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Sample records for cumulus-oocyte complex maturation

  1. Immature oocyte quality and maturational competence of porcine cumulus-oocyte complexes subpopulations.

    PubMed

    Alvarez, Gabriel Martin; Dalvit, Gabriel Carlos; Achi, María Verónica; Miguez, Marcelo Sergio; Cetica, Pablo Daniel

    2009-12-01

    Porcine immature oocyte quality (i.e., that of live oocytes at the germinal vesicle stage) was evaluated according to features of the surrounding cumulus, aiming to establish maturational competence of different subpopulations of such cumulus-oocyte complexes. Six subpopulations were identified: A1 (with a dense cumulus), A2 (with a translucent cumulus), B1 (with the corona radiata), B2 (partly naked oocytes), C (naked oocytes), D (with a dark cumulus). The percent incidence of live oocyte in these subpopulations changed significantly as related to cumulus features, however the occurrence of oocytes in the germinal vesicle stage was lower in class D only. Similar metaphase II rates achieved in A1, A2, B1 and B2 classes after in vitro maturation suggest that the nucleus may in fact mature in vitro, in spite of the different accompanying cumulus features which are typical of these classes. In contrast, a higher cytoplasmic maturation rate obtained in class A may indicate a stronger dependence of this variable upon cumulus features than that shown by nuclear maturation. When different types of cumulus expansion after in vitro maturation were considered (i.e., fully expanded cumulus, partly expanded cumulus, and partly naked oocyte), no differences were found in the percent of oocytes reaching metaphase II or cytoplasmic maturation. It is concluded that morphological features of the collected porcine cumulus-oocyte complexes (rather than cumulus behavior during culture) may be useful for selection of potentially competent oocytes for in vitro fertilization and embryo production. PMID:20067032

  2. Expression, regulation, and function of progesterone receptors in bovine cumulus oocyte complexes during in vitro maturation.

    PubMed

    Aparicio, I M; Garcia-Herreros, M; O'Shea, L C; Hensey, C; Lonergan, P; Fair, T

    2011-05-01

    Progesterone (P4) exerts its effects by binding to specific genomic (nPR-A/B) and non-genomic (mPRalpha/beta, PGRMC1/2) receptors. P4 has a role in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Therefore, the aim of the present study was to characterize the protein expression of P4 receptors (PRs) in bovine oocytes and cumulus cells during in vitro maturation (IVM) and to study the effect of P4 and its receptors on oocyte developmental competence. Cumulus-oocyte complexes (COCs) were subjected to IVM, in vitro fertilization, and in vitro culture. IVM was performed for 24 h in the presence or absence of P4, luteinizing hormone (LH), follicle-stimulating hormone (FSH), trilostane, promegestone (R5020), mifepristone (RU 486), or antibodies against mPRalpha or mPRbeta. Protein expression of PRs was studied by Western blotting and immunofluorescence. The results demonstrate the presence of both genomic and nongenomic PRs in bovine COCs. The dynamic changes observed in the protein expression of PRs following IVM or in response to supplementation with LH, FSH, or P4 suggest an important role during bovine oocyte maturation. Inhibition of P4 synthesis by cumulus cells or blocking of nPR and mPR alpha activity produced a decrease in bovine embryo development, indicating that P4 intracellular signaling is mediated by its interaction with nuclear and membrane PRs and is important for oocyte developmental competence. PMID:21228216

  3. Glucocorticoid metabolism in the bovine cumulus-oocyte complex matured in vitro.

    PubMed

    Tetsuka, Masafumi; Takagi, Ryo; Ambo, Nobuhiro; Myat, Thet Su; Zempo, Yuta; Onuma, Asuka

    2016-01-01

    Glucocorticoid action in target organs is regulated by relative activities of 11β-HSD type 1 (HSD11B1) that mainly converts cortisone to active cortisol and type 2 (HSD11B2) that inactivates cortisol to cortisone. HSD11Bs have been shown to be expressed in the ovary of various species. However, little is known about the expression and activity of HSD11Bs in the bovine cumulus-oocyte complex (COC). In the present study, we investigated the expression and activities of HSD11Bs in in vitro-matured (IVM) bovine COCs. Bovine COCs were matured in M199 supplemented with or without FSH and FCS. The expression of HSD11B1 and HSD11B2 was measured by using quantitative RT-PCR in denuded oocytes (DO) and cumulus cells (CC). Reductive and oxidative activities of HSD11Bs were determined by radiometric conversion assay using labeled cortisol, cortisone or dexamethasone in intact COCs, DO or CC in the presence or absence of 11-keto-progesterone (11kP), a selective inhibitor of HSD11B2. The presence of HSD11Bs in the oocyte was examined by immunofluorescence microscopy. Oocytes exclusively expressed HSD11B2 and its expression and activity were largely unchanged during IVM. CC, on the other hand, exclusively expressed HSD11B1 and its expression and activity were upregulated as IVM progressed. As a result, the net glucocorticoid metabolism shifted from inactivation to activation towards the end of IVM. These results indicate that the bovine COC is capable of modulating local glucocorticoid concentration and, by doing so, may create an environment that is favorable to ovulating oocyte for maturation, fertilization and subsequent development. PMID:26519454

  4. Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development.

    PubMed

    Dey, S R; Deb, G K; Ha, A N; Lee, J I; Bang, J I; Lee, K L; Kong, I K

    2012-04-01

    The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs. PMID:22153275

  5. 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.

  6. 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

  7. 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. PMID:25131826

  8. Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro.

    PubMed

    Torner, H; Heleil, B; Alm, H; Ghoneim, I M; Srsen, V; Kanitz, W; Tuchscherer, A; Fattouh, E M

    2003-09-15

    The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors

  9. Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes.

    PubMed

    Choi, Byung-Hyun; Bang, Jae-Il; Jin, Jong-In; Kim, Seong-Su; Jo, Hyun-Tae; Deb, Gautam Kumar; Ghanem, Nasser; Cho, Kyu-Woan; Kong, Il-Keun

    2013-12-01

    Oocyte quality is a key factor affecting success of in vitro embryo production in cattle. Improving the microenvironment of oocytes during in vitro maturation (IVM) can increase developmental rate and embryo quality. Therefore, the objective was to determine whether denuded oocytes (DO) affect embryo development and ultrastructure of the zona pellucida (ZP) in in vitro matured bovine oocytes. Intact immature cumulus-oocytes complexes (COC) obtained from a local abattoir or by ovum pick-up (OPU) were cocultured with and without abattoir-obtained DO at a COC:DO ratio of 1:5. After IVM, DO were removed and intact DO were either fertilized or observed by scanning electron microscopy. Blastocyst quality was evaluated using a TUNEL assay. The ZP pore size decreased after IVM in COC + DO coculture, regardless of their origin (OPU, 310.5 ± 92.5 vs. 428.9 ± 148.5 nm; abattoir, 317.5 ± 68.5 vs. 358.9 ± 128.5 nm; P < 0.05; mean values ± standard deviation). Moreover, the number of ZP pores in OPU COC + DO and COC + DO was greater than those in OPU COC and COC (control) groups (56 ± 4 and 55 ± 7 vs. 50 ± 6 and 42 ± 4; P < 0.05). The rate of blastocyst development in COC + DO and OPU COC + DO groups was greater those in control and OPU COC groups (36.6% and 55.5% vs. 28.1% and 40.0%; P < 0.05). Moreover, the total cell numbers of blastocysts in COC + DO group exceeded that of control (132.91 ± 30.90 vs. 115.44 ± 24.95; P < 0.05), with no significant between OPU COC + DO and OPU COC groups (139.31 ± 42.51 vs. 137.00 ± 61.34). In conclusion, in vitro embryo development competence and quality improved when oocytes were cocultured with DO. Furthermore, there more, but smaller, ZP pores. PMID:24084231

  10. 9-cis Retinoic acid inhibits cumulus cell apoptosis during the maturation of bovine cumulus-oocyte-complexes.

    PubMed

    Deb, G K; Dey, S R; Bang, J I; Lee, J G; Kong, I K

    2012-06-01

    Cumulus cell (CC) apoptosis is inversely correlated with embryonic development in vitro. Therefore, inhibition of CC apoptosis is important for proper embryonic development and quality. Retinoic acids (all-transRA and 9-cisRA) are natural components of retinoids, and 9-cisRA is the physiologically active metabolite of retinoic acid in vitro. During in vitro maturation, 9-cisRA enhances oocyte competence through multiple mechanisms affecting the oocyte and preimplantation embryo; however, the effect of 9-cisRA on CC apoptosis has yet to be elucidated. The aim of the present study was to evaluate the effect of 9-cisRA on CC apoptosis and to identify the molecular mechanism underlying that effect. Bovine slaughterhouse cumulus-oocyte complexes (COC) were matured in vitro in the absence or presence of 5 nM 9-cisRA. Cumulus cells were collected from immature and matured COC for the detection of apoptosis and gene expression analysis. Results showed that 9-cisRA reduced the number of apoptotic CC by about 2.7 fold (P < 0.023), compared with control. However, apoptosis is rare in CC of immature COC (0.01% ± 0.001). Transcripts involved in the caspase cascade were down-regulated upon exposure to 9-cisRA, including tumor necrosis factor alpha (TNF-α, 11.1 fold, P < 0.001), tumor necrosis factor alpha receptor 1 (TNFR1, 2.3 fold, P < 0.01), caspase 9 (CASP9, 2.0 fold, P < 0.031), caspase 8 (CASP8, 2.2 fold, P < 0.012), and caspase 3 (CASP3, 2.1 fold, P < 0.006), while antiapoptotic B-cell lymphoma 2 (BCL2) transcript was increased (3.1 fold, P < 0.004), compared with control. In addition, 9-cisRA inhibited mitogen activated protein kinase mRNA expression in CC, including extracellular signal-regulated kinase 1/2 (ERK1, 2.7 fold, P < 0.02; ERK2, 2.7 fold, P < 0.03), and c-Jun N-terminal kinase (JNK, 1.6 fold, P < 0.044), as well as the activator protein-1 (AP1) family members c-jun (1.6 fold, P < 0.041) and c-fos (2.0 fold, P < 0.06). The transcript abundances of TNF

  11. Recovery rate, morphological quality and nuclear maturity of canine cumulus-oocyte complexes collected from anestrous or diestrous bitches of different ages.

    PubMed

    Lopes, G; Sousa, M; Luvoni, G Cecilia; Rocha, A

    2007-10-01

    Canine cumulus-oocyte complexes (COC) were recovered from ovaries of post-pubertal animals (1-3, 4-6 and 7-10 years old) at different ovarian estrous phases (anestrus and diestrus). The number of COCs, and the number and nuclear maturity of high-quality (grade-1) oocytes were assessed. For all animals, no significant differences were found between the two reproductive phases relatively to the total number of COCs and grade-1 oocytes recovered. However, significant higher numbers of COCs were recovered from young than from elderly animals, and the proportion of grade-1 oocytes was also significantly higher in the younger group than in the other two age-groups. Of 226 grade-1 oocytes, 73% were at the germinal vesicle stage (GV), 10% had resumed meiosis (9% at germinal vesicle breakdown; 1% at metaphase-I) and 17% were degenerated. A significant effect of the reproductive phase on oocyte nuclear maturity was found only for adult animals, with a higher number of GV oocytes being found at anestrous (79%) due to higher rates of meiosis resumption (34%) at diestrous. The high number of grade-1 oocytes with meiosis resumption and fragmented or unidentified nuclear contents, indicates that current criteria for the selection of viable canine COCs are not optimized and need a new definition. PMID:17714773

  12. Effects of co-culture of cumulus oocyte complexes with denuded oocytes during in vitro maturation on the developmental competence of cloned bovine embryos.

    PubMed

    Ha, A-N; Fakruzzaman, M; Lee, K-L; Bang, J-I; Deb, G-K; Wang, Z; Kong, I-K

    2015-04-01

    This study evaluated the effects of co-culture of immature cumulus oocyte complexes (COCs) with denuded immature oocytes (DO) during in vitro maturation on the developmental competence and quality of cloned bovine embryos. We demonstrated that developmental competence, judged by the blastocyst formation rate, was significantly higher in the co-cultured somatic cell nuclear transfer (SCNT+DO, 37.1 ± 1.1%) group than that in the non-co-cultured somatic cell nuclear transfer (SCNT-DO, 25.1 ± 0.9%) group and was very similar to that in the control IVF (IVF, 38.8 ± 2.8%) group. Moreover, the total cell number per blastocyst in the SCNT+DO group (101.7 ± 6.2) was higher than that in the SCNT-DO group (81.7 ± 4.3), while still less than that in the IVF group (133.3 ± 6.0). Furthermore, our data showed that mRNA levels of the methylation-related genes DNMT1 and DNMT3a in the SCNT+DO group were similar to that in the IVF group, while they were significantly higher in the SCNT-DO group. Similarly, while the mRNA levels of the deacetylation-related genes HDAC2 and HDAC3 were significantly higher in the SCNT-DO group, they were comparable between the IVF and SCNT+DO groups. However, the mRNA levels of HDAC1 and DNMT3B were significantly higher in the SCNT+DO group than in the other groups. In conclusion, the present study demonstrated that co-culture of COCs with DO improves the in vitro developmental competence and quality of cloned embryos, as evidenced by increased total cell number. PMID:25605137

  13. Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus-oocyte complexes exposed to heat shock during in vitro maturation.

    PubMed

    Balboula, A Z; Yamanaka, K; Sakatani, M; Kawahara, M; Hegab, A O; Zaabel, S M; Takahashi, M

    2013-10-01

    Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM. PMID:23898216

  14. C-type natriuretic peptide improved vitrified-warmed mouse cumulus oocyte complexes developmental competence.

    PubMed

    Yang, Lei; Wei, Qiang; Li, Wei; Ge, Junbang; Zhao, Xiaoe; Ma, Baohua

    2016-04-01

    Cryopreservation of cumulus oocyte complexes (COCs) is important for reproductive medicine. However, the vitrified-warmed COCs have lower maturation rate and subsequent developmental competence compared with fresh COCs. The present study was aimed to evaluate the effects of supplementation of the maturation medium with C-type natriuretic peptide (CNP) on the developmental competence of vitrified-warmed mouse COCs. Addition of CNP to the maturation medium improved the maturation rate and enhanced the developmental competence of vitrified-warmed mouse COCs. The reason may be that vitrified COCs led to a decline in cyclic guanosine monophosphate (cGMP) levels. Furthermore, addition of CNP to the maturation medium elevated cGMP levels of the vitrified-warmed COCs. In conclusion, cryopreservation-associated lower maturation rate and developmental competence of COCs may be ameliorated by CNP during maturation culture after warming. PMID:26921772

  15. 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

  16. 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

  17. 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. PMID:26170094

  18. 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

  19. Effect of heat stress on the survival and development of in vitro cultured bovine preantral follicles and on in vitro maturation of cumulus-oocyte complex.

    PubMed

    Paes, V M; Vieira, L A; Correia, H H V; Sa, N A R; Moura, A A A; Sales, A D; Rodrigues, A P R; Magalhães-Padilha, D M; Santos, F W; Apgar, G A; Campello, C C; Camargo, L S A; Figueiredo, J R

    2016-09-01

    The deleterious effect of heat stress (HS) on competence of oocytes from antral follicles is well recognized, but there is a lack of data regarding its impact on the viability and growth of preantral follicles. In this study, we used in vitro preantral follicle cultures to investigate the effects of HS on the following parameters: survival and development of primordial follicles after in vitro culture of ovarian fragments (experiment I); growth and antrum formation of isolated advanced secondary follicles (experiment II); and maturation rates after in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) from antral follicles (>2-6 mm) grown in vivo (experiment III). Furthermore, the following end points were evaluated in all experiments: follicle/oocyte survival, reactive oxygen species (ROS), estradiol (E2) and progesterone (P4) production, as well as mRNA expression for select genes related to stress (HSP70) and apoptosis (MCL1 and BAX). In all experiments, HS consisted of exposing the structures (ovarian fragments, isolated preantral follicles and COCs) to 41 °C for 12 hours and then to 38.5 °C until the end of the culture (7 days for experiments I and II and 24 hours for experiment III). The temperature for the control group was held at 38.5 °C for the entire culture period. Heat stress increased (P < 0.05) the percentage of developing follicles (intermediate, primary, and secondary follicles) at 12 hours and increased levels of ROS at all evaluated time points (12, 24 hours, and D7), when compared to the control (experiment I). Heat stress did not affect (P > 0.05) any identified end points when preantral follicles were cultured in their isolated form (experiment II). However, in experiment III, HS decreased (P < 0.05) both the rates of metaphase II after 24 hours and E2 production at 12 hours of IVM. Moreover, HS increased (P < 0.0001) levels of P4 after IVM and ROS production at every evaluated time point, compared with the

  20. Associations between free fatty acids, cumulus oocyte complex morphology and ovarian function during in vitro fertilization

    PubMed Central

    Jungheim, Emily S.; Macones, George A.; Odem, Randall R.; Patterson, Bruce W.; Lanzendorf, Susan E.; Ratts, Valerie S.; Moley, Kelle H.

    2011-01-01

    Objective To determine if follicular free fatty acid (FFA) levels are associated with cumulus oocyte complex morphology Design Prospective cohort study Setting University in vitro fertilization (IVF) practice Patients 102 women undergoing IVF Interventions Measurement of FFAs in serum and ovarian follicular fluid Main Outcome Measures Total and specific follicular and serum FFA levels, correlations between follicular and serum FFAs, and associations between follicular FFA levels and markers of oocyte quality including cumulus oocyte complex (COC) morphology Results Predominant follicular fluid and serum FFAs were oleic, palmitic, linoleic and stearic acids. Correlations between follicular and serum FFA concentrations were weak (r=0.252, 0.288, 0.236, 0.309 respectively for specific FFAs; r=0.212 for total FFAs). A receiver operator characteristic curve determined total follicular FFAs ≥ 0.232 µmol/ml distinguished women with lower versus higher percentage COCs with favorable morphology. Women with elevated follicular FFAs (n=31) were more likely to have COCs with poor morphology than others (n=71) (OR 3.3, 95% CI:1.2–9.2). This relationship held after adjusting for potential confounders including age, BMI, endometriosis and amount of gonadotropin administered (β=1.2; OR 3.4, 95% CI:1.1–10.4). Conclusions Elevated follicular FFA levels are associated with poor COC morphology. Further work is needed to determine what factors influence follicular FFA levels and if these factors impact fertility. PMID:21353671

  1. Role of Fas-mediated apoptosis and follicle-stimulating hormone on the developmental capacity of bovine cumulus oocyte complexes in vitro.

    PubMed

    Rubio Pomar, Francisco J; Roelen, Bernard A J; Slot, Karin A; van Tol, Helena T A; Colenbrander, Ben; Teerds, Katja J

    2004-09-01

    Follicular atresia is believed to be largely regulated by apoptosis. To further understand how apoptosis can affect cumulus cells and oocytes we have evaluated the incidence and regulation of apoptosis affecting bovine cumulus oocyte complexes in vitro. Expression of components of the Fas signaling pathway was studied in both oocytes and cumulus cells by polymerase chain reaction after reverse transcription, immunoblotting, and indirect immunofluorescence. Furthermore, the Fas signaling pathway was activated in cumulus oocyte complexes with an agonistic anti-Fas antibody during in vitro maturation in the presence or absence of FSH. Viability and incidence of apoptosis in cumulus cells were evaluated by assessing membrane integrity and nuclear morphology. Oocyte nuclear maturation was also analyzed, as well as cleavage rates, blastocyst formation rates, and blastocyst quality, following in vitro fertilization. Fas mRNA and protein were expressed both in oocytes and cumulus cells. FasL protein was found in cumulus cells but could not be detected in oocytes, despite its mRNA expression. Both activation of the Fas pathway and presence of FSH during in vitro maturation increased the incidence of apoptosis in cumulus cells, affecting predominantly the middle and peripheral regions of the cumulus. The observed increase, however, had no effect on the developmental competence of the oocytes. PMID:15128594

  2. Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality.

    PubMed

    Lunn, Matthew O; Wright, Shirley J

    2013-09-15

    The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction. PMID:23790239

  3. 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. PMID:27172057

  4. 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. PMID:26658709

  5. In vitro effects of relaxin on gene expression in porcine cumulus-oocyte complexes and developing embryos

    PubMed Central

    2011-01-01

    Background Relaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig. Methods Immature cumulus-oocyte complexes (COCs) were obtained from ovarian follicles of sows. In experiment 1, COCs were matured in the presence of 0, 20, or 40 ng relaxin/ml, or 10% (v/v) porcine follicular fluid. In experiment 2, COCs were in vitro matured, fertilized and resulting embryos were cultured in the presence of 0, 20, or 40 ng relaxin/ml. In experiment 3, COCs were matured in the presence of 40 ng relaxin/ml, fertilized and zygotes were cultured as indicated in experiment 2. We evaluated the proportions of matured oocytes in experiment 1, cleaved and blastocysts on Day 2 and Day 7 post insemination in all experiments. The total cell number of blastocysts was also evaluated. In parallel, transcription levels of both relaxin and its receptors (RXFP1 and RXFP2), as well as a pro- (Bax) and anti- (Bcl2-like 1) apoptotic-related genes were determined. All data were analyzed by ANOVA and significant differences were fixed for P < 0.05. Results In experiment 1, relaxin significantly increased the proportions of matured oocytes and cleaved embryos, as well as the expression level of RXFP2 mRNA compared to RXFP1 (P < 0.05). There was no effect on endogenous expression of relaxin and Bcl2-like1/Bax ratios. In all experiments, relaxin did not affect the proportions of blastocysts, but did significantly increase their total cell numbers (P < 0.05). Furthermore, no effect of relaxin was observed on Bcl2-like1/Bax expression ratios, which were similar between groups. Conclusions Exogenous relaxin influences its own receptors expression, improves oocyte nuclear maturation. Its beneficial effect on total cell number of blastocysts appears to be through a Bcl2-like1

  6. Melatonin promotes the cumulus-oocyte complexes quality of vitrified-thawed murine ovaries; with increased mean number of follicles survival and ovary size following heterotopic transplantation.

    PubMed

    Hemadi, Masoud; Abolhassani, Farid; Akbari, Mohammad; Sobhani, Aligholi; Pasbakhsh, Parichehr; Ahrlund-Richter, Lars; Modaresi, Mohammad H; Salehnia, Mojdeh

    2009-09-15

    We have tested the protective effect of melatonin on neonate murine ovarian tissue after vitrification, thawing and heterotopic transplantation into ovariectomized recipient mice. Vitrified ovaries from neonate (CBA x C57Bl/6) F1 hybrid mice were thawed under standard condition with or without the addition of 100 microM melatonin. Following transplantation, melatonin (20 mg/kg/day) or saline solution (physiological saline) was injected i.p. to the treated and non-treated groups for 48 h respectively. Follicle survival and development, together with ovary size followed. Also, vaginal cytology was carried out for monitoring restored puberty. Histological and immunohistochemical studies showed that melatonin could promote the quality of the cumulus-oocyte complexes with uniform distribution of granulosa and stromal cells in the ovarian grafts. Furthermore, the mean follicles survival was improved and the ovary size increased (P< or = 0.001). The overall mean number of follicles entering the next maturation stage dramatically increased. However, the revascularization and restoration of puberty of ovarian grafts were similar between melatonin-treated and control groups. In conclusion, melatonin as a protection from ischemic injury and a reduce oxidative stress, was shown beneficial during the early days of transplantation. PMID:19622351

  7. Effects of aspiration vacuum and needle diameter on cumulus oocyte complex morphology and developmental capacity of bovine oocytes.

    PubMed

    Bols, P E; Van Soom, A; Ysebaert, M T; Vandenheede, J M; de Kruif, A

    1996-04-01

    The effects of aspiration vacuum and needle diameter on the morphology of the cumulusoocyte-complex (COC) and developmental capacity of the oocyte after IVF was studied in 2 experiments using a disposable ovum pick-up needle guidance system whose construction permits its use in vitro. In Experiment 1, the relationship was determined between the aspiration vacuum, expressed in millimetre of mercury, and the actual amount of water aspirated by the system, expressed in millilitre per minute. In Experiment 2, five different levels of aspiration vacuum for 3 different needle diameters (18g, 19g and 21g) were tested in slaughterhouse ovaries. The cumulus-oocyte complexes (COCs) were divided into 3 categories: 1) oocytes with a compact cumulus, 2) oocytes with an expanded cumulus and 3) naked oocytes. The results show that a change of needle diameter can triple the amount of fluid actually aspirated. The highest oocyte recovery rates are obtained when using the thickest needle (18-g), regardless of the aspiration vacuum. On the average, for all needle types, more oocytes are recovered at the highest aspiration vacuum. For all needle diameters, the proportion of oocytes surrounded by a compact cumulus decreases progressively as the vacuum increases. Regardless of the vacuum applied, thinner needles result in a higher proportion of recovered COCs with a compact cumulus. At a high aspiration vacuum, naked oocytes become predominant regardless of the needle diameter. The prevalence of blastocysts, expressed in proportion to the recovered COCs, decreases as the aspiration vacuum increases, being especially noticeable between 70 and 130 mm Hg. PMID:16727859

  8. Effects of needle tip bevel and aspiration procedure on the morphology and developmental capacity of bovine compact cumulus oocyte complexes.

    PubMed

    Bols, P E; Ysebaert, M T; Van Soom, A; de Kruif, A

    1997-04-15

    Effects of the needle tip bevel and the aspiration procedure on the morphology of cumulusoocyte-complexes (COCs) and the developmental capacity of the oocytes after IVF were studied in 2 in vitro oocyte pick-up (OPU) simulations using a disposable ovum pick-up needle guidance system. In Experiment 1, the influence of the length of the needle bevel was investigated using a short and a long bevelled 20-g disposable needle. After being aspirated from slaughterhouse ovaries, the retrieved COCs were divided into 3 categories: 1) oocytes surrounded by a compact cumulus, 2) oocytes with an expanded cumulus, 3) partially naked oocytes. In Experiment 2, the influence of 5 different levels of aspiration vacuum for 3 different needle diameters (18-g, 19-g, 20-g) and 2 different needle bevels (long, short) was tested on the recovery and on the morphology of the cumulus investment of a fixed number of previously scored compact cumulus oocytes complexes (CCOCs), retrieved after slicing slaughterhouse ovaries. The re-retrieved COCs were allocated to Categories 1 and 3. The results show that the length of the needle bevel has a significant effect on oocyte recovery, in favor of the long-bevelled needle. As soon as higher aspiration vacua are used, a decrease of the number of CCOCs can be observed, which is less prominent for the short-bevelled needle compared to the long-bevelled one. The final number of blastocysts is similar for both needle types. In Experiment 2, the disposable needle system proved to be highly effective since nearly 80% of the CCOCs were retrieved. At low aspiration vacuum, up to 90% of the CCOCs withstand the aspiration procedure undamaged. Increasing the aspiration vacuum results in a decrease of the number of CCOCs, which is less pronounced using thinner needles. Averaged over all needle types, the prevalence of blastocysts expressed relative to the number of recovered oocytes decreases with higher aspiration vacuum. PMID:16728071

  9. Transvaginal ultrasound-guided cumulus oocyte complexes aspiration and in vitro embryo production in suckled beef and lactating dairy cattle on pasture-based management conditions.

    PubMed

    Ratto, M H; Peralta, O A; Mogollon, G; Strobel, P; Correa, J

    2011-11-01

    This study was conducted to determine the use of repeated transvaginal ultrasound-guided cumulus oocyte complex (COC) aspiration on COC recovery rate, in vitro embryo production (IVP) and subsequent pregnancy rates in Holstein Friesian (HF) and Aberdeen Angus (AA) cows (Experiment 1), and in pregnant and non-pregnant Holstein Friesian cows (Experiment 2). Cycling, non-pregnant HF (n=17) and AA (n=32) cows with 40-70 days postpartum, between 3 and 5 years of age were used in the Experiment 1. All cows were submitted to repeated transvaginal ultrasound-guided COC aspiration twice a week for 5-7 weeks. Cumulus ooctye complexes (COC) were in vitro matured, fertilized and cultured for 8 days. An overall of 100 and 350 embryos from HF and AA cows respectively were cryopreserved using a conventional slow freezing (Experiment 1). A total of 81 and 285 frozen-thawed embryos from HF and AA cows respectively were transferred to recipient cows. Pregnancy diagnosis was performed at 60 and 150 days of gestation using transrectal ultrasonography. In Experiment 2, cycling non-pregnant (n=9) and pregnant (n=8) HF cows were submitted to repeated ultrasound-guided COC aspiration and COC were in vitro matured, fertilized and cultured as in Experiment 1, except that embryos were cryopreserved but not thawed and transferred as described for Experiment 1. The results of this study indicate that COC recovery rate and blastocyts production are affected by the breed of the donor cow. The quality of blastocyts produced from both breed did not differ in terms of pregnancy and calving rates (Experiment 1). The physiologic state of pregnancy did not affect COC recovery rate and blastocysts production per donor/session (Experiment 2). The use of ultrasound-guided COC aspiration and IVP could be a powerful technique to improve the genetic of beef and dairy cattle managed under pasture-based conditions management in the southern Chile. PMID:22030337

  10. 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. PMID:24599290

  11. 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

  12. Differential gene expression in cumulus oocyte complexes collected by ovum pick up from repeat breeder and normally fertile Holstein Friesian heifers.

    PubMed

    Puglisi, Roberto; Cambuli, Caterina; Capoferri, Rossana; Giannino, Laura; Lukaj, Aleksander; Duchi, Roberto; Lazzari, Giovanna; Galli, Cesare; Feligini, Maria; Galli, Andrea; Bongioni, Graziella

    2013-09-01

    The aim of this study was to establish whether perturbed gene expression during cumulus oocyte development causes repeat breeding in cattle. In this study, a repeat breeder was defined as a normal estrous cycling animal that did not become pregnant after three inseminations despite the absence of clinically detectable reproductive disorders. Transcripts of genes extracted from cumulus oocyte complexes (COC) that were collected from three repeat breeder and three normally fertile Holstein Friesian heifers were compared. Up to 40 COC were collected from each heifer by means of repeated sessions of ovum pick up in the absence of hormonal stimulation; immediately plunged into liquid nitrogen; and stored at -80°C until analysis. For each heifer, RNA was extracted from the pooled COC and hybridized on GeneChip(®) Bovine Gene Array (Affymetrix). Analysis of gene expression profiles of repeat breeder and control COC showed that 178 genes were differentially expressed (log2 fold change>1.5). Of these genes, 43 (24%) were up-regulated and 135 (76%) were down-regulated in repeat breeder relative to control heifers. This altered pattern of expression occurred in genes involved in several cellular biological processes and cellular components such as metabolism, angiogenesis, substrate/ion transport, regulation/signaling, cell adhesion and cytoskeleton. From these, 13 genes potentially involved in cumulus oocyte growth were subjected to validation by qRT-PCR and nine genes (annexin A1, ANXA1; lactoferrin, LTF; interferon stimulated exonuclease 20kDa, ISG20/HEM45; oxidized low density lipoprotein receptor 1, OLR1; fatty acid desaturase 2, FADS2; glutathione S-transferase A2 and A4, GSTA2 and GSTA4; glutathione peroxidase 1, GPX1; endothelin receptor type A, EDNRA) were confirmed to be differentially expressed. This study identified potential marker genes for fertility in dairy cattle. PMID:23911014

  13. The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol.

    PubMed

    Kempisty, B; Woźna, M; Piotrowska, H; Bukowska, D; Jackowska, M; Antosik, P; Jaśkowski, J M; Brüssow, K-P

    2012-02-01

    The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 μg/mL, 1.0 μg/mL, and 2.0 μg/mL of P4 (experiment 1), or with (2) 2.0 μg/mL E2, and with (3) a combination of E2 (2.0 μg/mL) and P4 (0.5, 1.0, or 2.0 μg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 μg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 μg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the

  14. Effect of temporary meiosis block during prematuration of bovine cumulus-oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production.

    PubMed

    Guemra, Samuel; da Silva Santo, Eriko; Zanin, Renato; Monzani, Paulo Sergio; Sovernigo, Tobias Canan; Ohashi, Otávio Mitio; Verde Leal, Cláudia Lima; Adona, Paulo Roberto

    2014-04-15

    Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus-oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199-HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 μL TCM 199 + 12 μM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO2 in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P < 0.05) and more embryos/cow (3.8 and 2.7, respectively, P

  15. The effects of superoxide dismutase addition to the transport medium on cumulus-oocyte complex apoptosis and IVF outcome in cats (Felis catus).

    PubMed

    Cocchia, Natascia; Corteggio, Annunziata; Altamura, Gennaro; Tafuri, Simona; Rea, Silviana; Rosapane, Isabella; Sica, Alessandro; Landolfi, Francesco; Ciani, Francesca

    2015-03-01

    The aim of the present study was to examine the effects of superoxide dismutase (SOD) addition to the ovary transport medium (4°C, 3-72 h) on ovarian cell viability and apoptosis and in vitro embryo production (IVEP) in domestic cats. The ovaries collected from 76 mixed-breed domestic queens were randomly assigned to the control or SOD-treated groups and incubated for 3, 24, 48 or 72 h. The ovaries were then subjected to the following: (1) fixed in formalin to assess the incidence of apoptosis (fragmented DNA in situ detection kit), (2) stored at -196°C in liquid nitrogen to evaluate the expression of the pro-apoptotic Bax gene and the anti-apoptotic Bcl-2 gene (RT-PCR), and (3) used to obtain the cumulus-oocyte complexes (COCs) in order to test the cell viability (carboxyfluorescein or trypan blue staining) and IVEP. The incidence of apoptosis appeared to be higher in the control compared with the SOD-treated ovaries. The ovarian expression of Bax was lower and the Bcl-2 expression was higher in the SOD-treated group compared with the control group. The presence of SOD in the transport medium increased the viability of COCs and IVEP compared with the control medium. In summary, the supplementation of the ovary transport medium with SOD reduced cellular apoptosis and enhanced COC survival and IVEP in domestic cats. PMID:25726378

  16. 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

  17. Expression of cumulus-oocyte complex genes and embryonic development in goats subjected to progestogen-based estrus synchronization.

    PubMed

    Thammasiri, Jiratti; Navanukraw, Chainarong; Uriyapongson, Suthipong; Khanthusaeng, Vilaivan; Kamollirt, Chuchat

    2016-07-15

    The objective was to investigate the effect of short-term (7 days) and long-term (14 days) progesterone-based estrus synchronization on number of follicles, progesterone concentrations, cumulus-oocyte complex (COC) gene expression, and embryonic development in goats. Nulliparous Thai-native goats (n = 45) were randomly assigned to one of two estrus synchronization treatments. Goats were treated with intravaginal sponges containing 60-mg medroxyprogesterone acetate (MAP; Synchrogest esponjas, Spain) during 7 or 14 days (short-term or long-term protocol, respectively). Multiple follicular development was induced by intramuscularly injections of 300-IU eCG in both groups (1 day before sponge withdrawal). An ovariectomy was performed at 24 hours after sponge removal to evaluate number of follicle and collect oocyte for IVF. Oocyte quality (healthy or nonhealthy) was determined by morphology of COCs before IVM. Recovery of COCs and total cellular RNA isolation were applied to determine apoptosis-related gene expression. After IVF, embryos were evaluated during the eight-day culture as numbers of cleaved oocyte, morula, and blastocyst embryo. Total numbers of follicles and oocytes were similar for both treatments. Plasma progesterone concentrations were not different during MAP insertion period (P > 0.05). However, goats that received the short-term protocol had a greater number of 4 to 6-mm follicle, healthy oocytes, cleaved oocytes, and morula embryos than goats that received the long-term protocol (P < 0.01). In addition, the expression of B-cell lymphoma 2 messenger RNA was greater (P < 0.05) in COCs derived from the 7 days MAP-treated when compared to the 14 days MAP-treated goats. These data highlight that the 7-day progestin-based treatment may contribute to quality of oocytes and embryonic development in goats. PMID:27056418

  18. 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

  19. Formulation and stability evaluation of 3D alginate beads potentially useful for cumulus-oocyte complexes culture.

    PubMed

    Dorati, Rossella; Genta, Ida; Ferrari, Michela; Vigone, Giulia; Merico, Valeria; Garagna, Silvia; Zuccotti, Maurizio; Conti, Bice

    2016-01-01

    Ovarian follicle encapsulation in synthetic or natural matrixes based on biopolymers is potentially a promising approach to in vitro maturation (IVM) process, since it maintains follicle 3D organisation by preventing its flattening and consequent disruption of gap junctions, preserving the functional relationship between oocyte and companion follicle cells. The aim of the work was to optimise physico-chemical parameters of alginate microcapsules for perspective IVM under 3D environments. On this purpose alginate and cross-linking agent concentrations were investigated. Alginate concentration between 0.75% and 0.125% w/w and Mg(2+), Ba(2+), Ca(2+ )at concentration between 100 and 20 mM were tested. Follicle encapsulation was obtained by on purpose modified diffusion setting gelation technique, and evaluated together with beads, chemical and mechanical stability in standard and stressing conditions. Beads permeability was tested towards albumin, fetuin, pyruvate, glucose, pullulan. Results demonstrated that 0.25% alginate cross-linked in 100 mM CaCl2 beads is suitable to follicle encapsulation. PMID:26791322

  20. Oocyte developmental competence after in vitro maturation depends on the persistence of cumulus-oocyte comunications which are linked to the intracellular concentration of cAMP.

    PubMed

    Modina, S; Luciano, A M; Vassena, R; Baraldi-Scesi, L; Lauria, A; Gandolfi, F

    2001-01-01

    Oocyte and cumulus cells are interconnected by an extensive network of gap junctions (GJ) formed by connexin 43. In the present study the functional and morphological status of GJ was investigated during in vitro maturation of bovine oocytes using media known to induce different rates of maturation and developmental competence as well as different levels of cumulus expansion and intracellular concentrations of cAMP. GJ functional condition was studied by microinjection of the fluorescent dye Lucyfer Yellow in cumulus-enclosed oocytes and the intracellular distribution of connexin 43 was examined by immunofluorescence immunocytochemistry. Both functional and morphological analysis of GJ between oocytes and cumulus cells indicated that high maturation and development rates are accompanied by the prolonged persistence of permeable communications, which, however, are independent from cumulus expansion. On the contrary, the premature interruption of such communications was linked to low maturation and development. When these results were correlated to the measurements of intracellular levels of cAMP it was observed that cumulus expansion, GJ permeability and good quality maturation require the highest levels of cAMP. Intermediate cAMP levels determine GJ permeability and good quality maturation in the absence of cumulus expansion while below a threshold level of cAMP, oocyte maturation is defective with a premature interruption of communications through GJ and lack of cumulus expansion which lead to poor development. PMID:11732583

  1. Increased progesterone production in cumulus-oocyte complexes of female mice sired by males with the Y-chromosome long arm deletion and its potential influence on fertilization efficiency.

    PubMed

    Kotarska, Katarzyna; Galas, Jerzy; Przybyło, Małgorzata; Bilińska, Barbara; Styrna, Józefa

    2015-02-01

    It was revealed previously that B10.BR(Y(del)) females sired by males with the Y-chromosome long arm deletion differ from genetically identical B10.BR females sired by males with the intact Y chromosome. This is interpreted as a result of different epigenetic information which females of both groups inherit from their fathers. In the following study, we show that cumulus-oocyte complexes ovulated by B10.BR(Y(del)) females synthesize increased amounts of progesterone, which is important sperm stimulator. Because their extracellular matrix is excessively firm, the increased progesterone secretion belongs presumably to factors that compensate this feature enabling unchanged fertilization ratios. Described compensatory mechanism can act only on sperm of high quality, presenting proper receptors. Indeed, low proportion of sperm of Y(del) males that poorly fertilize B10.BR(Y(del)) oocytes demonstrates positive staining of membrane progesterone receptors. This proportion is significantly higher for sperm of control males that fertilize B10.BR(Y(del)) and B10.BR oocytes with the same efficiency. PMID:24899473

  2. In vitro fertilization and development of bovine oocytes matured in serum-free medium.

    PubMed

    Saeki, K; Hoshi, M; Leibfried-Rutledge, M L; First, N L

    1991-02-01

    This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development. PMID:2009327

  3. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. 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. PMID:26249727

  6. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes

    PubMed Central

    Xie, Hong-Li; Wang, Yan-Bo; Jiao, Guang-Zhong; Kong, De-Ling; Li, Qing; Li, Hong; Zheng, Liang-Liang; Tan, Jing-He

    2016-01-01

    Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation. PMID:26857840

  7. Effects of glucose metabolism during in vitro maturation on cytoplasmic maturation of mouse oocytes.

    PubMed

    Xie, Hong-Li; Wang, Yan-Bo; Jiao, Guang-Zhong; Kong, De-Ling; Li, Qing; Li, Hong; Zheng, Liang-Liang; Tan, Jing-He

    2016-01-01

    Although there are many reports on the effect of glucose metabolism on oocyte nuclear maturation, there are few studies on its effect on ooplasmic maturation. By manipulating glucose metabolism pathways using a maturation medium that could support oocyte nuclear maturation but only a limited blastocyst formation without glucose, this study determined effects of glucose metabolism pathways on ooplasmic maturation. During maturation of cumulus-oocyte-complexes (COCs) with glucose, the presence of PPP inhibitor, DHEA or glycolysis inhibitor, iodoacetate significantly decreased blastocyst rates, intraoocyte glutathione and ATP. While blastocyst rates, GSH/GSSG ratio and NADPH were higher, ROS was lower significantly in COCs matured with iodoacetate than with DHEA. Fructose-6-phosphate overcame the inhibitory effect of DHEA on PPP. During maturation of COCs with pyruvate, electron transport inhibitor, rotenone or monocarboxylate transfer inhibitor, 4-CIN significantly decreased blastocyst rates. Cumulus-denuded oocytes had a limited capacity to use glucose or lactate, but they could use pyruvate to support maturation. In conclusion, whereas glycolysis promoted ooplasmic maturation mainly by supplying energy, PPP facilitated ooplasmic maturation to a greater extent by both reducing oxidative stress and supplying energy through providing fructose-6-phosphate for glycolysis. Pyruvate was transferred by monocarboxylate transporters and utilized through mitochondrial electron transport to sustain ooplasmic maturation. PMID:26857840

  8. 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. PMID:26411362

  9. 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. PMID:26165355

  10. Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation.

    PubMed

    Yokoo, Masaki; Kimura, Naoko; Abe, Hiroyuki; Sato, Eimei

    2008-11-01

    During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process. PMID:18838022

  11. Role of hyaluronic acid in maturation and further early embryo development of bovine oocytes.

    PubMed

    Marei, W F; Ghafari, F; Fouladi-Nashta, A A

    2012-08-01

    Hyaluronic acid (HA), an important component of the extracellular matrix, plays a crucial role for cumulus cell expansion. Genes and proteins involved in HA synthesis and its receptor CD44 are expressed in cumulus oocyte complexes (COCs) in different animal species and increase during maturation. Hyaluronidase enzymes (Hyal) degrade HA into smaller biologically active HA fragments. To investigate the effects of the molecular size and concentration of HA on oocyte maturation and further embryo development, bovine oocytes were matured in vitro in the presence or absence of HA, Hyal-2 or 4-methylumbelliferone (4-MU); an HA synthesis inhibitor. The rates of oocyte nuclear maturation to metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Hyal-2 inhibited cumulus cell expansion without affecting oocyte maturation and further embryo development. Whereas, 4-MU at 1 mm reduced cumulus cell expansion, oocyte maturation rate and further embryo development; an effect which was partially abrogated by exogenous HA supplementation. These data suggest that HA production by cumulus cells during maturation is essential not only for cumulus cell expansion, but also for oocyte maturation and further embryo development. This effect is not affected by HA-degradation by Hyal-2. PMID:22541325

  12. 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. PMID:20128947

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

    PubMed

    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

  14. Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin.

    PubMed

    Yasmin, Cut; Otoi, Takeshige; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani

    2015-03-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission. PMID:25655418

  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. 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. PMID:27233358

  17. Follicular 17β-estradiol and progesterone concentrations and degree of cumulus cell expansion as predictors of in vivo-matured oocyte developmental competence in superstimulated heifers.

    PubMed

    Aardema, Hilde; Roelen, Bernard A J; van Tol, Helena T A; Oei, Christine H Y; Gadella, Bart M; Vos, Peter L A M

    2013-10-01

    The quality of an oocyte is crucial for successful generation of offspring, but few selection parameters have been identified that reliably predict oocyte developmental competence. The objective of the present study was to determine whether the developmental competence of in vivo-matured oocytes derived from superstimulated heifers could be predicted by 17β-estradiol and progesterone concentrations in follicular fluid, degree of cumulus cell expansion, and follicular diameter. Cumulus oocyte complexes were individually collected from follicles ≥8 mm 22 hours after an induced LH peak and individually fertilized and cultured in vitro. Only oocytes that originated from follicles with 17β-estradiol ≤0.25 μM and progesterone ≥0.26 μM developed into blastocysts. When a combination of these cutoff values was evaluated as a predictor of oocyte competence, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 75%, 49%, and 100%, respectively. Hormone concentrations in follicular fluid were also associated with the degree of cumulus cell expansion and only cumulus oocyte complexes with full expansion developed into blastocysts; sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 71%, 45%, and 100%, respectively, when full expansion was used as the predictive criterion for blastocyst production. Follicular diameter was not a good predictor of oocyte competence. In conclusion, concentrations of 17β-estradiol and progesterone in the preovulatory follicle and the degree of cumulus cell expansion are predictors of blastocyst production in superstimulated heifers and can be used as selection markers for oocyte developmental competency. PMID:23831113

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

    PubMed

    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

    2015-11-27

    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. 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

  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. PMID:24420374

  1. In vitro fertilization and development of porcine oocytes matured in follicular fluid.

    PubMed

    Agung, Budiyanto; Otoi, Takeshige; Fuchimoto, Dai-ichiro; Senbon, Shoichiro; Onishi, Akira; Nagai, Takashi

    2013-01-01

    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 × 10(6) 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. In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes.

    PubMed

    Wu, J; Zhang, L; Wang, X

    2001-03-01

    The purpose of this study was to determine the rates of maturation, fertilization and embryo development of ultrarapidly frozen immature oocytes (immature cumulus-oocyte complexes; COCs) obtained from antral follicles in ovaries of patients with chocolate ovarian cysts. The COCs were cryopreserved by a vitrification method using 5.5 mol ethylene glycol l (-1) plus 1.0 mol sucrose l (-1) in Dulbecco's PBS (DPBS). The survival, maturation and fertilization rates, and the percentage of embryos developing to the two-cell stage were 59, 64, 70 and 71%, respectively. No significant differences were noted in the rates of maturation, fertilization and embryo development between control and cryopreserved oocytes. Two embryos that developed from cryopreserved oocytes of the oocyte donor programme were selected for transfer into the uterus of a recipient with premature ovarian failure, after the recipient had received steroid replacement. A biochemical pregnancy occurred in the recipient after embryo transfer. These results indicate that immature oocytes can survive after cryopreservation and subsequently can be cultured to mature oocytes that are capable of undergoing fertilization in vitro and developing into embryos. PMID:11226064

  3. 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…

  4. Ghrelin accelerates in vitro maturation of bovine oocytes.

    PubMed

    Dovolou, E; Messinis, I E; Periquesta, E; Dafopoulos, K; Gutierrez-Adan, A; Amiridis, G S

    2014-08-01

    Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes' expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process. PMID:24889518

  5. The effect of lysophosphatidic acid during in vitro maturation of bovine oocytes: embryonic development and mRNA abundances of genes involved in apoptosis and oocyte competence.

    PubMed

    Boruszewska, Dorota; Torres, Ana Catarina; Kowalczyk-Zieba, Ilona; Diniz, Patricia; Batista, Mariana; Lopes-da-Costa, Luis; Woclawek-Potocka, Izabela

    2014-01-01

    In the present study we examined whether LPA can be synthesized and act during in vitro maturation of bovine cumulus oocyte complexes (COCs). We found transcription of genes coding for enzymes of LPA synthesis pathway (ATX and PLA2) and of LPA receptors (LPAR 1-4) in bovine oocytes and cumulus cells, following in vitro maturation. COCs were matured in vitro in presence or absence of LPA (10(-5) M) for 24 h. Supplementation of maturation medium with LPA increased mRNA abundance of FST and GDF9 in oocytes and decreased mRNA abundance of CTSs in cumulus cells. Additionally, oocytes stimulated with LPA had higher transcription levels of BCL2 and lower transcription levels of BAX resulting in the significantly lower BAX/BCL2 ratio. Blastocyst rates on day 7 were similar in the control and the LPA-stimulated COCs. Our study demonstrates for the first time that bovine COCs are a potential source and target of LPA action. We postulate that LPA exerts an autocrine and/or paracrine signaling, through several LPARs, between the oocyte and cumulus cells. LPA supplementation of maturation medium improves COC quality, and although this was not translated into an enhanced in vitro development until the blastocyst stage, improved oocyte competence may be relevant for subsequent in vivo survival. PMID:24729661

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Effects of Aroclor 1254 on In Vivo Oocyte Maturation in the Mouse

    PubMed Central

    Liu, ShuZhen; Jiang, LiGang; Meng, XiaoQian; Han, XiaoYing; Cheng, Dong; Zhang, TianLiang; Miao, YiLiang

    2014-01-01

    Polychlorinated biphenyls (PCBs) are stable, lipophilic compounds that accumulate in the environment and in the food chain. Though some studies provided evidence that PCBs had adverse effects on reproductive function, most of these results were from in vitro models. Therefore we investigated the effect of Aroclor 1254 (a commercial PCBs mixture) treatments on in vivo maturation and developmental potential of mouse oocytes. In the present study, female ICR mice were treated with different doses (12.5, 25 and 50 mg/kg) of Aroclor 1254 (a commercial PCB mixture) once every 72 hours by intraperitoneal injection for 9 days. After three treatments of Aroclor 1254, the mice were superovulated to collect oocytes one day after the last exposure. The effects of Aroclor 1254 on oocyte maturation, fertilization, and preimplantation embryonic development were investigated. Immunofluorescence-stained oocytes were observed under a confocal microscope to assess the effects of Aroclor 1254 on spindle morphology. Parthenogenic activation and the incidence of cumulus apoptosis in cumulus-oocyte complexes were observed as well. Oocytes exposed to different doses of Aroclor 1254 in vivo were associated with a significant decrease in outgrowth potential, abnormal spindle configurations, and the inhibition of parthenogenetic activation of ovulated oocytes. Furthermore, the incidence of apoptosis in cumulus cells was increased after exposed to Aroclor 1254. These results may provide reference for the treatment of reproductive diseases such as infertility or miscarriage caused by environmental contaminants. PMID:25013911

  8. Leptin and ObRa/MEK signalling in mouse oocyte maturation and preimplantation embryo development.

    PubMed

    Ye, Yinghui; Kawamura, Kazuhiro; Sasaki, Mitsue; Kawamura, Nanami; Groenen, Peter; Sollewijn Gelpke, Maarten D; Kumagai, Jin; Fukuda, Jun; Tanaka, Toshinobu

    2009-08-01

    Recent studies indicate that LH stimulates production of ovarian paracrine factors that induce meiosis of the oocyte. DNA microarray analyses of ovarian transcripts were performed in mice and major increases of a short isoform of leptin receptor, ObRa, were identified by the preovulatory LH/human chorionic gonadotrophin (HCG) surge. In oocytes, the level of ObRa transcripts was increased shortly after HCG stimulation, whereas the level of ObRb transcripts was not changed. Leptin was produced by cumulus, granulosa, theca and interstitial cells of ovaries and its transcript level was not regulated during gonadotrophin treatment. Treatment with leptin promoted germinal vesicle breakdown (GVBD) in oocytes within preovulatory follicles, and enhance first polar body extrusion in both cumulus-oocyte complexes and denuded oocytes. The leptin-promoted GVBD and first polar body extrusion were blocked by a mitogen-activated protein kinase extracellular signal regulated kinase kinases (MEK)1/2 inhibitor, U0126, but not its inactive analogue U0124. Furthermore, leptin promoted fertilization of oocytes and the in-vitro development of zygotes to preimplantation embryos. These findings suggest paracrine roles of leptin in the enhancement of nuclear maturation of oocytes through MEK1/2 signalling, and in the promotion of cytoplasmic maturation essential for successful oocyte development to the preimplantation embryos. PMID:19712552

  9. Expression and cellular distribution of cyclin-dependent kinase 4 (Cdk4) and connexin 43 (Cx43) in porcine oocytes before and after in vitro maturation.

    PubMed

    Kempisty, Bartosz; Ziółkowska, Agnieszka; Piotrowska, Hanna; Antosik, Paweł; Bukowska, Dorota; Zawierucha, Piotr; Jaśkowski, Jędrzej M; Brüssow, Klaus-Peter; Nowicki, Michał; Zabel, Maciej

    2014-03-01

    It is recognised that connexin 43 (Cx43) and cyclin-dependent kinase 4 (Cdk4) are involved in the cumulus cell-oocyte communication via gap junctions and the control of cell cycle progress. However, little is known about their mRNA expression pattern and encoded proteins distribution in porcine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were collected from 31 puberal crossbred Landrace gilts and analysed for their Cdk4 and Cx43 mRNA expression using RQ-PCR and for the respective protein expression by confocal microscopic observations. An increased Cdk4 and Cx43 mRNA expression was found in oocytes after IVM (P < 0.001 and P < 0.05, respectively). Confocal microscopic observations revealed a significant increase of Cdk4 protein expression in the cytoplasm of oocytes during the maturation process. The localisation of Cx43 changed from zona pellucida before to cytoplasm of oocytes after IVM. It is supposed that the increased expression of Cdk4 and Cx43 mRNA in oocytes after IVM is linked with the accumulation of a large amount of templates during the process of oocyte maturation. The translocation especially of Cx43 from the zona pellucida into the cytoplasm may be associated with a decrease in gap junction activity in fully grown porcine oocytes. Both Cdk4 and Cx43 can be used as 'checkpoints' of oocyte maturation. PMID:24334079

  10. 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. PMID:26781547

  11. 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.

    2016-01-01

    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.

  12. Maturation of pig oocytes in vitro in a medium with pyruvate.

    PubMed

    Gonzales-Figueroa, H; Gonzales-Molfino, H M

    2005-06-01

    The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC) and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouse and transported to the laboratory within 2 h at 35-39 degrees C,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM) medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF) and in SM + pFF + gonadotropins (eCG and hCG) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9%) and SM + pFF (42.9%) showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6%) showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 +/- 3.6%) and SM + pFF (42.9 +/- 3.7%). These results indicate that pig oocytes can be successfully matured in a chemically defined medium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment. PMID:15933780

  13. 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

  14. Effect of different activators on development of activated in vitro matured caprine oocytes

    PubMed Central

    Sharma, J. R.; Agarwal, S.; Kharche, S. D.; Goel, A. K.; Jindal, S. K.; Agarwal, S. K.

    2015-01-01

    This study was designed to compare the effectiveness of different activation treatments for activation of in vitro matured oocytes and their developmental potency in mCR2aa medium so as to obtain maximum number of embryos. A total of 1090 cumulus oocyte complexes (COC’s) were collected from 480 ovaries. In vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n=226) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Group 2 in vitro matured oocytes (n=294) were exposed to 7% ethanol for 5 min followed by treatment with 10 µg/ml CHX for 4 h in mCR2aa medium. Group 3 in vitro matured oocytes (n=325) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 µg/ml CHX for 4 h in mCR2aa medium. Group 4 in vitro matured oocytes (n=108) were cultured for 4 h without any chemical treatment in mCR2aa medium (control). The cleavage rate in groups 1, 2, 3 and 4 was 54.42%, 44.55%, 51.69% and 0.00%, respectively. The percentage of morula and blastocyst production in group 1, group 2 and group 3 was 26.01%, 29.77% and 29.76% and 2.43%, 1.52% and 1.78%, respectively. These results suggest that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa is most favorable for parthenogenetic caprine embryos production. PMID:27175149

  15. Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro.

    PubMed

    Warzych, Ewelina; Pers-Kamczyc, Emilia; Krzywak, Anna; Dudzińska, Stanisława; Lechniak, Dorota

    2013-03-01

    Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM. PMID:23522075

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity.

    PubMed

    Held, Eva; Mertens, Eva-Maria; Mohammadi-Sangcheshmeh, Abdollah; Salilew-Wondim, Dessie; Besenfelder, Urban; Havlicek, Vitezslav; Herrler, Andreas; Tesfaye, Dawit; Schellander, Karl; Hölker, Michael

    2012-01-01

    In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence. PMID:22541545

  18. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs.

    PubMed

    Kim, Jin-Woo; Park, Hyo-Jin; Chae, Sung-Kyu; Ahn, Jae-Hyun; DO, Geon-Yeop; Choo, Young-Kug; Park, Joung Jun; Jung, Bae Dong; Kim, Sun-Uk; Chang, Kyu-Tae; Koo, Deog-Bon

    2016-06-17

    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

  19. A comparative study of parthenogenetic activation and in vitro fertilization of in vitro matured caprine oocytes

    PubMed Central

    Kouamo, J; Kharche, S. D.

    2015-01-01

    The aim of the study was to compare the parthenogenetic activation and in vitro fertilization (IVF) of in vitro matured caprine oocytes. A total of 881 cumulus-oocyte complexes (COC’s) were collected from 243 ovaries. Oocytes were matured in TCM-199 medium containing eCG (20 IU/ml), hCG (20 IUµg/ml), oestradiol-17β (1 µg/ml), BSA embryo tested (3 mg/ml) supplemented with 10% fetal bovine serum at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. Based on cumulus expansion, the maturation rate was 86.86%. Morphological matured oocytes (n=749) were selected, denuded and randomly divided into two groups. Group 1 (n=223) in vitro matured oocytes activated with 5 µm calcium ionophore for 5 min and cultured in mCR2aa medium containing 5 mM DMAP for 4 h. After 4 h of DMAP treatment, the presumptive zygotes were washed and cultured in the embryo culture medium. Group 2 (n=526) in vitro matured oocytes processed for IVF in mTALP using fresh semen of a fertile pure bred adult Sirohi buck and in vitro culture in mCR2aa medium. Development of putative zygotes was observed every 24 h till day 9 post activation or fertilization under inverted phase contrast microscope. The cleavage rate, morula and blastocyst percentage in groups 1 and 2 were 67.36%, 23.07% and 9.23%, and 30.99%, 19.63% and 9.82%, respectively. The results indicated that the cleavage rate was comparatively higher following parthenogenetic activation with ionomycin/6-DMAP than IVF. PMID:27175145

  20. Ganglioside GD1a promotes oocyte maturation, furthers preimplantation development, and increases blastocyst quality in pigs

    PubMed Central

    KIM, Jin-Woo; PARK, Hyo-Jin; CHAE, Sung-Kyu; AHN, Jae-Hyun; DO, Geon-Yeop; CHOO, Young-Kug; PARK, Joung Jun; JUNG, Bae Dong; KIM, Sun-Uk; CHANG, Kyu-Tae; KOO, Deog-Bon

    2016-01-01

    Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs. PMID:26860251

  1. A comparative study of parthenogenetic activation and in vitro fertilization of in vitro matured caprine oocytes.

    PubMed

    Kouamo, J; Kharche, S D

    2015-01-01

    The aim of the study was to compare the parthenogenetic activation and in vitro fertilization (IVF) of in vitro matured caprine oocytes. A total of 881 cumulus-oocyte complexes (COC's) were collected from 243 ovaries. Oocytes were matured in TCM-199 medium containing eCG (20 IU/ml), hCG (20 IUµg/ml), oestradiol-17β (1 µg/ml), BSA embryo tested (3 mg/ml) supplemented with 10% fetal bovine serum at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. Based on cumulus expansion, the maturation rate was 86.86%. Morphological matured oocytes (n=749) were selected, denuded and randomly divided into two groups. Group 1 (n=223) in vitro matured oocytes activated with 5 µm calcium ionophore for 5 min and cultured in mCR2aa medium containing 5 mM DMAP for 4 h. After 4 h of DMAP treatment, the presumptive zygotes were washed and cultured in the embryo culture medium. Group 2 (n=526) in vitro matured oocytes processed for IVF in mTALP using fresh semen of a fertile pure bred adult Sirohi buck and in vitro culture in mCR2aa medium. Development of putative zygotes was observed every 24 h till day 9 post activation or fertilization under inverted phase contrast microscope. The cleavage rate, morula and blastocyst percentage in groups 1 and 2 were 67.36%, 23.07% and 9.23%, and 30.99%, 19.63% and 9.82%, respectively. The results indicated that the cleavage rate was comparatively higher following parthenogenetic activation with ionomycin/6-DMAP than IVF. PMID:27175145

  2. 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. PMID:23986966

  3. Effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine

    PubMed Central

    Amini, Ebrahim; Asadpour, Reza; Roshangar, Leila; Jafari-Joozani, Razi

    2016-01-01

    Background: Linoleic acid (LA) is a polyunsaturated fatty acid present in high concentrations in follicular fluid, when added to maturation culture media, it affects oocyte competence. Objective: In the present study, we investigated effect of linoleic acid supplementation on in vitro maturation, embryo development and apoptotic related gene expression in ovine Materials and Methods: The experiments conducted on 450 ovine Cumulus-oocyte complexes (COCs) with homogenous ooplasm and more than two compact layers of cumulus cells. For in vitro maturation COCs were randomly allocated into four treatment groups for 24 hr period. Treatment groups were as follow: control maturation media, 0 µM LA, 50 µM LA, 100 µM LA and 200 µM LA. The cumulus cell expansion and blastocysts rates were recorded. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to apoptotic gene expression by real-time PCR. Results: Highest concentration (200 µM/mL) of LA significantly decreased the rate of fully expanded cumulus cells 24 hr after in vitro maturation (IVM) and the percentage of blastocyste rate compared with the control (p<0.05). These inhibitory effects were associated with an increased in relative mRNA expression of Bax (Bcl-2- associated X) gene compared with controls. Conclusion: Data obtained in present study suggest that low concentration of LA used for maturation had no deleterious effect on subsequent embryonic development compared to high concentration of LA. Relative expression of Bcl-2 (B-cell lymphoma 2) and Bax in embryos seems to be associated with LA concentration. PMID:27351027

  4. Effects of Crocin Supplementation during In Vitro Maturation of Mouse Oocytes on Glutathione Synthesis and Cytoplasmic Maturation

    PubMed Central

    Mokhber Maleki, Elham; Eimani, Hussein; Bigdeli, Mohammad Reza; Golkar Narenji, Afsane; Abedi, Reyhane

    2016-01-01

    Background Crocin is an active ingredient of saffron (Crocus sativus L.) and its antioxidant properties have been previously investigated. This carotenoid scavenges free radicals and stimulates glutathione (GSH) synthesis; consequently, it may protect cells against oxidative stress. The aim of this research is to protect oocytes from oxidative stress by the addition of a natural source antioxidant. Materials and Methods In the present in vitro experimental study, we collected cumulus oocyte complexes (COCs) from mouse ovaries of euthanized, 6-8 week-old female Naval Medical Research Institute (NMRI) mice. Oocytes were subjected to in vitro maturation (IVM) in the presence of either crocin (5 or 10 μg/ml), 5 mM buthionine-[S-R]- sulfoximine (BSO), or the combination of crocin plus BSO. Oocytes that matured in vitro in a medium without crocin or BSO supplements were considered as controls. Following 16-18 hours of IVM, matured oocytes (n=631) were fertilized by capacitated sperm from NMRI male mice, and cultured in vitro for up to 96 hours to assess preimplantation embryonic development. The levels of GSH in metaphase II (MII) oocytes after IVM (n=240) were also assessed by the 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay. Results Supplementation of IVM media with 10 µg/ml crocin significantly (P<0.05) increased nuclear maturation, preimplantation development and GSH concentrations compared with the control group. Maturation of oocytes in IVM medium supplemented with BSO alone or the combination of 5 µg/ml crocin and BSO drastically decreased GSH concentrations and subsequently resulted in low rates of maturation, fertilization and blastocyst development. However, the combination of 10 µg/ml crocin with 5 mM BSO increased the level of nuclear maturation which was comparable to the control group. Conclusion Supplementation of IVM media with crocin can improve nuclear maturation rates and subsequent developmental potential of mouse

  5. The Effects of cAMP-elevating Agents and Alpha Lipoic Acid on In Vitro Maturation of Mouse Germinal Vesicle Oocytes

    PubMed Central

    Rahnama, Ali; Zavareh, Saeed; Ghorbanian, Mohammad Taghi; Karimi, Isaac

    2013-01-01

    Background In spite of extensive efforts to improve in vitro oocyte maturation, the obtained results are not very efficient. This study was conducted to assess impacts of cAMP elevating agents and alpha lipoic acid (ALA) on in vitro oocyte maturation and fertilization. Methods Mouse germinal vesicle (GV) oocytes were categorized into cumulus denuded oocytes (DOs; n=896) and cumulus oocyte complexes (COCs; n=1077) groups. GV oocytes were matured in vitro with or without ALA; (I) without the meiotic inhibitors; (II) supplemented with cilostamide; (III) supplemented with forskolin and (IV) supplemented with Forskolin plus cilostamide. The obtained metaphase II (MII) oocytes were subjected to in vitro fertilization. Independent-samples t-testand ANOVA were used for data analysis. A p-value less than 0.05 was considered to be statistically significant. Results The COCs maturation, fertilization and two cell embryo rates were higher than those of DOs in the control group, while no significant difference was observed between relevant COCs and DOs when they were cultured with cilostamide meiotic inhibitors in two step manner. Combined treatment of cilostamide and forskolin significantly elevated the developmental rates in both COCs and DOs as compared to other groups. The developmental rates of COCs and DOs in the presence of ALA were similar to their respective groups without ALA. Conclusion cAMP elevating agents were more effective on DOs than COCs with regard to rates of maturation and fertilization. However, ALA did not affect the developmental rates of both COCs and DOs in in vitro maturation in one or two step manner. PMID:24551571

  6. Cumulus cell features and nuclear chromatin configuration of in vitro matured canine COCs and the influence of in vivo serum progesterone concentrations of ovary donors.

    PubMed

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

    2009-02-01

    Phenotype integrity is viewed as an indicator of cumulus-oocyte complex (COC) viability. The objectives of this study were: (a) to observe the influence of cumulus investment expansion on the nuclear chromatin configuration of canine oocytes matured in vitro; (b) to examine the relationship between cumulus cell (CC) expansion and its morphology after in vitro maturation (IVM); (c) to ascertain the influence of in vivo serum progesterone (SP) concentrations of ovary donors on oocyte nuclear maturation, CC phenotypes and degrees of CC expansion of in vitro matured COCs. After 48 h of IVM in modified TCM 199, CCs from grade 1 and 2 COCs were stained with propidium iodide. Oocyte chromatin configuration was visualized by Hoechst 33342 stain. Results showed that oocyte IVM was not influenced by degree of CC expansion (D1, D2 and D3) in COCs. From the CC types (C1, C2 and C3), number of C1 types was higher at D1 expansion and differed from those observed at D2 and D3 expansions. Additionally, rates of apoptosis in D1 CCs were lower than those observed in D2 CCs (p < 0.05). Oocyte nuclear maturation was not influenced by in vivo SP concentrations of ovary donors. On the other hand, D3 expansion prevailed in COCs from bitches at SP > 2.5 ng/ml (p < 0.001). Moreover, in vitro CC apoptosis was associated both with low (0-1 ng/ml) and with high (>5 ng/ml) in vivo SP levels. These findings indicate that morphology of CCs from in vitro matured dog oocytes gives valuable information on viability of COCs and could possibly be used as a parameter in predicting the quality of oocytes destined for in vitro fertilization (IVF) and their outcomes. PMID:19126265

  7. Vitrification and Subsequent In Vitro Maturation of Mouse Preantral Follicles in Presence of Growth Factors

    PubMed Central

    Oryan Abkenar, Zahra; Ganji, Roya; Eghbal Khajehrahimi, Amir; Bahadori, Mohammad Hadi

    2014-01-01

    Objective Cryopreservation of ovarian tissue or follicles has been proposed as an alternative method for fertility preservation. Although successful vitrification of follicles has been reported in several mammalian species, the survival rate is generally low. The aim of this study was to investigate the effects of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on in vitro preantral follicle development after vitrification. Materials and Methods In this experimental study, preantral follicles with diameter of 150-180 µm were mechanically isolated from ovaries of 18-21 days old NMRI mice. Follicles were vitrified and warmed, then cultured in a-minimal essential medium (α-MEM) without growth factor supplementation as control group (group I), while supplemented with 20 ng/ml FGF (group II), 20 ng/ml EGF (group III), and 20 ng/ml FGF +20 ng/ml EGF (group IV). After 12 days, human chorionic gonadotrophin (hCG)/EGF was added to culture medium, and after 18-20 hours, the presence of cumulus oocyte complexes (COCs) and oocyte maturation were assessed. The chi-square (Χ2) test was used to analyze survival and ovulation rates of the follicles. Results Our results showed that the rate of metaphase II (MII) oocytes in FGF group increased in comparison with control and other treatment groups (p<0.027), but there was no difference between control with EGF and EGF+FGF groups in oocyte maturation rate (p>0.05). There was a significant decrease in survival rate of follicles in EGF+FGE group in comparison with other groups (p<0.008). After in vitro ovulation induction, the follicles in EGF group showed a higher ovulation rate (p<0.008) than those cultured in other groups. Conclusion FGF has beneficial effect on oocyte maturation, and EGF increases COCs number in vitro. Combination of EGF and FGE decreases the number of survived follicles. PMID:24611145

  8. Disruption of Bidirectional Oocyte-Cumulus Paracrine Signaling During In Vitro Maturation Reduces Subsequent Mouse Oocyte Developmental Competence1

    PubMed Central

    Yeo, Christine X.; Gilchrist, Robert B.; Lane, Michelle

    2009-01-01

    Oocyte-cumulus cell bidirectional communication is essential for normal development of the oocyte and cumulus cells (CCs) within the follicle. We showed recently that addition of recombinant growth differentiation factor 9 (GDF9), which signals through the SMAD2/3 pathway, during mouse oocyte in vitro maturation (IVM) increased fetal viability. This study thus aimed to observe the effects of disrupting oocyte-CC bidirectional communication during IVM on oocyte developmental competence and fetal outcomes. Cumulus-oocyte complexes (COCs) from equine chorionic gonadotropin-primed prepubertal (CBA/C57BL6) mice were cultured with or without 50 mIU/ml follicle-stimulating hormone (FSH) and 10 ng/ml epidermal growth factor (EGF) or 4 μM SMAD2/3 inhibitor SB-431542. Cumulus expansion and first polar body extrusion were then assessed, or COCs were fertilized and stained to evaluate sperm entry or cultured to the blastocyst stage. Embryo development and blastocyst quality were assessed, and Day 4.5 blastocysts were transferred to pseudopregnant recipients to analyze fetal outcomes. SMAD2/3 inhibition or FSH/EGF absence during IVM resulted in decreased cumulus expansion. First polar body extrusion and sperm entry were decreased in the absence of FSH/EGF, whereas only sperm entry was affected in SB-431542-matured COCs. Embryo development and blastocyst rates were unaffected; however, blastocyst quality was significantly altered, with reduced inner cell mass cell numbers in embryos derived from COCs matured in both treatments. When COCs were matured with SB-431542 in the absence of FSH/EGF, cumulus expansion was reduced, but fertilization, embryo development, and embryo quality were not. Inhibition of SMAD2/3 signaling in the presence of FSH/EGF significantly reduced fetal survival but had no effect on implantation or fetal and placental dimensions and morphology. PMID:19144958

  9. Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation.

    PubMed

    Kempisty, Bartosz; Piotrowska, Hanna; Bukowska, Dorota; Woźna, Magdalena; Ciesiółka, Sylwia; Wojtanowicz-Markiewicz, Katarzyna; Włodarczyk, Renata; Jopek, Karol; Jeseta, Michal; Bruska, Małgorzata; Nowicki, Michał; Jaśkowski, Jędrzej M; Brüssow, Klaus-Peter; Zabel, Maciej

    2015-12-01

    This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus-oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization. PMID:25315095

  10. 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

  11. In vitro maturation and development of porcine oocytes cultured in a straw or dish using a portable incubator with a CO2 chamber.

    PubMed

    Fujii, A; Kaedei, Y; Tanihara, F; Ito, A; Hanatate, K; Kikuchi, K; Nagai, T; Otoi, T

    2010-08-01

    We investigated the effects of a portable incubator with a CO(2) chamber on the viability and development of porcine oocytes/embryos for their transportation and examined the operational suitability of a straw or dish as a container for culturing the oocytes or embryos in the portable incubator. In the first experiment, the cumulus-oocyte complexes (COCs) were placed either in a dish or straw; and they were then cultured for 44 h in a standard CO(2) incubator, in the CO(2) chamber in an incubator, or in the CO(2) chamber in a portable incubator. The matured oocytes were fertilized with frozen-thawed spermatozoa and then cultured in a dish in the standard CO(2) incubator for 8 days. There were no differences in the proportions of oocytes reaching metaphase II stage among the groups. However, the proportions of cleavage and development to blastocysts derived from oocytes matured in a straw were lower than those from oocytes matured in a dish, irrespective of the type of incubator used. In the second experiment, the COCs were matured in a dish in the standard CO(2) incubator, and the matured oocytes were fertilized and then placed either in a dish or straw. These were then cultured for 8 days in the standard CO(2) incubator or portable incubator. Some zygotes cultured in the portable incubator developed to the blastocyst stage. The proportions of cleavage and development to blastocysts were significantly lower for putative zygotes cultured in straw than for those cultured in dish, irrespective of the type of incubator used. Our results indicate that a portable incubator with a CO(2) chamber can maintain the viability and development of oocytes/embryos, but the straw is not a suitable system for in vitro culture of the oocytes/embryos during transportation. PMID:19144033

  12. Successful piglet production by IVF of oocytes matured in vitro using NCSU-37 supplemented with fetal bovine serum.

    PubMed

    Suzuki, Misae; Misumi, Koji; Ozawa, Manabu; Noguchi, Junko; Kaneko, Hiroyuki; Ohnuma, Katsuhiko; Fuchimoto, Dai-ichiro; Onishi, Akira; Iwamoto, Masaki; Saito, Norio; Nagai, Takashi; Kikuchi, Kazuhiro

    2006-01-20

    Recently, piglets have been obtained from in vitro-produced blastocysts by using in vitro maturation systems in which oocytes have been matured in North Carolina State University (NCSU) solution supplemented with porcine follicular fluid (PFF). However, PFF is not available commercially. To prepare PFF from the ovaries required time and effort and there is substantial variation in quality among batches. Furthermore, PFF is considered a potential source of infectious agents. We evaluated another commercially available potential protein source, fetal bovine serum (FBS), for in vitro maturation, to produce embryos and piglets. Cumulus-oocyte complexes were matured in NCSU-37 with PFF or with one of four batches of FBS. The proportions of oocytes with expanded cumulus cells were lower in all FBS batch groups (P < 0.05, 15-41%) than that in the PFF group (74%). The proportions of oocytes that matured were also lower in all FBS batch groups (P < 0.05, 26-41%) than in the PFF group (73%), irrespective of cumulus expansion. However, the proportions of oocytes that underwent germinal vesicle breakdown were almost the same in all groups (76-96%). After in vitro fertilization, the rate of sperm penetration into matured oocytes was higher in the PFF group (P < 0.05, 63%) than in one batch of FBS (22%) and removal of the compacted cumulus cells after maturation did not affect fertilization status (21%). Subsequent in vitro embryo culture of the PFF and FBS groups for 6 day resulted in similar rates of blastocyst formation (17 and 19%, respectively) and similar numbers of cells per blastocyst (43 and 46 cells, respectively). When blastocysts obtained from oocytes matured with FBS were transferred into two recipients, one became pregnant and farrowed seven piglets. Transfer of blastocysts obtained from oocytes matured with PFF into two other recipients resulted in one pregnancy and production of four piglets. These data suggested that porcine in vitro maturation in NCSU-37

  13. Influence of selected (pre-)maturational parameters on in vitro development and sex distribution of bovine embryos.

    PubMed

    Abele, Eva; Stinshoff, Hanna; Hanstedt, Ana; Wilkening, Sandra; Meinecke-Tillmann, S; Wrenzycki, Christine

    2014-02-01

    The objectives of this research were to study the influence of a reduced oxygen concentration during in vitro maturation (IVM) and examine the effect of follicular glucose concentration on bovine in vitro development and sex distribution. In the first experiment, abattoir-derived cumulus-oocyte complexes (COC) were matured under 5% O2 or 20% O2. Secondly, COC were isolated and the glucose (G) concentration of each follicle was determined. COC were pooled in groups (G (< 1.1 mMol) or G (≥ 1.1 mMol)) according to the glucose content before being subjected to in vitro production (IVP). Cleavage and development rates were assessed on days 3, 7 and 8 post insemination. Blastocysts of each group were sexed by polymerase chain reaction (PCR). Expanded blastocysts were stained to assess total cell numbers and live-dead cell ratio. Cleavage and development rates stayed similar after reducing the O2 concentration during IVM. The sex ratio of embryos generated from oocytes matured under 5% O2 was shifted in favour of the female (♀: 61.9%), whereas the sex ratio of embryos belonging to the IVM 20% O2 group did not differ significantly from the expected 50:50 ratio. Neither a 'higher' nor a 'lower' intrafollicular glucose concentration influenced cleavage and development rates, cell numbers or live-dead cell ratio. Eighty five per cent (G (<1.1)) and 63.6% (G (≥ 1.1)) of the analysed embryos were female. In summary, neither a reduced O2 concentration during IVM nor selection based on follicular glucose concentrations affected the morphological quality of embryos. Although the sex distribution was shifted in favour of female embryos in all three experimental groups, more male embryos could be seen in the G (≥ 1.1) group compared with the G(<1.1) group. PMID:22784675

  14. Nitric Oxide Synthase (NOS) Inhibition during Porcine In Vitro Maturation Modifies Oocyte Protein S-Nitrosylation and In Vitro Fertilization

    PubMed Central

    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. PMID:25542028

  15. 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. PMID:23701210

  16. 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. PMID:25628423

  17. Leptin enhances maturation and development of calf oocytes in vitro.

    PubMed

    Jia, Z; Zhang, J; Wu, Z; Tian, J

    2012-10-01

    We investigated the effects of leptin on the in vitro maturation (IVM) and development of calf oocytes. Cumulus-oocyte complexes were matured in IVM medium containing 0-100 ng/ml leptin. Experiment 1 showed that exposure of calf oocytes to IVM medium containing 1 or 10 ng/ml leptin significantly increased rates of development to the metaphase II stage compared with the control (81.7 ± 3.0% and 83.3 ± 2.1% for 1 and 10 ng/ml leptin, respectively, vs 64.1 ± 5.1% for control; p < 0.05). Experiment 2 showed that 1 or 10 ng/ml leptin significantly improved cleavage rates after in vitro fertilization when compared to control (58.6 ± 3.3% and 59.3 ± 2.9% for 1 and 10 ng/ml leptin, respectively, vs 48.5 ± 2.6% for control; p < 0.05); in addition, when compared to control medium, the addition of 10 ng/ml leptin to the IVM medium resulted in more presumptive zygotes reaching the 4- to 8-cell stage after 48 h of in vitro culture (30.3 ± 2.3% vs 20.1 ± 2.3%; p < 0.05) and developing into blastocysts after 8 days of culture (20.4 ± 1.6% vs 11.7 ± 1.7%; p < 0.05). Experiment 3 showed that the addition of 1 or 10 ng/ml leptin significantly increased the total number of blastocyst cells on day 8 of culture (114.6 ± 7.8 and 117.4 ± 5.9 for 1 and 10 ng/ml leptin, respectively, vs 92.7 ± 8.3 for control; p < 0.05) and trophectoderm (TE) cells (88.5 ± 5.5 and 90.6 ± 3.7 for 1 and 10 ng/ml leptin, respectively, vs 70.1 ± 5.9 for control; p < 0.05). In summary, these results indicate that the addition of leptin to IVM medium enhances meiotic maturation and embryo development from calf oocytes and improves the quality of embryos derived from these oocytes. PMID:22136278

  18. 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

  19. Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes.

    PubMed

    Maalouf, W E; Lee, J-H; Campbell, K H S

    2009-04-15

    The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24h post-onset of maturation (hpm), control cumulus oocyte complexes (COC's) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO's (30 hpm) vs. COC's (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% (P<0.05)), however, cleavage increased in the DO group on aging (73.4%) and this was not affected by caffeine (73.0%). The percentage of COC's and DO's developing to the blastocyst stage significantly decreased on aging, caffeine treatment of DO's prevented this (31.3%, 12.7% and 29.4% respectively (P<0.05)) but had no effect on COC's (4.2% vs. 3.9%). Total cell numbers in blastocysts were not statistically different (92.4+/-5.2, 84.7+/-3.7 and 80.4+/-5.8 (P>0.05)). In summary caffeine treatment of aged COC's had no significant effect on the frequency of development, however, in aged DO's caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy. PMID:19185338

  20. The exosome complex establishes a barricade to erythroid maturation.

    PubMed

    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; Bresnick, Emery H

    2014-10-01

    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

  1. 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

  2. Effects of cumulus cells on rabbit oocyte in vitro maturation.

    PubMed

    Tao, Y; Cao, C; Zhang, M; Fang, F; Liu, Y; Zhang, Y; Ding, J; Zhang, X

    2008-08-01

    Cumulus cells (CCs) are of great importance in oocyte development and maturation in many species, but detailed influence of CCs has not been extensively examined, especially on rabbit. The present study was designed to investigate the effects of CCs and the elongation of in vitro maturation (IVM) time on rabbit oocyte nuclear and ooplasmic maturation and survival. Cumulus oocyte complexes (COCs) and naked oocytes (NOs) were recovered directly from rabbits super-ovulated with eCG. Corona-enclosed oocytes (COs) and denuded oocytes (DOs) were obtained from COCs after removing a part or whole of CCs. The oocytes were cultured in the following seven groups. (i) Cumulus cell enclosed oocytes (CEOs) were cultured alone (CEOs); (ii) COs were cultured alone (COs); (iii) DOs were cultured alone (DOs); (iv) NOs were cultured alone; (v) DOs were co-cultured with COCs [DOs(COCs)]; (vi) DOs were co-cultured with CCs [DOs(CCs)]; (vii) NOs were co-cultured with CCs [NOs(CCs)]. After the oocytes were cultured for 24 and 30 h, the nuclear maturation was evaluated by first polar body (PB1) extrusion while the ooplasmic maturation was evaluated by the cleavage rate after parthenogenetic activation. The results showed that the nuclear maturation rate of CEOs, COs, DOs(COCs) and DOs(CCs) after 24 h incubation were significantly different from each other (p < or = 0.05), the rate of DOs(CCs) was similar to that of DOs (p > or = 0.05). The cleavage rates in the first two groups were significantly higher than those of the others (p < 0.05). For oocytes cultured for 30 h, the nuclear maturation rates were significantly different for each culture model (p < 0.05). The cleavage rates in first two groups were significantly higher than those of others (p < 0.05). Both the nuclear and cleavage rates significantly increased when the culture time of DOs(COCs) was prolonged from 24 to 30 h. DOs(CCs) nuclear maturation was significantly improved when the culture time was prolonged from 24 to 30 h

  3. Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes.

    PubMed

    Carneiro, G; Lorenzo, P; Pimentel, C; Pegoraro, L; Bertolini, M; Ball, B; Anderson, G; Liu, I

    2001-09-01

    The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39 degrees C in air with 5% CO(2), 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage. PMID:11514356

  4. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Effect of addition of FSH, LH and proteasome inhibitor MG132 to in vitro maturation medium on the developmental competence of yak (Bos grunniens) oocytes

    PubMed Central

    2014-01-01

    Background The competence for embryonic development after IVF is low in the yak, therefore, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF. Methods In Experiment 1, yak cumulus-oocyte complexes (COCs) were in vitro matured (IVM) in TCM-199 with 20% fetal calf serum (FCS), 1 microg/mL estradiol-17beta, and different combinations of LH (50 or 100 IU/mL) and FSH (0, 1, 5, 10 microg/mL) at 38.6 degrees C, 5% CO2 in air for 24 h. Matured oocytes were exposed to frozen–thawed, heparin-capacitated yak sperm. Presumptive zygotes were cultured in SOF medium containing 6 mg/ml BSA, 0.5 mg/mL myoinositol, 3% (v/v) essential amino acids, 1% nonessential amino acids and 100 μg/mL L-glutamine (48 h, 38.5 degrees C, 5% CO2, 5% O2, and 90% N2). In Experiment 2, cumulus cells were collected at the end of IVM to determine FSHR and LHR mRNA expression by real-time PCR. In Experiment 3 and 4, COCs were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 18–24 h after initiation of maturation. Results The optimum concentration of FSH and LH in IVM media was 5 microg/mL FSH and 50 IU/mL LH which resulted in the greatest cleavage (79.1%) and blastocyst rates (16.1%). Both FSHR and LHR mRNA were detected in yak cumulus cells after IVM. Treatment with MG132 early in maturation reduced (P < 0.05) cleavage and blastocyst rates. Conversely, treatment with MG132 late in maturation improved (P < 0.05) blastocyst rate. Optimal results with MG132 were achieved at a concentration of 10 microM. Conclusions An optimum concentration of FSH and LH in IVM medium, and treatment with MG132 late in maturation can improve yak oocytes competence for development after IVF. PMID:24754924

  6. Effect of oocyte-secreted factors on porcine in vitro maturation, cumulus expansion and developmental competence of parthenotes.

    PubMed

    Gomez, Ma Ninia L; Kang, Jung Taek; Koo, Ok Jae; Kim, Su Jin; Kwon, Dae Kee; Park, Sol Ji; Atikuzzaman, Mohammad; Hong, So Gun; Jang, Goo; Lee, Byeong Chun

    2012-05-01

    The oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus-oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up

  7. Holding equine oocytes in a commercial embryo-holding medium: New perspective on holding temperature and maturation time.

    PubMed

    Dini, Pouya; Bogado Pascottini, Osvaldo; Ducheyne, Kaatje; Hostens, Miel; Daels, Peter

    2016-09-15

    In the present study, we examined the effect of holding equine oocytes in Syngro embryo holding medium (EHM) overnight at either 4 °C, 17 °C, or 22 °C to 25 °C, on the time to maturation and developmental competence. We also examined the effect of placing denuded oocyte without extruded polar body back in maturation condition on subsequent maturation rate. In experiment 1, cumulus-oocyte complexes (COCs) were recovered postmortem and placed in EHM at 22 °C to 25 °C for 18 to 20 hours (OH) or placed directly in maturation (DM). The maturation rate was assessed after 22, 24, or 28 hours of culture. After denuding cumulus cells at 22 or 24 hours, oocytes without obvious polar body were placed back into culture and reassessed at subsequent time points. At 22 hours, a higher proportion of oocytes placed in OH achieved nuclear maturation than those placed in DM (63% and 37%, respectively, P = 0.008). At 24 and 28 hours, no significant differences in the % MII stage oocytes were observed between OH and DM. The nuclear maturation rate for OH oocytes was similar at 22, 24, and 28 hours, indicating that the maximum maturation rate was reached at an earlier time than that in DM. Oocytes fertilized by intracytoplasmic sperm injection resulted in a 7.1% and 6.3% blastocyst rate for OH and DM, respectively. Denuding oocytes after 22 hours or more of culture did not have an adverse effect on the final nuclear maturation rate. After 28 hours of culture, the same nuclear maturation rate (MII) was reached for nondenuded oocytes and oocytes denuded after 22 hours of 24 hours of culture. In experiment 2, COCs were held overnight at room temperature in EHM, then placed in maturation for 20, 22, and 28 hours. Nuclear maturation rate was significantly lower at 20 hours than 22 and 28 hours of culture and was similar at 22 and 28 hours, suggesting that at least 22 hours of culture is required to reach maximal maturation rate for stored oocytes (43%, 62%, and 65% at 20, 22

  8. 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…

  9. 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. PMID:27226011

  10. The effects of season and moderate nutritional restriction on ovarian function and oocyte nuclear maturation in cycling gilts.

    PubMed

    Swinbourne, A M; Kelly, J M; Kind, K L; Kennaway, D J; van Wettere, W H E J

    2014-12-01

    The fertility of female pigs is impaired during summer and in response to restriction of feed intake, resulting in reduced productivity of the breeding herd. This study determined the effect of season and moderate nutritional restriction on ovarian function and oocyte developmental competence of cycling gilts. Eighty prepubescent gilts were used across two seasons-summer (S: January to March) and winter (W: June to August)-and received either a high (2.5× maintenance) or a moderately restricted (1.5× maintenance) feeding level for the first 19 days of their second estrous cycle. On Day 19, ovaries were collected post-slaughter. Diameters of all surface follicles over 1 mm were measured. All follicles ≥4 mm were aspirated and cumulus-oocyte complexes underwent in vitro maturation for ∼44 hours to assess oocyte developmental competence on the basis of metaphase II (MII) attainment. Moderate dietary nutrition reduced daily liveweight gain but did not affect the ovarian follicle population or oocyte developmental competence. The number of large follicles (≥6 mm) was lower during summer (S: 10.7 ± 1.74 vs. W: 15.5 ± 1.15, P < 0.05), as was the proportion of oocytes at the germinal vesicle stage of meiosis (S: 0.06 ± 0.02 vs. W: 0.08 ± 0.02, P < 0.05). However, the proportion of oocytes attaining MII was similar in summer and winter (S: 0.72 ± 0.04 and W: 0.69 ± 0.06, P > 0.05). Intrafollicular concentrations of luteinizing hormone were higher in summer (S: 43.05 ± 6.44 vs. W: 12.05 ± 5.12 ng/mL, P < 0.001), whereas estradiol was lower (S: 1.27 ± 0.36 vs. W: 27.52 ± 5.59 ng/mL, P < 0.001). In conclusion, our data demonstrated that in summer, follicle growth beyond 6 mm is impaired during the periovulatory period, without affecting oocyte meiotic competence. Importantly, these data also demonstrated that ovarian follicle growth and the capacity of oocytes to reach MII in vitro appear unaffected by moderate nutritional restriction during the preceding

  11. Melatonin enhances the in vitro maturation and developmental potential of bovine oocytes denuded of the cumulus oophorus.

    PubMed

    Zhao, Xue-Ming; Min, Jiang-Tao; Du, Wei-Hua; Hao, Hai-Sheng; Liu, Yan; Qin, Tong; Wang, Dong; Zhu, Hua-Bin

    2015-08-01

    This study was designed to determine the effect of melatonin on the in vitro maturation (IVM) and developmental potential of bovine oocytes denuded of the cumulus oophorus (DOs). DOs were cultured alone (DOs) or with 10-9 M melatonin (DOs + MT), cumulus-oocyte complexes (COCs) were cultured without melatonin as the control. After IVM, meiosis II (MII) rates of DOs, and reactive oxygen species (ROS) levels, apoptotic rates and parthenogenetic blastocyst rates of MII oocytes were determined. The relative expression of ATP synthase F0 Subunit 6 and 8 (ATP6 and ATP8), bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9) mRNA in MII oocytes and IFN-tau (IFN-τ), Na+/K+-ATPase, catenin-beta like 1 (CTNNBL1) and AQP3 mRNA in parthenogenetic blastocysts were quantified using real-time polymerase chain reaction (PCR). The results showed that: (1) melatonin significantly increased the MII rate of DOs (65.67 ± 3.59 % vs. 82.29 ± 3.92%; P < 0.05), decreased the ROS level (4.83 ± 0.42 counts per second (c.p.s) vs. 3.78 ± 0.29 c.p.s; P < 0.05) and apoptotic rate (36.99 ± 3.62 % vs. 21.88 ± 2.08 %; P < 0.05) and moderated the reduction of relative mRNA levels of ATP6, ATP8, BMP-15 and GDF-9 caused by oocyte denudation; (2) melatonin significantly increased the developmental rate (24.17 ± 3.54 % vs. 35.26 ± 4.87%; P < 0.05), and expression levels of IFN-τ, Na+/K+-ATPase, CTNNBL1 and AQP3 mRNA of blastocyst. These results indicated that melatonin significantly improved the IVM quality of DOs, leading to an increased parthenogenetic blastocyst formation rate and quality. PMID:24869483

  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. 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. PMID:25531035

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

    PubMed Central

    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. PMID:25531035

  15. The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation.

    PubMed

    Fendrych, Matyás; Synek, Lukás; Pecenková, Tamara; Toupalová, Hana; Cole, Rex; Drdová, Edita; Nebesárová, Jana; Sedinová, Miroslava; Hála, Michal; Fowler, John E; Zársky, Viktor

    2010-09-01

    Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein-tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall. PMID:20870962

  16. Role for a Novel Usher Protein Complex in Hair Cell Synaptic Maturation

    PubMed Central

    Zallocchi, Marisa; Meehan, Daniel T.; Delimont, Duane; Rutledge, Joseph; Gratton, Michael Anne; Flannery, John; Cosgrove, Dominic

    2012-01-01

    The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1−/− mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzerav3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well. PMID:22363448

  17. MET Receptor Tyrosine Kinase Controls Dendritic Complexity, Spine Morphogenesis, and Glutamatergic Synapse Maturation in the Hippocampus

    PubMed Central

    Lu, Zhongming; Levitt, Pat

    2014-01-01

    The MET receptor tyrosine kinase (RTK), implicated in risk for autism spectrum disorder (ASD) and in functional and structural circuit integrity in humans, is a temporally and spatially regulated receptor enriched in dorsal pallial-derived structures during mouse forebrain development. Here we report that loss or gain of function of MET in vitro or in vivo leads to changes, opposite in nature, in dendritic complexity, spine morphogenesis, and the timing of glutamatergic synapse maturation onto hippocampus CA1 neurons. Consistent with the morphological and biochemical changes, deletion of Met in mutant mice results in precocious maturation of excitatory synapse, as indicated by a reduction of the proportion of silent synapses, a faster GluN2A subunit switch, and an enhanced acquisition of AMPA receptors at synaptic sites. Thus, MET-mediated signaling appears to serve as a mechanism for controlling the timing of neuronal growth and functional maturation. These studies suggest that mistimed maturation of glutamatergic synapses leads to the aberrant neural circuits that may be associated with ASD risk. PMID:25471559

  18. Scrapie affects the maturation cycle and immune complex trapping by follicular dendritic cells in mice.

    PubMed

    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 (PrP(d)) 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 PrP(d) 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 PrP(d) plasmalemmal accumulations. Our data suggest this previously unrecognised morphology represents the initial stage of an abnormal FDC maturation cycle. Alterations to the FDCs included PrP(d) accumulation, abnormal cell membrane ubiquitin and excess immunoglobulin accumulation. Regressing FDCs, in contrast, appeared to lose their membrane-attached PrP(d). Together, these data suggest that TSE infection adversely affects the maturation and regression cycle of FDCs, and that PrP(d) accumulation is causally linked to the abnormal pathology observed. We therefore support the hypothesis that TSEs cause an abnormality in immune function. PMID:19997557

  19. Maturation of the HIV reverse transcription complex: putting the jigsaw together.

    PubMed

    Warrilow, David; Tachedjian, Gilda; Harrich, David

    2009-11-01

    Upon HIV attachment, fusion and entry into the host cell cytoplasm, the viral core undergoes rearrangement to become the mature reverse transcription complex (RTC). Reduced infectivity of viral deletion mutants of the core proteins, capsid and negative factor (Nef), can be complemented by vesicular stomatitis virus (VSV) pseudotyping suggesting a role for these viral proteins in a common event immediately post-entry. This event may be necessary for correct trafficking of the early complex. Enzymatic activation of the complex occurs either before or during RTC maturation, and may be dependent on the presence of deoxynucleotides in the host cell. The RTC initially becomes enlarged immediately after entry, which is followed by a decrease in its sedimentation rate consistent with core uncoating. Several HIV proteins associated with the RTC and recently identified host-cell proteins are important for reverse transcription while genome-wide siRNA knockdown studies have identified additional host cell factors that may be required for reverse transcription. Determining precisely how these proteins assist the RTC function needs to be addressed. PMID:19750561

  20. Crystal structures of the HypCD complex and the HypCDE ternary complex: transient intermediate complexes during [NiFe] hydrogenase maturation.

    PubMed

    Watanabe, Satoshi; Matsumi, Rie; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2012-12-01

    [NiFe] hydrogenase maturation represents one of the most dynamic and sophisticated processes in metallocenter assembly. The Fe(CN)(2)CO moiety of [NiFe] hydrogenases is assembled via unknown transient interactions among specific maturation proteins HypC (metallochaperone), HypD (redox protein), and HypE (cyanide synthesis/donor). Here, we report the structures of the HypC-HypD and HypC-HypD-HypE complexes, providing a view of the transient interactions that take place during the maturation process. HypC binds to the conserved region of HypD through extensive hydrophobic interactions. The ternary complex formation between HypE and the HypCD complex involves both HypC and HypD, rendering the HypE conformation favorable for cyanide transfer. In the complex, the conserved cysteines of HypC and HypD form an Fe binding site. The conserved C-terminal cysteine of HypE can access the thiol redox cascade of HypD. These results provide structural insights into the Fe atom cyanation in the HypCDE complex. PMID:23123111

  1. Progression of meiotic recombination requires structural maturation of the central element of the synaptonemal complex.

    PubMed

    Hamer, Geert; Wang, Hong; Bolcun-Filas, Ewelina; Cooke, Howard J; Benavente, Ricardo; Höög, Christer

    2008-08-01

    The synaptonemal complex is an elaborate meiosis-specific supramolecular protein assembly that promotes chromosome synapsis and meiotic recombination. We inactivated the meiosis-specific gene Tex12 and found that TEX12 is essential for progression of meiosis in both male and female germ cells. Structural analysis of the synaptonemal complex in Tex12-/- meiocytes revealed a disrupted central element structure, a dense structure residing between the synapsed homologous chromosomes. Chromosome synapsis is initiated at multiple positions along the paired homologous chromosomes in Tex12-/- meiotic cells, but fails to propagate along the chromosomes. Furthermore, although meiotic recombination is initiated in Tex12-/- meiotic cells, these early recombination events do not develop into meiotic crossovers. Hence, the mere initiation of synapsis is not sufficient to support meiotic crossing-over. Our results show that TEX12 is a component of the central element structure of the synaptonemal complex required for propagation of synapsis along the paired homologous chromosomes and maturation of early recombination events into crossovers. PMID:18611960

  2. Maturation of suprathreshold auditory nerve activity involves cochlear CGRP-receptor complex formation.

    PubMed

    Dickerson, Ian M; Bussey-Gaborski, Rhiannon; Holt, Joseph C; Jordan, Paivi M; Luebke, Anne E

    2016-07-01

    In adult animals, the neuropeptide calcitonin gene-related peptide (CGRP) is contained in cochlear efferent fibers projecting out to the cochlea, and contributes to increased suprathreshold sound-evoked activity in the adult auditory nerve. Similarly, CGRP applied to the lateral-line organ (hair cell organ) increases afferent nerve activity in adult frogs (post-metamorphic day 30), yet this increase is developmentally delayed from post-metamorphic day 4-30. In this study, we discovered that there was also a developmental delay in increased suprathreshold sound-evoked activity auditory nerve between juvenile and adult mice similar to what had been observed previously in frog. Moreover, juvenile mice with a targeted deletion of the αCGRP gene [CGRP null (-/-)] did not show a similar developmental increase in nerve activity, suggesting CGRP signaling is involved. This developmental delay is not due to a delay in CGRP expression, but instead is due to a delay in receptor formation. We observed that the increase in sound-evoked nerve activity is correlated with increased formation of cochlear CGRP receptors, which require three complexed proteins (CLR, RAMP1, RCP) to be functional. CGRP receptor formation in the cochlea was incomplete at 1 month of age (juvenile), but complete by 3 months (adult), which corresponded to the onset of suprathreshold enhancement of sound-evoked activity in wild-type animals. Taken together, these data support a model for cochlear function that is enhanced by maturation of CGRP receptor complexes. PMID:27440744

  3. 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.

  4. Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts.

    PubMed

    Khazaie, Niusha; Massumi, Mohammad; Wee, Ping; Salimi, Mahdieh; Mohammadnia, Abdulshakour; Yaqubi, Moein

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs. PMID:26938987

  5. Involvement of Polycomb Repressive Complex 2 in Maturation of Induced Pluripotent Stem Cells during Reprogramming of Mouse and Human Fibroblasts

    PubMed Central

    Khazaie, Niusha; Massumi, Mohammad; Wee, Ping; Salimi, Mahdieh; Mohammadnia, Abdulshakour; Yaqubi, Moein

    2016-01-01

    Induced pluripotent stem cells (iPSCs) provide a reliable source for the study of regenerative medicine, drug discovery, and developmental biology. Despite extensive studies on the reprogramming of mouse and human fibroblasts into iPSCs, the efficiency of reprogramming is still low. Here, we used a bioinformatics and systems biology approach to study the two gene regulatory waves governing the reprogramming of mouse and human fibroblasts into iPSCs. Our results revealed that the maturation phase of reprogramming was regulated by a more complex regulatory network of transcription factors compared to the initiation phase. Interestingly, in addition to pluripotency factors, the polycomb repressive complex 2 (PRC2) members Ezh2, Eed, Jarid2, Mtf2, and Suz12 are crucially recruited during the maturation phase of reprogramming. Moreover, we found that during the maturation phase of reprogramming, pluripotency factors, via the expression and induction of PRC2 complex members, could silence the lineage-specific gene expression program and maintain a ground state of pluripotency in human and mouse naïve iPSCs. The findings obtained here provide us a better understanding of the gene regulatory network (GRN) that governs reprogramming, and the maintenance of the naïve state of iPSCs. PMID:26938987

  6. Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

    PubMed Central

    Takemori, T; Mizuguchi, J; Miyazoe, I; Nakanishi, M; Shigemoto, K; Kimoto, H; Shirasawa, T; Maruyama, N; Taniguchi, M

    1990-01-01

    Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2114976

  7. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    PubMed

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

  8. 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.

  9. Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

    PubMed Central

    Lange Consiglio, Anna; Dell'Aquila, Maria Elena; Fiandanese, Nadia; Ambruosi, Barbara; Cho, Yoon S; Bosi, Giampaolo; Arrighi, Silvana; Lacalandra, Giovanni M; Cremonesi, Fausto

    2009-01-01

    Background The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI). Methods Compact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml) of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R) was investigated by immunocytochemical staining. Results In expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P < 0.05) and fertilization after ICSI (56% vs 23% for 10 ng/ml leptin-treated and control groups, respectively; P < 0.05). However, the developmental rate and quality of 8-cell stage embryos derived from leptin-treated oocytes (100 ng/ml) was significantly reduced, in contrast to previous data in other species where leptin increased embryo cleavage. Ob and Ob-R proteins were detected up to the 8-cell stage with cortical and cytoplasmic granule-like distribution pattern in each blastomere. Conclusion Leptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin. PMID:19835605

  10. Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

    PubMed Central

    Burri, Lena; Strahm, Yvan; Hawkins, Christine J.; Gentle, Ian E.; Puryer, Michelle A.; Verhagen, Anne; Callus, Bernard; Vaux, David; Lithgow, Trevor

    2005-01-01

    DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX5P in Imp1 and NX5S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. PMID:15814844

  11. 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. PMID:21939078

  12. Fenestrated stent grafts for the treatment of complex aortic aneurysm disease: A mature treatment paradigm.

    PubMed

    Georgiadis, George S; van Herwaarden, Joost A; Antoniou, George A; Giannoukas, Athanasios D; Lazarides, Miltos K; Moll, Frans L

    2016-06-01

    The introduction of fenestrated stent grafts (SGs) to treat abdominal aortic aneurysms (AAAs) with short proximal necks began in 1999. Nowadays, the whole visceral aorta can be treated totally by endovascular means. The established use of fenestrated devices to treat complex AAAs as a first-line management option has been previously reported. An up-to-date evaluation of the literature was performed including all types of publications regarding the use of fenestrated technology to repair complex AAAs. Fenestrated repair is now an established alternative to hybrid/chimney/snorkel repairs. However, specific criteria and prerequisites are required for the use and improvement of this method. Multiple device morphologies have been used incorporating the visceral arteries in various combinations. This modular strategy connects different devices (bridging covered stents and bifurcated SGs) with the aortic main body, thus excluding the aneurysm from the circulation. Precise deployment of the fenestrated SG is mandatory for successful visceral vessel revascularization. Accurate SG sizing and customization, a high level of technical skill, and facilities with modern imaging techniques including 3D road mapping and dedicated hybrid rooms are required. Most experience has been with the custom-made Zenith Cook platform, although off-the-shelf devices have been recently implanted. More complex repairs have been performed over the last few years, but device complexity has also increased. Perioperative, mid-term, and a few recently reported long-term results are encouraging. Secondary interventions remain the main problem, similar to that observed after traditional endovascular abdominal aortic aneurysm repair (EVAR). PMID:27013644

  13. Replacement of Rbpj with Rbpjl in the PTF1 complex controls the final maturation of pancreatic acinar cells

    PubMed Central

    Masui, Toshihiko; Swift, Galvin H.; Deering, Tye; Shen, Chengcheng; Coats, Ward S.; Long, Qiaoming; Elsässer, Hans-Peter; Magnuson, Mark A.; MacDonald, Raymond J.

    2010-01-01

    Background & Aims The mature pancreatic acinar cell is dedicated to the production of very large amounts of digestive enzymes. The early stages of pancreatic development require the Rbpj-form of the trimeric transcription factor complex PTF1 (PTF1-J). As acinar development commences, Rbpjl gradually replaces Rbpj; in the mature pancreas, PTF1 contains Rbpjl (PTF1-L). We investigated whether PTF1-L controls the expression of genes that complete the final stage of acinar differentiation. Methods We analyzed acinar development and transcription in mice with disrupted Rbpjl (Rbpjlko/ko mice). We performed comprehensive analyses of the mRNA population and PTF1 target genes in pancreatic acinar cells from these and wild-type mice. Results In Rbpjlko/ko mice, acinar differentiation was incomplete and characterized by decreased expression (as much as 99%) of genes that encode digestive enzymes or proteins of regulated exocytosis and mitochondrial metabolism. Whereas PTF1-L bound regulatory sites of genes in normal adult pancreatic cells, the embryonic form (PTF1-J) persisted in the absence of Rbpjl and replaced PTF1-L; the extent of replacement determined gene expression levels. Loss of PTF1-L reduced expression (>2-fold) of only about 50 genes, 90% of which were direct targets of PTF1-L. The magnitude of the effects on individual digestive enzyme genes correlated with the developmental timing of gene activation. Absence of Rbpjl increased pancreatic expression of liver-restricted mRNAs. Conclusions Replacement of Rbpj by Rbpjl in the PTF1 complex drives acinar differentiation by maximizing secretory protein synthesis, stimulating mitochondrial metabolism and cytoplasmic creatine-phosphate energy stores, completing the packaging and secretory apparatus, and maintaining acinar-cell homeostasis. PMID:20398665

  14. 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.

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

    SciTech Connect

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

    1996-01-01

    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%.

  16. 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%.

  17. 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).

  18. Follicle Structure Influences the Availability of Oxygen to the Oocyte in Antral Follicles

    PubMed Central

    Clark, A. R.; Stokes, Y. M.

    2011-01-01

    The ability of an oocyte to successfully mature is highly dependent on intrafollicular conditions, including the size and structure of the follicle. Here we present a mathematical model of oxygen transport in the antral follicle. We relate mean oxygen concentration in follicular fluid of bovine follicles to the concentration in the immediate vicinity of the cumulus-oocyte complex (COC). The model predicts that the oxygen levels within the antral follicle are dependent on the size and structure of the follicle and that the mean level of dissolved oxygen in follicular fluid does not necessarily correspond to that reaching the COC. PMID:22162722

  19. Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex.

    PubMed

    James, H E; Bartling, D; Musgrove, J E; Kirwin, P M; Herrmann, R G; Robinson, C

    1989-11-25

    The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases. PMID:2684958

  20. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    PubMed Central

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  1. Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.

    PubMed Central

    Hammond, S A; Cook, S J; Lichtenstein, D L; Issel, C J; Montelaro, R C

    1997-01-01

    Equine infectious anemia virus (EIAV) provides a natural model system by which immunological control of lentivirus infections may be studied. To date, no detailed study addressing in parallel both the humoral and cellular immune responses induced in horses upon infection by EIAV has been conducted. Therefore, we initiated the first comprehensive characterization of the cellular and humoral immune responses during clinical progression from chronic disease to inapparent stages of EIAV infection. Using new analyses of antibody avidity and antibody epitope conformation dependence that had not been previously employed in this system, we observed that the humoral immune response to EIAV required a 6- to 8-month period in which to fully mature. During this time frame, EIAV-specific antibody evolved gradually from a population characterized by low-avidity, nonneutralizing, and predominantly linear epitope specificity to an antibody population with an avidity of moderate to high levels, neutralizing activity, and predominantly conformational epitope specificity. Analyses of the cell-mediated immune response to EIAV revealed CD4+ and CD8+ major histocompatibility complex-restricted, EIAV-specific cytotoxic T-lymphocyte (CTL) activity apparent within 3 to 4 weeks postinfection, temporally correlating with the resolution of the primary viremia. After resolution of the initial viremia, EIAV-specific CTL activity differed greatly among the experimentally infected ponies, with some animals having readily detectable CTL activity while others had little measurable CTL activity. Thus, in contrast to the initial viremia, it appeared that no single immune parameter correlated with the resolution of further viremic episodes. Instead, immune control of EIAV infection during the clinically inapparent stage of infection appears to rely on a complex combination of immune system mechanisms to suppress viral replication that effectively functions only after the immune system has evolved to a

  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. PMID:26318856

  4. An Orange Ripening Mutant Links Plastid NAD(P)H Dehydrogenase Complex Activity to Central and Specialized Metabolism during Tomato Fruit Maturation[C][W

    PubMed Central

    Nashilevitz, Shai; Melamed-Bessudo, Cathy; Izkovich, Yinon; Rogachev, Ilana; Osorio, Sonia; Itkin, Maxim; Adato, Avital; Pankratov, Ilya; Hirschberg, Joseph; Fernie, Alisdair R.; Wolf, Shmuel; Usadel, Björn; Levy, Avraham A.; Rumeau, Dominique; Aharoni, Asaph

    2010-01-01

    In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of OrrDs, a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in OrrDs, as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly β-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, OrrDs leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism. PMID:20571113

  5. Effects of selected endocrine disruptors on meiotic maturation, cumulus expansion, synthesis of hyaluronan and progesterone by porcine oocyte-cumulus complexes.

    PubMed

    Mlynarcíková, Alzbeta; Nagyová, Eva; Ficková, Mária; Scsuková, Sona

    2009-04-01

    In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100pM-100microM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte-cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100microM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors. PMID:19162163

  6. 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

  7. The Cytochrome c Maturation Components CcmF, CcmH, and CcmI Form a Membrane-integral Multisubunit Heme Ligation Complex*

    PubMed Central

    Sanders, Carsten; Turkarslan, Serdar; Lee, Dong-Woo; Onder, Ozlem; Kranz, Robert G.; Daldal, Fevzi

    2008-01-01

    Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus. PMID:18753134

  8. Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2*

    PubMed Central

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle; van Agthoven, Jan; Raynal, Bertrand; Hoos, Sylviane; Kragelund, Birthe B.; Kelly, Paul A.; Ducruix, Arnaud; England, Patrick; Goffin, Vincent

    2010-01-01

    We report the first crystal structure of a 1:2 hormone·receptor complex that involves prolactin (PRL) as the ligand, at 3.8-Å resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL·PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 (“three-pin plug”): the conserved glycine 129 of helix α3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL·PRLR2 structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer. PMID:20053995

  9. 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

  10. 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. PMID:27010650

  11. The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3′ ends

    PubMed Central

    Xie, Mingyi; Zhang, Wei; Shu, Mei-Di; Xu, Acer; Lenis, Diana A.; DiMaio, Daniel; Steitz, Joan A.

    2015-01-01

    Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3′ end processing signal (3′ box), generates the 5′ ends of HVS pre-miRNA hairpins. Here, we identify a novel 3′ box-like sequence (miRNA 3′ box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3′ end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3′ box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA–protein interactions inside cells. Surprisingly, in contrast to snRNA 3′ end processing, HVS pre-miRNA 3′ end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology. PMID:26220997

  12. 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.

  13. An ER Complex of ODR-4 and ODR-8/Ufm1 Specific Protease 2 Promotes GPCR Maturation by a Ufm1-Independent Mechanism

    PubMed Central

    Chen, Changchun; Itakura, Eisuke; Weber, Katherine P.; Hegde, Ramanujan S.; de Bono, Mario

    2014-01-01

    Despite the importance of G-protein coupled receptors (GPCRs) their biogenesis is poorly understood. Like vertebrates, C. elegans uses a large family of GPCRs as chemoreceptors. A subset of these receptors, such as ODR-10, requires the odr-4 and odr-8 genes to be appropriately localized to sensory cilia. The odr-4 gene encodes a conserved tail-anchored transmembrane protein; the molecular identity of odr-8 is unknown. Here, we show that odr-8 encodes the C. elegans ortholog of Ufm1-specific protease 2 (UfSP2). UfSPs are cysteine proteases identified biochemically by their ability to liberate the ubiquitin-like modifier Ufm1 from its pro-form and protein conjugates. ODR-8/UfSP2 and ODR-4 are expressed in the same set of twelve chemosensory neurons, and physically interact at the ER membrane. ODR-4 also binds ODR-10, suggesting that an ODR-4/ODR-8 complex promotes GPCR folding, maturation, or export from the ER. The physical interaction between human ODR4 and UfSP2 suggests that this complex's role in GPCR biogenesis may be evolutionarily conserved. Unexpectedly, mutant versions of ODR-8/UfSP2 lacking catalytic residues required for protease activity can rescue all odr-8 mutant phenotypes tested. Moreover, deleting C. elegans ufm-1 does not alter chemoreceptor traffic to cilia, either in wild type or in odr-8 mutants. Thus, UfSP2 proteins have protease- and Ufm1-independent functions in GPCR biogenesis. PMID:24603482

  14. 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

  15. Market maturity

    SciTech Connect

    Meade, B.; Bowden, S.; Ellis, M

    1995-02-01

    The power sector in the Philipines provides one of the most mature independent power markets in Asia. Over the past five years, National Power Corp. (NPC), the government owned utility, has actively invited the power sector into power generation. Distribution has remained in the hands of private and rural cooperative utilities. Private utilities have been operating as full requirements customers of NPC while the growth in capacity additions by independent power producers (IPPs) has outpaced NPC`s for the second year in a row. With a recovering economy and regulatory reform proceeding, the outlook for independent power remains strong through the end of the decade. The Philipine Congress is now reviewing draft legislation that will decentralize NPC and begin the process of privatization and market-based reforms throughout the country`s power sector.

  16. Meiotic maturation of incompetent prepubertal sheep oocytes is induced by paracrine factor(s) released by gonadotropin-stimulated oocyte-cumulus cell complexes and involves mitogen-activated protein kinase activation.

    PubMed

    Cecconi, Sandra; Mauro, Annunziata; Capacchietti, Giulia; Berardinelli, Paolo; Bernabò, Nicola; Di Vincenzo, Anna Rita; Mattioli, Mauro; Barboni, Barbara

    2008-01-01

    In this study, sheep oocyte-cumulus cell complexes (OCC) derived from medium (M) antral follicles (M-OCC) were in vitro matured alone or in coculture with OCC derived from small (S) antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage, and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to metaphase II, and S-CC expanded after coculture with gonadotropin-stimulated M-OCC and M-CC but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were cocultured with M-denuded oocytes, or when S-denuded oocytes were cocultured with M-CC. The capacity of these paracrine factor(s) to activate the MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MAPK kinase/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, first in CC and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes cocultured with M-OCC or M-CC. These results demonstrate that soluble factors specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via CC. These factors can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding CC to respond to gonadotropin stimulation. PMID:17884935

  17. 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).

  18. Studying the Functions of TGF-β Signaling in the Ovary.

    PubMed

    Yu, Chao; Zhou, Jian-Jie; Fan, Heng-Yu

    2016-01-01

    In mammals, ovulation is a multistep physiological process that includes preovulatory follicle growth, oocyte meiotic maturation, cumulus-oocyte complex (COC) expansion, follicle rupture, and luteinization. TGF-β signaling pathway has multiple functions in mammalian ovary, as its complexity in ovarian function has been demonstrated by mouse models with knockouts of TGF-β receptors and SMADs. We describe the protocol that we use to study functions of TGF-β signaling pathway in follicle development and ovulation. Because total knockout of TGF-β pathway components often causes embryonic lethality, which prevents further investigation of these genes in ovarian functions, people have generated ovarian cell type-specific knockout mouse strains for TGF-β signaling pathway genes. These mouse models are also described. PMID:26520133

  19. Examination of potential measures of vine maturity in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant maturity is a complex physiological trait. The criteria used to characterize plant maturity are not universal among crop plants. In potato, vine characteristics are typically used to estimate plant maturity. This study investigates several reported measures of vine maturity in potato cultivars...

  20. Improved Oocyte Isolation and Embryonic Development of Outbred Deer Mice

    PubMed Central

    Kyu Choi, Jung; He, Xiaoming

    2015-01-01

    In this study, we improved the protocol for isolating cumulus-oocyte complexes (COCs) from the outbred deer mice by using only one hormone (instead of the widely used combination of two hormones) with reduced dose. Moreover, we identified that significantly more metaphase II (MII) oocytes could be obtained by supplementing epidermal growth factor (EGF) and leukemia inhibition factor (LIF) into the previously established medium for in vitro maturation (IVM) of the COCs. Furthermore, we overcame the major challenge of two-cell block during embryonic development of deer mice after either in vitro fertilization (IVF) or parthenogenetic activation (PA) of the MII oocytes, by culturing the two-cell stage embryos on the feeder layer of inactivated mouse embryonic fibroblasts (MEFs) in the medium of mouse embryonic stem cells. Collectively, this work represents a major step forward in using deer mice as an outbred animal model for biomedical research on reproduction and early embryonic development. PMID:26184014

  1. The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells.

    PubMed

    Pepermans, Elise; Michel, Vincent; Goodyear, Richard; Bonnet, Crystel; Abdi, Samia; Dupont, Typhaine; Gherbi, Souad; Holder, Muriel; Makrelouf, Mohamed; Hardelin, Jean-Pierre; Marlin, Sandrine; Zenati, Akila; Richardson, Guy; Avan, Paul; Bahloul, Amel; Petit, Christine

    2014-07-01

    Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/-) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects. PMID:24940003

  2. The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells

    PubMed Central

    Pepermans, Elise; Michel, Vincent; Goodyear, Richard; Bonnet, Crystel; Abdi, Samia; Dupont, Typhaine; Gherbi, Souad; Holder, Muriel; Makrelouf, Mohamed; Hardelin, Jean-Pierre; Marlin, Sandrine; Zenati, Akila; Richardson, Guy; Avan, Paul; Bahloul, Amel; Petit, Christine

    2014-01-01

    Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/−) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects. PMID:24940003

  3. 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.

  4. 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…

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

    PubMed Central

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

    2014-01-01

    SUMMARY Cytochrome c maturation (ccm) in many bacteria, archaea, and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches heme covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of heme 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 heme. 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. PMID:24397552

  6. Cone Early Maturity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hop cone early maturity is thought to be caused by diffuse infections of cone, just prior to harvest, by Podosphaera macularis. The disease is best managed by limiting the amount of leaf infection by P. macularis prior to bloom. The yield and quality reductions associated with Hop cone early matur...

  7. 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

  8. The asymmetry in the mature amino-terminus of ClpP facilitates a local symmetry match in ClpAP and ClpXP complexes

    PubMed Central

    Bewley, Maria C.; Graziano, Vito; Griffin, Kathleen; Flanagan, John M.

    2013-01-01

    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 Å 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 ∼ 12–15 Å 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

  9. 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

  10. 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. PMID:26986845

  11. Data Product Maturity

    Atmospheric Science Data Center

    2013-03-25

    ... document, maturity levels are provided separately for each scientific data set (SDS) included with the data files. The data product ... indiscriminate use of these data products as the basis for research findings, journal publications, and/or presentations.   ...

  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. 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

  14. 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.

  15. Structure, function and dynamics in adenovirus maturation

    DOE PAGESBeta

    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

  16. 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…

  17. 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

  18. Bluetongue virus capsid assembly and maturation.

    PubMed

    Mohl, Bjorn-Patrick; Roy, Polly

    2014-08-01

    Maturation is an intrinsic phase of the viral life cycle and is often intertwined with egress. In this review we focus on orbivirus maturation by using Bluetongue virus (BTV) as a representative. BTV, a member of the genus Orbivirus within the family Reoviridae, has over the last three decades been subjected to intense molecular study and is thus one of the best understood viruses. BTV is a non-enveloped virus comprised of two concentric protein shells that encapsidate 10 double-stranded RNA genome segments. Upon cell entry, the outer capsid is shed, releasing the core which does not disassemble into the cytoplasm. The polymerase complex within the core then synthesizes transcripts from each genome segment and extrudes these into the cytoplasm where they act as templates for protein synthesis. Newly synthesized ssRNA then associates with the replicase complex prior to encapsidation by inner and outer protein layers of core within virus-triggered inclusion bodies. Maturation of core occurs outside these inclusion bodies (IBs) via the addition of the outer capsid proteins, which appears to be coupled to a non-lytic, exocytic pathway during early infection. Similar to the enveloped viruses, BTV hijacks the exocytosis and endosomal sorting complex required for trafficking (ESCRT) pathway via a non-structural glycoprotein. This exquisitely detailed understanding is assembled from a broad array of assays, spanning numerous and diverse in vitro and in vivo studies. Presented here are the detailed insights of BTV maturation and egress. PMID:25196482

  19. 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

  20. 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.

  1. Retroperitoneal Mature Cystic Teratoma in a Neonate: A Case Report.

    PubMed

    Patil, Prashant Sadashiv; Kothari, Paras; Gupta, Abhaya; Dikshit, Vishesh; Kamble, Ravi; Kekre, Geeta; Deshmukh, Shahaji

    2016-01-01

    We report a case of retroperitoneal mature cystic teratoma in a 2-day-old neonate. Diagnostic and surgical procedure including its complexity and relevant literature review has been discussed. PMID:27123399

  2. Retroperitoneal Mature Cystic Teratoma in a Neonate: A Case Report

    PubMed Central

    Patil, Prashant Sadashiv; Kothari, Paras; Gupta, Abhaya; Dikshit, Vishesh; Kamble, Ravi; Kekre, Geeta; Deshmukh, Shahaji

    2016-01-01

    We report a case of retroperitoneal mature cystic teratoma in a 2-day-old neonate. Diagnostic and surgical procedure including its complexity and relevant literature review has been discussed. PMID:27123399

  3. 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…

  4. A Socioanalytic Model of Maturity

    ERIC Educational Resources Information Center

    Hogan, Robert; Roberts, Brent W.

    2004-01-01

    K0 describes a point of view on maturity that departs from earlier treatments in two ways. First, it rejects the popular assumption from humanistic psychology that maturity is a function of self-actualization and stipulates that maturity is related to certain performance capacities--namely, the ability to form lasting relationships and to achieve…

  5. An Unexpected Twist in Viral Capsid Maturation

    PubMed Central

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

    2009-01-01

    Lambda-like dsDNA 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 packaging1. The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring 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. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å2. 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 demonstrates that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy3. We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many ds

  6. Intrafallopian transfer of gametes and early stage embryos for in vivo culture in cattle.

    PubMed

    Wetscher, F; Havlicek, V; Huber, T; Gilles, M; Tesfaye, D; Griese, J; Wimmers, K; Schellander, K; Müller, M; Brem, G; Besenfelder, U

    2005-07-01

    It may be possible to avoid inadequate in vitro culture conditions by incubating gametes or embryos in the oviducts for a short time. Ideally, an optimized procedure should be devised, combining in vitro and in vivo systems, in order to achieve synchronization in cattle. We transferred gametes as well as embryos in various stages of development and placed them into the oviducts. Embryos were recovered on Day 7 by flushing of oviducts and uterine horns. Blastocyst rates were determined on Day 7 and on Day 8. Experimental designs included transfer of in vitro matured cumulus oocyte complexes into previously inseminated heifers (COCs group), transfer of in vitro matured COCs simultaneously with capacitated spermatozoa (GIFTs group), transfer of four to eight cell stage embryos developed in vitro after IVM/IVF (Cleaved Stages group) and a group of solely in vitro produced embryos (IVP control group). Our results indicate that in vivo culture of IVM/IVF embryos in the homologous bovine oviduct has a positive influence on subsequent pre-implantation development. In addition, we have evidence that in vitro maturation and in vivo fertilization cannot be synchronized. PMID:15935840

  7. 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.

  8. 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.

  9. Transcriptional Landscape of Cardiomyocyte Maturation

    PubMed Central

    Uosaki, Hideki; Cahan, Patrick; Lee, Dong I.; Wang, Songnan; Miyamoto, Matthew; Fernandez, Laviel; Kass, David A.; Kwon, Chulan

    2015-01-01

    SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation. PMID:26586429

  10. Sperm Proteome Maturation in the Mouse Epididymis

    PubMed Central

    Skerget, Sheri; Rosenow, Matthew A.; Petritis, Konstantinos; Karr, Timothy L.

    2015-01-01

    In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm. PMID:26556802

  11. 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…

  12. In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer.

    PubMed

    Pope, C E; Gómez, M C; Kagawa, N; Kuwayama, M; Leibo, S P; Dresser, B L

    2012-02-01

    We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients. PMID:22015162

  13. 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.

  14. 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

  15. Fine Structures of the Oocyte in Relation to Serum, Follicular Fluid Steroid Hormones and IGF-I in the Ovulatory-Sized Follicles in One-Humped Camel (Camelus dromedarius)

    PubMed Central

    Kafi, Mojtaba; Mesbah, Seyed Fakhroddin; Davoodian, Najmeh; Kadivar, Ali

    2014-01-01

    Background The following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles in relation to concentrations of steroids and IGF-I in the Follicular Fluid (FF) and serum in the dromedary camel. Methods Camel follicles with a clear and healthy appearance were categorized into three classes: follicles 10 to 13.9, 14-17.9 and 18-30 mm diameter. The FF and serum samples were assayed for estradiol-17β, progesterone and IGF-I. Recovered Cumulus-Oocyte Complexes (COCs) were prepared for transmission electron microscopy. Results The mean (±SD) FF concentrations of progesterone and IGF-I was significantly (p < 0.05) higher in follicles 18 to 30 mm diameter compared to other groups of follicles. There was no difference in the mean (±SD) serum estradiol-17β, progesterone and IGF-I concentrations between camels with different ovulatory-sized follicles (p > 0.05). Oocytes from follicles 18 to 30 mm diameter (group 3) showed more advanced signs of maturation including the disappearance of the nuclear envelope, increased number of microvilli in erect position, the increase in number and size of vesicles and more even distribution of the mitochondria throughout the ooplasm. Conclusion The final stages of oocyte maturation in dromedary camel is associated with increasing progesterone and IGF-I concentrations and constant high estradiol concentration in the follicular fluid which are paralleled with well-defined ultrastructural changes in oocytes. PMID:24551433

  16. Amphiregulin co-operates with bone morphogenetic protein 15 to increase bovine oocyte developmental competence: effects on gap junction-mediated metabolite supply.

    PubMed

    Sugimura, Satoshi; Ritter, Lesley J; Sutton-McDowall, Melanie L; Mottershead, David G; Thompson, Jeremy G; Gilchrist, Robert B

    2014-06-01

    This study assessed the participation of amphiregulin (AREG) and bone morphogenetic protein 15 (BMP15) during maturation of bovine cumulus-oocyte complexes (COCs) on cumulus cell function and their impact on subsequent embryo development. AREG treatment of COCs enhanced blastocyst formation and quality only when in the presence of BMP15. Expression of hyaluronan synthase 2 was enhanced by follicle-stimulating hormone (FSH) but not by AREG, which was reflected in the level of cumulus expansion. Although both FSH and AREG stimulated glycolysis, AREG-treated COCs had higher glucose consumption, lactate production and ratio of lactate production to glucose uptake. Autofluorescence levels in oocytes, indicative of NAD(P)H and FAD(++), were increased with combined AREG and BMP15 treatment of COCs. In contrast, these treatments did not alter autofluorescence levels when cumulus cells were removed from oocytes, even in the presence of other COCs, suggesting that oocyte-cumulus gap-junctional communication (GJC) is required. FSH contributed to maintaining GJC for an extended period of time. Remarkably, BMP15 was equally effective at maintaining GJC even in the presence of AREG. Hence, AREG stimulation of COC glycolysis and BMP15 preservation of GJC may facilitate efficient transfer of metabolites from cumulus cells to the oocyte thereby enhancing oocyte developmental competence. These results have implications for improving in vitro oocyte maturation systems. PMID:24557840

  17. 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. PMID:25084396

  18. 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…

  19. Effect of trans-10 cis-12 conjugated linoleic acid on bovine oocyte competence and fatty acid composition.

    PubMed

    Lapa, M; Marques, C C; Alves, S P; Vasques, M I; Baptista, M C; Carvalhais, I; Silva Pereira, M; Horta, A E M; Bessa, R J B; Pereira, R M

    2011-10-01

    The reproductive performance of dairy cows may be improved by feeding conjugated linoleic acid (CLA) supplements during early lactation. The mechanism of action of t10,c12 CLA is not clearly known. Our objective was to investigate the effect of t10,c12 CLA on oocyte maturation and lipid composition of cumulus oocyte complexes (COC). The developmental potential of oocytes incubated in in vitro maturation (IVM) medium supplemented with t10,c12 CLA to the blastocyst stage and embryo quality were also assessed. In experiment 1, abattoir-derived oocytes were matured in TCM199 + 10% serum supplemented with 100 μM t10,c12 CLA (t10,c12 CLA n = 672) or without it (control n = 672). Mature oocytes were either stained for chromatin configuration or inseminated and cultured for embryo development assessment. In experiment 2, COC and IVM culture media were subjected to fatty acid (FA) analysis prior and after maturation with t10,c12 CLA or without it (control). Total lipids and FA profiles in oocytes, cumulus cells and culture media were determined by gas chromatography. t10,c12 CLA supplementation to IVM medium improved (p = 0.05) embryo quality evaluated morphologically. This effect was associated with t10,c12 CLA presence (3.1 ± 0.7%, p = 0.04) and lower levels of arachidonic acid in FA profile of t10,c12 CLA mature oocytes (immature oocytes = 4.4 ± 1.9%, t10,c12 CLA mature oocytes = 1.0 ± 0.7%, p = 0.05). Differences in myristic and eicotrienoic acids, saturated and unsaturated FA concentrations between oocytes and cumulus cells were detected (p ≤ 0.05). In conclusion, the presence of t10,c12 CLA during maturation interfered on lipid metabolism improving bovine oocyte competence to develop into higher quality embryos. PMID:21366717

  20. Ovum pick up in swine: the influence of aspiration vacuum pressure on oocyte recovery from preovulatory follicles.

    PubMed

    Brüssow, K P; Torner, H; Rátky, J; Hunter, M G; Nürnberg, G

    1997-01-01

    Endoscopical ovum pick up (OPU) in swine is a minimal invasive and reliable technique to aspirate oocytes from preovulatory follicles for studying intrafollicular development and oocyte maturation as well as for IVM/IVF-programs. The aim of the present study was to determine the effect of different aspiration vacuum pressures on oocyte recovery and on the morphology of cumulus oocyte complexes (COCs). Oestrus of crossbred Landrace gilts (n = 33) was synchronized by feeding altrenogest and follicular growth was stimulated with 1,000 IU PMSG 24 h after the last altrenogest application. On day 4 after PMSG application preovulatory healthy follicles of > 5 mm diameter were aspirated laparoscopically. Aspiration was carried out using a two-way cannula and an electronic aspiration pump. Five different vacuum pressures were used: 10, 17, 32, 47 and 66 ml water/min, corresponding to 30, 60, 125, 250 and 375 mm Hg, respectively. Fluids from different follicles were pooled per ovary and the morphology of cumulus oocyte complexes (COCs) was determined microscopically immediately after aspiration. COCs were classified into oocytes with intact cumulus (i.e. compact or slightly expanded COC), oocytes with corona radiata and denuded oocytes. A total of 695 follicles were aspirated and 501 COCs recovered. Increasing the aspiration pressure stepwise from 10 ml water/min up to 66 ml water/min resulted in a decrease in oocyte recovery rate. A significant higher (P < 0.05) rate of oocyte recovery (77.4% v.s. 59.8%) was achieved using a vacuum pressure of 17 ml water/min compared to 66 ml water/min, respectively. There was a tendency to reduce the portion of COCs with intact cumulus from 82 to 88% to 77% if the vacuum pressure increased to more than 47 ml water/min. A higher aspiration pressure provoked an increase (P < 0.05) in the number of denuded oocytes: 0 to 3% at 10 to 32 ml water/min, respectively, compared to 10% at 47 ml water/min to 17% at 66 ml water/min. These results

  1. Effect of estrus synchronization with norgestomet on the integrity of oocytes from persistent follicles in beef cattle.

    PubMed

    Borchert, K M; Farin, C E; Washburn, S P

    1999-10-01

    Our objective was to determine whether oocyte integrity is compromised when oocytes are recovered from progestogen-induced persistent follicles. Beef cows were presynchronized using PGF2alpha (PGF). Cows detected in estrus after PGF were assigned to either NOR (one 6-mg norgestomet implant for 10 d starting on d 16 of cycle; day 0 = estrus; n = 112) or CON (control, no implant [n = 128] and presynchronized 8 d later than NOR). All cows received 25 mg of PGF at the end of treatment (NOR, d 26; CON, d 18). Treatments produced persistent preovulatory follicles (NOR) or normal preovulatory-size follicles (CON), which were measured via ultrasonography 1 d before slaughter. Ovaries were collected from all animals (NOR, d 27; CON, d 19) along with random (RAN) ovaries from cattle slaughtered on the same days. Cumulus oocyte complexes (COC) were aspirated from the preovulatory follicles with recovery rates of 63% across treatments. Small follicles (2 to 7 mm diameter) from NOR, CON, and RAN cows were also aspirated to recover COC. Preovulatory follicles were larger (19.5+/-.9 vs. 13.6+/-.4 mm, P<.05), serum P4 was lower (.4+/-.1 vs. 3.9+/-.2 ng/mL, P<.05), and serum E2 was higher (28.7+/-1.6 vs. 7.6+/-.8 pg/mL, P<.05) in NOR than in CON cows. Cumulus oocyte complexes recovered from preovulatory follicles (62 NOR, 64 CON) were matured, fertilized, and cultured in vitro for comparison of embryonic development. A subset (24 NOR, 34 CON) of COC were assigned morphological quality grades. A separate set of recovered COC (10 NOR, 15 CON) was fixed within 1 h after recovery for assessment of the stage of meiosis. Treatments did not differ for oocyte quality grade or stage of meiosis. However, COC from NOR cows had more layers of cumulus cells (P<.05), and more of those COC had undergone cumulus expansion (29.2 vs. 5.9%, P<.05 for NOR vs. CON, respectively). Development of cleaved embryos to the morula and blastocyst stages from preovulatory follicles (22.6% NOR, 18.9% CON) or

  2. 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. PMID:8914080

  3. 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.

  4. Cerebellar mature teratoma in adulthood.

    PubMed

    Zavanone, M; Alimehmeti, R; Campanella, R; Ram-Pini, P; Locatelli, M; Egidi, M; Righini, A; Bauer, D

    2002-03-01

    Mature teratoma of the posterior cranial fossa in adults is extremely rare. We report a particularly rare case of medio-lateral cerebellar mature teratoma that became symptomatic in a middle-aged man. The CT revealed the lesion of heterogeneous density with calcifications in the solid medial portion. Only the MRI could reliably define the borders of the cystic component extending into the left cerebellar lobe. Histologically the presence of fully matured representative tissues of the 3 germ layers ensured the diagnosis of mature teratoma. We suggest that the cyst formation from progressive latent hemorrhage and/or secretion from the gland cells of the tumor, may be responsible for the clinical decompensation even in adulthood. PMID:12118223

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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...

  14. Disparate maturation adaptations to size-dependent mortality

    PubMed Central

    Gårdmark, Anna; Dieckmann, Ulf

    2006-01-01

    Body size is an important determinant of resource use, fecundity and mortality risk. Evolution of maturation size in response to size-dependent selection is thus a fundamental part of life-history theory. Increased mortality among small individuals has previously been predicted to cause larger maturation size, whereas increased mortality among large individuals is expected to have the opposite effect. Here we use a continuously size-structured model to demonstrate that, contrary to these widespread expectations, increased mortality among small individuals can have three alternative effects: maturation size may increase, decrease or become evolutionarily bistable. We show that such complex responses must be reckoned with whenever mortality is size-dependent, growth is indeterminate, reproduction impairs growth and fecundity increases with size. Predicting adaptive responses to altered size-dependent mortality is thus inherently difficult, since, as demonstrated here, such mortality cannot only reverse the direction of adaptation, but also cause abrupt shifts in evolutionarily stable maturation sizes. PMID:16901838

  15. 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. PMID:27441263

  16. 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.

  17. AMPK: a master energy regulator for gonadal function.

    PubMed

    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

  18. Cats cloned from fetal and adult somatic cells by nuclear transfer.

    PubMed

    Yin, X J; Lee, H S; Lee, Y H; Seo, Y I; Jeon, S J; Choi, E G; Cho, S J; Cho, S G; Min, W; Kang, S K; Hwang, W S; Kong, I K

    2005-02-01

    This work was undertaken in order to study the developmental competence of nuclear transfer (NT) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning. PMID:15695619

  19. Comparison of hCG triggering versus hCG in combination with a GnRH agonist: a prospective randomized controlled trial

    PubMed Central

    Decleer, W.; Osmanagaoglu, K.; Seynhave, B.; Kolibianakis, S.; Tarlatzis, B.; Devroey, P.

    2014-01-01

    A prospective randomized controlled trial comparing two groups of ICSI (intra-cytoplasmatic sperm injection) patients with a different form of triggering the final oocyte maturation has been performed. All patients received an ovarian stimulation for in vitro fertilisation (IVF) using an antagonist protocol using recombinant-FSH (rec-FSH) and Ganirelix. 120 Patients were randomized into two groups with similar clinical parameters. The first group had triggering with hCG, whereas the second group received a combination of hCG + GnRH agonist (Gonadotropin Releasing Hormone). As the primary endpoint, the number of metaphase II oocytes were analysed, the secondary endpoints were the number of cumulus oocyte complexes (COC), the number of fertilized oocytes, embryo morphology, pregnancy rate and the number of cryopreserved embryos. The mean number of MII oocytes in the hCG triggered group was 9.2 compared with 10.3 in the hCG-GnRH agonist group. There was no statistically significant difference in the number of COCs or pregnancy rates. However, the number of patients who received at least one embryo of excellent quality was significantly higher (p = 0.001) in the group with the combined triggering (45 out of 61 patients or 73.8%) versus the group with hCG triggering alone (28 out of 59 patients or 47.5%). The number of cryopreserved embryos was also higher in this group. PMID:25593695

  20. Advanced studies on ovary physiology in China in the past 30 years.

    PubMed

    Liu, Yi-Xun

    2016-08-25

    Follicular development and differentiation are sequential events which are tightly regulated by endocrine hormones, intraovarian regulators and cell-cell interactions. Balanced cell proliferation and apoptosis play an important role in the selection of dominant follicle. Primordial germ cell migration and homing within the gonadal ridge requires regulation by integrated signals, such as the oocyte-secreted polypeptide growth factors, the growth and differentiation factor 9, the bone morphogenetic proteins, stem cell factor (SCF), basic fibroblast growth factor (bFGF), the transcription factor Wilms' tumour 1 (Wt1), and involves the contact of primordial germ cells with extra-cellular matrix proteins and cellular substrates and attraction by the developing gonads. Maturation of cumulus-oocyte complexes and ovulation are directly controlled by luteinizing hormone (LH) and require activation of mitogen-activated protein kinase in granulosa cells. In this review, the key molecules involved in the cell-cell interaction and signal transduction during follicular development, differentiation and ovulation will be summarized, mainly focusing on the signaling factors produced by oocyte and the somatic cells. PMID:27546498

  1. Reactive oxygen species in bovine embryo in vitro production.

    PubMed

    Dalvit, G C; Cetica, P D; Pintos, L N; Beconi, M T

    2005-08-01

    Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39 degrees C in 5% CO2: 95% humidified air. In vitro fertilization was carried out in IVF-mSOF with frozen-thawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90% N2: 5% CO2: 5% O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2',7'-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P < 0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time. PMID:16187501

  2. 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. PMID:23226944

  3. Expression and Cellular Distribution of INHA and INHB before and after In Vitro Cultivation of Porcine Oocytes Isolated from Follicles of Different Size

    PubMed Central

    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. PMID:23226944

  4. Cumulus cell gene expression associated with pre-ovulatory acquisition of developmental competence in bovine oocytes.

    PubMed

    Bunel, A; Nivet, A L; Blondin, P; Vigneault, C; Richard, F J; Sirard, M A

    2014-01-01

    The final days before ovulation impact significantly on follicular function and oocyte quality. This study investigated the cumulus cell (CC) transcriptomic changes during the oocyte developmental competence acquisition period. Six dairy cows were used for 24 oocyte collections and received FSH twice daily over 3 days, followed by FSH withdrawal for 20, 44, 68 and 92 h in four different oestrous cycles for each of the six cows. Half of the cumulus-oocyte complexes were subjected to in vitro maturation, fertilisation and culture to assess blastocyst rate. The other half of the CC underwent microarray analysis (n=3 cows, 12 oocyte collections) and qRT-PCR (n=3 other cows, 12 oocyte collections). According to blastocyst rates, 20 h of FSH withdrawal led to under-differentiated follicles (49%), 44 and 68 h to the most competent follicles (71% and 61%) and 92 h to over-differentiated ones (51%). Ten genes, from the gene lists corresponding to the three different follicular states, were subjected to qRT-PCR. Interestingly, CYP11A1 and NSDHL gene expression profiles reflected the blastocyst rate. However most genes were associated with the over-differentiated status: GATM, MAN1A1, VNN1 and NRP1. The early period of FSH withdrawal has a minimal effect on cumulus gene expression, whereas the longest period has a very significant one and indicates the beginning of the atresia process. PMID:23827322

  5. Amino acid and fatty acid composition of follicular fluid as predictors of in-vitro embryo development.

    PubMed

    Sinclair, K D; Lunn, L A; Kwong, W Y; Wonnacott, K; Linforth, R S T; Craigon, J

    2008-06-01

    The value of using the amino acid and fatty acid composition of follicular fluid as predictors of embryo development was assessed in a bovine model of in-vitro maturation (IVM), IVF and blastocyst culture (IVC). A total of 445 cumulus-oocyte complexes (COC) aspirated from visually healthy follicles underwent IVM and IVF singly (n = 138) or in groups (n = 307). Of these COC, 349 cleaved (78%) following IVF and 112 went on to form blastocysts (32% of cleaved) following IVC. Culture method (singly or in groups) had no effect on development. In contrast to fatty acids, which had no predictive value, the amino acid composition of follicular fluid was associated with morphological assessments of COC quality and with post-fertilization development to the blastocyst stage. Principal component analysis identified two amino acids (i.e. alanine and glycine) that had the highest value for predicting early post-fertilization development. The predictive value of these two amino acids, in terms of the percentage of oocytes that cleaved following IVF, was greatest for COC with the poorest morphological grades but, with respect to blastocyst yields, was independent of morphological grade, and so may serve as a useful additional non-invasive measure of COC quality. PMID:18549697

  6. Lipopolysaccharide initiates inflammation in bovine granulosa cells via the TLR4 pathway and perturbs oocyte meiotic progression in vitro

    PubMed Central

    Bromfield, John J.; Sheldon, I. Martin

    2012-01-01

    Infections of the reproductive tract or mammary gland with Gram-negative bacteria perturb ovarian function, follicular growth and fecundity in cattle. We hypothesised that lipopolysaccharide (LPS) from Gram-negative bacteria stimulates an inflammatory response by ovarian granulosa cells that is mediated by TLR4. The present study tested the capacity of bovine ovarian granulosa cells to initiate an inflammatory response to pathogen associated molecular patterns (PAMPs), and determined subsequent effects on the in vitro maturation of oocytes. Granulosa cells elicited an inflammatory response to PAMPs (LPS, lipoteichoic acid, peptidoglycan or Pam3CSK4) with accumulation of the cytokine IL-6, and the chemokine IL-8, in a time- and dose-dependent manner. Granulosa cells responded acutely to LPS with rapid phosphorylation of TLR signaling components, p38 and ERK, and increased expression of IL6 and IL8 mRNA, although nuclear translocation of p65 was not evident. Targeting TLR4 with siRNA, attenuated granulosa cell accumulation of IL-6 in response to LPS. Endocrine function of granulosa cells is regulated by FSH, but here FSH also enhanced responsiveness to LPS, increasing IL-6 and IL-8 accumulation. Furthermore, LPS stimulated IL-6 secretion and expansion by cumulus-oocyte complexes (COCs), and increased rates of meiotic arrest and germinal vesicle breakdown failure. In conclusion, bovine granulosa cells initiate an innate immune response to LPS via the TLR4 pathway leading to inflammation and to perturbation of meiotic competence. PMID:21990308

  7. 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

  8. Combination effects of epidermal growth factor and glial cell line-derived neurotrophic factor on the in vitro developmental potential of porcine oocytes.

    PubMed

    Valleh, Mehdi Vafaye; Rasmussen, Mikkel Aabech; Hyttel, Poul

    2016-06-01

    The developmental potential of in vitro matured porcine oocytes is still lower than that of oocytes matured and fertilized in vivo. Major problems that account for the lower efficiency of in vitro production include the improper nuclear and cytoplasmic maturation of oocytes. With the aim of improving this issue, the single and combined effects of epidermal growth factor (EGF) and glial cell line-derived neurotrophic factor (GDNF) on oocyte developmental competence were investigated. Porcine cumulus-oocyte cell complexes (COCs) were matured in serum-free medium supplemented with EGF (0, 10 or 50 ng/ml) and/or GDNF (0, 10 or 50 ng/ml) for 44 h, and subsequently subjected to fertilization and cultured for 7 days in vitro. The in vitro-formed blastocysts derived from selected growth factor groups (i.e. EGF = 50 ng/ml; GDNF = 50 ng/ml; EGF = 50 ng/ml + GDNF = 50 ng/ml) were also used for mRNA expression analysis, or were subjected to Hoechst staining. The results showed that the addition of EGF and/or GDNF during oocyte maturation dose dependently enhanced oocyte developmental competence. Compared with the embryos obtained from control or single growth factor-treated oocytes, treatment with the combination of EGF and GDNF was shown to significantly improve oocyte competence in terms of blastocyst formation, blastocyst cell number and blastocyst hatching rate (P < 0.05), and also simultaneously induced the expression of BCL-xL and TERT and suppressed the expression of caspase-3 in resulting blastocysts (P < 0.05). These results suggest that both GDNF and EGF may play an important role in the regulation of porcine in vitro oocyte maturation and the combination of these growth factors could promote oocyte competency and blastocyst quality. PMID:26350562

  9. Paraoxonase 1 and HDL maturation.

    PubMed

    Gugliucci, Alejandro; Menini, Teresita

    2015-01-15

    Understanding the kinetics and function of paraoxonase 1 (PON1) is becoming an important issue in atherosclerosis. Low PON1 activity has been consistently linked with an increased risk of major cardiovascular events in the setting of secondary prevention of coronary artery disease. Recent studies have shown that there is a specific interaction of myeloperoxidase (MPO)-apoAI-PON1 on HDL surface that seems to be germane to atherogenesis. MPO specifically inhibits PON1 and PON1 mitigates MPO effects. Surprisingly, very little is known about the routes by which PON1 gets integrated into HDL or its fate during HDL remodeling in the intravascular space. We have developed a method that assesses PON1 activity in the individual HDL subclasses with the aid of which we have shown that PON1 is present across the HDL particle range and preferentially in HDL3, confirming data from ultracentrifugation (UC) studies. Upon HDL maturation ex vivo PON1 is activated and it shows a flux to both smaller and larger HDL particles as well as to VLDL and sdLDL. At the same time apoE, AI and AII are shifted across particle sizes. PON1 activation and flux across HDL particles are blocked by CETP and LCAT inhibitors. In a group of particles with such a complex biology as HDL, knowledge of the interaction between apo-lipoproteins, lipids and enzymes is key for an increased understanding of the yet multiple unknown features of its function. Solving the HDL paradox will necessitate the development of techniques to explore HDL function that are practical and well adapted to clinical studies and eventually become useful in patient monitoring. The confluence of proteomic, functional studies, HDL subclasses, PON1 assays and zymogram will yield data to draw a more elaborate and comprehensive picture of the function of HDL. It must be noted that all these studies are static and conducted in the fasting state. The crucial phase will be achieved when human kinetic studies (both in the fasting and post

  10. Cheese maturity assessment using ultrasonics.

    PubMed

    Benedito, J; Carcel, J; Clemente, G; Mulet, A

    2000-02-01

    The relationship between Mahon cheese maturity and ultrasonic velocity was examined. Moisture and textural properties were used as maturity indicators. The ultrasonic velocity of the cheese varied between 1630 and 1740 m/s, increasing with the curing time mainly because of loss of water, which also produced an increase of the textural properties. Because of the nature of low-intensity ultrasonics, velocity was better related to those textural parameters that involved small displacements. Ultrasonic velocity decreased with increasing temperature because of the negative temperature coefficient of the ultrasonic velocity of fat and the melting of fat. These results highlight the potential use of ultrasonic velocity measurements to rapidly and nondestructively assess cheese maturity. PMID:10714857

  11. 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…

  12. 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…

  13. 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…

  14. 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)

  15. Chyloabdomen in a mature cat.

    PubMed Central

    Nelson, K L

    2001-01-01

    A mature, castrated male cat presented with progressive lethargy and a severely distended abdomen. Abdominal radiographs, abdominocentesis, and evaluation of the fluid obtained led to a diagnosis of chyloabdomen. The underlying pathology, etiology, diagnosis, and treatment associated with this disease are discussed. PMID:11360862

  16. Mature Cystic Teratoma of Liver

    PubMed Central

    Gupta, Richa; Bansal, Kalpana; Manchanda, Vivek

    2013-01-01

    A four-year-old boy presented with constipation and mild abdominal distention for one year. Radiologic investigations showed a multiloculated cystic lesion in the caudate lobe of liver with focal calcification in the wall. The child underwent laparotomy with marsupialization of the cystic lesion. Histopathologic examination showed mature teratoma of liver. PMID:24040591

  17. 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. PMID:26280670

  18. Gene expression in bovine oocytes and cumulus cells after meiotic inhibition with the cyclin-dependent kinase inhibitor butyrolactone I.

    PubMed

    Leal, C L V; Mamo, S; Fair, T; Lonergan, P

    2012-08-01

    The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulus-oocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 μm BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation. PMID:22034924

  19. 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

  20. Thermal Maturation of Gas Shale Systems

    NASA Astrophysics Data System (ADS)

    Bernard, Sylvain; Horsfield, Brian

    2014-05-01

    Shale gas systems serve as sources, reservoirs, and seals for unconventional natural gas accumulations. These reservoirs bring numerous challenges to geologists and petroleum engineers in reservoir characterization, most notably because of their heterogeneous character due to depositional and diagenetic processes but also because of their constituent rocks' fine-grained nature and small pore size -- much smaller than in conventional sandstone and carbonate reservoirs. Significant advances have recently been achieved in unraveling the gaseous hydrocarbon generation and retention processes that occur within these complex systems. In addition, cutting-edge characterization technologies have allowed precise documentation of the spatial variability in chemistry and structure of thermally mature organic-rich shales at the submicrometer scale, revealing the presence of geochemical heterogeneities within overmature gas shale samples and, notably, the presence of nanoporous pyrobitumen. Such research advances will undoubtedly lead to improved performance, producibility, and modeling of such strategic resources at the reservoir scale.

  1. 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

  2. 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.

  3. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    PubMed

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  4. Rest represses maturation within migrating facial branchiomotor neurons

    PubMed Central

    Love, Crystal E.; Prince, Victoria E.

    2015-01-01

    The vertebrate brain arises from the complex organization of millions of neurons. Neurogenesis encompasses not only cell fate specification from neural stem cells, but also the terminal molecular and morphological maturation of neurons at correct positions within the brain. RE1-silencing transcription factor (Rest) is expressed in non-neural tissues and neuronal progenitors where it inhibits the terminal maturation of neurons by repressing hundreds of neuron-specific genes. Here we show that Rest repression of maturation is intimately linked with the migratory capability of zebrafish facial branchiomotor neurons (FBMNs), which undergo a characteristic tangential migration from hindbrain rhombomere (r) 4 to r6/r7 during development. We establish that FBMN migration is increasingly disrupted as Rest is depleted in zebrafish rest mutant embryos, such that around two-thirds of FBMNs fail to complete migration in mutants depleted of both maternal and zygotic Rest. Although Rest is broadly expressed, we show that de-repression or activation of Rest target genes only within FBMNs is sufficient to disrupt their migration. We demonstrate that this migration defect is due to precocious maturation of FBMNs, based on both morphological and molecular criteria. We further show that the Rest target gene and alternative splicing factor srrm4 is a key downstream regulator of maturation; Srrm4 knockdown partially restores the ability of FBMNs to migrate in rest mutants while preventing their precocious morphological maturation. Rest must localize to the nucleus to repress its targets, and its subcellular localization is highly regulated: we show that targeting Rest specifically to FBMN nuclei rescues FBMN migration in Rest-deficient embryos. We conclude that Rest functions in FBMN nuclei to inhibit maturation until the neurons complete their migration. PMID:25769695

  5. 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...

  6. Young Children's Social Maturity and Their Drawings

    ERIC Educational Resources Information Center

    Hendrickson, Norejane J.; And Others

    1974-01-01

    The relationship between preschool children's level of social maturity and their drawing expressions was tested; findings stregthened the contention that the drawings of preschoolers are not appropriate indicators of their social maturity. (SDH)

  7. Cloacal Exstrophy with Mature Teratoma: A Rare Association in a Neonate.

    PubMed

    Patil, Prashant Sadashiv; Kothari, Paras; Gupta, Abhaya; Gupta, Rahul; Kekre, Geeta; Dikshit, Vishesh; Kamble, Ravi

    2016-01-01

    Cloacal exstrophy is a very rare and complex malformation. We report a neonate of cloacal exstrophy with mature teratoma presenting as a component of exstrophy. To our knowledge this has not been reported in the literature. PMID:27123401

  8. Cloacal Exstrophy with Mature Teratoma: A Rare Association in a Neonate

    PubMed Central

    Patil, Prashant Sadashiv; Kothari, Paras; Gupta, Abhaya; Gupta, Rahul; kekre, Geeta; Dikshit, Vishesh; Kamble, Ravi

    2016-01-01

    Cloacal exstrophy is a very rare and complex malformation. We report a neonate of cloacal exstrophy with mature teratoma presenting as a component of exstrophy. To our knowledge this has not been reported in the literature. PMID:27123401

  9. Increased NK Cell Maturation in Patients with Acute Myeloid Leukemia

    PubMed Central

    Chretien, Anne-Sophie; Granjeaud, Samuel; Gondois-Rey, Françoise; Harbi, Samia; Orlanducci, Florence; Blaise, Didier; Vey, Norbert; Arnoulet, Christine; Fauriat, Cyril; Olive, Daniel

    2015-01-01

    Understanding immune alterations in cancer patients is a major challenge and requires precise phenotypic study of immune subsets. Improvement of knowledge regarding the biology of natural killer (NK) cells and technical advances leads to the generation of high dimensional dataset. High dimensional flow cytometry requires tools adapted to complex dataset analyses. This study presents an example of NK cell maturation analysis in Healthy Volunteers (HV) and patients with Acute Myeloid Leukemia (AML) with an automated procedure using the FLOCK algorithm. This procedure enabled to automatically identify NK cell subsets according to maturation profiles, with 2D mapping of a four-dimensional dataset. Differences were highlighted in AML patients compared to HV, with an overall increase of NK maturation. Among patients, a strong heterogeneity in NK cell maturation defined three distinct profiles. Overall, automatic gating with FLOCK algorithm is a recent procedure, which enables fast and reliable identification of cell populations from high-dimensional cytometry data. Such tools are necessary for immune subset characterization and standardization of data analyses. This tool is adapted to new immune cell subsets discovery, and may lead to a better knowledge of NK cell defects in cancer patients. Overall, 2D mapping of NK maturation profiles enabled fast and reliable identification of NK cell subsets. PMID:26594214

  10. Oocyte Maturation: The Coming of Age of a Germ Cell

    PubMed Central

    Jamnongjit, Michelle; Hammes, Stephen R.

    2006-01-01

    Normal female fertility relies on proper development of the oocyte. This growth culminates just prior to ovulation, when oocyte maturation occurs. Oocyte maturation refers to a release of meiotic arrest that allows oocytes to advance from prophase I to metaphase II of meiosis. This precisely regulated meiotic progression is essential for normal ovulation and subsequent fertilization, and involves changes in the delicate balance between factors promoting meiotic arrest and others that are stimulating maturation. Most of the inhibitory mechanisms appear to involve the upregulation of intracellular cyclic adenosine monophosphate levels. These processes may include direct transport of the nucleotide into oocytes via gap junctions, G protein-mediated stimulation of adenylyl cyclase, and inhibition of intracellular phosphodiesterases. In contrast, potential factors that play roles in triggering oocyte maturation include gonadotropins (e.g., follicle-stimulating factor and luteinizing hormone), growth factors (e.g., amphiregulin and epiregulin), sterols (e.g., follicular fluid-derived meiosis-activating sterol), and steroids (e.g., testosterone progesterone, and estradiol). Delineating the complex interactions between these positive and negative components is critical for determining the role that oocyte maturation plays in regulating follicle development and ovulation, and may lead to novel methods that can be used to modulate these processes in women with both normal and aberrant fertility. PMID:16059829