Roura, M; Catalá, M G; Paramio, M T; Mogas, T; Izquierdo, D

    2013-04-01

    The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre-pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus-oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 μm and ≥ 125 μm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post-warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre-pubertal goat small oocytes in GM would be useful to improve the quality of in vitro-produced blastocysts. © 2012 Blackwell Verlag GmbH.

  16. Novel maturity parameters for mature to over-mature source rocks and oils based on the distribution of phenanthrene series compounds.

    PubMed

    Wang, Zixiang; Wang, Yongli; Wu, Baoxiang; Wang, Gen; Sun, Zepeng; Xu, Liang; Zhu, Shenzhen; Sun, Lina; Wei, Zhifu

    2016-03-01

    Pyrolysis experiments of a low-mature bitumen sample originated from Cambrian was conducted in gold capsules. Abundance and distribution of phenanthrene series compounds in pyrolysis products were measured by GC-MS to investigate their changes with thermal maturity. Several maturity parameters based on the distribution of phenanthrene series compounds have been discussed. The results indicate that the distribution changes of phenanthrene series compounds are complex, and cannot be explained by individual reaction process during thermal evolution. The dealkylation cannot explain the increase of phenanthrene within the EasyRo range of 0.9% ∼ 2.1%. Adding of phenanthrene into maturity parameters based on the methylphenanthrene isomerization is unreasonable, even though MPI 1 and MPI 2 could be used to some extent. Two additional novel and an optimized maturation parameters based on the distribution of phenanthrene series compounds are proposed and their relationships to EasyRo% (x) are established: log(MPs/P) = 0.19x + 0.08 (0.9% < EasyRo% < 2.1%); log(MPs/P) = 0.64x - 0.86 (2.1% < EasyRo% < 3.4%); log(DMPs/TMPs) = 0.71x - 0.55 (0.9% < EasyRo% < 3.4%); log(MTR) = 0.84x - 0.75 (0.9% < EasyRo% < 3.4%). These significant positive correlations are strong argument for using log(MPs/P), log(DMPs/TMPs) and log(MTR) as maturity parameters, especially for mature to over-mature source rocks.

  17. Thermal and hydrocarbon maturation models for coastal California

    SciTech Connect

    Heasler, H.P.; Surdam, R.C.

    1985-02-01

    Hydrocarbon maturation models for coastal California must consider thermal and geochemical constraints imposed by plate tectonics, diagenetic reactions, and the sedimentation history of the region. Plate tectonism drastically effects the thermal history of California basins in many ways. Initially, temperatures in the crust of coastal California are suppressed during subduction of the Farallon plate. With the passage of the Mendocino triple junction, subduction ceases and a void is created into which asthenosphere moves. This elevates temperatures in the basins in a complex manner depending on the time of passage of the Mendocino triple junction and the location of a specific basin. Finite-difference numerical models were developed to approximate the thermal effects of subduction and lithospheric upwelling. Diagenetic reactions and sedimentation history affect both the maturation model and thermal history of a basin. Diagenetic reactions through time in the Miocene Monterey Formation may change thermal conductivity values by 70%. Facies changes also have an important effect on sediment thermal conductivity and hence sediment temperatures. Maturation models indicate varying levels of maturity depending on the method used. Models using the Time Temperature Index of Lopatin indicate the lowest level of maturity. Tissot and Espitalie's method, which uses multiple activation energies and varying constants for the kerogen types, results in an intermediate level of maturity. The highest level of maturity results in an intermediate level of maturity. The highest level of maturity results from the use of the Tissot and Espitalie method modified by using a single activation energy of 178.69 kJ mole/sup -1/ and a constant of 4.92 x 10/sup 13/ hour/sup -1/ as reported by M.D. Lewan for shale from the Phosphoria Formation.

  18. Ultrastructure of human oocytes after in vitro maturation.

    PubMed

    Coticchio, Giovanni; Dal Canto, Mariabeatrice; Fadini, Rubens; Mignini Renzini, Mario; Guglielmo, Maria Cristina; Miglietta, Selenia; Palmerini, Maria Grazia; Macchiarelli, Guido; Nottola, Stefania Annarita

    2016-02-01

    How does the ultrastructure of human oocytes matured in vitro compare with oocytes collected from women after full hormonal stimulation? The ultrastructure of human oocytes matured in vitro is largely, but not entirely, similar to those matured in vivo. Embryos derived from in vitro-matured oocytes often have limited developmental potential, possibly as an effect of inappropriate in vitro maturation (IVM) conditions. Transmission electron microscopy (TEM) is a valuable research tool to compare in vivo and in vitro matured oocytes. However, previous studies on the ultrastructure of human IVM oocytes were done with inadequate material or inappropriate IVM conditions, and have limited significance. Immature cumulus cell-enclosed oocytes, retrieved from mid-sized antral follicles of women requiring IVM treatment, were matured in vitro for 30 h. No leftover germinal vesicle-stage oocytes collected from fully stimulated cycles were used. Control in vivo matured oocytes were obtained from age-matched women undergoing full ovarian stimulation. In vitro and in vivo matured oocytes were analysed by TEM and compared according to previously established morphometric criteria of oocyte quality. All oocytes had normal ooplasm showing uniform distribution of organelles. Mitochondrial morphology appeared similar between the maturation conditions. Cortical granules were found typically stratified in a single, mostly continuous row just beneath the ooplasm in all oocytes. Microvilli were well preserved after IVM. Vacuoles were only occasionally found in all oocytes and, if present, they were frequently associated with lysosomes. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and mitochondria-vesicles (MV) complexes were commonly found in in vivo matured oocytes. However, large MV complexes partially replaced M-SER aggregates in IVM oocytes. As a note of caution it should be noticed that, being laborious and technically demanding, TEM cannot be applied to a large number

  19. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  20. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  1. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  2. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  3. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  4. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2011-07-01 2011-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  5. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  6. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  7. Career Maturity and Physically Disabled College Students.

    ERIC Educational Resources Information Center

    Burkhead, E. Jane; Cope, Corrine S.

    1984-01-01

    Examined the relationships between career maturity, sex, physical disability, and grades in 40 disabled and 46 nondisabled college students. Results showed disabled students were more vocationally mature than nondisabled students and female students were more vocationally mature than males. Type of disability was not related to career maturity.…

  8. 7 CFR 51.2841 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.2841 Section 51.2841 Agriculture... Creole Types) Definitions § 51.2841 Mature. Mature means well cured. Midseason onions which are not customarily held in storage shall be considered mature when harvested in accordance with good commercial...

  9. 7 CFR 51.2841 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.2841 Section 51.2841 Agriculture... Mature. Mature means well cured. Midseason onions which are not customarily held in storage shall be considered mature when harvested in accordance with good commercial practice at a stage which will not result...

  10. 7 CFR 51.484 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.484 Section 51.484 Agriculture Regulations..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Cantaloups 1 Definitions § 51.484 Mature. Mature means that the cantaloup has reached the stage of maturity which will insure the proper completion...

  11. 7 CFR 51.484 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.484 Section 51.484 Agriculture Regulations..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Cantaloups 1 Definitions § 51.484 Mature. Mature means that the cantaloup has reached the stage of maturity which will insure the proper completion...

  12. 7 CFR 51.2841 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.2841 Section 51.2841 Agriculture... Creole Types) Definitions § 51.2841 Mature. Mature means well cured. Midseason onions which are not customarily held in storage shall be considered mature when harvested in accordance with good commercial...

  13. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  14. In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone.

    PubMed

    Magalhães, D M; Duarte, A B G; Araújo, V R; Brito, I R; Soares, T G; Lima, I M T; Lopes, C A P; Campello, C C; Rodrigues, A P R; Figueiredo, J R

    2011-01-01

    The objective was to evaluate the effects of growth hormone (GH) on the survival, growth, maturation, and fertilization of oocytes derived from caprine preantral ovarian follicles cultured in vitro. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the absence (control) or presence of bovine GH at concentrations of 10 or 50 ng/mL (GH10 and GH50, respectively). Follicle development was evaluated on the basis of survival, antral cavity formation, diameter increase, and the presence of healthy cumulus-oocyte complexes and mature oocytes. After culture, oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of antrum formation after Day 6 of culture was higher in both GH10 and GH50 than in the control (81.0, 92.7, and 47.6%, respectively, P < 0.05). Percentages of grown oocytes that were acceptable for IVM were also higher (P < 0.05) in GH-treated groups than in the control (54.8, 48.8, and 11.9% for GH10, GH50, and Control). A higher percentage of oocytes in the GH50 treatment underwent meiotic resumption (50.0%), produced mature oocytes, and enabled production of an embryo after IVF than in the control group (0.0%; P < 0.05). In conclusion, GH promoted in vitro growth and maturation of goat preantral follicle oocytes and enabled production of an embryo. Furthermore, this study was apparently the first to produce a caprine embryo by in vitro fertilization of oocytes derived from preantral follicles grown in vitro.

  15. Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

    PubMed

    Li, R; Wu, H; Zhuo, W W; Mao, Q F; Lan, H; Zhang, Y; Hua, S

    2015-10-01

    Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation. © 2015 Blackwell Verlag GmbH.

  16. Developmental Issues in Career Maturity and Career Decision Status.

    ERIC Educational Resources Information Center

    Patton, Wendy; Creed, Peter A.

    2001-01-01

    Reports cross-sectional data from 1,971 Australian adolescents who completed the Career Decision Scale and the Career Development Inventory. Results illustrate a developmental progression in career maturity, although a less uniform pattern emerged with gender differences. Findings regarding career indecision also presented a complex picture and…

  17. Enticing Mature Females into College.

    ERIC Educational Resources Information Center

    Loseth, Lexie; Moreau, Linda

    Following a review of the literature on mature female students, this paper examines enrollment trends in a selection of colleges in Alberta (Canada) and presents the findings of a survey of returning women students at Red Deer College. The literature review highlights factors related to the personal and professional development of women graduates…

  18. Psychosocial Maturity or Social Desirability?

    ERIC Educational Resources Information Center

    Greenberger, Ellen

    The psychosocial maturity scale (PSM) described in several earlier papers is a self-report questionnaire. It is vulnerable, as are other questionnaires of this type, to respondents' wishes to present themselves in a socially desirable light. In this study, scores on two social desirability scales are examined in relation to PSM. Correlations…

  19. Human oocyte maturation in vitro.

    PubMed

    Coticchio, Giovanni; Dal-Canto, Mariabeatrice; Guglielmo, Maria-Cristina; Mignini-Renzini, Mario; Fadini, Rubens

    2012-01-01

    Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

  20. Vineland Social Maturity Scale Profile.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    The Vineland Social Maturity Scale (VSMS), despite its limitations, is an excellent clinical technique and includes psychometric and questionnaire characteristics. It is a good single measure of adaptive behavior. The VSMS Profile in this paper uses content categories different from the original Scale, but based upon the same items. It lends…

  1. The People Capability Maturity Model

    ERIC Educational Resources Information Center

    Wademan, Mark R.; Spuches, Charles M.; Doughty, Philip L.

    2007-01-01

    The People Capability Maturity Model[R] (People CMM[R]) advocates a staged approach to organizational change. Developed by the Carnegie Mellon University Software Engineering Institute, this model seeks to bring discipline to the people side of management by promoting a structured, repeatable, and predictable approach for improving an…

  2. Enticing Mature Females into College.

    ERIC Educational Resources Information Center

    Loseth, Lexie; Moreau, Linda

    Following a review of the literature on mature female students, this paper examines enrollment trends in a selection of colleges in Alberta (Canada) and presents the findings of a survey of returning women students at Red Deer College. The literature review highlights factors related to the personal and professional development of women graduates…

  3. Adolescent Maturation in Transitioning Cultures.

    ERIC Educational Resources Information Center

    Mulroy, Kevin; Palacios, Anna; Reid, Robert E.

    This is a theoretical study of adolescent maturation within a cultural context. Personality development and disintegration due to the pressure of a dominant culture on a minority culture is considered. An attempt is made to understand how teachers might assist students to work out their psychological growth by story telling. The need for cultural…

  4. Financial maturity of paper birch

    Treesearch

    Joseph J. Mendel

    1969-01-01

    One objective in forestry is to earn the greatest possible return on the capital invested in growing timber. To do this, the forester not only must know which silvicultural methods to use, but also ought to know the methods of economic analysis that will help him make the decisions that will lead to the greatest return. The financial maturity concept provides a method...

  5. Motivational Maturity and Helping Behavior

    ERIC Educational Resources Information Center

    Haymes, Michael; Green, Logan

    1977-01-01

    Maturity in conative development (type of motivation included in Maslow's needs hierarchy) was found to be predictive of helping behavior in middle class white male college students. The effects of safety and esteem needs were compared, and the acceptance of responsibility was also investigated. (GDC)

  6. Megakaryopoiesis: transcriptional insights into megakaryocyte maturation.

    PubMed

    Kostyak, John Creigh; Naik, Ulhas Pandurang

    2007-01-01

    Platelets are small anucleate cells that travel near the vessel wall during laminar flow. In response to vascular injury, platelets undergo alterations in morphology which allow them to aggregate and cover the injured site. Platelets are produced by megakaryocytes in a process that involves the formation of platelet precursors called proplatelets and subsequent release of these proplatelets into the circulation. By forming a demarcation membrane system within the cytosol, megakaryocytes contain a membrane reservoir which allows for the production of thousands of platelets per mature megakaryocyte. Interestingly, the above process known as megakaryopoiesis is not yet fully understood. However, several groups have contributed evidence to unveil the role of thrombopoietin (TPO), the principal regulator of megakaryopoiesis in vivo. TPO is necessary for megakaryocyte maturation in that TPO deficient mice display greatly reduced megakaryocyte production as well as reduced numbers of mature megakaryocytes. Several transcription factors have also been implicated in megakaryopoiesis including, GATA-1, friend of GATA-1 (FOG-1), nuclear factor-erythroid 2 (NF-E2), and Fli-1. In fact, interactions among some of the transcription factors have been reported to produce synergistic effects. GATA-1 and Fli-1 interactions result in heightened GPIX and GPIb (2 components of von Willebrand Factor (vWF) receptor) expression, while GATA-1, RUNX1 and core-binding factor beta interactions result in improved alphaIIb promoter activity. Mutations in the vWF complex and alphaIIb beta3 have been linked to disorders such as Bernard-Soulier syndrome and Glazmann thrombasthenia respectively. Therefore, a more comprehensive understanding of the transcriptional control of megakaryopoiesis may lead to more effective treatments of platelet-related disorders.

  7. A Regulatory Network for Coordinated Flower Maturation

    PubMed Central

    Ploense, Sara E.; Wu, Miin-Feng; Yadav, Vandana; Tholl, Dorothea; Chételat, Aurore; Haupt, Ina; Kennerley, Brian J.; Hodgens, Charles; Farmer, Edward E.; Nagpal, Punita; Reed, Jason W.

    2012-01-01

    For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs. PMID:22346763

  8. A regulatory network for coordinated flower maturation.

    PubMed

    Reeves, Paul H; Ellis, Christine M; Ploense, Sara E; Wu, Miin-Feng; Yadav, Vandana; Tholl, Dorothea; Chételat, Aurore; Haupt, Ina; Kennerley, Brian J; Hodgens, Charles; Farmer, Edward E; Nagpal, Punita; Reed, Jason W

    2012-02-01

    For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.

  9. Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine.

    PubMed

    Chen, Jun-Yi; Li, Xiao-Xia; Xu, Ya-Kun; Wu, Hua; Zheng, Jun-Jun; Yu, Xue-Li

    2014-12-01

    The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P<0.05) than that of the liquid nitrogen group (81.1%). When the vitrified-thawed oocytes were matured in vitro for 24h, the maturation rate in liquid helium group (50.6%) was higher (P<0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P<0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified-thawed oocytes were matured 24h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Generation of live piglets from cryopreserved oocytes for the first time using a defined system for in vitro embryo production.

    PubMed

    Somfai, Tamás; Yoshioka, Koji; Tanihara, Fuminori; Kaneko, Hiroyuki; Noguchi, Junko; Kashiwazaki, Naomi; Nagai, Takashi; Kikuchi, Kazuhiro

    2014-01-01

    We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.

  11. Obox4-silencing-activated STAT3 and MPF/MAPK signaling accelerate nuclear membrane breakdown in mouse oocytes.

    PubMed

    Lee, Hyun-Seo; Kim, Kyeoung-Hwa; Kim, Eun-Young; Lee, Su-Yeon; Ko, Jung-Jae; Lee, Kyung-Ah

    2016-04-01

    Mouse oocytes begin to mature in vitro once liberated from ovarian follicles. Previously, we showed that oocyte-specific homeobox 4 (Obox4) is critical for maintaining the intact nuclear membrane of the germinal vesicle (GV) in oocytes and for completing meiosis at the metaphase I-II (MI-MII) transition. This study further examines the molecular mechanisms of OBOX4 in regulating GV nuclear membrane breakdown. Maturation-promoting factor (MPF) and MAPK are normally inactive in GV stage oocytes but were activated prematurely in arrested GV stage oocytes by 3-isobutyl-1-metyl-xanthine (IBMX) in vitro after Obox4 RNA interference (RNAi). Furthermore, signal transducer and activator of transcription 3 (STAT3) was significantly activated by Obox4 RNAi. We confirmed that this Obox4 RNAi-induced premature STAT3 and MPF/MAPK activation at the GV stage provoked subsequent GV breakdown (GVBD) despite the opposing force of high cAMP in the IBMX-supplemented medium to maintain intact GV. When cumulus-oocyte complexes were exposed to interferon α (IFNA), a STAT3 activator, oocytes matured and cumulus cells expanded to resume nuclear maturation in IBMX-supplemented medium, suggesting that STAT3 activation is sufficient for stimulating the continuation of meiosis. Using Stattic, a specific STAT3 inhibitor, we confirmed that GVBD involves STAT3 activation in Obox4-silenced oocytes. Based on these findings, we concluded that i) Obox4 is an important upstream regulator of MPF/MAPK and STAT3 signaling, and ii) Obox4 is a key regulator of the GV arrest mechanism in oocytes.

  12. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-01-01

    Only a fraction of oocytes present in the ovaries at birth are ever ovulated during the lifetime of a female mammal. In vitro maturation (IVM) offers the possibility to exploit what is a largely untapped biological resource. Although IVM is used routinely for the in vitro production of embryos in domestic species, especially cattle, its clinical use in human-assisted reproduction is still evolving. The successful recapitulation in vitro of the events associated with successful oocyte maturation is not always achieved, with the majority of immature oocytes typically failing to develop to the blastocyst stage. Evidence suggests that although culture conditions throughout in vitro embryo production may have a modest influence on the developmental potential of the early embryo, the quality of the oocyte at the start of the process is the key factor determining the proportion of oocytes developing to the blastocyst stage.

  13. PERSISTENT WRIST PAIN IN A MATURE GOLFER

    PubMed Central

    Hazle, Charles

    2012-01-01

    Clients presenting with ulnar-sided wrist pain can provide diagnostic and management challenges for physical therapists. Symptoms in this region may originate from multiple structures. Integration of clinical examination and diagnostic imaging results is often required for optimal decision-making and patient management. To obtain the most informative imaging results, practitioners need an understanding of injury patterns and their detection by various imaging modalities. This case describes a mature golfer who presented with persistent ulnar-sided wrist pain and was eventually determined to have a fracture of the hook of the hamate accompanied by neighboring soft tissue involvement also contributing to his symptom complex. His history and the diagnostic process are detailed along with a brief discussion of his subsequent management post-operatively. Level of Evidence: 5 (Single Case Report) PMID:22893862

  14. Maturation of the adolescent brain

    PubMed Central

    Arain, Mariam; Haque, Maliha; Johal, Lina; Mathur, Puja; Nel, Wynand; Rais, Afsha; Sandhu, Ranbir; Sharma, Sushil

    2013-01-01

    Adolescence is the developmental epoch during which children become adults – intellectually, physically, hormonally, and socially. Adolescence is a tumultuous time, full of changes and transformations. The pubertal transition to adulthood involves both gonadal and behavioral maturation. Magnetic resonance imaging studies have discovered that myelinogenesis, required for proper insulation and efficient neurocybernetics, continues from childhood and the brain’s region-specific neurocircuitry remains structurally and functionally vulnerable to impulsive sex, food, and sleep habits. The maturation of the adolescent brain is also influenced by heredity, environment, and sex hormones (estrogen, progesterone, and testosterone), which play a crucial role in myelination. Furthermore, glutamatergic neurotransmission predominates, whereas gamma-aminobutyric acid neurotransmission remains under construction, and this might be responsible for immature and impulsive behavior and neurobehavioral excitement during adolescent life. The adolescent population is highly vulnerable to driving under the influence of alcohol and social maladjustments due to an immature limbic system and prefrontal cortex. Synaptic plasticity and the release of neurotransmitters may also be influenced by environmental neurotoxins and drugs of abuse including cigarettes, caffeine, and alcohol during adolescence. Adolescents may become involved with offensive crimes, irresponsible behavior, unprotected sex, juvenile courts, or even prison. According to a report by the Centers for Disease Control and Prevention, the major cause of death among the teenage population is due to injury and violence related to sex and substance abuse. Prenatal neglect, cigarette smoking, and alcohol consumption may also significantly impact maturation of the adolescent brain. Pharmacological interventions to regulate adolescent behavior have been attempted with limited success. Since several factors, including age, sex

  15. Maturation of the adolescent brain.

    PubMed

    Arain, Mariam; Haque, Maliha; Johal, Lina; Mathur, Puja; Nel, Wynand; Rais, Afsha; Sandhu, Ranbir; Sharma, Sushil

    2013-01-01

    Adolescence is the developmental epoch during which children become adults - intellectually, physically, hormonally, and socially. Adolescence is a tumultuous time, full of changes and transformations. The pubertal transition to adulthood involves both gonadal and behavioral maturation. Magnetic resonance imaging studies have discovered that myelinogenesis, required for proper insulation and efficient neurocybernetics, continues from childhood and the brain's region-specific neurocircuitry remains structurally and functionally vulnerable to impulsive sex, food, and sleep habits. The maturation of the adolescent brain is also influenced by heredity, environment, and sex hormones (estrogen, progesterone, and testosterone), which play a crucial role in myelination. Furthermore, glutamatergic neurotransmission predominates, whereas gamma-aminobutyric acid neurotransmission remains under construction, and this might be responsible for immature and impulsive behavior and neurobehavioral excitement during adolescent life. The adolescent population is highly vulnerable to driving under the influence of alcohol and social maladjustments due to an immature limbic system and prefrontal cortex. Synaptic plasticity and the release of neurotransmitters may also be influenced by environmental neurotoxins and drugs of abuse including cigarettes, caffeine, and alcohol during adolescence. Adolescents may become involved with offensive crimes, irresponsible behavior, unprotected sex, juvenile courts, or even prison. According to a report by the Centers for Disease Control and Prevention, the major cause of death among the teenage population is due to injury and violence related to sex and substance abuse. Prenatal neglect, cigarette smoking, and alcohol consumption may also significantly impact maturation of the adolescent brain. Pharmacological interventions to regulate adolescent behavior have been attempted with limited success. Since several factors, including age, sex, disease

  16. Maturity model for enterprise interoperability

    NASA Astrophysics Data System (ADS)

    Guédria, Wided; Naudet, Yannick; Chen, David

    2015-01-01

    Historically, progress occurs when entities communicate, share information and together create something that no one individually could do alone. Moving beyond people to machines and systems, interoperability is becoming a key factor of success in all domains. In particular, interoperability has become a challenge for enterprises, to exploit market opportunities, to meet their own objectives of cooperation or simply to survive in a growing competitive world where the networked enterprise is becoming a standard. Within this context, many research works have been conducted over the past few years and enterprise interoperability has become an important area of research, ensuring the competitiveness and growth of European enterprises. Among others, enterprises have to control their interoperability strategy and enhance their ability to interoperate. This is the purpose of the interoperability assessment. Assessing interoperability maturity allows a company to know its strengths and weaknesses in terms of interoperability with its current and potential partners, and to prioritise actions for improvement. The objective of this paper is to define a maturity model for enterprise interoperability that takes into account existing maturity models while extending the coverage of the interoperability domain. The assessment methodology is also presented. Both are demonstrated with a real case study.

  17. Asymmetric conformational maturation of HIV-1 reverse transcriptase.

    PubMed

    Zheng, Xunhai; Perera, Lalith; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

    2015-06-03

    HIV-1 reverse transcriptase utilizes a metamorphic polymerase domain that is able to adopt two alternate structures that fulfill catalytic and structural roles, thereby minimizing its coding requirements. This ambiguity introduces folding challenges that are met by a complex maturation process. We have investigated this conformational maturation using NMR studies of methyl-labeled RT for the slower processes in combination with molecular dynamics simulations for rapid processes. Starting from an inactive conformation, the p66 precursor undergoes a unimolecular isomerization to a structure similar to its active form, exposing a large hydrophobic surface that facilitates initial homodimer formation. The resulting p66/p66' homodimer exists as a conformational heterodimer, after which a series of conformational adjustments on different time scales can be observed. Formation of the inter-subunit RH:thumb' interface occurs at an early stage, while maturation of the connection' and unfolding of the RH' domains are linked and occur on a much slower time scale.

  18. Sterols in spermatogenesis and sperm maturation

    PubMed Central

    Keber, Rok; Rozman, Damjana; Horvat, Simon

    2013-01-01

    Mammalian spermatogenesis is a complex developmental program in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. One intriguing aspect of sperm production is the dynamic change in membrane lipid composition that occurs throughout spermatogenesis. Cholesterol content, as well as its intermediates, differs vastly between the male reproductive system and nongonadal tissues. Accumulation of cholesterol precursors such as testis meiosis-activating sterol and desmosterol is observed in testes and spermatozoa from several mammalian species. Moreover, cholesterogenic genes, especially meiosis-activating sterol-producing enzyme cytochrome P450 lanosterol 14α-demethylase, display stage-specific expression patterns during spermatogenesis. Discrepancies in gene expression patterns suggest a complex temporal and cell-type specific regulation of sterol compounds during spermatogenesis, which also involves dynamic interactions between germ and Sertoli cells. The functional importance of sterol compounds in sperm production is further supported by the modulation of sterol composition in spermatozoal membranes during epididymal transit and in the female reproductive tract, which is a prerequisite for successful fertilization. However, the exact role of sterols in male reproduction is unknown. This review discusses sterol dynamics in sperm maturation and describes recent methodological advances that will help to illuminate the complexity of sperm formation and function. PMID:23093550

  19. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a...

  20. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a...

  1. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a...

  2. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  3. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND THE EGG PRODUCTS INSPECTION ACT FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION... Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  4. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND THE EGG PRODUCTS INSPECTION ACT FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1 2 (INSPECTION... Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  5. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  6. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a stage of growth which will insure a proper completion of...

  7. Career Maturity: The Construct and its Measurement.

    ERIC Educational Resources Information Center

    Savickas, Mark L.

    1984-01-01

    Describes vocational maturity and assists counselors in identifying what the various career maturity instruments measure. Discusses task variable measures, intervening variable measures, response variable measures, and methods of choosing an instrument. (JAC)

  8. Career Maturity: The Construct and its Measurement.

    ERIC Educational Resources Information Center

    Savickas, Mark L.

    1984-01-01

    Describes vocational maturity and assists counselors in identifying what the various career maturity instruments measure. Discusses task variable measures, intervening variable measures, response variable measures, and methods of choosing an instrument. (JAC)

  9. Surfactin induces maturation of dendritic cells in vitro

    PubMed Central

    Xu, Wenwen; Liu, Haofei; Wang, Xiaoqing; Yang, Qian

    2016-01-01

    Surfactin has multiple immune activities, such as triggering immune-related defense responses and enhancing humoral and cellular immune responses. Although, the mechanisms are still unclear. The maturation of dendritic cells (DCs) is essential for inducing downstream immune response. To shed light on the mechanisms of surfactin-induced immune activities, we verified the influences of surfactin on DCs maturation. The results showed that after stimulated with 20 μg/ml surfactin for 24 h, DCs were conferred morphologic and phenotypic characteristics of a mature state, showing an increased shape index and up-regulated expressions of major histocompatibility complex II (MHCII) and CD40. Moreover, surfactin also induced DCs to release IL-6 and tumour necrosis factor-α (TNF-α), indicating that DCs were functionally mature. In addition, the IκB-α level in surfactin-treated DCs was significantly reduced whereas the nuclear p65 level was notably increased, preliminarily indicating that nuclear factor-kappa B (NF-κB) signalling pathway might play an important role in surfactin-induced DCs maturation. PMID:27534429

  10. Evaluating the Maturity of Cybersecurity Programs for Building Control Systems

    SciTech Connect

    Glantz, Clifford S.; Somasundaram, Sriram; Mylrea, Michael E.; Underhill, Ronald M.; Nicholls, Andrew K.

    2016-08-29

    The cyber-physical security threat to buildings is complex, non-linear, and rapidly evolving as operational and information technologies converge and connect buildings to cyberspace. Cyberattacks on buildings can exploit smart building controls and breach corporate networks, causing financial and reputational damage. This may result in the loss of sensitive building information or the disruption of, or damage to, the systems necessary for the safe and efficient operation of buildings. For the buildings and facility infrastructure, there is a need for a robust national cybersecurity strategy for buildings, guidance on the selection and implementation of appropriate cybersecurity controls for buildings, an approach to evaluate the maturity and adequacy of the cybersecurity programs. To provide an approach for evaluating the maturity of the cybersecurity programs for building control systems, the US Department of Energy’s widely used Cybersecurity Capability and Maturity Model (C2M2) has been adapted into a building control systems version. The revised model, the Buildings-C2M2 (B-C2M2) provides maturity level indicators for cybersecurity programmatic domains. A “B-C2M2 Lite” version allows facility managers and building control system engineers, or information technology personnel to perform rapid self-assessments of their cybersecurity program. Both tools have been pilot tested on several facilities. This paper outlines the concept of a maturity model, describes the B-C2M2 tools, presents results and observations from the pilot assessments, and lays out plans for future work.

  11. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1554 Section 51.1554 Agriculture...-tenth of the skin missing or “feathered.” ...

  12. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature means that the outer skin (epidermis... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

  13. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature means that the outer skin (epidermis... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

  14. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.1554 Section 51.1554 Agriculture...-tenth of the skin missing or “feathered.” ...

  15. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  16. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  17. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  18. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  19. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  20. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  1. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Loan maturities. 201.11 Section 201... PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities. (a... the original loan to the final maturity of the refinanced loan shall not exceed: (i) In the case of a...

  2. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  3. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  4. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  5. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  6. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  7. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  8. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  9. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  10. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  11. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  12. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  13. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  14. 7 CFR 1710.115 - Final maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Final maturity. 1710.115 Section 1710.115 Agriculture... Basic Policies § 1710.115 Final maturity. (a) RUS is authorized to make loans and loan guarantees with a final maturity of up to 35 years. The borrower may elect a repayment period for a loan not longer than...

  15. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  16. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 13 Business Credit and Assistance 1 2012-01-01 2012-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  17. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  18. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  19. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  20. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  1. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  2. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 13 Business Credit and Assistance 1 2014-01-01 2014-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  3. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  4. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  5. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  6. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 13 Business Credit and Assistance 1 2013-01-01 2013-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  7. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These orange maturity requirements are contained in the Florida Citrus Code, Chapter...

  8. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These tangerine maturity requirements are contained in the Florida Citrus Code, Chapter...

  9. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These orange maturity requirements are contained in the Florida Citrus Code, Chapter...

  10. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These tangerine maturity requirements are contained in the Florida Citrus Code, Chapter...

  11. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These grapefruit maturity requirements are contained in the Florida Citrus Code, Chapter...

  12. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These grapefruit maturity requirements are contained in the Florida Citrus Code, Chapter...

  13. Career Maturity in High School Age Females.

    ERIC Educational Resources Information Center

    Pedro, Joan Daniels

    1982-01-01

    Examined career maturity in high school females by using a set of general career-maturity and gender-specific, career-related measures, and an alternate career-maturity criterion measure, career-planning involvement. Results indicated significant relationships between achievement orientation and occupational information and knowledge of women's…

  14. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Pears for Canning Definitions § 51.1351 Mature. Mature means that the pear has reached the...

  15. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.1238 Mature. Mature means that the shells are firm and well developed. ...

  16. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.1238 Mature. Mature means that the shells are firm and well developed. ...

  17. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1554 Section 51.1554 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes are generally firmly set and not more than 5 percent of the...

  18. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Standards for Pears for Canning Definitions § 51.1351 Mature. Mature...

  19. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Cleaned Virginia Type Peanuts in the Shell Definitions § 51.1238 Mature. Mature means that the...

  20. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Standards for Pears for Canning Definitions § 51.1351 Mature. Mature...

  1. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1554 Section 51.1554 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes...

  2. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature...

  3. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.3203 Mature. Mature means that the onion is fairly well cured, and at least fairly firm. ...

  4. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Bermuda-Granex-Grano Type Onions Definitions § 51.3203 Mature. Mature means that the...

  5. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature means that the outer skin (epidermis...

  6. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.3203 Mature. Mature means that the onion is fairly well cured, and at least fairly firm. ...

  7. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.693 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  8. 7 CFR 51.1907 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1907 Section 51.1907 Agriculture..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Fresh Tomatoes Definitions § 51.1907 Mature. Mature means that the tomato has reached the stage of development which will insure a proper completion...

  9. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.631 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  10. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Pears for Canning Definitions § 51.1351 Mature. Mature means that the pear has reached the...

  11. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature...

  12. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.2930 Section 51.2930 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of development which will insure a proper completion of the...

  13. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Bermuda-Granex-Grano Type Onions Definitions § 51.3203 Mature. Mature means that the...

  14. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Florida, California, and Arizona) Definitions § 51.631 Mature. Mature shall have the same meaning...

  15. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.2930 Section 51.2930 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of development which will insure a proper completion of the...

  16. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1554 Section 51.1554 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes are generally firmly set and not more than 5 percent of the...

  17. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.2930 Section 51.2930 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of...

  18. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.2930 Section 51.2930 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of...

  19. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.693 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  20. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.631 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  1. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., California, and Arizona) Definitions § 51.693 Mature. Mature shall have the same meaning currently assigned...

  2. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Cleaned Virginia Type Peanuts in the Shell Definitions § 51.1238 Mature. Mature means that the...

  3. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Florida, California, and Arizona) Definitions § 51.631 Mature. Mature shall have the same meaning...

  4. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.631 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  5. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.693 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  6. 7 CFR 51.1907 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1907 Section 51.1907 Agriculture..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Fresh Tomatoes Definitions § 51.1907 Mature. Mature means that the tomato has reached the stage of development which will insure a proper completion...

  7. 7 CFR 51.32